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Biomedical subjects

R Kaiser

Publications and source records attributed to R Kaiser.

At least 127 records · Page 7Linked to original sources
PubMed1996

Biological testing versus sequence analysis for determination of drug resistance of human immunodeficiency virus.

Resistance of HIV-1 to 3'-azido-3'-deoxythymidine (AZT) is associated with one or more of five mutations in the reverse transcriptase (RT) segment of the polymerase gene ("AZT-specific mutations"). Therefore, sequence analysis of the proviral DNA, derived directly from the blood, is considered to replace the biological test. Additional arguments are non-cultivatable viral strains, the universality of the sequence analysis in combination therapy, and the suspicion that the cultivated virus does not represent the predominant viral variant in the blood. In this investigation, 21 strains of human immunodeficiency virus type 1 (HIV-1) were comparatively analysed by molecular and biological testing. For 12 strains, the homology of the RT gene segment between the predominant provirus in the blood and the cultivated virus was ascertained (99.72% homology). 11 strains from untreated patients or patients treated no longer than 5 months were free from AZT-specific mutations and proved to be sensitive. 10 strains from patients treated for 17 to 57 months displayed 2-4 AZT-specific mutations. However, it was not possible to correlate the degree of sensitivity to the number or the pattern of the mutations. Suppression of AZT resistance by strain-specific sequences in other parts of the gene are discussed as the reason for that discrepancy. Remarkably, the productivity of resistant virus strains could be drastically enhanced by non-inhibiting concentrations of the drug.

DNA, Viral↗
PubMed1996

Polymorphic enzymes of xenobiotic metabolism as modulators of acquired P53 mutations in bladder cancer.

Occurrence or specific types of mutations found in oncogenes or tumor suppressor genes may partially be determined by activities of toxifying or detoxifying enzymes, such as glutathione S-transferases (GST) M1 and T1, arylamine N-acetyltransferase (NAT2), microsomal epoxide hydrolase, and the cytochrome P-450 enzymes 2D6, 1A1, 2A6, and 2E1. In an explorative observational study, 69 bladder cancer patients were analysed for acquired mutations in the p53 tumor suppressor gene. The same patients were studied for the polymorphic traits of xenobiotic metabolism given above which were characterized from blood cell DNA by molecular methods. In 20 patients, single point mutations in p53 were detected whereas five patients carried two mutations; thus in total 25 mutations were detected. Twelve of these were G:C-->A:T transitions, six were A:T-->G:C transitions and seven were transversions (three G:C-->T:A, two A:T-->T:A, one G:C-->C:G, and one A:T-->C:G). There was no correlation between the types of p53 mutations and lifetime smoking or occupational history. In correlation with xenobiotic metabolism, 86% of the seven transversion mutations were found in homozygously deficient individuals for GSTM1 compared to only 44% of GSTM1 deficiency in the carriers of the 18 transition mutations of p53 (p = 0.06). A similar trend was seen for NAT2: six of the seven carriers of transversion mutations had two slow NAT2 alleles. No apparent associations were seen for the other polymorphic traits which were studied. In conclusion, low or deficient activities of two conjugating enzymes of foreign compound metabolism, GSTM1 and NAT2, may influence types of acquired mutations in p53 in bladder cancer.

Arylamine N-Acetyltransferase↗
PubMed1995

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues.

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His5,Trp7,Leu8]-GnRH, dogfish (df) GnRH; [His5,Asn8]-GnRH, catfish (cf) GnRH; and [His5,Trp7,Tyr8]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro (D)-isomers D-arginine (D-Arg) or D-naphthylalanine (D-Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of D-Nal6 into [His5,Asn8]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of D-Nal6 or D-Arg6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [D-Nal6,Pro9NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [D-Nal6,Pro9NEt]-mGnRH (kd = 0.40 +/- 0.04 and 0.55 +/- 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.

Amino Acid Sequence↗
PubMed1995

Isolation, characterization and structure of subtilisin from a thermostable Bacillus subtilis isolate.

A serine protease has been isolated and characterized from Bacillus subtilis, strain RT-5 (a thermostable soil isolate from the Tharparkar desert of Pakistan) able to grow at 55 degrees C. The primary structure was established by a combination of protein and DNA-sequence analyses. The amino-acid sequence, inhibition pattern and solubility properties identify the enzyme as a subtilisin. It has 43 amino-acid replacements toward subtilisin BPN' and as much as 83 replacements toward another subtilisin, confirming that strain variabilities are extensive between different subtilisin forms. However, the structure is identical to one of unknown functional properties deduced from DNA and is closely related to mesentericopeptidase but that homologue is not thermostable. From comparisons with that form and with subtilisin BPN', it is concluded that replacements of Ala --> Ser at positions 85 and 89, Ser --> Ala at position 88 and Asp or Ser --> Asn at position 259 may promote thermostability.

