Biomedical subjects

S Blumberg

Publications and source records attributed to S Blumberg.

At least 19 recordsLinked to original sources

PubMed2006

Effects of CCK-8 on independent ingestion and central c-Fos-like immunoreactivity in rats on postnatal days 10 and 11.

Controls of the independent ingestion of food in the preweanling rat emerge in the second postnatal week. We investigated the effects of CCK-8 (0, 1, 5, or 10 microg/kg IP) on intake and c-Fos-like immunoreactive (CFLI) cells in hindbrain and forebrain on postnatal days 10 and 11. Five micrograms per kilogram decreased intake and increased the number of CFLI cells in four subnuclei of the nucleus tractus solitarius (NTS), in arcuate nucleus (ARC), and in central nucleus of the amygdala (CeA). Ten micrograms per kilogram decreased intake and increased CFLI in three NTS subnuclei as much as 5 microg/kg did, but was more potent than 5 microg/kg in the medial NTS subnucleus. Ten micrograms per kilogram increased CFLI in paraventricular (PVN) and supraoptic (SON) nuclei, but 5 microg/kg did not. Thus, reduction of intake by CCK-8 on days 10 and 11 is associated with increased hindbrain and forebrain CFLI.

Animals↗

PubMed2005

Independent ingestion and microstructure of feeding patterns in infant rats lacking CCK-1 receptors.

Otsuka Long-Evans Tokushima fatty (OLETF) rats are a strain of Long-Evans Tokushima Otsuka (LETO) rats that do not express CCK-1 receptors, developing in adulthood, hyperphagia, obesity, and non-insulin-dependent diabetes mellitus (NIDDM). We examined weight gain and meal patterns during a 30-min independent ingestion test on postnatal days 2-4 and again on days 9-11 in OLETF and LETO rat pups. OLETF pups were significantly heavier compared with their LETO controls at both ages, and they consumed significantly more of the sweet milk diet. The difference in intake can be attributed to a significant increase in meal size and duration. Number of clusters and bursts of licking within a meal were greater in OLETF rat pups, with no difference between strains in burst and cluster size. Interlick interval (ILI) was not significantly different between OLETF and LETO pups. This measure decreased on days 9-11 compared with days 2-4 in both strains. Latency to start feeding was significantly shorter on days 2-4 in OLETF vs. LETO pups, but this difference disappeared at the second test at the older age. Two- to four-day-old OLETF pups consumed a larger volume of milk during the first minute of feeding, and their initial lick rate and decay of lick rate were significantly larger compared with their LETO controls. Lack of CCK-1 receptors, or other OLETF-related abnormalities, therefore, resulted in a satiation deficit, leading to increased meal size, hyperphagia, and increased weight gain as early as 2-4 postnatal days.

Aging↗

PubMed2004

Binding of inhibitory aromatic amino acids to Streptomyces griseus aminopeptidase.

The bacterial aminopeptidase isolated from the extracellular extract of Streptomyces griseus (SGAP) is a double-zinc exopeptidase with a high preference for large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), is heat-stable, displays a high and efficient catalytic turnover and its activity is modulated by calcium ions. Several free amino acids were found to inhibit the activity of SGAP in the millimolar concentration range and can therefore serve for the study of binding of both inhibitors and reaction products. The current study is focused on the X-ray crystallographic analysis of the SGAP complexes with L-tryptophan and p-iodo-L-phenylalanine, both at 1.30 A resolution. These two bulky inhibitory amino acids were found to bind to the active site of SGAP in very similar positions and orientations. Both of them bind to the two active-site zinc ions via their free carboxylate group, while displacing the zinc-bound water/hydroxide that is present in the native enzyme. Further stabilization of the binding of the amino-acid carboxylate group is achieved by its relatively strong interactions with the hydroxyl group of Tyr246 and the carboxylate group of Glu131. The binding is also stabilized by three specific hydrogen bonds between the amine group of the bound amino acid and enzyme residues Glu131, Asp160 and Arg202. These consistent interactions confirm the key role of these residues in the specific binding of the free amine of substrates and products, as proposed previously. The phenyl ring of Phe219 of the enzyme is involved in stacking interactions with the corresponding aromatic ring of the bound affector. This interaction seems to be important for the binding and orientation of the aromatic side chain within the specificity pocket. These structural results correlate well with the results obtained for the complexes of SGAP with other inhibitory amino acids and support the general catalytic mechanism proposed for this and related enzymes.

