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Biomedical subjects

R Tobias

Publications and source records attributed to R Tobias.

At least 19 recordsLinked to original sources
PubMed1997

Removal of endotoxin from recombinant protein preparations.

OBJECTIVES: To develop an effective method to remove endotoxin from large scale E. coli recombinant protein purifications. DESIGN AND METHODS: Triton X-114 phase separation, affinity chromatography utilizing immobilized polymyxin B or immobilized histidine, were used to remove endotoxin from purified preparations of recombinant CK-BB, CK-MB, CK-MM, myoglobin, and cardiac troponin I. Endotoxin levels were measured by a Limulus Amebocyte Lysate gel-clot assay. The immunoactivity of these protein preparations was determined by BIAcore analysis using a panel of in-house generated monoclonal antibodies and by a Stratus Fluorometric Analyzer. In the case of troponin I, the BIAcore was also utilized to measure troponin C interactions. RESULTS: Phase separation with Triton X-114 was the most effective method in reducing the amount of endotoxin present in the protein preparations compared to either polymyxin B or histidine affinity chromatography. With Triton X-114, the reduction in endotoxin levels was greater than 99% and recovery of the proteins after endotoxin removal was greater than 90%. All three procedures for removing endotoxin had no deleterious effects on the immunoactivity of majority proteins when tested with a panel of monoclonal antibodies. Troponin I also retained its ability to bind to troponin C in the presence of Ca2+. Recombinant CK-BB and CK-MM which were expressed in the soluble fraction of E. coli cell lysates, contained significantly higher endotoxin levels than recombinant CK-MB, myoglobin and cardiac troponin I which were expressed in the form of inclusion bodies. CONCLUSION: Of the three methods tested, Triton X-114 phase separation was the most effective way of removing endotoxin from recombinant proteins.

Biochemistry↗
PubMed1997

Association mapping of disease loci, by use of a pooled DNA genomic screen.

Genomic screening to map disease loci by association requires automation, pooling of DNA samples, and 3,000-6,000 highly polymorphic, evenly spaced microsatellite markers. Case-control samples can be used in an initial screen, followed by family-based data to confirm marker associations. Association mapping is relevant to genetic studies of complex diseases in which linkage analysis may be less effective and to cases in which multigenerational data are difficult to obtain, including rare or late-onset conditions and infectious diseases. The method can also be used effectively to follow up and confirm regions identified in linkage studies or to investigate candidate disease loci. Study designs can incorporate disease heterogeneity and interaction effects by appropriate subdivision of samples before screening. Here we report use of pooled DNA amplifications-the accurate determination of marker-disease associations for both case-control and nuclear family-based data-including application of correction methods for stutter artifact and preferential amplification. These issues, combined with a discussion of both statistical power and experimental design to define the necessary requirements for detecting of disease loci while virtually eliminating false positives, suggest the feasibility and efficiency of association mapping using pooled DNA screening.

Case-Control Studies↗
PubMed1997

Unravelling a complex trait: the genetics of insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM), also known as type 1 or juvenile diabetes, is one of the first disorders with a complex genetic basis that researchers have begun to unravel. More than 20 years ago, the HLA region was found to contain a major locus that influences predisposition to IDDM, and a decade ago a locus with a smaller effect was identified in the insulin-gene region. With the advent of numerous microsatellite markers suitable for genome screening, an additional 6 loci that influence susceptibility to IDDM have been reported since late 1994. This paper summarizes that progress, with particular emphasis on research conducted by Field and associates. Some of the new loci appear to predispose people to IDDM independently of HLA and may be important factors in families with IDDM who lack strong HLA susceptibility. Other loci may interact to cause susceptibility, and specific combinations may be especially diabetogenic. Although isolating the actual predisposing genes in IDDM is more difficult than isolating those involved in single-locus genetic disorders, the fact that the genes can be identified with the use of a reasonable number of families is very encouraging for future research on other genetically complex disorders.

Diabetes Mellitus, Type 1↗
PubMed1996

Susceptibility to insulin-dependent diabetes mellitus maps to a locus (IDDM11) on human chromosome 14q24.3-q31.

