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Biomedical subjects

J Pohl

Publications and source records attributed to J Pohl.

At least 109 records · Page 6Linked to original sources
PubMed1992

Multichannel electromyographic observations in spasmodic dysphonia patients and normal control subjects.

Spasmodic dysphonia is primarily a disorder of vocalization. Increasing evidence, however, suggests that individuals with this disorder comprise a heterogeneous population characterized by abnormal motor control throughout the vocal tract. Multichannel simultaneous electromyography was performed on 11 spasmodic dysphonia patients and 10 normal awake subjects to investigate both the distribution of neuromotor abnormality within the vocal tract (eg, intrinsic and extrinsic laryngeal muscles, tongue, and palate) and the contribution of activation of higher central nervous system centers to observed abnormality. Experimental tasks ranged from vegetative (quiet breathing) to simple linguistic (short sentences). Digitized electromyographic signals were analyzed to compute the amplitude envelope and extract a set of parameters that represent amplitude characteristics. Electrode insertions were cross-validated by quantitative analysis of patterns of activation across selected reference tasks and by traditional qualitative methods. Between-group differences were found for measures of normalized median and peak token amplitudes. These differences are both task- and measure-dependent. Results highlight the complex and interactive effects of muscle, task, and quantitative measures on between-group differences.

Adult↗
PubMed1992

[Arterial hypertension. Prevention in infants with congenital urinary tract malformations].

Eighty-nine newborns and infants with congenital urinary tract malformations were treated in the childrens' hospital of the Westfälische Wilhelms-University from 1986 to 1989. Twenty patients of this group (22.5%) developed severe hypertension requiring treatment within the first year of life. Mean age of diagnosis of hypertension was 5 months (range 0.5-12 months). Median values for blood pressure at time of diagnosis were 138 mmHg (range 120-170) for systolic and 92 mmHg (range 80-110) for diastolic values. Six patients showed characteristic symptoms for hypertension such as restlessness, sweating and sleep disorders. Plasma levels of renin were obtained in 12 of 20 patients. Five patients had raised plasma renin levels. All patients with a severe hypertension were treated with one to several antihypertensive drugs. Risk factors for the development of severe renal hypertension in early infancy are cystic renal malformation, vesico-ureteral reflux, obstructive uropathy and to our experience also short term percutaneous nephrostomy in obstructive uropathy in particular in connection with pyelonephritis. Hypertension can still appear after the successful surgical correction of urinary obstruction. We describe the group of patients with severe hypertension in our study group; diagnostic principles and our therapeutic approach are explained. We conclude that early diagnosis of severe hypertension and consecutive treatment are important in infants with congenital urinary malformations.

Antihypertensive Agents↗
PubMed1991

Peptide-binding specificity of the molecular chaperone BiP.

Members of the heat-shock protein family (hsp70s) can distinguish folded from unfolded proteins. This property is crucial to the role of hsp70s as molecular chaperones and is attributable to the amino-acid specificity of the peptide-binding site. The specificity for peptide ligands is investigated using a set of peptides of random sequence but defined chain length. The peptide-binding site selects for aliphatic residues and accommodates them in an environment energetically equivalent to the interior of a folded protein.

Adenosine Triphosphatases↗
PubMed1991

Human lysosomal cathepsin G and granzyme B share a functionally conserved broad spectrum antibacterial peptide.

Human neutrophil lysosomal cathepsin G (cat G) exerts broad-spectrum antibacterial action in vitro against Gram-negative and -positive bacteria independent of its serine protease activity. We recently determined that an internal peptide of cat G (HPQYNQR), obtained after digestion of cat G with clostripain, possessed broad-spectrum antibacterial action in vitro, displaying an ED50 of 5 x 10(-5) M. In order to evaluate the structure-antibacterial properties of this peptide, synthetic variants with single alanine substitutions at each position were prepared and tested for antibacterial action. We found that alanine substitution for His-1 or Tyr-4, or certain modifications of the His-1 side chain, produced nonbactericidal peptides. A hexapeptide lacking the COOH-terminal Arg-7 but not a pentapeptide lacking both Gln-6 and Arg-7 possessed in vitro bactericidal activity. Interestingly, the cat G bactericidal peptide displays similarity to sequences within other serine proteases, notably the proposed cytotoxic granzymes present in the cytolytic granules of human and mouse cytotoxic T lymphocytes. We now report that an internal peptide of one human granzyme (granzyme B) with the sequence of HPAYNPK also displays bactericidal action in vitro. Our results suggest that an internal antibacterial domain among human serine proteases cat G and granzyme B has been functionally conserved through evolution perhaps for the purpose of host defense against microbial pathogens and targets of cytotoxic T lymphocyte killing.

