CORRECTION

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Direct measurement of papillary collecting duct sodium transport in the rat. Evidence for heterogeneity of nephron function during Ringer loading.

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It has been suggested that collecting duct sodium transport was inhibited by extracellular volume expansion. To directly evaluate this possibility, micropuncture of the papillary collecting duct of young rats was performed during hydropenia and Ringer loading. The possibility of heterogeneity of nephron function was evaluated during Ringer and hyperoncotic albumin loading by comparing the delivery of sodium to the end of the distal tubule of superficial nephrons with papillary base delivery. During hydropenia (n = 14), sodium delivery to the base averaged 0.95% of the filtered sodium load and reabsorption along the collecting duct was noted from base to tip in each collection pair averaging 0.80% of the filtered load. During Ringer loading, sodium delivery to the base was markedly greater than in hydropenia, 11.8 vs. 0.95% of the filtered load (P less than 0.001). Yet, sodium reabsorption was also much greater, 6 vs. 0.8% (P less than 0.001). In 13 paired collections, during Ringer loading, sodium delivery to the papillary base, 12.2% of the filtered load, was consistently greater than late distal tubular delivery from superficial nephrons. 8% (P less than 0.005). In contrast, reabsorption of sodium from late distal tubule to papillary base was found during albumin infusion, 6.2 vs. 3.1% (P less than 0.001). Therefore, these studies demonstrate that: (a) the delivery of sodium to and reabsorption along the papillary collecting duct were markedly greater during Ringer loading than in hydropenia; (b) the amount of sodium delivered to the papillary base was greater than the delivery to the end of the distal tubule of superficial nephrons during Ringer loading, suggesting that deeper nephrons deliver more sodium to the collecting duct in this setting; and (c) the difference in sodium excretion between Ringer loading and hyperoncotic albumin infusion is due to events occurring between the late distal tubule of superficial nephrons and the base of the papillary collecting duct.

Authors

J H Stein, R W Osgood, R T Kunau Jr

Further characterization of NADPH oxidase activity of human polymorphonuclear leukocytes.

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Mn2+ was shown to catalyze a nonenzymatic oxidation of NADPH in the presence of superoxide anion by means of an isotopic assay for measurement of the oxidation of NADPH to NADP+. Human polymorphonuclear leukocyte granule NADPH oxidase activity was evaluated in the absence of Mn2+ and was found to be higher in granules from phagocytizing cells than in granules from resting cells. The drug phorbol myristate acetate, which affects the oxidative metabolism of the neutrophil like phagocytosis, was found to activate granule NADPH oxidase activity. Superoxide dismutase was shown to inhibit NADPH oxidase activity both in the presence and absence of added Mn2+. The NADPH oxidase reaction in the absence of Mn2+ was optimal at pH 5.5, and was more linear with increasing time and protein concentration than in the presence of Mn2+. No activity was measurable in granules isolated from resting cells until the level of NADPH added was above 0.25 mM. Activity was present in granules isolated from cells challenged with opsonized zymosan, even at 0.05 mM NADPH, and was higher than the activity found in granule fractions from resting cells at all levels of NADPH tested. The addition of as little as 0.1 muM NADH to the reaction mixture was found to inhibit granular NADPH oxidase activity, indicating a possible regulatory role for NADH. These results suggest that NADPH oxidase may be the enzyme that initiates the metabolic events accompanying phagocytosis.

Authors

L C McPhail, L R DeChatelet, P S Shirley

Selective uptake of intact parathyroid hormone by the liver: differences between hepatic and renal uptake.

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Hepatic and renal extraction of immunoreactive parathyroid hormone (i-PTH) was studied in awake dogs with explanted kidneys and chronic indwelling hepatic vein catheters. After a single injection of bovine PTH 1-84 (b-PTH 1-84), hepatic arteriovenous (A-V) differences for immunoreactive PTH (i-PTH) was 39% at 2 min after injection but decreased to 0% by 25 min, despite high levels of i-PTH in the arterial circulation. Gel filtration of arterila and hepatic venous samples obtained when hepatic A-V differences for i-PTH were demonstrable revealed hepatic uptake of the intact hormone and addition of a smaller COOH-terminal fragment, eluting just after the intact hormone, to the hepatic venous blood. Gel filtration of samples obtained 20-30 min after injection of b-PTH was demonstrable) revealed no detectable intact hormone in the circulation. Levels of COOH-terminal fragments of the hormone at the time were identical in arterial and hepatic venous samples. In additional experiemtns no hepatic A-V difference was observed after the injection of the synthetic bovine PTH 1-34 (syn b-PTH 1-34). By comparison there was a demonstrable A-V difference of 20% across the kidney for both intact PTH and COOH-terminal fragments that persisted until i-PTH disappeared from the circulation. The kidney also demonstrated an A-V difference of 22% after injection of syn b-PTH 1-34. These studies demonstrate selective extraction of intact PTH but not of its fragments by the liver. The kidney, on the other hand, extracted the intact hormone and both COOH and NH2 terminal fragments. The studies demonstrate that the kidney was the only organ of those examined that detectably removed the fragments of PTH from the circulation.

Authors

K Martin, K Hruska, A Greenwalt, S Klahr, E Slatopolsky

The origin of the chemiluminescence of phagocytosing granulocytes.

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Granulocytes engaged in the phagocytosis of opsonized zymosan emit light by a process that is inhibited by superoxide dismutase and catalase. In the present report is is shown that light emission is the result of reactions between certain unspecified constituents of the ingested particles and some or all of the oxidizing agents (H2O2, O2),and possibly the hydroxyl radical and singlet oxygen) produced by the activated cells. This conclusion is based on a study of light emission by both activated cells ans artificial O2 generating system containing xanthine oxidase and purine. With these two systems light production required the presence of both zymosan and oxidizing agent, suggesting that the oxidation of particle components is necessary for luminescence to occur. The characteristics of the emission spectrum as well as the finding that granulocytes activated by a nonparticulate agent (F-) fail to liminesce show that light emission by the relaxation of singlet oxygen to the ground state does not contribute in a major way to the chemiluminescence of phagocytosing granulocytes; whether singlet oxygen contributes to chemiluminescence in other ways cannot be decided from the data available. Inasmuch as the oxidation of constituents of ingested particles is an important bacterial killing mechanism in the granulocyte, chemiluminescence may be viewed as a manifestation of the microbicidal activity of the cell.