Amino Acid Sequence↗
PubMed1995

Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase.

BACKGROUND: Candida cylindracea cholesterol esterase (CE) reversibly hydrolyzes cholesteryl linoleate and oleate. CE belongs to the same alpha/beta hydrolase superfamily as triacylglycerol acyl hydrolases and cholinesterases. Other members of the family that have been studied by X-ray crystallography include Torpedo californica acetylcholinesterase, Geotrichum candidum lipase and Candida rugosa lipase. CE is homologous to C. rugosa lipase 1, a triacylglycerol acyl hydrolase, with which it shares 89% sequence identity. The present study explores the details of dimer formation of CE and the basis for its substrate specificity. RESULTS: The structures of uncomplexed and linoleate-bound CE determined at 1.9 A and 2.0 A resolution, respectively, reveal a dimeric association of monomers in which two active-site gorges face each other, shielding hydrophobic surfaces from the aqueous environment. The fatty-acid chain is buried in a deep hydrophobic pocket near the active site. The positioning of the cholesteryl moiety of the substrate is equivocal, but could be modeled in the hydrophobic core of the dimer interface. CONCLUSIONS: The monomer structure is the same in both the complexed and uncomplexed crystal forms. The dimers differ in the relative positions of the two monomers at the dimer interface. Of the 55 residues that are different in CE from those in C. rugosa lipase 1, 23 are located in the active site and at the dimer interface. The altered substrate specificity is a direct consequence of these substitutions.

Animals↗
PubMed1995

Myopic intrastromal photorefractive keratectomy with the neodymium-yttrium lithium fluoride picosecond laser in the cat cornea.

OBJECTIVE: To evaluate the refractive effects of myopic intrastromal photorefractive keratectomy (IPRK) with the neodymium-yttrium lithium fluoride (Nd-YLF) picosecond laser. METHODS: A 1053-nm Nd-YLF picosecond laser was used to produce myopic IPRK in cat corneas at threshold energy densities. Preoperative corneal topography and ultrasonic pachymetry were performed followed by IPRK in one eye each of 21 cats. No anti-inflammatory medications were administered. Postoperative corneal topography, ultrasonic pachymetry, and slit-lamp examination were performed at scheduled intervals. RESULTS: An average topographic flattening of 11.4 diopters and pachymetric thinning of 50 microns at the center of the treatment zone were observed by 6 weeks following treatment. The average diameter of the zone of flattening was 5.1 mm at 6 weeks and 6.0 mm at 12 weeks postoperatively. At 6 months postoperatively, the average central corneal power was unchanged, while the average central thickness had returned to the preoperative value. A mild subepithelial haze was detected at 1 month following IPRK, which was barely perceptible at the second through sixth months. Regression of the topographic effects of PRK was not observed. CONCLUSION: Intrastromal PRK with the Nd-YLF picosecond laser produces central flattening of the cat cornea with barely perceptible stromal haze and no regression of topographic effect after 6 months in the absence of treatment with anti-inflammatory agents.

Animals↗
PubMed1995

Intrathecal immune response in patients with neuroborreliosis: specificity of antibodies for neuronal proteins.

Cerebrospinal fluid (CSF) and serum samples of 47 patients with serologically proven neuroborreliosis were examined by Western blotting for antibodies to a crude extract of human cortex (CNS) comprising a multitude (> 40) of protein bands. Intrathecal synthesis of total immunoglobulins was determined by the Reiber formula and of autoantibodies to CNS proteins by enzyme-linked immunoassay (ELISA) and by Western blotting. Employing ELISA, intrathecal synthesis of autoantibodies (IgG, IgM and/or IgA) was demonstrated in 40 of 47 patients with neuroborreliosis (85%), in 5 of 40 with multiple sclerosis (12%), and in 22 of 40 with viral meningoencephalitis (55%). Of 40, 35 and 15 patients with neuroborreliosis and an intrathecal synthesis of total IgG, IgM or IgA, 20 revealed an intrathecal production of IgG antibodies (50%), 24 of IgM antibodies (68%) and 6 of IgA autoantiodies (40%) in the CSF. The specificity of autoantibodies differed greatly between most patients. Of 24 different CNS proteins which elicited an immune response in various patients, identities could be determined only for the myelin basic protein (5 of 40) and for the three neurofilament proteins (NF-68, NF-150, NF-200) (13 of 40 patients). In this limited number of patients no significant correlation between individual clinical symptoms and certain autoantiodies could be detected. The higher frequency of intrathecally produced autoantibodies in patients with neuroborreliosis is assumed to result from mitogenic rather than specific activation of autoreactive B-cell clones by Borrelia burgdorferi. The pathogenic relevance of these autoantibodies remains to be determined.

