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Biomedical subjects

S Blumberg

Publications and source records attributed to S Blumberg.

At least 37 records · Page 2Linked to original sources
PubMed1994

Differential effects of retinoic acid and growth factors on osteoblastic markers and CD10/NEP activity in stromal-derived osteoblasts.

The effects of retinoic acid (RA) on the expression of osteoblastic-related cell markers was examined. A marrow stromal osteogenic cell line, MBA-15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constitutively express mRNA encoding for procollagen alpha 2 (I), osteonectin, osteopontin, biglycan, and alkaline phosphatase (ALK-P). Gene expression was unchanged in response to RA triggering for 24 hr. Furthermore, cell growth and enzymatic activities of ALK-P and neutral endopeptidase (CD10/NEP) were studied. These parameters were examined in MBA-15 and clonal populations representing different stages of differentiation. The cell's growth rate was unchanged, while ALK-P activity was greatly increased during the culture period under RA treatment in MBA-15 and in the clonal cell lines examined while CD10/NEP activity displayed a different pattern. MBA-15.4, a preosteoblast cell line, exhibited an inhibition in CD10/NEP activity at the beginning of the culture period, reaching basal level with time. This activity was greatly increased over control level in MBA-15.6, a mature stage of osteoblasts. Furthermore, the response of cell lines to various growth factors was tested subsequent to priming the cultures with RA. A synergistic effect was monitored for ALK-P activity in MBA-15.4 and MBA-15.6 cells under rh-bone morphogenic protein (BMP-2) and purified osteogenin (BMP-3), and an antagonist effect was measured when cells were exposed to transforming growth factor beta (TGF beta). Contrarily, BMP-2 and BMP-3 inhibited the CD10/NEP activity that had remained unchanged following priming of the cell with RA. Insulin-like growth factor I (IGF-I) and basic fibroblast growth factors (bFGF) did not affect either ALK-P nor CD10/NEP activities in both cloned cells. Cellular response to bone-seeking hormone, parathyroid hormone (PTH), and prostaglandin E2 (PGE2) was monitored by activation of intracellular cAMP. Treatment with RA caused a dramatic decrease in MBA-15.6 cell responses to PTH and PGE2, but no significant effects could be observed in other clonal lines.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Chemotaxis and chemokinesis of human spermatozoa to follicular factors.

Human spermatozoa accumulate in vitro in diluted follicular fluids obtained from follicles from which the eggs have been fertilized. Using capillary assays under a variety of experimental conditions (ascending or descending gradients of follicular fluid, or no gradient at all) and microscopic assays in which individual spermatozoa could be followed, we found that the sperm accumulation in follicular fluid was the result of both sperm chemotaxis and chemokinesis and eventually hyperactivation-like motility. We determined the optimal conditions for sperm accumulation, which involved sperm preincubation (possibly to induce sperm capacitation) and proper dilution of follicular fluid. In all the assays, the net accumulation was low, probably reflecting the chemotactic responsiveness of only a small fraction of the sperm population at any given time. We partially fractionated follicular fluid in a Centricon microconcentrator (Amicon, Danvers, MA) and by acetone precipitation, and found that at least one of the chemotactic factors is a small (< 10-kDa) molecule that is probably nonhydrophobic. This is the first time that sperm chemotaxis and chemokinesis in response to a follicular factor(s) in mammals has been established and has been distinguished from other processes that might cause sperm accumulation. The physiological significance of these findings is discussed.

Capillary Action↗
PubMed1993

Atrial natriuretic peptide attracts human spermatozoa in vitro.

Here we report that atrial natriuretic peptide (ANP), a known activator of particulate guanylate cyclase, induces attraction and swimming speed enhancement of human spermatozoa in vitro. Using capillary assays under a variety of experimental conditions (ascending or descending gradients of ANP, or no gradient at all) and microscopic assays in which individual spermatozoa could be followed, we found that spermatozoa followed the gradient of ANP and accumulated in it. Speed enhancement was detected in the presence of ANP without a gradient. These observations suggest either that an ANP-like substance is the physiological attractant for human spermatozoa, or, more likely, that ANP directly affects guanylate cyclase in a manner similar to that caused by the physiological attractant.

Atrial Natriuretic Factor↗
PubMed1993

Variability of cardiac output as determined by impedance cardiography in pacemaker patients.