Authors

B D Cheson, R L Christensen, R Sperling, B E Kohler, B M Babior

Mechanisms of methylene blue stimulation of the hexose monophosphate shunt in erythrocytes.

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The response of the hexose monophosphate shunt in erythrocytes was studied with the ionization chamber-electrometer apparatus to measure continuously 14CO2 derived from 14C-labeled substrates. The effect of methylene blue at high (0.1 mM) and low (1 muM) concentrations was evaluated under different gas mixtures; air, carbon monoxide, and 6% carbon monoxide in air. The latter gas mixture results in nearly 100% carboxyhemoglobin but provides a physiologic partial pressure of oxygen. The extent to which pentose is recycled through the shunt in response to methylene blue stimulation was examined with radioactive glucose substrates labeled on the first, second, and third carbon positions. Generation of hydrogen peroxide after stimulation of erythrocytes with methylene blue was evaluated by the catalase-aminotriazole trapping technique, [14C]formate oxidation, and oxidation of reduced glutatione. Stimulation of the shunt with 1 muM methylene blue was markedly impaired in the absence of oxyhemoglobin, but stimulation with 0.1 mM methylene blue was only slightly impaired under the carbon monoxide-air mixture. The higher concentration of methylene blue produced evidence of hydrogen peroxide generation of all three techniques. Despite the evidence for the involvement of oxygen, oxyhemoglobin, and hydrogen peroxide in the response to methylene blue, cells containing methemoglobin induced by sodium nitrite or from a patient with congenital methemoglobinemia responded normally to methylene blue in the absence of oxygen. These experiments indicate that the reactions induced by methylene blue in erythrocytes are more complex than generally thought and that high concentrations are associated with production of peroxide.

Authors

E N Metz, P Balcerzak, A L Sagone Jr

Long term effects of radiation of T and B lymphocytes in peripheral blood of patients with Hodgkin's disease.

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Total lymphocyte counts, and the percentage of T and B lymphocytes and monocytes in untreated patients with Hodgkin's disease were not significantly different from those observed in normal donors. At the completion of radiotherapy, the mean total lymphocyte count of 503/mm3 was 4 SD below the mean for normal controls. Although a group of 26 patients in continuous complete remission from 12 to 111 mo after radiation treatment regained normal total numbers of lymphocytes and monocytes, they exhibited a striking T lymphocytopenia and B lymphocytosis. Concomitantly, there was a significant increase of null (neither T nor B) lymphocytes. The response of peripheral blood lymphocytes to phytohemagglutinin, concanavalin A, and tetanus toxoid before treatment was significantly impaired. 1-10 yr after completion of treatment there seemed to be little or no recovery of these responses. The capacity of peripheral blood lymphocytes to respond to allo-antigens on foreign lymphocytes in vitro (mixed lymphocyte reaction) was normal in nine untreated patients. However, the mixed lymphocyte reaction was markedly impaired during the first 2 yr after treatment. There was a partial and progressive restoration of the mixed lymphocyte reaction during the next 3 yr, and normal responses were observed in patients in continuous complete remission for 5 yr or more. The in vivo response to dinitrochlorobenzene was also examined. 88% (15/17) of patients initially sensitive to dinitrochlorobenzene were anergic to the allergen at the completion of a course of radiotherapy, but nine of these regained their hypersensitivity response during the 1st yr after treatment. This data suggests that there is a sustained alteration in both the number and function of circulating T cells after radiation therapy in patients with Hodgkin's disease which may persist for as long as 10 yr after treatment. The restoration of cell mediated immune functions after radiotherapy is time dependent and its kinetics may differ for various T-cell functions. The implications of these findings with respect to the state of immunological competence after radiotherapy are discussed.

Authors

Z Fuks, S Strober, A M Bobrove, T Sasazuki, A McMichael, H S Kaplan

Urate crystal-induced chemotactic factor: isolation and partial characterization.

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A factor with chemotactic properties for neutrophils and mononuclear cells was extracted from the lysosomal fraction of both human and rabbit neutrophils that had been allowed to phagocytose monosodium urate crystals. The chemotactic factor was found to be a glycoprotein with a mol wt of 8,400 daltons. The factor is heat labile and has chemotactic activity for human as well as rabbit cells. Preincubation of the cells with the urate induced chemotactic factor or with complement activated plasma prevents the cell from migrating chemotactically when challenged with either factor in the chemotactic chamber. The chemotactic factor induces release of lysosomal enzymes for cytochalasin B treated human neutrophils.

Authors

I Spilbert, A Gallacher, J M Mehta, B Mandell

Red cell age-related changes of hemoglobins AIa+b and AIc in normal and diabetic subjects.

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The minor hemoglobin components, hemoglobin AIa+b and hemoglobin AIc, were measured in the 10% youngest and 10% oldest erythrocytes of 15 normal and 14 diabetic subjects. Erythrocyte fractions were obtained by centrifugation in isopyknic concentrations of dextran: 28.5% of 40,000-mol wt dextran yeilded the 10% lightest of young cells, and 30.5% dextran provided the 10% heaviest or old erythrocytes. Both normal and diabetic erythrocytes contain increased amounts of Hb AIa+b and Hb AIc in old as compared to young cells. In normal subjects, young cells contained 1.2+/-0.2%, and old cells contained 1.8+/-0.4% Hb AIa+b. Corresponding values for diabetic cells were 1.7+/-0.6 and 2.6+/-0.9%. Hb AIc increased from 3.1+/-0.8 to 6.0+/-1.1% in normals and from 5.1+/-2.1 to 10.1+/-3.7% in diabetics. The results indicate that both cell age and diabetes are significant determinants of the amounts of Hb AIa+b and Hb AIc.