Amino Acids↗

PubMed2001

Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism.

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.

Amino Acids↗

PubMed2000

Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue: a structural study at 1.53 A resolution.

SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces griseus cultures. It is a double-zinc enzyme with a strong preference for large hydrophobic amino-terminus residues. It is a monomeric (30 kDa) heat-stable enzyme, with a high and efficient catalytic activity modulated by calcium ions. The small size, high activity and heat stability make SGAP a very attractive enzyme for various biotechnological applications. Only one other related aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed to date and its structure was shown to be considerably similar to SGAP, despite the low sequence homology between the two enzymes. The motivation for the detailed structural analysis of SGAP originated from a strong mechanistic interest in the family of double-zinc aminopeptidases, combined with the high potential applicability of these enzymes. The 1.75 A crystallographic structure of native SGAP has been previously reported, but did not allow critical mechanistic interpretations owing to inconclusive structural regions around the active site. A more accurate structure of SGAP at 1.58 A resolution is reported in this paper, along with the 1.53 A resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process. These two high-resolution structures enable a better understanding of the SGAP binding mode of both substrates and products. These studies allowed the tracing of the previously disordered region of the enzyme (Glu196-Arg202) and the identification of some of the functional groups of the enzyme that are involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and Phe219). These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The structural results are compared with a recent structure of AAP with an hydroxamate inhibitor in order to draw general functional conclusions which are relevant for this family of low molecular-weight aminopeptidases.

Aeromonas↗

PubMed1999

Renal effects of 2-mercaptoacetyl-L-leucyl-L-phenylalanine, a novel selective inhibitor of neutral endopeptidase 24.11 (Neprilysin): comparison with SQ 28,603.

The effects of 2-mercaptoacetyl-L-leucyl-L-phenylalanine (MA-LF) on the activity of neutral endopeptidase and on renal hemodynamics and excretory function were investigated in experiments in vitro and in vivo. In vitro studies showed that the compound effectively inhibited purified bovine kidney neutral endopeptidase (Ki = 0.012 microM), while having slight influence on the activity of angiotensin I converting enzyme (Ki = 0.14 microM). In experiments on normal anesthetized rats (thiobutabarbital sodium salt, 100 mg/kg), IV administration of MA-LF (20 and 60 mg/kg) produced a dose-dependent increase in absolute rate and fractional excretion of sodium (+324% and +299%, respectively) and urinary flow rate (+261%), but did not change renal and systemic hemodynamics. Renal excretory effects of the new compound were comparable to those of the selective neutral endopeptidase inhibitor SQ 28,603. These results demonstrate that MA-LF is a potent neutral endopeptidase inhibitor with prominent natriuretic and diuretic properties.

Alanine↗

PubMed1998

Inhibition of Streptomyces griseus aminopeptidase and effects of calcium ions on catalysis and binding--comparisons with the homologous enzyme Aeromonas proteolytica aminopeptidase.

Streptomyces griseus aminopeptidase is a zinc metalloenzyme containing 2 mol zinc/mol protein, similar to the homologous enzyme Aeromonas proteolytica aminopeptidase. In addition, a unique Ca2+-binding site has been identified in the Streptomyces enzyme, which is absent in the Aeromonas enzyme. Binding of Ca2+ enhances stability of the Streptomyces enzyme and modulates its activity and affinity towards substrates and inhibitors in a structure-dependent manner. Among the three hydrophobic 4-nitroanilides of alanine, valine and leucine, the latter displays the largest overall activation (increase in k(cat)/Km). Large enhancements in affinity (1/Ki) upon Ca2+ binding have been observed for inhibitors with flexible (leucine-like) residues at their N-termini and smaller enhancements for inhibitors with rigid (phenylalanine-like) residues.

Aeromonas↗

PubMed1998

Increased tissue neutral endopeptidase 24.11 activity in spontaneously hypertensive hamsters.