To locate genes predisposing to insulin-dependent diabetes mellitus (IDDM), an autoimmune disorder resulting from destruction of the insulin-producing pancreatic cells, we are testing linkage of IDDM susceptibility to polymorphic markers across the genome using families with two or more IDDM children. A new susceptibility locus (IDDM11) has been localized to chromosome 14q24.3-q31 by detection of significant linkage to microsatellite D14S67, using both maximum likelihood methods (LODmax = 4.0 at Theta = 0.20) and affected sib pair (ASP) methods (P = 1 x 10(-5)). This represents the strongest reported evidence for linkage to any IDDM locus outside the HLA region. The subset of families in which affected children did not show increased sharing of HLA genes (HLA sharing </= 50%) provided most of the support for D14S67 linkage (LODmax 4.6 at Theta = 0.12; ASP P < 5 x 10(-6)). There was significant linkage heterogeneity between the HLA-defined subsets of families (P = 0.009), suggesting that IDDM11 may be an important susceptibility locus in families lacking strong HLA region predisposition.

Base Sequence↗
PubMed1996

[A case of necrotizing meningoencephalitis in a pug dog (pug dog encephalitis--PDE)].

A three-year-old female pug dog was euthanized because of recurrent seizures. Pathological examination revealed severe multifocal necrosis confined to the cerebrum. Histologically, areas of malacia of different stages, with prominent gitter cell infiltration were observed. Furthermore, there was severe rarefication resulting in cavities separated by tissue bridges and blood vessels. In the adjacent tissue and in the meninges a moderate to severe non-purulent meningoencephalitis was evident. The lesions are consistent with those reported for pug dog encephalitis (PDE). In the present paper, the first case of PDE in Germany is described and an overview of the clinical symptoms, the neuropathological findings of this etiologically unknown disease, the differential diagnoses and the possible pathogenesis is given.

Animals↗
PubMed1994

A locus on chromosome 15q26 (IDDM3) produces susceptibility to insulin-dependent diabetes mellitus.

To identify new loci predisposing to insulin-dependent diabetes mellitus (IDDM), we have investigated 250 families with more than one diabetic child. Affected sibling pair linkage analysis revealed strong evidence for an IDDM susceptibility locus near D15S107 on chromosome 15q26 (P = 0.0010) termed IDDM3. Families less predisposed through genes in the HLA region provided most of the evidence for linkage. In these families, discordant sibling pairs also showed linkage (P = 0.0052), and sibling pair disease concordance or discordance was strongly related to the proportion of genes the pair shared at D15S107 (P = 0.0003). Our study also revealed evidence for an IDDM locus on chromosome 11q13 (IDDM4) using affected siblings (P = 0.0043), but no evidence using discordant siblings.

Canada↗
PubMed1993

Polygalacturonase isozymes from Botrytis cinerea grown on apple pectin.

Five isozymes (four acidic and one basic) of polygalacturonase were separated by chromatofocusing from the culture filtrate of Botrytis cinerea grown on apple pectin. The isozymes, designated as Polygalacturonase I to V, have isoelectric points of 9.7, 4.9, 4.6, 3.7, and 2.7, respectively, with Polygalacturonase III exhibiting the highest specific activity. Polygalacturonase I appeared to function as an endo-polygalacturonase while the other four isozymes act as exo-polygalacturonases. The pH optima of the isozymes range from pH 4.5 to 5.5 with Polygalacturonase V being less sensitive to higher pH compared with the rest of the isozymes.

Chromatography, Thin Layer↗
PubMed1987

Nocturnal dosage regimen of sucralfate in maintenance treatment of gastric ulcer.

Sixty-six patients with recently healed gastric ulcers were entered into a double-blind, placebo-controlled, six-month maintenance trial to determine whether sucralfate 2 g at night reduces the liability to recurrent ulceration. Thirty-three patients were randomly assigned to treatment with sucralfate and 33 were assigned to placebo. Endoscopy was performed at the time of entry into the study and at 24 weeks, or earlier if clinical relapse occurred during this period. Of the patients available for analysis, endoscopic recurrences were found in eight of the 29 patients (28 percent) randomly assigned to sucralfate and in 15 of the 27 patients (56 percent) assigned to placebo. Eight of the recurrences noted at 24 weeks were asymptomatic and, of these, five were in the placebo-treated group. The cumulative relapse rate at 24 weeks was significantly lower in the sucralfate-treated group (p less than 0.05), and the Cox-Mantel text showed a significant difference between the cumulative relapse curves of the two treatment groups over the 24-week period (p less than 0.05). The results indicate that a single maintenance dose of sucralfate 2 g at night reduces the relapse rate in patients with recently healed gastric ulceration.

Antacids↗
PubMed1986

Prospective controlled trial of transhepatic biliary endoprosthesis versus bypass surgery for incurable carcinoma of head of pancreas.