Hemodynamic assessment of pacemaker patients is necessary for gauging responses to changes in programming or other conditions affecting circulation. Impedance cardiography permits noninvasive determinations of cardiac output at short intervals but data regarding variability of this method in patients with pacemakers is unavailable. Thirty-eight patients with pacemakers (24 with DDD and 14 with VVI devices) and 6 normal subjects were studied. Each patient was studied in the supine position and repeated impedance measurements were obtained. Fourteen patients were studied during sinus rhythm, 24 were studied during DDD pacing, and 32 patients were studied during VVI pacing. Variability was assessed by methods that analyzed both serial measurements and variability between 2 consecutive and nonconsecutive measurements. The mean indexes and coefficients of variation of 2 and serial measurements in sinus rhythm and DDD were 4%; in VVI it was 6%. The precision of impedance cardiography in all pacing modes, as demonstrated by analysis of variability, indicates that detected changes of stroke volume and cardiac output > 7% on serial (2 and more) measurements, performed by the same operator and during the same session, represent true hemodynamic alterations with 95% confidence. The precision of impedance cardiography demonstrated may be comparable or superior to other frequently used techniques, and the data obtained are valuable both investigationally and clinically.

Adult↗
PubMed1993

Crystallization and preliminary crystallographic analysis of Streptomyces griseus aminopeptidase.

Streptomyces griseus excretes a small molecular mass (30 kDa) aminopeptidase that could be used for various biotechnological applications. This enzyme was isolated from an extracellular protease mixture of Streptomyces griseus (Pronase E. Sigma) and single crystals were obtained by the vapor diffusion method using polyethylene glycol 4000 as the precipitant. The crystals belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2), with cell dimensions of a = b = 61.82(3) A and c = 145.88(4) A. These crystals are mechanically strong, they are stable in the X-ray beam and they diffract to better than 1.8 A resolution. The cell dimensions and the cell symmetry are consistent with one molecule in the asymmetric unit and the crystals are suitable for a detailed high-resolution crystallographic analysis. A complete native data set to 1.9 A resolution has been collected on a Rigaku R-AXIS-IIC Imaging Plate Detector system and a heavy-atom derivative search is in progress.

Aminopeptidases↗
PubMed1993

Specificity of Streptomyces griseus aminopeptidase and modulation of activity by divalent metal ion binding and substitution.

Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloenzyme characterized by a high enzymic reactivity, high thermal stability and low molecular mass [Spungin, A. and Blumberg, S. (1989) Eur. J. Biochem. 183, 471-477]. A study of the specificity of S. griseus aminopeptidase using amino acid 4-nitroanilide substrates shows that the leucine derivative is the best substrate. Derivatives of other hydrophobic amino acids, methionine and phenylalanine, are also excellent substrates for the enzyme. The 4-nitroanilides of alanine, valine, proline and lysine are good substrates whereas those of the small size glycine and the acidic amino acids are very poor. No hydrolysis of a terminal Xaa residue can be detected with Xaa-proline N-terminal sequences. Calcium ions bind to the enzyme and modulate its activity in a substrate-dependent manner. The catalytically essential zinc of S. griseus aminopeptidase is removed by dialysis against 1,10-phenanthroline and replaced by manganese or cobalt ions, resulting in enzyme derivatives of altered specificities. Thus, whereas the zinc enzyme hydrolyzes leucine 4-nitroanilide at a 10-fold faster rate than the manganese or cobalt enzymes, the cobalt enzyme hydrolyzes alanine 4-nitroanilide at a more than 20-fold faster rate than the zinc or manganese enzymes.

Aminopeptidases↗
PubMed1992

Precision of impedance cardiography measurements of cardiac output in pacemaker patients.

Objective hemodynamic assessment of pacemaker patients is necessary for gauging responses to changes in programming or other conditions that affect the circulation. Impedance cardiography permits noninvasive, repetitive determinations of cardiac output at short intervals, but data regarding variability of this method in patients with pacemakers is unavailable. Thirty-eight patients with implanted pacemakers (24 DDD, 14 VVI) and six normal subjects were studied. A computerized impedance cardiograph was used to calculate cardiac output from the product of the first derivative of the thoracic impedance signal (dZ/dt), the ventricular ejection time, and heart rate. Each patient was studied while supine after a period of at least 15 minutes of rest and repeated impedance measurements (about ten) were performed. Fourteen patients were studied in sinus rhythm, 24 were studied during DDD pacing, and 32 patients were studied during VVI pacing. Cardiac and stroke indices were calculated 706 times on the basis of 11,296 accepted beats. Variability was assessed by methods that analyzed serial measurements and variability between two consecutive and nonconsecutive measurements. The mean indices and coefficients of variation of two measurements and of serial measurements in sinus rhythm and during DDD pacing were 4%; in VVI it was 6%. The precision of impedance cardiography in all pacing modes, as demonstrated by analysis of variability, indicates that detected changes of stroke volume and cardiac output > 7% on serial (two and more) measurements, performed by the same operator and during the same session, represent true hemodynamic alterations with 95% confidence.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1992

Orthostatic responses in patients with DDD pacemakers: signs of autonomic dysfunction.

