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Biomedical subjects

R I Roth

Publications and source records attributed to R I Roth.

32 records · Page 2Linked to original sources
PubMed1992

Ability of gonococcal and meningococcal lipooligosaccharides to clot Limulus amebocyte lysate.

We investigated whether the striking difference in severity of coagulopathy observed between bacterial sepsis involving Neisseria meningitidis and Neisseria gonorrhoeae species is related to species-dependent abilities to directly activate coagulation. Using lipooligosaccharide (LOS)-activated gelation of Limulus amebocyte lysate, we compared the relative abilities of outer membrane LOS of 10 N. meningitidis and 10 N. gonorrhoeae strains to initiate coagulation. A wide range of procoagulant potencies was observed for each species, and there was significant overlap of potencies between species. Relative biological activities did not correlate with the oligosaccharide components as defined by LOS molecular weight or specific antigenic epitopes. Purified lipid A of two LOS strains of different potency demonstrated relative procoagulant biological activities similar to those of their parent LOSs. When these lipid A preparations were further separated by thin-layer chromatography, the most polar component of each lipid A possessed the majority of the procoagulant activity. We concluded that the ability of neisserial LOS to initiate coagulation of Limulus lysate is a property of the lipid A portion of the molecule and is most likely determined by fine structural differences in the lipid A which are independent of species.

Animals↗
PubMed1992

Selective cutaneous hyperpigmentation in mice following zidovudine administration.

BACKGROUND AND DESIGN: C57BL/6N mice fed zidovudine in their drinking water develop selective hyperpigmentation of the tails and footpads. Zidovudine-fed and identical control mice were observed and sequential biopsy specimens were obtained. Routine light microscopy, electron microscopy, and image analysis of unstained biopsy specimens were used to evaluate the extent, nature, and amount of cutaneous hyperpigmentation. RESULTS: Beginning at day 14 selective hyperpigmentation of the tails and footpads of the mice was noted. Histologic evaluation revealed a gradual increase in melanin, beginning in the lower levels of the epidermis, with eventual pigmentation of the stratum corneum. Electron microscopy demonstrated a sixfold increase in melanosomes in the tail skin of the zidovudine-fed mice. Using image cytometry, melanin was quantitatively shown to increase, paralleling the clinically apparent hyperpigmentation. The hyperpigmentation was reversible on discontinuation of zidovudine. CONCLUSIONS: This animal model parallels the human in developing reversible and selective hyperpigmentation on administration of zidovudine. In this model the increased pigmentation is due to increased numbers of melanosomes within epidermal keratinocytes. Image cytometry may be useful in semiquantitatively studying the pathogenesis of various disorders of hyperpigmentation.

Animals↗
PubMed1991

An animal model for the study of azidothymidine-induced hyperpigmentation.

Mice fed azidothymidine demonstrated dramatic hyperpigmentation of their tails. Histologic examination demonstrated large quantities of melanin pigment throughout the entire epidermal layer. Control mice had scant melanin pigment localized to the basal layer. Our findings demonstrate that the mouse is a useful model for the investigation of drug-induced hyperpigmentation.

Animals↗
PubMed1990

Optimization of detection of bacterial endotoxin in plasma with the Limulus test.

Detection and quantification of bacterial endotoxin in plasma by the Limulus amebocyte lysate test (or other assays for endotoxins) is hindered by the presence of inhibitors. Treatment of plasma to overcome inhibitory activities is required before plasma can be successfully assayed for endotoxin. We have conducted an investigation comparing the three most commonly used procedures (dilution-heating, trifluoroacetic acid oxidation, and chloroform extraction) for treatment of plasma before its assay for endotoxin with the chromogenic Limulus test. Initially, conditions were optimized for treatment of plasma by each of these methods. Subsequently, a direct comparison of the three plasma treatment procedures was performed with plasma spiked with known concentrations of endotoxin. The optimized dilution-heating procedure resulted in the most sensitive detection of endotoxin, with sensitivity approximately 10 times greater than the optimized trifluoroacetic acid oxidation procedure and approximately 100 times greater than treatment of plasma by chloroform extraction. Maximal detection of low concentrations of endotoxin by the chromogenic Limulus test was obtained by dilution of plasma fourfold with 0.15 mol/L NaCl followed by heating at 60 degrees C for 30 minutes. This procedure was simple, rapid, and did not involve addition of any reagents to plasma that could potentially add contaminating endotoxin.

Animals↗
PubMed1989

Stability of gels formed following coagulation of Limulus amebocyte lysate: lack of covalent crosslinking of coagulin.