Amino Acid Sequence↗
PubMed1991

Investigation into the immunological effects of miltefosine, a new anticancer agent under development.

Miltefosine is the prototype of alkylphosphocholines, a new class of anticancer agents related to alkyl-lysophospholipids. These agents were considered to possess potent immunomodulatory properties. Miltefosine was highly active against the human KB tumour xenograft in nude mice, leading to growth inhibition as well as regression of large established tumours, which suggested that its mode of action was not mediated by the T cell system. In vivo natural killer cell activity was measured by chromium release of YAC-1 cells using spleen cells from treated animals as effector cells. Miltefosine had no significant effect on YAC-1 cytolysis. Similarly, the compound did not induce cytotoxic spleen cells against KB target cells. The results were identical when spleen cells from tumour-bearing animals were used. Humoral antibody production in rats following sheep red blood cell immunization was not changed by miltefosine pretreatment. Finally, the in vitro phagocytic activity of mouse bone marrow macrophages was not stimulated but rather inhibited in a dose-dependent manner. In conclusion, there is no experimental evidence that the miltefosine action is mediated by the host immune system and no major immunotoxicity was observed.

Animals↗
PubMed1991

Child sexual abuse prevention: evaluation and one-year follow-up.

Because of the high incidence and negative psychological consequences of child sexual abuse, prevention programs have been developed and implemented nationwide. Few programs, however, have been comprehensively evaluated. In this multimodal study, a 3-session adaptation of the Feeling Yes, Feeling No curriculum was provided to 286 third and fourth graders from four schools whose responses were compared to 113 delayed-treatment control children from two schools. Program impact was assessed using a knowledge scale, the State-Trait Anxiety Inventory for Children, a videotape vignettes measure, a parent questionnaire, and disclosure data. Treatment children exhibited significantly greater knowledge and better ability to discriminate safe from unsafe situations on the video measure than control children at posttesting. These gains were maintained at 6-week follow-up testing. There were no differences in treatment and control children's self-reported anxiety or parents' reports of negative emotional/behavioral consequences, which were minimal. Over 5% of participating children reported ongoing or past sexual abuse. In the One-Year Follow-Up Study, children's knowledge gains and prevention skills scores on the video measure were maintained at one-year follow-up. A 1-session "booster shot" program further enhanced children's safety discrimination skills on the video measure.

Adaptation, Psychological↗
PubMed1991

In vivo measurement of force in the ureter. I. Basics of a new measurement principle.

A new device for the simultaneous in vivo measurement of pressure and developed force in the ureter is introduced. The 5-French catheter was cystoscopically inserted into the ureters of 12 dogs under anesthesia. The forms of the peristaltic waves for pressure and force show considerable differences. A basal force analogous to the basal pressure is only rarely measurable. During the systole, the developed force describes the pure muscle contraction process. The results are dependent on the location of the probe. Our new method is able to give information about the extent to which a ureter possesses the capacity to effectively close its lumen. This function, which is so decisive in urinary transport, can be determined not only qualitatively but also quantitatively.

Animals↗
PubMed1991

In vivo measurement of force in the ureter. 2. Influence of urinary flow.

By means of a newly developed measuring catheter the occluding force of the ureter under changing diuresis conditions was investigated using 12 canine ureters. Even under extremely high flow rates of some 50 times normal values, the ureter returned after short phases of noncoaptive transport to active bolus transport. No direct connection between flow rate and basal pressure on the one hand and the form of transport on the other hand was observable. Only after prolonged continuous loading on the ureter with high flow rates was a loss of occluding forces observed with the noncoaptive form of transport, then taking on more significance.

Animals↗
PubMed1991

S-S bridges of cathepsin B and H from bovine spleen: a basis for cathepsin B model building and possible functional implications for discrimination between exo- and endopeptidase activities among cathepsins B, H and L.