53 patients with obstructive jaundice due to incurable carcinoma of the head of the pancreas were randomly allocated to percutaneous transhepatic placement of a permanent biliary endoprosthesis (PTE) or bypass surgery. After exclusions 25 patients in each group were treated. Technical success was achieved in 21 patients (84%) in the PTE group and 19 (76%) in the surgery group. The incidence of postprocedural complications (PTE 7, surgery 8) and 30-day mortality (PTE 2, surgery 5) were similar. Recurrent jaundice occurred more often in the PTE (8/21) than the surgery group (3/19). Duodenal obstruction developed in 3 patients in the PTE group. Although the initial median postprocedural hospital stay was significantly shorter in the PTE than the surgery group, the difference was no longer significant when readmissions for blocked endoprosthesis and gastric outlet obstruction were taken into account. There was no difference in the median survival time in the two groups (PTE 19 weeks, surgery 15 weeks).

Aged↗
PubMed1986

A comparison of sucralfate dosage schedule in duodenal ulcer healing. Two grams twice a day versus one gram four times a day.

The conventional dosage schedule for sucralfate is 1 g 4 i.d., but a dose of 2 g 2 i.d. may be equally effective in duodenal ulcer healing. We compared the efficacy of these two regimens in duodenal ulcer healing. Seventy-seven patients with endoscopically proven duodenal ulceration were entered into a double-blind, controlled study and randomized to treatment with sucralfate 2 g 2 i.d. (on waking and at bedtime) or 1 g 4 i.d. (1/2 h before meals and at bedtime). The patients were endoscoped before entry into the study, after 4 weeks, and after 8 weeks if unhealed at 4 weeks. Of the patients considered suitable for analysis at 4 weeks, 79% (26/33) of those taking 2 g 2 i.d. had healed ulcers in comparison to 72% (23/32) of those taking 1 g 4 i.d. After 8 weeks, cumulative healing rates were 85% (28/33) and 80% (24/30), respectively. The results suggest that the more convenient dosage schedule of 2 g 2 i.d. is as effective as the 1 g 4 i.d. regimen in the short-term treatment of duodenal ulcer.

Adult↗
PubMed1983

Primary sclerosing cholangitis associated with inflammatory bowel disease in Cape Town, 1975 - 1981.

Patients with inflammatory bowel disease and serum alkaline phosphatase persistently raised to more than twice the normal level were investigated to assess the frequency of primary sclerosing cholangitis (PSC) in the Gastro-intestinal Clinic from 1975 to 1981. Twelve patients had a persistently raised alkaline phosphatase level of hepatic origin, 9 out of 250 with ulcerative colitis and 3 out of 164 with Crohn's disease. PSC was demonstrated in 8(3%) of the patients with ulcerative colitis, and carcinoma of the pancreas in the remaining 1. Three of the patients with PSC also had gallstones. The colitis antedated the biliary symptoms and signs in all but 1 patient. There was no correlation between the duration, extent and activity of the colitis and the development and outcome of the liver involvement. Investigations in the 3 patients with Crohn's disease revealed the presence of PSC in 2 (1,2%) and chronic active hepatitis in the 3rd. Of the 2 with PSC, one had cholelithiasis and has had recurrent episodes of cholangitis. The other has had only mild symptoms.

Adult↗
PubMed1982

Tuberculosis of the distal colon. A case report.

A case of tuberculosis of the distal colon is described. The presenting features and the sites of the lesions are unusual even in a country where gastrointestinal tuberculosis is common. Attention is drawn to the diagnostic difficulty encountered, as the lesion is often clinically and radiologically indistinguishable from amoebiasis, carcinoma or Crohn's disease.

Adult↗
PubMed1982

Preoperative external biliary drainage in obstructive jaundice. A prospective controlled clinical trial.

57 patients with obstructive jaundice were randomly allocated to surgery with preoperative external biliary drainage (29 patients) and without preoperative external biliary drainage (28 patients). 22 patients ultimately underwent laparotomy after a mean of 11.7 days of drainage and 25 had surgery without preoperative drainage. The postoperative complication rate was low and similar in both groups but complications associated with the drainage procedure were substantial. Perioperative mortality was 4/28 (14%) in the drainage group and 4/27 (15%) in the non-drainage group. There seems to be no advantage associated with routine preoperative external biliary drainage before surgery for obstructive jaundice.

Acute Kidney Injury↗