UNLABELLED: Responses to orthostasis may be altered in states associated with autonomic dysfunction. Computerized impedance cardiography, a noninvasive method for continuous assessment of stroke volume and mean blood pressure, was utilized to study the postural hemodynamic changes in eight normal and 27 patients with DDD pacemakers. Twenty patients with complete heart block (five with heart failure) were studied in the VDD mode and seven patients with sick sinus syndrome were assessed in DVI (four) or VDD (three). The results with pacemaker patients are significantly different from those observed in normal. Pacemaker patient responses to standing included: (1) a reduction in systolic, diastolic, and mean blood pressure; (2) an increase in heart rate in patients with intact sinus node function and no change in patients with sick sinus syndrome; and (3) stroke volume was unchanged in patients with sick sinus syndrome or heart failure and only modest reduction occurred in the remaining patients. CONCLUSIONS: (1) No reduction in stroke volume during upright posture occurs in DDD patients with sick sinus syndrome and this appears to be a compensatory reaction to an inadequate heart rate response to standing; (2) The hemodynamic response of DDD patients to the assumption of an upright posture is consistent with autonomic dysfunction; and (3) The primary cause for autonomic dysfunction in DDD patients may be the asynchronous ventricular depolarization caused by right ventricular pacing.

Aged↗
PubMed1991

Hydrolysis of thymic humoral factor gamma 2 by neutral endopeptidase (EC 3.4.24.11).

A search for the natural substrates for neutral endopeptidase (NEP; EC 3.4.24.11) in the immune system led to investigation of the enzyme's action on thymic humoral factor gamma 2 (THF). The ectoenzyme rapidly and efficiently hydrolyses the Lys6-Phe7 bond of the octapeptide. The site of cleavage was confirmed by h.p.l.c. analysis, amino acid analysis and sequence determination of the products. Phosphoramidon (3.6 microM), a potent inhibitor of the enzyme, prevents this cleavage even during prolonged incubation. The high efficiency of hydrolysis of THF by NEP is similar to that reported for [Leu5]enkephalin, and the dipeptide Phe-Leu is the C-terminal product in the hydrolysis of both peptides. The presence of NEP, reportedly identified as the common acute lymphoblastic leukaemia antigen (CALLA), in bone-marrow cells and other cells of the immune system raises the possibility that it may play a role in modulating the activity of peptides such as THF.

Amino Acid Sequence↗
PubMed1991

Common regulation of feeding and mating in Aplysia fasciata: pheromones released by mating and by egg cordons increase feeding behavior.

We examined whether pheromones released by reproductive behaviors (mating and egg-laying) affect feeding behavior. A preliminary experiment demonstrated that the quantity of food eaten can be used to measure the effects of pheromones on feeding. Using this measure, we then showed that Aplysia that were prevented from mating, but that were in the same aquarium as mating conspecifics, eat more food than do Aplysia in a medium lacking mating animals. Mating and feeding were not temporally correlated, indicating that pheromones released by mating probably do not initiate feeding, but rather modulate feeding after it has begun. Aplysia that were in the same aquarium as freshly deposited egg cordons also ate more than did animals in a medium lacking eggs.

Animals↗
PubMed1990

Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types.

A series of cultured mouse marrow stromal cell lines of different phenotype characteristics were examined for their neutral endopeptidase (NEP) activity using the highly selective chromophoric substrate of enzyme, 3-carboxypropanoyl-alanyl-alanyl-leucine 4-nitroanilide. All the cell lines tested contained appreciable amounts of NEP activity, but the cells expressing an osteoblastic phenotype, MBA-15, showed the high levels. Other non-stromal osteoblastic cell lines, MC-3T3-E1, ROS 17/2.8 cells derived from mouse long bone explants, were also rich in NEP. A four-fold stimulation of NEP activity was observed when certain MBA-15 clones were cultured in the presence of 1 alpha,25-dihydroxyvitamin D3. Since NEP is an effective cell-surface endopeptidase, it may play a role in the dynamics of bone formation, via interaction with biologically active polypeptides.

Amino Acid Sequence↗
PubMed1990

Endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance.