The purpose of this study was to determine whether tissue neutral endopeptidase (NEP) 24.11 activity, a membrane-bound metalloenzyme widely distributed in the peripheral circulation that cleaves and inactivates vasodilator peptides, is increased in spontaneously hypertensive hamsters relative to genetically/age-matched normotensive hamsters. Mean arterial pressure and heart rate were 163 +/- 11 mm Hg and 312 +/- 7 beats/min in spontaneously hypertensive hamsters and 99 +/- 3 mm Hg and 302 +/- 10 beats/min in normotensive hamsters, respectively (mean +/- SEM). NEP 24.11 activity is significantly increased in the kidney, cheek pouch, and spinotrapezius muscle, and significantly decreased in the heart and aorta of spontaneously hypertensive hamsters relative to controls (P < .05). Lung and brain NEP 24.11 activity is similar in both groups. Renal NEP 24.11 activity increases and to a similar extent in spontaneously hypertensive and normotensive hamsters as chloride anion concentration in the assay buffer is increased. Substituting citrate for chloride anion significantly attenuates renal NEP 24.11 activity. Taken together, these data indicate that NEP 24.11 activity in spontaneously hypertensive hamsters is increased in two organs that contribute appreciably to peripheral vascular resistance, skeletal muscle, and kidney. We suggest that the spontaneously hypertensive hamster is a suitable model to study the role of skeletal muscle and renal NEP 24.11 in regulating vasomotor tone in essential hypertension.

Animals↗

PubMed1998

Social isolation blocks the expression of memory after training that a food is inedible in Aplysia fasciata.

Isolating a sexually mature Aplysia fasciata for either 1 or 24 hr immediately after training that a food is inedible blocks the subsequent expression of memory measured 24 hr later. Isolation that is delayed for 1 hr after training, but not for 12 hr after training, is also effective in blocking memory. Isolation affects memory because of a specific effect caused by the absence of pheromones secreted by conspecifics rather than by a nonspecific change in the chemical environment, because transferring animals to a novel environment (120% seawater) that contains a conspecific does not affect memory. Isolation also does not affect memory in sexually immature Aplysia, even though immature animals are able to sense one another's presence. Isolation may affect memory because social (and sexual) isolation is a form of stress in mature A. fasciata, and stress after training affects retention in many animals.

Age Factors↗

PubMed1997

The rhinophores sense pheromones regulating multiple behaviors in Aplysia fasciata.

Pheromones released during mating and egg laying in Aplysia facilitate various aspects of behavior. We now show that the chemosensory rhinophores sense these pheromones. Ablating the rhinophores causes a significant decrease in the time spent mating. In addition, the lesion blocks the increases of feeding in response to pheromones released by egg cordons and by mating conspecifics. Respiratory pumping is significantly increased in response to egg cordons, mating conspecifics and egg laying hormone (ELH). The increase in response to egg cordons is blocked by ablating the rhinophores, but not by lesioning the osphradium, a second chemosensory organ.

Animals↗

PubMed1997

Streptomyces griseus aminopeptidase: X-ray crystallographic structure at 1.75 A resolution.

The X-ray crystal structure of the enzyme Streptomyces griseus aminopeptidase (SGAP) has been determined in its double zinc form to 1.75 A resolution, in its apo-enzyme from (zinc removed) to 2.1 A resolution, and as a mercury replaced derivative to 2.1 A resolution. The structure solution was achieved by single isomorphous replacement with phasing from anomalous scattering (SIRAS), followed by density modification with histogram matching. The protein consists of a central beta-sheet made up of eight parallel and antiparallel strands, surrounded by helices on either side. The active site is located at the carbonyl ends of two middle strands of the beta-sheet region. Two sections of the chain that could not be traced were Glu196 to Arg202, which borders the active site, and the final seven C-terminal residues starting with Gly278. The active site contains two zinc cations, each with similar ligands, at a distance of 3.6 A from each other. An unknown molecule appears to be bound to both zinc ions in the active site at partial occupancy and has been modelled as a phosphate ion. A calcium binding site has also been identified, consistent with the observations that calcium modulates the activity of the enzyme, and increases its heat stability. The mechanism by which the calcium cation modulates enzyme activity is not apparent, since the location of the calcium binding site is approximately 25 A distant from the active site zinc ions. Comparison of the structure of SGAP to other known aminopeptidases shows that the enzyme is most similar to Aeromonas proteolytica aminopeptidase (AAP). Both enzymes share a similar topology, although the overall sequence identity is very low (24% in aligned regions). The coordination of the two active site zinc cations in SGAP resembles that of AAP. These two microbial enzymes differ from bovine lens leucine aminopeptidase (LAP) in both overall structure and in coordination of the two zinc ions.