Authors

J F Fitzgibbons, R D Koler, R T Jones

Idiopathic scoliosis. Gas exchange and the age dependence of arterial blood gases.

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The aims were to examine the gas exchange and arterial blood gas abnormalities among patients with scoliosis, and the correlation of these abnormalities with age and severity of deformity. Means among 51 patients were as follows: age 25.4 +/- 17.5 yr, angle of scoliosis 80.2 +/- 29.9 (SD), vital capacity 1.94 +/- 0.91 (SD) (i.e. 60.6 +/- 19.2% of predicted), PaO2 85.8 +/- 12.0 (SD), PaCO2 42.4 +/- 8.0, physiological dead space to tidal volume ratio 0.438 +/- 0.074 (SD), and alveolar-arterial oxygen difference breathing air 14.9 +/- 8.9 (SD). Statistically significant correlations were as follows: the PaCO2 and physiological dead space to tidal volume ratio increased with age, and the PaO2 and alveolar ventilation decreased with age. The PaO2, alveolar ventilation, and tidal volume were inversely related to the angle of scoliosis and directly related to the vital capacity, precent predicted vital capacity, and the compliance of the respiratory system. The physiological dead space to tidal volume ratio and the alveolar-arterial oxygen difference were inversely related to the vital capacity, percent predicted vital capacity, and the compliance of the respiratory system. PaCO2 was directly related to the elastance of the respiratory system. We conclude that ventilation-blood flow maldistribution as a result of deformity of the rib cage was the primary abnormality in gas exchange, and that with age there was progressive deterioration in gas exchange. The age-dependent increase in PaCO2 and decrease in alveolar ventilation were due to the increasing physiological dead space to tidal volume ratio and failure of a compensatory increase in ventilation.

Authors

E R Kafer

Regulation of angiotensin II receptors in the rat adrenal cortex by dietary electrolytes.

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The binding affinity and concentration of specific angiotensin II receptor sites of rat adrenal cortical cells and homogenates were determined after 1 and 6 wk of altered sodium and potassium intake. Sodium deprivation caused marked increases in plasma renin, blood angiotensin II, and plasma aldosterone, and was accompanied by a significant increase (+74%) in the number of specific angiotensin II receptor sites per adrenal cortical cell. High potassium intake was followed by increased serum potassium and markedly elevated plasma aldosterone, with subnormal levels of renin and angiotensin II and a 170% increase in the number of angiotensin II receptors per cell after 1 wk. Sodium loading and potassium deprivation were followed by the opposite effect upon adrenal receptors, with reduction of the angiotensin II-binding capacity. None of the dietary electrolyte changes were accompanied by an ancrease in receptor affinity above the control value of 2 nM-1. A decrease in receptor affinity was noted after 6 wk of either low sodium or low potassium intake, when the renin and angiotensin II levels were increased by 104-129%. The adrenals of normal rats infused acutely with synthetic angiotensin II, or anesthetized with ether or sodium pentobarbital, which markedly increased plasma renin activity, contained fewer angiotensin receptors. These reductions in binding site concentration were not accompanied by changes in affinity and were attributed to occupancy by angiotensin II. These studies have demonstrated that chronic changes in sodium or potassium balance and acute changes in blood angiotensin II levels can exert modulating effects upon the adrenal content and/or affinity of specific receptor sites for angiotensin II.

Authors

J Douglas, K J Catt

Glomerular basement membrane: biosynthesis and chemical composition in the streptozotocin diabetic rat.

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To study the effect of streptozotocin induced diabetes on glomerular basement membrane (GBM) synthesis, an isolated rat glomerular preparation has been developed, and its metabolic properties have been defined. The chemical composition of normal rat GBM isolated from this preparation closely resembles human GBM. Incubation with [U-14C] lysine leads to prompt incorporation of label into GBM and the subsequent appearance of labeled hydroxylysine. A 1-h lag before detection of labeled hydroxylysine in GBM suggests a delay in the release of GBM precursors. Significantly lower counts appeared in the nondialyzable fraction of the medium than in insoluble GBM during pulse-chase experiments, and labeled hydroxylysine accounted for a lower portion of the total counts in the medium (0.85%) than in the GBM (1.98%). Isolated glomeruli were prepared from streptozotocin diabetic rats of 4-6 wks duration. After incubation with [ U-14C] lysine recovery of label in diabetic GBM (88.98+/-8.26 nmol/g GBM) did not differ from age matched controls (82.52 +/- 8.26 nmol/g GBM). In pulse-chase experiments recovery of label in hydroxylysine of diabetic GBM (o.473 +/- 0.082 nmol/g GBM) did not differ from age matched controls (0567+/-0.065 nmol/g GBM). These findings indicate normal rates of GBM synthesis and hydroxylation of lysine residues in animals with streptozotocin diabetes.

Authors

P J Beisswenger

Inherited deficiency of the second component of complement. Rheumatic disease associations.

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The prevalence of homozygous and heterozygous deficiency of the second component of complement (C2) was determined in patients with rheumatic disease including 137 with systemic lupus erythematosus (SLE), 274 with juvenile rheumatoid arthritis, and 134 with rheumatoid arthritis. 1 C2 homozygous deficient and 19 possible heterozygous deficient individuals were identified by using both immunochemical and functional assays to determine C2 levels. Of the 20, 8 had SLE (5.9%), 10 had juvenile rheumatoid arthritis (3.7%), and 2 had rheumatoid arthritis (1.4%), the homozygous deficient individual having SLE. The prevalence of C2 deficiency in the SLE and juvenile rheumatoid arthritis patients was significantly increased (P = 0.0009 and P = 0.02, respectively) when compared with controls, 6 (1.2%) of 509 blood donors having C2 levels consistent with heterozygous deficiency. 15 of the 20 C2 deficient patients were HLA typed and found to have antigens A10(Aw25), B18, or both. The patients with C2 deficiency and SLE had earlier age of onset of disease and less antinuclear antibody when compared with the C2 normal SLE patients. 11 families of the propositi were studied and found to have one or more C2 heterozygous deficient individuals. The family members had an equal distribution of rheumatic disease and antinuclear antibody in the C2 deficient and C2 normal groups. C2 deficient individuals were found to have significantly lower levels of properdin Factor B (242 mug/ml+/-54) when compared with the non-C2 deficient family members (282 mug/ml+/-73). These data support the concept that inherited deficiency of C2 is significantly associated with both SLE and juvenile rheumatoid arthritis.