Incubation of lysates prepared from amebocytes of the horseshoe crab (Limulus polyphemus) with bacterial endotoxin results in coagulation and formation of a solid gel. Although Limulus gels remained solid indefinitely, if undisturbed, they were easily disrupted by mechanical agitation. Chemical solubility studies of gelled lysates demonstrated rapid solubilization of gels in monochloroacetic acid, a property of clots that have not been covalently stabilized; but in contrast demonstrated resistance to solubilization by urea, a property of stabilized clots. Analysis of solubilized proteins by polyacrylamide gel electrophoresis in SDS demonstrated coagulin, the designation for the activated form of coagulogen (the clottable protein) that forms a gel, only in samples derived from clotted lysate that had been previously incubated with monochloroacetic acid, but not in samples following incubation with urea, confirming the results of the chemical solubility studies. Enzymatic assays for transpeptidase (Factor XIII-like) activity in either native or gelled Limulus lysates were negative. Furthermore, analysis for covalently crosslinked peptides in gelled coagulin confirmed the absence of intermolecular gamma-glutamyl-epsilon-lysyl bonds. Therefore, the stable gels formed following coagulation of Limulus lysate by bacterial endotoxin are not covalently crosslinked.

Animals↗
PubMed1989

A modified Limulus amebocyte lysate test with increased sensitivity for detection of bacterial endotoxin.

We have developed a simple modification of the chromogenic Limulus amebocyte lysate test that increases the sensitivity for the detection of bacterial endotoxins. In this assay, free paranitroaniline, cleaved from synthetic chromogenic substrates by proteases that were generated by Limulus lysate after incubation with endotoxin, was then derived. Derivation was with p-dimethylaminocinnamaldehyde in the presence of strong acid, forming a stable Schiff base end product with much greater molar absorbancy than the parent chromogen. Conditions (times and temperatures of incubations, concentrations of reagents) for the augmented chromogenic procedure were optimized. A ten-fold or greater increase in sensitivity for bacterial endotoxin was obtained with the modified assay as compared with the standard chromogenic Limulus test, with unequivocal detection of endotoxin concentrations of less than 100 pg/ml. The greater sensitivity of this modified Limulus test increases its usefulness for a wide range of research applications and clinical investigations.

Colorimetry↗
PubMed1985

Intestinal infection with Mycobacterium avium in acquired immune deficiency syndrome (AIDS). Histological and clinical comparison with Whipple's disease.

At endoscopy, a 30-year-old man with acquired immune deficiency syndrome (AIDS), Kaposi's sarcoma, diarrhea, and unexplained malabsorption showed erythematous macular duodenal lesions consistent with Whipple's disease by histology and electron microscopy. Symptoms did not respond to tetracycline. Subsequent cultures revealed systemic Mycobacterium avium (M. avium) infection. Tissue from this patient, from patients with Whipple's disease and from a macaque with M. avium were compared. All contained PAS-positive macrophages but M. avium could be distinguished by positive acid-fast stains and a difference in pattern of indirect immunofluorescence staining with bacterial typing antisera. PAS-positive macrophages in the intestinal lamina propria are no longer pathognomonic of Whipple's disease. Ultrastructural and histological similarities between Whipple's disease and M. avium infection suggest that both are manifestations of immune deficits limiting macrophage destruction of particular bacteria after phagocytosis. M. avium must be considered in the differential diagnosis of diarrhea in patients with AIDS and other immunosuppressed conditions.

Acquired Immunodeficiency Syndrome↗
PubMed1983

Effect of cholesterol feeding on the distribution of plasma lipoproteins and on the metabolism of apolipoprotein E in the rabbit.

Changes in lipoprotein distribution and in apolipoprotein E metabolism were studied in rabbits fed a diet containing 1% cholesterol. Lipoprotein distribution was monitored by rate zonal ultracentrifugation, gel filtration chromatography, and electrophoretic techniques. Normolipidemic rabbit plasma contained VLDL, IDL, LDL, and HDL. Within 1 week on the 1% cholesterol diet, the d less than 1.006 g/ml material was greatly elevated, and the lipoproteins of higher density (LDL and HDL) decreased below levels of detection. Cholesteremic d less than 1.006 g/ml material was cholesteryl ester-rich, triglyceride-poor, and contained particles of Sf 20 to greater than 400. Upon diet normalization, the LDL and HDL reappeared within 2-4 weeks accompanied by a decrease in the d less than 1.006 g/ml material. The metabolism of apoE was studied by injecting purified and 125I-labeled apoE into rabbits and following the clearance of the tracer. ApoE in the normolipemic rabbit demonstrated a fractional catabolic rate (FCR) of 0.132 hr-1 and a half-life (t1/2) of 10.3 hr. ApoE in the hypercholesterolemic rabbit demonstrated an FCR of 0.055 hr-1 and a t1/2 of 49.5 hr. ApoE concentrations in the plasma as estimated by electroimmunoassay were 19.5 mg/dl in the control rabbit and 199 mg/dl in the hypercholesterolemic rabbit. From these data, absolute synthetic rates of 20.3 mg/kg per day and 86.1 mg/kg per day were calculated for the control and the hypercholesterolemic rabbit, respectively. We conclude that the cholesterol-supplemented diet caused pronounced elevation of apoE in the plasma due to overproduction of the protein.