Antibody Specificity↗
PubMed1995

Rat corticostatin R4: synthesis, disulfide bridge assignment, and in vivo activity.

We have synthesized significant amounts of the most potent member of the rat corticostatins that inhibits ACTH-induced corticosteroid and compared its structure to that of the natural hormone. The cystine bridging arrangement that corresponds to that reported for a human defensin (3-31, 5-20, 10-30) was determined. The in vitro corticostatic activity of the synthetic rat corticostatin R4 paralleled that of the natural R4. Biological studies in vivo showed that doses of 8 or 12 mg corticostatin/kg effectively interfered with corticosterone release in stressed rats. We conclude that in the assays that were used, the biological activity of the synthetic and natural molecules was identical. The availability of significant amounts of synthetic material will make possible studies investigating the physiological role played by corticostatins in modulating the activity of the hypothalamic-pituitary-adrenal axis.

Adrenal Glands↗
PubMed1995

Antibody reactivity to individual structural proteins of measles virus in the CSF of SSPE and MS patients.

BACKGROUND: Chronic progressive disorders of the central nervous system (CNS) impose diagnostic problems, particularly in younger patients. The demonstration of antibodies against measles virus (MV) in the cerebrospinal fluid (CSF) plays a major role in the laboratory diagnosis of subacute sclerosing panencephalitis (SSPE) as well as multiple sclerosis (MS). OBJECTIVES: Because intrathecally synthesized antibodies against MV can be found in both diseases, it is necessary to establish easy and reliable methods to improve the differential diagnosis. STUDY DESIGN: Seventy-one paired serum/CSF samples obtained from patients with the diagnosis of SSPE (n = 23), MS (n = 14), or acute postinfectious measles encephalitis (APME, n = 8) have been examined. The reactivity of intrathecally synthesized immunoglobulin to individual recombinant MV structural proteins was assessed using Western blot analysis, ELISA as well as isoelectric focusing (IEF). RESULTS: All CSF samples obtained from patients suffering from SSPE showed a strong antibody response to MV-nucleocapsid (N) and phosphoprotein (P). Sera from 15 of the 23 SSPE patients were reactive to MV-fusion protein (F). Faint reactivity was obtained against MV-matrix (M) or hemagglutinin protein (H) in the minority of samples (40 and 20%, respectively). CSF samples of MS patients only revealed a clear response to N, and in two cases to F. The other proteins were not recognized in the CSF samples of MS patients. In contrast to SSPE, the IEF of CSF from MS patients revealed only few MV-specific oligoclonal bands. In the CSF samples from APME patients, intrathecal MV antibodies were not detected. CONCLUSIONS: This study shows that discrimination between SSPE and MS can be achieved in doubtful cases by IEF using MV-N, P and F proteins.

Journal Article↗
PubMed1995

Intrathecal immune response in neuroborreliosis: importance of cross-reactive antibodies.

The intrathecal IgG response to Borrelia burgdorferi was evaluated in 35 patients with neuroborreliosis (NB). Samples were tested with and without preabsorption with Treponema phagedenis. Specific antibody concentrations in the CSF and serum were determined by ELISA. The antibody index (AIBb = QBb/QIgG) was calculated from the ratio between the CSF/serum quotients for specific antibodies (QBb) and total IgG (QIgG). Intrathecal synthesis of B. burgdorferi antibodies was demonstrated in 31 samples before and in 24 samples after preabsorption with T. phagedenis. The clonal distribution of intrathecally produced IgG antibodies was determined by isoelectric focusing combined with affinity blotting. B. burgdorferi-specific oligoclonal IgG bands occurring predominantly in the CSF were demonstrated in 32/35 patients. In 29/32 samples, the major proportion of these bands also reacted with T. phagedenis. Preabsorption of samples with T. phagedenis removed a considerable share of bands reacting with B. burgdorferi. In patients with neurosyphilis, intrathecal synthesis of antibodies with specificity for B. burgdorferi was demonstrated in 7/10 samples before, and in no sample, after preabsorption of cross-reactive antibodies. Due to the lower sensitivity in determining the AIBb (-20%), preabsorption of cross-reactive antibodies cannot be generally recommended. In all patients with suspected neuroborreliosis, but uncommon neurological symptoms and missing anamnestic data concerning a tick bite or erythema migrans, neurosyphilis should be excluded by a negative TPHA test.