Authors

D Glass, D Raum, D Gibson, J S Stillman, P H Schur

Renal prostaglandin synthesis in the spontaneously hypertensive rat.

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The precise role of the kidney in spontaneous experimental hypertension is unknown. We have analyzed the rates of renal prostaglandin synthesis by utilizing a spontaneously hypertensive rat model. The synthetic rate of prostaglandin E2, prostaglandin F2alpha, and prostaglandin A2-like products was measured in vitro with renal microsomes. In the rabbit and rat there is a steep gradient of microsomal prostaglandin synthetase from papilla to cortex with highest activities in the papilla. Comparison of the activity of prostaglandin synthetase in medullary microsomes form normotensive and hypertensive rats showed accelerated synthesis in the spontaneously hypertensive rat. These differences appeared after several months of age, were statistically significant from 3 mo of age and, on the average, represented at least a twofold increase of in vitro activity. All classes of prostaglandins were involved with increased synthesis of prostaglandin E2, prostaglandin F2alpha and prostaglandin A2-like material. These data reenforce and extend previous work showing alterations of granularity and presumably prostaglandin synthesis in renal medullary intersitital cells in various experimental hypertensions. We also measured renal tissue content of prostaglandin E and prostaglandin A-prostaglandin B by radioimmunoassay. Swift and careful handling of the tissue was necessary to avoid extensive postmortem synthesis of prostaglandins. In rapidly-frozen medullary tissue only prostaglandin E was detectable in concentrations ranging from 10 to 200 pg/mg tissue. No significant differences were found in the medullary content of prostaglandin E in the control and hypertensive rats despite the increased rates of enzymatic synthesis. We conclude that renal prostaglandin synthesis is increased in renal medullary microsomes obtained from spontaneously hypertensive rat. This apparently occurs in response to the progressive development of hypertension since young animals did not show an increase Renal tissue prostaglandin E content did not increase and therefore appears to be a poor index of enhanced prostaglandin synthesis.

Authors

M J Dunn

The effect of D-penicillamine on polymorphonuclear leukocyte function.

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D-Penicillamine, a reducing and chelating agent used in the treating of rheumatoid arthritis, was tested for its effects of polymorphonuclear leukocyte chemotaxis, phagocytosis, and lysosomal enzymes. beta-Glucuronidase release from polymorphonuclear leukocytes after phagocytosis of latex particles was not affected by D-penicillamine at concentrations ranging from 25 to 400 mg/liter. No direct effect was seen on enzyme activity at the maximum concentration of the drug. There was no inhibition of latex particle ingestion. No cell damage was found at 400 mg/liter penicillamine as measured by lactic dehydrogenase release. At this drug concentration there was only a 15% reduction in hemolytic complement levels. Chemotaxis was significantly decreased at concentrations of 50 mg/liter with a dose-dependent effect at higher concentrations which showed a plateau from 200 to 400 mg/liter. The parent compound D-cysteine was also tested in these systems. The same lack of effect of phagocytosis and enzyme release was found. D-Cysteine did inhibit chemotaxis but to a lesser degree than D-penicillamine. This dicotomy of drug effect may indicate that the beneficial action of D-penicillamine in the treatment of rheumatoid arthritis is due to the decreased chemotaxis of polymorphonuclear leukocytes into the joint, while the absence of an effect of phagocytosis and lysosomal enzymes shows the cells can still function to ingest and destroy bacteria. This latter effect correlates with the absence of infection in patients treated with this compound.

Authors

H Chwalinska-Sadowska, J Baum

Maximal oxygen consumption in patients with lung disease.

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A theoretical model for oxygen transport assuming a series linkage of ventilation, diffusion, oxygen uptake by erythrocytes, cardiac output, and oxygen release was used to calculate expected values for maximal oxygen intake (VO2max) of patients with various pulmonary disorders 22 patients with either restrictive or obstructive ventilatory impairment were studied at rest and maximal exercise. When exercise measurements of maximal pulmonary blood flow (QCmax), oxygen capacity, membrane diffusing capacity for CO, pulmonary capillary blood volume, alveolar ventilation, and mixed venous oxygen saturation were employed as input values, predictions of VO2max from the model correlated closely with measured values (r = 0.978). Measured VO2max was 976+/-389 ml/min (45.3+/-13% of predicted normal), and VO2max predicted from the model was 1,111+/-427 ml/min. The discrepancy may in part reflect uneven matching of alveolar ventilation, pulmonary capillary blood flow, and membrane diffusing capacity for CO within the lung; uniform matching is assumed in the model so that mismatching will impair gas exchange beyond our predictions. Although QCmax was less than predicted in most patients (63.6+/-19.6% of predicted) the model suggests that raising QCmax to normal could have raised VO2max only 11.6+/-8.8% in the face of existent impairment of intrapulmonary gas exchange. Since pulmonary functions measured at rest correlated well with exercise parameters needed in the model to predict VO2max we developed a nomogram for predicting VO2max from resting CO diffusing capacity, the forced one second expired volume, and the resting ratio of dead space to tidal volume. The correlation coefficient between measured and predicted VO2max, by using this nomogram, was 0.942.

Authors

K L Wehr, R L Johnson Jr

In vitro action of bombesin and bombesin-like peptides on amylase secretion, calcium efflux, and adenylate cyclase activity in the rat pancreas: a comparison with other secretagogues.