Bovine spleen cathepsin B contains 7 disulfide bridges. Using different chemical and enzymatic cleavage methods we isolated fragments representing the individual disulfides: Cys14-Cys43, Cys26-Cys71, Cys62-Cys128, Cys63-Cys67, Cys100-Cys132, Cys108-Cys119, and Cys148-Cys252. A similar line of approach was applied to determine the S-S bridges of bovine spleen cathepsin H: Cys23-Cys66, Cys57-Cys99, Cys157-Cys207, and Cys212-Cys5A, where Cys5A is located in the propart portion of the procathepsin H chain. On the basis of the knowledge of the S-S bridges of cathepsin B a novel sequence alignment of papain and cathepsin B has been proposed. This enabled us to construct a reasonable 3D-model of cathepsin B and propose the region (a 18 residue insertion between Glu89 and Gly90 of papain) responsible for the carboxypeptidase activity of cathepsin B functioning as a "closure". A similar approach was applied to explain the aminopeptidase activity of cathepsin H. A general model of steric regulation of accessibility of the preformed "endopeptidase-like" binding cleft by distant parts of the polypeptide chain of the proteinases discussed is proposed as a factor determining the mode of binding and thus cleavage of polypeptide substrates.

Amino Acid Sequence↗
PubMed1990

Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin.

Comparison of the three-dimensional structure of bovine chymosin with the structures of homologous aspartic proteinases complexed with peptide inhibitors shows that Val111 in chymosin occupies a position between the specificity subsites S1 and S3. A mutation corresponding to Val111 to Phe has been introduced in an intermediary plasmid construct of prochymosin by bridging its unique restriction sites by a synthetic mutant oligonucleotide duplex. A prochymosin fusion product was expressed in Escherichia coli in such a way that the extension and substitution of the propart does not interfere with the activation of the zymogen. After activation of the crude prochymosin, the enzyme was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as ligand. This procedure provided large amounts of pure protein as judged by FPLC, the activity/protein ratio, and SDS-PAGE. The enzymatic properties were determined by using a variety of peptide substrates and inhibitors; KM values for the mutant enzyme were approximately twice those of the wild type, but the kcat values were little changed. The mutant enzyme was crystallized, X-ray data were collected to 2.0-A resolution by using a FAST area detector, and the structure was solved by using difference Fourier methods and refined to an R factor of 19.5%. The mutation leads to only local changes in conformation, with the phenylalanine side chain occupying part of the S1 and S3 pockets. This accounts for the increased KM of this mutant for a substrate with a large phenylalanine side chain at P1. It is also consistent with the higher affinity of the mutant for an inhibitor with small side chains at P1 and P3 when compared with the wild-type enzyme.

Amino Acid Sequence↗
PubMed1990

Amino acid sequence of CAP37, a human neutrophil granule-derived antibacterial and monocyte-specific chemotactic glycoprotein structurally similar to neutrophil elastase.

We report the amino acid sequence of CAP37, a human neutrophil granule protein with antibacterial and monocyte-specific chemotactic activity. CAP37 is a single-chain protein consisting of 222 amino acid residues. It has three N-glycosylation sites, at Asn residues 100, 114 and 145. Some species of CAP37 are glycosylated at all three sites; some at Asn-114 alone, others at Asn-114 and Asn-110 or Asn-145. CAP37 has 45% sequence identity to human neutrophil elastase, and 30-37% identity to several other granule serine proteinases. Despite these similarities, CAP37 is not a serine proteinase because the active site residues serine and histidine are replaced.

Amino Acid Sequence↗
PubMed1990

Distinct chromatographic forms of human hemi-myeloperoxidase obtained by reductive cleavage of the dimeric enzyme. Evidence for subunit heterogeneity.