Aeromonas↗

PubMed1996

Aminopeptidase from Streptomyces griseus: primary structure and comparison with other zinc-containing aminopeptidases.

The aminopeptidase from Streptomyces griseus is a calcium-activated metalloenzyme, which contains 2 mol tightly bound zinc/mol protein. This aminopeptidase rapidly hydrolyzes peptide bonds formed by N-terminal hydrophobic amino acids, such as leucine, methionine and phenylalanine. We have determined the complete primary structure of the protein, which contains 284 amino acid residues, yielding a molecular mass of 29723 Da. A search in the Swiss-Prot database for sequence similarities revealed a low degree of identity (26-34%) to Saccharomyces cerevisiae aminopeptidase Y, Aeromonas proteolytica aminopeptidase, and a hypothetical 49.5-kDa protein from Bacillus subtilis, which is supposed to belong to the aminopeptidase Y family. In all these proteins, the residues that are known to be involved in zinc coordination are conserved.

Aeromonas↗

PubMed1996

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases.

Tripeptide derivatives like 3-carboxypropanoylalanyl-alanyl-leucine 4-nitroanilide or 3-carboxypropanoyl-alanyl-alanyl-phenylalanine 4-nitroanilide are very sensitive substrates for neprilysin (k cat > 10(2)s(-1); k cat/Km > or = 10(6) s(-1) x M(-1)) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (k cat approximately 1s(-1)-34s(-1)). By substituting the N-terminal alanine of the substrates with proline, the catalytic efficiency of the enzymic reaction, by the serine proteases, is diminished by 2-3 orders of magnitude, whereas that by neprilysin and theromlysin decreases only slightly. These effects demonstrate that structural alterations in peptide substrates that impair secondary sub-site interactions with one class of peptidases may enhance the selectivity of the substrates towards another class of peptidases.

Amino Acid Sequence↗

PubMed1996

Activity of neutral endopeptidase and aminopeptidase N in mouse thymic stromal cells which bind double-positive thymocytes.

The activity of two peptidases was determined in immortalized lines of thymic stromal cells. A line of total stromal cells (T-TG-St) was grown from transgenic mouse expressing temperature-sensitive SV40 T antigen under the control of the regulatory elements of the mouse major histocompatibility complex class I gene. From these cells we isolated a subset (DP-TG-St) that binds thymocytes which are mainly CD4+8+. We also assayed a clone of fetal thymic epithelial cells (BA/10) that binds CD4+8+ thymocytes. Both lines of double -positive cell-binding stroma exhibited strong activity of two peptidases, neutral endopeptidase (NEP; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). In contrast, the activity of both enzymes was very low in the total thymic stromal line. Use of the specific inhibitors confirmed that these two enzymes were responsible for the activity observed but also suggested the presence of additional unidentified aminopeptidase(s) in the same stromal cells. The high activity of the two peptidases on stromal cells that bind thymocytes at the double-positive stage raises the possibility that they might contribute to the microenvironment of the developing thymocytes.

Amino Acid Sequence↗

PubMed1996

The effect of human recombinant erythropoietin on the growth of a human neuroblastoma cell line.

Erythropoietin is a growth factor. Cancer can be described as disturbance of the fine balance of positive and negative growth control mechanisms. The effect of human recombinant erythropoietin (EPO) was studied on the cell growth and differentiation of a human neuroblastoma cell line (h-NMB). Cell growth curves, trypan blue staining and thymidine uptake were used to assess cell proliferation and death. To assess cell differentiation, neutral endopeptidase (cell membrane enzyme marker), creatine kinase (cytosolic enzyme marker), dopamine uptake (dopamine transporter marker) and cell morphology were determined. Specific EPO receptor mRNA, by RT-PCR technique, was demonstrated. The incubation of erythropoietin with the tumor cell line resulted in inhibition of cell proliferation as evidenced in a diminished cell growth. EPO was shown to have induced a differentiation process as seen from the two different enzymatic markers, membranal and cytosolic, and from the cells dopamine uptake studies. However, the morphological changes did not document a full differentiation effect. EPO specific antibodies blocked the effects of EPO on cell proliferation and creatine kinase activity. In this study, EPO did not produce any sign of proliferation in the nervous tumor cell line used.