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Abstract

Bombesin (a tetradecapeptide), the C-terminal nonapeptide of bombesin (bombesin-NP), and litorin (a parent nonapeptide), each stimulated amylase secretion from rat pancreatic fragments. These responses were not affected by atropine. The concentrations that produced half-maximal stumulation of secretion were 0.25 nM for bombesin, 0.30 nM for bombesin-NP, and 0.07 nM for litorin, as compared to 0.12 nM for caerulein and 0.80 muM for the cholinergic agent carbamylcholine. When used at maximal concentrations, bombesin, bombesin-NP, and litorin showed no action on cyclic AMP levels in the presence of 5 mM theophylline. By contrast, caerulein and secretin increased cyclic AMP levels by 27 and 208%, respectively. Bombesin, bombesin-NP, and litorin did not activate adenylate cyclase in a purified pancreatic plasma membrane preparation, whereas caerulein and secretin increased this activity 20 and 16-times, respectively...

Authors

M Deschodt-Lanckman, P Robberecht, P De Neef, M Lammens, J Christophe

Acute effects of antiglomerular basement membrane antibody on the process of glomerular filtration in the rat.

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Abstract

Nehron filtration rate (sngfr) and the factors controlling filtration were examined before and with 60 min of the intravenous infusion of 225-450 mug of antiglomerular basement membrane antibody (AGBM Ab) (greater than 50% antigenic saturation) in plasma-expanded (2.5% body wt) Munich-Wistar rats. Pressures in glomerular capillaries (PG) and bowman's space (Pt) were measured with a servo-nulling device, systemic (piA) and efferent arteriolar oncotic pressures (piE) were measured by microprotein methods, and nephron plasma flow (rpf) and sngfr were measured by micropuncture techniques in both control and post-AGBM Ab conditions in each rat. The sngfr fell from 52.7+/-2.9 to 24.1+/-1.9 nl/min per g kidney wt (n = 7, P less than 0.001). Both afferent and efferent arteriolar resistances increased and rpf fell from 221+/-25 to 90+/-9 nl/min per g kidney wt (P less than 0.001) but the hydrostatic pressure gradient across the glomerular membrane deltaP = PG - Pt) increased from 37+/- 1 to 50+/-2 mm Hg (P less than 0.001). The increase in deltaP and a numerical decrease in piA both acted to maintain sngfr after AGBM Ab and effectively nullified the influence of decreased rpf upon sngfr. The mean effective filtration pressure (EFP = deltaP - pi) increased from 14+/-2 to 30+/-3 mm Hg (P less than 0.001) while sngfr decreased. The major and critical reason for this reduction in sngfr was a decrease in the glomerular permeability coefficient from 0.077+/-0.017 to 0.014+/-0.001 nl/s per g kidney wt per mm Hg P less than 0.001) where sngfr=EFP-LpA.

Authors

R C Blantz, C B Wilson

The steady state concentration gradients of an electron-dense marker (ferritin in the three-layered hemochorial placenta of the rabbit.

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Ferritin was injected into the fetal or the maternal circulation of 27-29-day-pregnant rabbits. After the occurrence of a quasi-steady state, the placentas were prepared for electron microscopy. Ferritin particles were counted in the electron micrographs in the fetal capillaries, in the maternal blood spaces, and in the two interstitial compartments of the three-layered placenta. Under the circumstances of the experiments (excessively elevated plasma ferritin concentrations), no evidence was found for nondiffusional transport of radiolabeled ferritin. Comparison of the standing concentration gradients in the placentas, recorded after maternal and after fetal injection, showed that the interstitial spaces "excluded" ferritin; the plasma-interstitial space ferritin partition coefficients were 10 in the basement membrane space and 3 in the space between the cyto- and syncytiotrophoblasts. 55% of the total concentration gradient across the rabbit placenta occurred across the fetal endothelium, about 45% across the cytotrophoblast, and less than 5% across the syncytiotrophoblast. These figures are believed to reflect the relative contributions of these three layers to the total diffusional resistance in the rabbit placenta. When compared to previous data on the relative contributions of these three layers for small ions and molecules, the present data lead to the conclusion that discrimination of molecular size is a function of the fetal capillary endothelium alone.

Authors

K L Thornburg, J J Faber

Lead poisoning: association with hemolytic anemia, basophilic stippling, erythrocyte pyrimidine 5'-nucleotidase deficiency, and intraerythrocytic accumulation of pyrimidines.

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Lead intoxication is accompanied by an acquired deficiency of erythrocyte pryimidine-specific, 5'-nucleotidase. Genetically determined deficiency of this enzyme is associated with chronic hemolysis, marked basophilic stippling of erythrocytes on stained blood films, and unique intraerythrocytic accumulations of pyrimidine-containing nucleotides. The present report documents that lead-induced deficiency when sufficiently severe gives rise to findings similar to the hereditary disorder. Whereas pyrimidine-containing nucleotides are virutally absent in the erythrocytes of normal and reticulocyte-rich blood, 12% of erythrocyte nucloetides in the blood of a patient with lead intoxication contained cytidine. Nucleotidase activity was about 25% that in normal erythrocytes and 15% or less of that expected in comparable reticulocyte-rich blood. The distribution of nucleotidase activity in patient erythrocytes is unknown, and much more severe deficiency could have been present in subsets of the cell populations analyzed. The findings indicate that the hemolytic anemia and increased basophilic stippling characteristic of certain cases of lead intoxication may share a common etiology with essentially identical features of the genetically determined disorder.

Authors

W N Valentine, D E Paglia, K Fink, G Madokoro

Rheumatoid factor-producing cells detected by direct hemolytic plaque assay.