The enzyme myeloperoxidase (MPO) is a functionally important glycoprotein of neutrophilic granulocytes and occurs in three major isoforms (forms 1, 2, and 3) that are dimeric structures composed of two heavy subunit-light subunit protomers, each of which is associated with a chlorine-like prosthetic group. In the present study, highly purified MPO isoforms were obtained from the cells of a single normal donor, and each protein was subjected to reductive alkylation under nondenaturing conditions. The resulting enzymatically active protomers were separated from unreacted dimer using gel filtration chromatography. Use of a fast protein liquid chromatography cation exchange system with a Mono S matrix revealed heterogeneity of the protomers, and allowed essentially complete resolution of the protomers of MPO form 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two resolved protomeric species under reducing conditions revealed small but reproducible differences in the Mr of their heavy subunits (59,000 and 57,000). Treatment with either endo-beta-N-acetylglucosaminidase or peptide N-glycohydrolase F reduced the Mr of each heavy subunit by approximately 3000 but did not change their relative electrophoretic mobilities. Heavy and light subunits were prepared from each of the MPO isoforms by reductive alkylation under conditions that allowed full retention of the prosthetic group with the heavy subunit. Reverse-phase chromatography and amino-terminal sequencing showed that each MPO isoform contained one major species of light subunit and several minor species. No differences in peroxidatic activity or inhibition by salicylhydroxamic acid were observed among any of the MPO isoforms or resolved protomers, but the latter were considerably more heat labile than dimeric forms of the enzyme and a monomeric form isolated from HL-60 cells. This is the first report of the isolation and partial characterization of distinct protomers from a single isoform of human MPO and suggests that the structure of MPO is more complex than considered previously.

Anti-Infective Agents↗
PubMed1990

Identification of the primary antimicrobial domains in human neutrophil cathepsin G.

Lysosomal cathepsin G from human neutrophils is a chymotrypsin-like protease which also possesses antimicrobial activity. The antimicrobial activity, however, is independent of protease activity, because treatment of this enzyme with the irreversible serine protease inhibitor diisopropylfluorophosphate has no effect on its antimicrobial action. In this study, we found that digestion of cathepsin G with clostripain caused a loss of proteolytic activity in this neutrophil proteinase. However, bactericidal activity in in vitro assays against Staphylococcus aureus and Neisseria gonorrhoeae was retained. Fractionation of the clostripain-digested cathepsin G mixture yielded two distinct antimicrobial peptides. The sequences of these peptides were IIGGR and HPQYNQR (residues 1-5 and 77-83 in cathepsin G, respectively). Synthetic peptides corresponding to these sequences were also prepared and found to exert broad-spectrum antimicrobial activity in vitro, displaying conditions of temperature- and pH-dependent optima for antimicrobial action resembling that of the full-length enzyme. Depending on the target bacterial strain, these peptides exhibited antimicrobial activity between 5.0 x 10(-5) and 4.0 x 10(-4) M. Significantly, replacement of certain residues within these peptides with either alanine or valine significantly reduced their antibacterial capacities. Our studies suggest that cathepsin G has two antimicrobial sequences, either or both of which may contribute to its bactericidal activity.

Amino Acid Sequence↗
PubMed1990

The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population.

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of heme oxygenase from chick liver. Comparison of the avian and mammalian enzymes.

A major inducible form of heme oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pretreated with cadmium chloride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE-Sephacel, carboxymethyl-cellulose (CM-52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver-stained SDS/polyacrylamide gels (Mr = 33,000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase, NADPH-cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 +/- 0.5 microM; for heme in the presence of bovine serum albumin (5 mol heme/3 mol albumin) the Km was 5.0 +/- 0.8 microM; and the Km for NADPH was 6.1 +/- 0.4 microM (all values mean +/- SD, n = 3). Oxygen concentration as low as 15 microM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4; at 37 degrees C, the apparent Vmax was 580 +/- 44 nmol biliverdin.(mg protein)-1.min-1 and the molecular activity was 19.2 min-1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heme to non-biliverdin products and led to nearly stoichiometric conversion of heme to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin greater than tin protoporphyrin greater than zinc protoporphyrin greater than manganese protoporphyrin greater than cobalt protoporphyrin. Protoporphyrin (3.3 or 6.6 microM) (and several other porphyrins) and metallic ions (100 microM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 microM and totally at 15 microM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from cDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase-1 and the residues (Lys128-Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase-2.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

CAP 37, a 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases.

We have previously shown that a major granule-associated cationic protein CAP 37 (Mr = 37 kD) derived from human PMN is a monocyte-specific chemoattractant. The N-terminal amino acid sequence of this novel chemotactic protein shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, a critical substitution of a serine for a histidine at position 41, results in its lack of serine protease activity.

Amino Acid Sequence↗