Adult↗
PubMed1995

The genetic diversification of the HIV type 1 gag p17 gene in patients infected from a common source.

An evolutionary analysis was undertaken of HIV-1 gag p17 sequences taken from a small cohort of hemophilia B patients infected from a common batch of clotting factor concentrate. The sequence population found at seroconversion was highly homogeneous, suggesting that the infecting batch also contained little sequence variation. Genetic diversification was found in follow-up sequences taken approximately 3 years later and was generally found to be complex. Greater rates of synonymous to nonsynonymous substitution were observed, especially when comparing distantly related isolates, and the rate of synonymous substitution was used to estimate times of divergence for a number of isolates of HIV-1 including the origin of the subtypes A to H. The p17 region is therefore proposed as a useful marker for future epidemiological studies.

Amino Acid Sequence↗
PubMed1995

Preclinical evaluation of HBY 097, a new nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 replication.

HBY 097 [(S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3, 4-dihydroquinoxaline-2(1H)-thione] was selected from a series of quinoxalines as a nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (NNRTI). HBY 097 was shown to be a highly potent inhibitor of HIV-1 induced cell killing and HIV-1 replication in a variety of human cell lines as well as in fresh human peripheral blood lymphocytes and macrophages. The compound was also active against a variety of clinical isolates of HIV-1 including different HIV-1 subtypes and viruses resistant to 3'-deoxy-3'-azidothymidine. Mutant reverse transcriptases which arise as a consequence of treatment with other nonnucleoside inhibitors of HIV-1 reverse transcriptase were still inhibited by HBY 097 at relatively low concentrations. An HIV-1MN variant resistant to inhibition by HBY 097 displayed in the reverse transcriptase gene a mutation causing a substitution at position 190 of a glutamic acid for a glycine residue (G190 --> E), which is characteristic for quinoxaline derivatives. The drug was demonstrated to possess a favorable toxicity profile and to show good oral bioavailability in both mice and dogs. As a consequence of its outstanding properties, HBY 097 was selected for further development and is at present undergoing clinical trials.

Animals↗
PubMed1995

Ligand requirements of the human corticotropin-releasing factor-binding protein.

CRF-binding protein (CRF-BP), identified as a 37-kilodalton human serum protein, binds human (h) CRF (Kd = 0.17 +/- 0.01 nM) and blocks hCRF's ability to stimulate ACTH release by pituitary cells in vitro. The present study examines ligand requirements of CRF-BP by testing the affinity of recombinant CRF-BP for synthetic analogs of CRF and peptides in the CRF family. The relative affinities of various fragments of hCRF or related peptides for CRF-BP indicate that residues 9-28 are crucial for ligand binding. CRF-BP binds human/rat CRF and urotensin-I with high affinity, sauvagine with moderate affinity, and ovine (o) CRF with low affinity. The marked difference in the affinity of CRF-BP for oCRF (Ki = 1100 +/- 97 nM) compared to hCRF (Ki = 0.17 +/- 0.01 nM), when considered with the importance of the central domain, suggests that amino acids 22, 23, and/or 25 are critical for binding. Altering oCRF residues 22, 23, or 25 individually or collectively to match those of hCRF increases the affinity of CRF-BP for these ligands; [Ala22, Arg23, Glu25]oCRF, in which all three of these central amino acids are substituted by their hCRF counterparts, binds CRF-BP with an affinity equal to that of hCRF. CRF-BP has differential affinities for CRF receptor antagonists, binding alpha-helical CRF-(9-41) with high affinity and [D-Phe12, Nle21,38]hCRF-(12-41) with low affinity. Thus, the structural requirements for binding to CRF-BP can clearly be distinguished from those for CRF receptor recognition of both agonists and antagonists. Peptides such as hCRF-(9-33), with low biological activity but which retain high affinity for the binding protein, can competitively override the effects of CRF-BP to block CRF-induced ACTH secretion, raising the possibility that whereas endogenous CRF-BP serves to limit the distribution or duration of action of CRF, specific pharmacological inhibitors of the ligand-binding protein interaction might be used to therapeutically elevate free CRF levels.

Amino Acid Sequence↗