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Abstract

Lymphocytes secreting anti-IgC antibodies, rheumatoid factors (RF), can be detected in the peripheral bloods, synovial fluids, and bone marrows of patients with seropositive rheumatoid arthritis by using a direct plaque-forming cell (PFC) assay with sheep erythrocytes sensitized with reduced and alkylated rabbit IgG hemolysin. The autospecific nature of the RF produced by RF-PFC was indicated by inhibition studies in which the order of patency was human IgG greater than rabbit IgG greater than bovine IgG. In metabolic studies puromycin, cycloheximide, and venblastine suppressed RF-PFC. Cyclic AMP and cyclic GMP were without effect. A need was recognized for using full tissue culture media during the cell separation and plaquing procedures to optimize detection of the RF-PFC. RF-PFC may appear in the blood of patients intermittently despite their continuing presence in the bone marrow. They have been found in the peripheral blood, especially during acutely exacerbating polyarticular synovitis, generalized vasculities, or generally active, aggressive disease. RF-PFC were found in synovial effusions of new or recrduescent acute synovitis. RF-PFC were observed to disappear from the peripheral circulation and the bone marrow during therapy with cytotoxic drugs. The data are consistent with the hypothesis that the appearance of RF-PFC in the peripheral blood represents an anamnestic response to transiently appearing antigen. The nature of the antigen is not specified. The bone marrow may be a site of origin of RF-PFC.

Authors

J H Vaughan, T Chihara, T L Moore, D L Robbins, K Tanimoto, J S Johnson, R McMillan

The role of complement in the clearance of cold agglutinin-sensitized erythrocytes in man.

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Abstract

To define the pathophysiologic mechanisms of cold agglutinin disease, we investigated a human model of this syndrome in normal volunteers and in patients with diminished levels of serum complement. Subjects received intravenous injections of autologous, chromated (51Cr) erythrocytes which had been exposed in vitro to purified cold agglutinin preparations and to fresh autologous serum (as a source of complement). In vitro tests confirmed that such cells were coated with activated complement components (C3b), but not with immunoglobulin. Studies of erythrocyte clearance and simultaneous organ scanning showed that erythrocytes sensitized with low levels of cold agglutinin primarily undergo reticuloendothelial sequestration by the liver rather than intravascular hemolysis. After the initial sequestration of C3b-coated erythrocytes, a fraction of the cells are released back into the circulation and survive normally thereafter. Both phenomena are dose dependent and closely follow the sequestration and release pattern observed with IgM isoagglutinin sensitization. Experiments that used heated autologous serum as a source of B3 inactivator demonstrated that functionally intact C3b is required for hepatic sequestration. Erythrocytes coated with C3d were not cleared from the circulation. In vitro assays that sued human macrophage monolayers suggested that the intrahepatic conversion of C3b to C3d is responsible for the release of sensitized erythrocytes back into the circulation. The clearance of cold agglutinin-sensititzed erythrocytes was compared to the clearance mediated by IgM isoagglutinin. We found that the rate of complement fixation by an IgM antibody proceeds rapidly in vivo that the time for complement activation is not a factor in limiting the rate of hepatic sequestration. The major limiting factor appears to be the rate of liver blood flow. Maximal in vitro coating of erythrocytes with C3d conferred protection from further cold agglutinin sensitization but not from IgM isoagglutinin-mediated clearance. This suggests a mechanism for the resistance to lysis observed in cells obtained from patients with the cold agglutinin syndrome and confirms the marked dependence of the site of C3 attachment on the site of membrane localization of the sensitizing antibody.

Authors

C J Jaffe, J P Atkinson, M M Frank

Inhibition of lymphoproliferation by hyperlipoproteinemic plasma.

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Abstract

Plasma for patients with primary type IV or V hyperlipoproteinemia inhibited [3H]thymidine incorporation by cultured mononuclear leukocytes. This previously unreported abnormality affected mononuclear leukocytes from patients with type IV or V hyperlipoproteinemia and from normal subjects. Patient cells incorporated [3H]thymidine normally when washed and incubated in medium containing normal plasma. Both spontaneous incorporation and stimulated incorporation in response to various mitogens and antigens were inhibited. The inhibitory effect was identified with the chylomicron and very low density lipoprotein fractions isolated from plasma and was concentration-dependent. Lectin used to stimulate cultured cells and [3H]thymidine used to measure responses were not bound to the lipoproteins in appreciable amounts. [3H]-Thymidine incorporation correlated well with morphologic evidence of lymphoproliferation. The mechanism of the inhibitory effect of type IV or V hyperlipoproteinemic plasma upon the response tested was not identified by may be related to interaction between lipoproteins and the cell membranes. We suggest that these lipoproteins may also interfere with the function of other cells.

Authors

C C Waddell, O D Taunton, J J Twomey

Irreversible deformation of the spectrin-actin lattice in irreversibly sickled cells.

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Abstract

Irreversibly sickled cells (ISC's) are circulating erythrocytes in patients with sickle cell disease that retain a sickled shape even when oxygenated. Evidence points to a membrane defect that prevents the return of these cells to the normal biconcave shape. The erythrocyte membrane protein spectrin is believed to help control erythrocyte shape and deformability. Recent studies suggest that normally spectrin and an erythrocyte actin form a self-supporting, fibrillar, lattice-like network on the cytoplasmic membrane surface. When normal erythrocyte ghosts are extracted with Triton X-100 all the integral membrane proteins and most of the membrane lipids are removed, leaving a ghost-shaped residue composed principally of spectrin and actin. We concentrated ISC's from patients with sickle cell anemia and compared the morphology and protein composition of ghosts and Triton-extracted ghost residues prepared from these ISC's with similar preparations of reversibly sickable cells and normal cells. (a) Many ISC's formed ISC-shaped ghosts. (b) All ISC-shaped ghosts formed ISC-shaped Triton residues. (c) Spectrin, erythrocyte actin (Band 5), an unidentified Band 3 component, and Band 4.1 were the major protein components of the Triton residues. All membrane-associated sickle hemoglobin was removed by the Triton treatment. (d) No ISC-shaped ghosts or ISC-shaped Triton residues were formed when deoxygenated, sickled RSC's were lysed or Triton-extracted. ISC-shaped ghosts and Triton residues were never formed from normal cells. These observations suggest that a defect of the "spectrin-actin lattice" may be the primary abnormality of the ISC membrane. Since ISC's are rigid cells, the data support the postulate that spectrin is a major determinant of membrane deformability. Finally, they provide direct evidence that spectrin is important in determining erythrocyte shape.