Base Sequence↗

PubMed1996

The Utility of Unipolar Electrogram Recording for Catheter Ablation of Accessory Pathways During Atrial Fibrillation.

INTRODUCTION: Atrial fibrillation, a commonly occurring rhythm in patients with manifest accessory pathways, may prevent the usual mapping criteria for successful catheter ablation from being obtained. Unipolar electrogram recordings may be of value in this situation. METHODS: Unipolar recordings were obtained during atrial fibrillation in one patient with a manifest left-sided accessory pathway, and another patient with a septal accessory pathway with Mahaim-like properties. The timing of the intrinsic deflection, and the presence of a QS complex, were utilized as criteria to define the successful ablation site. RESULTS: Successful ablation of the accessory pathways was achieved during atrial fibrillation. CONCLUSIONS: The use of unipolar recordings can aid successful catheter ablation of the accessory pathways during atrial fibrillation, giving added information to the bipolar electrograms.

Journal Article↗

PubMed1995

Use of P-wave-triggered, P-wave signal-averaged electrocardiogram to predict atrial fibrillation after coronary artery bypass surgery.

Atrial fibrillation occurs commonly after coronary artery bypass surgery. However, despite numerous attempts at prediction, no accurate and generally accepted method exists to predict its occurrence. P-wave-triggered P-wave signal averaging was performed on 54 patients before coronary artery bypass surgery to evaluate the utility of this method to predict atrial fibrillation after coronary artery bypass surgery. After excluding six patients with unevaluable P-wave signal averages and three patients with postoperative arrhythmias other than atrial fibrillation, the P-wave signal averages of 45 patients were analyzed. Sixteen patients had postoperative atrial fibrillation and 29 did not. The mean P-wave duration of the filtered, signal-averaged P wave was 163 +/- 19 msec in the 16 patients with atrial fibrillation and 144 +/- 16 msec in the 29 patients without (p < 0.005). Left atrial enlargement on the surface electrocardiogram (ECG) was the only other statistically significant variable that correlated weakly with the onset of postoperative atrial fibrillation (p = 0.04). Other clinical variables such as P-wave duration in ECG lead II, left ventricular hypertrophy on ECG, age, sex, hypertension, and left ventricular ejection fraction were not significantly different between the two groups. With a cut point of 155 msec, chi-squared analysis revealed a p value of < 0.005, yielding a sensitivity of 69%, a specificity of 79%, a positive predictive value of 65%, and a negative predictive value of 82%. Signal-averaging of the P wave in patients before coronary artery bypass surgery provides a good predictor of postoperative atrial fibrillation.

Aged↗

PubMed1995

Urinary neutral endopeptidase 24.11 activity: modulation by chronic salt loading.

Neutral endopeptidase (NEP) 24.11 is a zinc-metallopeptidase involved in the metabolism of several biologically active peptides including enkephalin, atrial natriuretic peptide, bradykinin, and endothelin. The enzyme is found in abundant amounts in the brush border of renal proximal epithelial cells. A soluble form of NEP was previously identified in human urine with characteristics similar to the renal enzyme. The present study further characterized the excreted form of NEP activity in urine of normal rats using a sensitive two-stage enzymatic assay. The response of urinary NEP to known inhibitors such as phosphoramidon and thiorphan, and its dependence on pH and salt concentration was studied. In addition, we evaluated the effects of acute and chronic changes in salt balance, induced by i.v. saline infusion and drinking of saline solution, on urinary NEP and on the activity of the enzyme in isolated proximal tubules. Our findings demonstrated that abundant NEP activity was detected in the urine of normal rats. Furthermore, chronic salt loading, but not acute salt infusion, was associated with increased activity of NEP in urine and in isolated proximal tubules, suggesting that the enzyme may be regulated by salt balance. Finally, the data suggest that urinary NEP may be used as an index of enzyme activity in the kidney.

Animals↗