Incubation of endothelins (ETs) with bovine kidney neutral endopeptidase (NEP) resulted in a selective two-step degradation with loss of biochemical activity. The Km of the enzyme indicated high-affinity binding, and hydrolysis was completely inhibited by phosphoramidon. The first step was nicking of the Ser5-Leu6 bond, followed by cleavage at the amino side of Ile19. The nicked peptide exhibited biochemical activities comparable to those of the intact peptide--i.e., binding to the ET receptor, induction of inositol phospholipid hydrolysis, and toxicity. The twice-cleaved product was inactive. The sarafotoxins (SRTXs) were more resistant than the ETs to NEP: for example, the half-time for ET-1 was approximately 1 hr, while it was approximately 4 hr for SRTX-b and even higher for SRTX-c. These in vitro findings may indicate a regulatory role of NEP (or similar enzymes) in the physiological inactivation of ETs. They might also help to explain why under certain physiological conditions ETs may be less toxic than SRTXs.

Amino Acid Sequence↗
PubMed1989

Investigation of neutral endopeptidases (EC 3.4.24.11) and of neutral proteinases (EC 3.4.24.4) using a new sensitive two-stage enzymatic reaction.

A sensitive two-stage enzymatic reaction for mammalian and bacterial metalloendopeptidases has been developed using the substrate 3-carboxypropanoyl-alanyl-alanyl-leucine-4-nitroanilide supplemented with Streptomyces griseus amino-peptidase. Neutral endopeptidase EC 3.4.24.11 from bovine kidney hydrolyzes the substrate (pH 7.5, 25 degrees C) with a catalytic efficiency (kcat = 1.2 x 10(2) s-1, Km = 0.15 mM) of the highest ever reported for the enzyme acting on synthetic chromophoric and fluorogenic substrates. Thermolysin hydrolyzes the substrate at a faster rate (kcat = 1.2 x 10(3) s-1) but the overall efficiency is diminished by a higher Km (4.2 mM). Suspensions of human neutrophil cells and culture filtrates of Bacillus cereus have been assayed sensitively for their neutral endopeptidases and neutral proteinase activities, respectively. The assay provides a convenient tool for the kinetic investigation of neutral endopeptidases and neutral proteinases and for assessing their function in biological systems.

Animals↗
PubMed1989

Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloprotein. Purification and properties of the enzyme.

A heat-stable aminopeptidase with an N-terminal Ala-Pro-Asp-Ile-Pro-Leu sequence has been purified from Streptomyces griseus by heat treatment followed by gel-exclusion and anion-exchange chromatographic procedures. The enzyme is a monomeric zinc metalloenzyme showing an apparent molecular mass of 33 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 21 kDa by gel filtration on Superose 12. Calcium ions bind to the enzyme, pKCa 4.5, and activate it about sixfold when the substrate is leucine-4-nitroanilide (0.4 mM in 50 mM Tris/HCl pH 8.0, 25 degrees C). Binding of Ca2+ also contributes to the thermal stability of the protein. This aminopeptidase may be useful for two-stage assays of bacterial and mammalian metalloendopeptidases; it may also serve in studies of proteolytic enzyme activation by calcium ions.

Amino Acid Sequence↗
PubMed1989

Effects of hydroxyethyl rutosides upon the permeability of single capillaries in the frog mesentery.

1. We have investigated the effects of a standardised mixture of hydroxyethyl rutosides (HR) upon the permeability of the walls of single capillaries and venules of the frog mesentery. 2. In each experiment a single vessel was perfused via a micropipette with frog Ringer solutions containing bovine serum albumin (10 mg ml-1) and Ficoll 70 (40 mg ml-1) first in the absence of HR and then with HR added to the perfusate. The permeability of the vessel walls was assessed during each perfusion by using a development of the Landis micro-occlusion technique to estimate their hydraulic permeability (Lp) and the effective osmotic pressure (sigma delta pi) exerted across them by the perfusate macromolecules. 3. Measurements were made both in vessels which appeared to be healthy and in vessels showing signs of stasis or inflammation before perfusion. 4. HR at concentrations of 1.0, 0.1 and 0.01 mg ml-1 reduced hydraulic permeability to approximately half of its value in the absence of HR. It increased sigma delta pi to macromolecules at concentrations of 10, 1.0, 0.1, 0.01 and 0.001 mg ml-1. The effects of HR upon permeability were not reversed within 10 min of perfusion with an HR-free solution. 5. Ultrastructural examination of a number of vessels in which initial high values of permeability were reduced to values within the normal range of permeabilities by HR, showed clear signs of damage to the endothelium, with large gaps between adjacent endothelial cells. 6. These observations suggest that HR does reduce microvascular permeability both in healthy vessels and vessels showing of inflammation. The reduction in permeability of inflamed vessels does not appear to be the result of closure of the gaps between adjacent endothelial cells.

Animals↗