Authors

S E Lux, K M John, M J Karnovsky

Mechanisms of portal hypertension-induced alterations in renal hemodynamics, renal water excretion, and renin secretion.

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Abstract

Clinical states with portal venous hypertension are frequently associated with impairment in renal hemodynamics and water excretion, as well as increased renin secretion. In the present investigation, portal venous pressure (PVP) was increased in anesthetized dogs undergoing a water diuresis. Renal arterial pressure was maintained constant in all studies. As PVP was increased from 6 to 20 mm Hg, decreases in cardiac output (2.5-2.0 liter/min, P less than 0.05) and mean arterial pressure (140-131 mm Hg, P less than 0.05) were observed. Increases in PVP were also associated with decreases in glomerular filtration rate (GFR, 40-31 ml/min, P less than 0.001), renal blood flow (RBF, 276-193 ml/min, P less than 0.001), and increases in renin secretion (232-939 U/min, P less than 0.025) in innervated kidneys. No significant change in either GFR or RBF and a decrease in renin secretion occurred with increases in PVP in denervated kidneys. To dissociate the changes in cardiac output and mean arterial pressure induced by increase PVP from the observed decreases in GFR and RBF, studies were performed on animals undergoing constriction of the thoracic inferior vena cava. In these studies, similar decreases in cardiac output and mean arterial pressure were not associated with significant changes in GFR or RBF. Increases in PVP also were associated with an antidiuresis as urine osmolality increased from 101 to 446 mosmol/kg H2O (P less than 0.001). This antidiuresis was significantly blunted but not abolished by acute hypophysectomy. In hypophysectomized animals, changes in free water clearance and urine flow were linearly correlated as PVP was increased. These studies indicate that increases in PVP result in decreases in GFR and RBF and increases in renin secretion mediated by increased renal adrenergic tone. Increased PVP is also associated with antidiuresis; this antidiuresis is mediated both by vasopressin release and by diminished tubular fluid delivery to the distal nephron.

Authors

R J Anderson, R E Cronin, K M McDonald, R W Schrier

The role of lysosomal elastase in the digestion of Escherichia coli proteins by human polymorphonuclear leukocytes: experiments with living leukocytes.

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Abstract

Human polymorphonuclear leukocyte (PMN) elastase has been implicated in various pathological conditions. However, its physiological role remains undefined. One possible function of this enzyme may be digestion of bacterial proteins after phagocytosis. To test this hypothesis, we prepared Escherichia coli labeled with [3H]arginine and treated these bacteria with a lipid-soluble, active-site-directed chloromethyl ketone inactivator of pancreatic and granulocyte elastases (carbobenzoxy-L-glycyl-L-leucyl-L-alanine chloromethyl ketone, dissolved in methanol). Control bacteria were treated with methanol alone. When E. coli pretreated with the inactivator were incubated with solutions of porcine pancreatic elastase or with PMN granule extract, release of trichloroacetic acid-soluble radioactivity was significantly lower than in the control E. coli. Similar results were obtained when treated and control E. coli were fed to viable human PMN. In contrast, release of trichloroacetic acid-soluble radioactivity from E. coli containing [3H]thymidine was not affected by pretreatment of bacteria with elastase inactivator before feeding them to PMN, suggesting that phagocytosis of E. coli had not been inhibited by the chloromethyl ketone. When treated and control bacteria were fed to PMN, no significant difference was observed in the activity of lysosomal beta-glucuronidase recovered from post-granule supernatant fractions of homogenized leukocytes, suggesting that lysosomal degranulation had not been suppressed by the inactivator. However, elastase activity of the same fractions was depressed if the leukocytes had phagocytized chloromethyl ketone-treated E. coli, suggesting that inhibition of PMN elastase had occurred. We conclude that PMN elastase participates in digestion of E. coli proteins by human PMN.

Authors

J Blondin, A Janoff

Selective inhibition of osmotic water flow by general anesthetics to toad urinary bladder.

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Abstract

Vasopressin increases the permeability of the total urinary bladder, an analogue of the mammalian renal collecting duct, to water and small solutes, especially the amide urea. We have observed that three general anesthetic agents of clinical importance, the gases methoxyflurane and halothane and the ultrashortacting barbiturate methohexital, reversibly inhibit vasopressin-stimulated water flow, but do not depress permeability to urea, or the the lipophilic solute diphenylhydantoin. In contrast to their effects in vasopressin-treated bladders, the anesthetics do not inhibit cyclic AMP-stimulated water flow, consistent with an effect on vasopressin-responsive adenylate cyclase. The selectivity of the anesthetic-induced depression of water flow suggests that separate adenylate cyclases and cyclic AMP pools may exist for control of water and urea permeabilities in to toad bladder. Furthermore, theophylline's usual stimulatory effect on water flow, but not its effect on urea permeability, was entirely abolished in methoxyflurane-treated bladders, suggesting that separate phosphodiesterases that control water and urea permeabilities are present as well. We conclude that the majority of water and urea transport takes place via separate pathways across the rate-limiting luminal membrane of the bladder cell, and that separate vasopressin-responsive cellular pools of cyclic AMP appear to control permeability to water and to urea.

Authors

S D Levine, R D Levine, R E Worthington, R M Hays

The particulate superoxide-forming system from human neutrophils. Properties of the system and further evidence supporting its participation in the respiratory burst.

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Abstract

Studies were performed to characterize the previously reported particulate O2--forming system from human neutrophils. Of eight reducing agents examined, including glutathione, ascorbic acid, and intermediates of the glycolytic and hexose monophosphate shunt pathways, only the pyridine nucleotides could serve as electron donors. At 0.1 mM pyridine nucleotide, O2- production was relatively independent of pH. The Km for NADH was approximately 0.7 mM regardless of pH, while with NADPH the Km varied from 0.02 mM at pH 6.0 to 0.3 mM at pH 7.5. The molar ratio of NADPH oxidized to O2- produced was consistent with the reaction: NADPH + 2 O2- leads to NADP+ H+; the product nucleotide was shown enzymatically to be NADP. O2- production was not inhibited by CN-, Na-, EDTA, or 1,10-phenanthroline. Particulate O2- production accounted for 35% of the oxygen taken up during the respiratory burst by an equivalent number of intact neutrophils. Greatly diminished O2- production was seen with particles prepared from cells obtained from three patients with chronic granulomatous disease, with 2.5 mM NADPH as electron donor. With 5.0 mM NADH similar observations were made with particles from two of the patients, but with this nucelotide, O2- production was only slightly reduced in the third case. The evidence available suggests that this particulate O2- -forming system is the one responsible for the respiratory burst in activated neutrophils. The relationship between this system and other O2- -forming system found in human neutrophils is discussed.