Animals↗
PubMed1980

Influence of an atherogenic diet on the structure of swine low density lipoproteins.

Five groups of three swine each were fed a basal diet supplemented with 15% tallow and either 0.0, 1.0, 1.5, 2.0%, or 2.5% cholesterol. The animals were studied over a period of 9 weeks to observe changes in plasma lipids and low density lipoproteins (LDL). At the end of the study period, LDL was analyzed by rate zonal ultracentrifugation, characterized chemically, and examined by differential scanning calorimetry. Within 3 weeks of initiation of the cholesterol-supplemented diets, there was an increase in the plasma levels of cholesterol and total LDL. LDL from swine fed the basal diet and the basal diet plus 1.0% cholesterol appeared in two LDL populations (LDL1 and LDL2) when analyzed by rate zonal ultracentrifugation. After diets containing 1.5, 2.0, and 2.5% dietary cholesterol, there was an increase in the mean flotation rate of total LDL which shifted to a lower density. LDL1 from the high cholesterol diets had a decreased triglyceride content when compared to those of the low cholesterol diets. When examined by differential scanning calorimetry, the LDL1 from the animals fed at least 1.5% cholesterol had phase transitions above body temperature, whereas the LDL from those fed 0 and 1.0% cholesterol had phase transitions below 37 degrees C. In contrast, the thermal behavior and fatty acid compositions of the extracted cholesteryl esters of the LDL obtained after the five different diets were not remarkably different. Since LDL triglyceride and cholesteryl esters are predicted to coexist in a common phase in the LDL core, the different thermal behavior of the LDL obtained after diets with different cholesterol contents is due to differences in triglyceride content which are a secondary effect of cholesterol-feeding. From these data we conclude that dietary cholesterol increases plasma LDL content, decreases LDL triglyceride content, and alters the particle structure. These changes in lipoprotein structure may contribute to the known development of atherosclerosis in cholesterol-fed swine.

Animals↗
PubMedDate not supplied

Amplification of chromogenic staining of proteases within electrophoretic gels.

A method is described for amplification of bands generated by chromogenic substrates following their reaction with proteases within electrophoretic gels. Chromogenic substrates, consisting of synthetic peptides containing the chromophore 4-nitroaniline (paranitroaniline, PNA), are applied directly to the surface of agarose or acrylamide gels. Protease activity within the gel results in the enzymatic amidolysis of the chromogenic substrates, releasing free PNA. The yellow PNA chromogen is then derivatized by reaction with p-dimethylaminocinnamaldehyde (DACA) to form a purple Schiff base compound. The resultant complex has a significantly higher molar absorbancy than the original PNA chromogen, thus increasing the sensitivity for low levels of amidolytic activity. The derivatized chromogen is easily visualized for photography.

Aniline Compounds↗
PubMedDate not supplied

Production of modified crosslinked cell-free hemoglobin for human use: the role of quantitative determination of endotoxin contamination.

In vivo toxicity remains a major barrier to the successful use of cell-free hemoglobin (Hb) as an oxygen carrier in humans. Bacterial endotoxin (lipopolysaccharide, LPS) is known to contribute to the in vivo toxicity of Hb preparations, and the prevention of LPS contamination is a critical aspect of the effort to create an efficacious Hb blood substitute. Limulus amebocyte lysate assays for endotoxin were performed on multiple Hb samples from 26 independent production runs for the preparation of human crosslinked cell-free hemoglobin (alpha alpha Hb). High levels of LPS contamination (1- > 100 ng/mL) of alpha alpha Hb solutions were detected in multiple samples during many of the initial production runs. It was observed that LPS contamination of alpha alpha Hb solutions could occur at any step during the production sequence. Substantial enhancement by alpha alpha Hb of the biologic effects of LPS was demonstrated by two independent assays for endotoxin (the Limulus amebocyte lysate test and a mononuclear cell procoagulant assay), whereas LPS biologic activity was only slightly increased by human serum albumin and substantially diminished by IgG. These results suggest that the prevention of LPS-related toxicities in vivo may be more important to the clinical use of Hb solutions than to the use of other intravenous protein products. Therefore, it was encouraging to note that, with the careful monitoring for LPS in the production facility and in multiple samples during cell-free Hb production, sources of LPS contamination were recognized and the appropriate sites were made endotoxin-free.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