Authors

B M Babior, J T Curnutte, B J McMurrich

Molecular organization of the cholesteryl ester droplets in the fatty streaks of human aorta.

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Abstract

X-ray diffraction patterns from human arterial specimens containing atherosclerotic fatty streak lesions exhibited a single sharp reflection, corresponding to a structural spacing of about 35 A. Specimens without lesions did not. When specimens with fatty streaks were heated, an order-to-disorder phase transition was revealed by the disappearance of the sharp reflection. The transition was thermally reversible and its temperature varied from aorta to aorta over a range from 28 degrees to 42 degrees C. Since cholesteryl ester droplets are a major component of fatty streaks, comparison studies were made of the diffraction behavior from pure cholesteryl esters. We found that the diffraction patterns of the fatty streak material could be accounted for by the organization of the cholesteryl esters into a liquid-crystalline smectic phase that melts from the smectic to a less ordered phase upon heating. When combined with the conclusions of others from polarized light microscopy, our study shows that a droplet in the smectic phase has well-defined concentric layers of lipid molecules. In each layer, the long axes of the molecules have a net radial orientation with respect to the droplet, but the side-to-side organization is disordered. We suggest that the accessibility of portions of the lipids for specific binding to enzymes or transport proteins may be restricted when they are in the smectic state, and that exchange of lipids with surrounding membranes or other potential binding sites may likewise be inhibited. The restriction in the smectic phase should be greater than in the less ordered phases that exist at higher temperatures.

Authors

D M Engelman, G M Hillman

Compositional mapping of cholesteryl ester droplets in the fatty streaks of human aorta.

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Abstract

Frozen sections prepared from human aortic tissue containing fatty streak lesions were examined on a thermally controlled stage with a polarizing light microscope. Distinct birefringent droplets, 0.5-5 mum in diameter, were observed, many apparently aggregated into clusters. The clusters were about 20 X 20 mum in diameter (the approximate size of foam cells). Upon being heated, each smectic droplet exhibited a sudden change of birefringence, indicating a change of state. The transition temperatures were compared to assess compositional distributions in the tissue. We found that for 52% of the clusters the standard deviation of the cluster's droplet melting point distribution was less than half that observed in the surrounding microscopic field. If clusters were intracellular lipid inclusions, this observation indicates that the lipid composition within a foam cell is more homogeneous than that of the overall field. However, using statistical methods, we compared droplet melting populations from cluster to cluster and found significant heterogeneity. The observations can be interpreted to suggest that many foam cells modify the cholesteryl ester fatty acid composition of their accumulations be selective uptake, temporal sampling, or chemical reaction. Furthermore, the intercellular heterogeneity suggests that different cells in the lesion may have different metabolic and transport enzyme affinities or be in different states.

Authors

G M Hillman, D M Engelman

The paradox of Hemophilus infuenzae type B bacteremia in the presence of serum bactericidal activity.

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Abstract

We investigated the role of serum bactericidal activity in Hemophiplus influenzae type b infections in infants with meningitis and in a rat model. In infected infants, 13/22 admission sera had bactericidal activity against the infecting strain, and bacteremia was as frequent in those with bactericidal activity (54%) as those without (56%). The coexistence of bactericidal activity and bacteremia was reproduced and studied in experimentally infected weanling rats. Serum from such rats kills in vitro 95% of conventionally broth-grown bacteria within 10 min, but does not kill organisms obtained from the infected animals. Thus bactericidal activity as conventionally determined for H. influenzae b may have no relevance in vivo, Incubation of broth-grown bacteria in normal rat serum for 30 min at 37 degrees C produces a resistance like that of in vivo organisms. This phenotypic conversion depends on factors that are of molecular weight less than 1,000, stable to 100 degrees C, but destroyed by ashing. When injected intravenously into nonimmune animals, broth-grown bacteria are quickly cleared, while serum-preincubated bacteria are not. The latter, however, are cleared when injected into bacteremic rats (half-life 30 min). Bacteremia in the rats may persist despite this capacity for clearance because bacteria are entering the blood from extravascular fluids, which contain greater than 90% of the total bacterial burden.

Authors

S Shaw, A L Smith, P Anderson, D H Smith

Metalloproteases of human articular cartilage that digest cartilage proteoglycan at neutral and acid pH.

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Abstract

Extracts of human articular cartilage contain proteases capable of degrading the proteoglycan component of cartilage matrix at neutral and acid pH. These enzymes have been partially purified by ion exchange chromotography and characterized by disc electrophoresis, inhibition patterns, and action of proteoglycan. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit optimally at pH 7.25 has been purified up to 900-fold. It is strongly inhibited by o-phenanthroline, alpha-2-macroglobulin, and egg white, and to a lesser extent by D-penicillamine and EDTA. Inhibition by chelating agents is reversed by cobalt, zinc, and ferrous ions. Two acid metalloproteases, distinct from cathespins B1, D, and F, digest proteoglycan subunit at pH 4.5 and 5.5. Both are inhibited by o-phenanthroline and activity is restored by cobalt, zinc, or ferrous ions. With electron microscopy, it was found that cartilage slices were depleted of ruthenium red-staining matrix proteoglycan after incubation in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immuno-diffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteoglycan subunit, producing fragments with 5 to 12 chondroitin sulfate chains. The link proteins were not digested.

Authors

A I Sapolsky, H Keiser, D S Howell, J F Woessner Jr