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Biomedical subjects

R D Isaacs

Publications and source records attributed to R D Isaacs.

18 recordsLinked to original sources
PubMed1994

Borrelia burgdorferi bind to epithelial cell proteoglycans.

Borrelia burgdorferi adhere to mammalian cells in vitro but neither the ligand(s) nor the receptor(s) has (have) been clearly established. Using an in vitro attachment-inhibition assay, a B. burgdorferi attachment mechanism has been identified. Heparin, heparan sulfate, and dermatan sulfate reduced the attachment of virulent B. burgdorferi strain 297 to HeLa cells by approximately 60%. In addition, virulent, but not avirulent, B. burgdorferi strains B31, N40, and HB19 demonstrated heparin attachment-inhibition. Attachment to Chinese hamster ovary cells deficient in heparan sulfate proteoglycans was reduced by 68% compared to attachment to wild-type cells and was identical to attachment at maximum heparin inhibition to the wild-type cells. Pretreatment of HeLa cell monolayers with heparitinase, heparinase, and chondroitinase ABC, but not with chondroitinase AC, reduced borrelial attachment by approximately 50%. A moderately high affinity, low copy number, promiscuous B. burgdorferi glycosaminoglycan receptor was demonstrated by equilibrium binding studies. A 39-kD polypeptide, purified by heparin affinity chromatography from Triton X-100 extracts derived from virulent borrelia, was a candidate for this receptor. These studies indicate that one mode of B. burgdorferi attachment to eukaryotic cells is mediated by a borrelial glycosaminoglycan receptor attaching to surface-exposed proteoglycans on mammalian cells.

Animals↗
PubMed1991

Lipoproteins of Borrelia burgdorferi and Treponema pallidum activate cachectin/tumor necrosis factor synthesis. Analysis using a CAT reporter construct.

Lipoproteins from two pathogenic spirochetes (Borrelia burgdorferi and Treponema pallidum) induced the biosynthesis of TNF in murine macrophages and in permanently transformed macrophages of the cell line RAW 264.7. Induction was studied by measuring the secretion of biologically active TNF and by measuring the activity of the reporter enzyme chloramphenicol acetyltransferase (CAT) produced within macrophages transfected with an endotoxin-responsive CAT construct. Several lines of evidence indicated that the induction of TNF and CAT was attributable to the spirochete lipoproteins rather than to contaminating or endogenous LPS: 1) the dose response curves observed for the lipoproteins were markedly different from those obtained with LPS; 2) lipoprotein-mediated activation was unaffected by amounts of polymyxin B that completely neutralized the induction of TNF and CAT by LPS, 3) low concentrations of the lipoproteins induced TNF in macrophages from endotoxin-unresponsive C3H/HeJ mice as effectively as in macrophages from normal C3H/HeN mice, and 4) isolated spirochete lipoproteins, but not a non-lipoprotein immunogen, were potent inducers of CAT in the transformed macrophages. Moreover, LPS was not detected in the B. burgdorferi lipoprotein mixtures by Limulus amebocyte lysate assay. Proteolytic digestion of the intact bacterial protein preparations only modestly diminished their ability to activate the cells, suggesting that small lipopeptides comprise the biologically active portions of the molecules, as is the case with the murein lipoprotein of Escherichia coli. Through their ability to induce TNF production by macrophages, spirochete lipoproteins may play important roles in the development of the local inflammatory changes and the systemic manifestations that characterize syphilis and Lyme disease.

Animals↗
PubMed1990

Expression in Escherichia coli of the 37-kilodalton endoflagellar sheath protein of Treponema pallidum by use of the polymerase chain reaction and a T7 expression system.

We previously reported the complete primary structure of the 37-kilodalton endoflagellar sheath protein (FlaA) of Treponema pallidum. However, we were unable to determine the nucleotide sequence of flaA upstream of amino acid 10. The desired nucleotide sequence was obtained by use of a strategy based upon the polymerase chain reaction and was found to contain a consensus Escherichia coli promoter, a ribosomal binding site, and a 20-amino-acid signal peptide. Expression of FlaA in E. coli was achieved by cloning polymerase chain reaction-derived constructs lacking the native T. pallidum promoter into a temperature-inducible T7 expression system. Pulse-chase and ethanol inhibition analyses of protein processing in E. coli cells and minicells, respectively, indicated that processing of the FlaA precursor was incomplete. Native and recombinant FlaA were identical as assessed by antibody reactivity and sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoretic mobilities. Soluble FlaA was not detected in either the cytoplasmic or the periplasmic fractions of E. coli transformants. Fractionation of E. coli cell envelopes unexpectedly revealed that FlaA precursor and FlaA were associated with both the cytoplasmic and outer membranes. This is the first report of expression in E. coli of a T. pallidum protein which could not be cloned or expressed with its native promoter. Our data also indicate that information obtained in E. coli regarding the subcellular location of cloned treponemal proteins must be cautiously extrapolated to T. pallidum.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum.

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflagellar outer layer, or sheath, protein. Initially, a lambda gt11 clone (designated lambda A34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein. The insert from lambda A34 provided a probe with which a chimeric plasmid (pR14) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. pallidum(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pR14 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.

Amino Acid Sequence↗
PubMed1988

Wound infection with aerogenic Aeromonas strains: a review of twenty-seven cases.

Aeromonas spp. have been implicated in a wide spectrum of human disease including wound infection. A review is made of local experience of 27 wound infections from which aerogenic strains of Aeromonas were recovered; of these, Aeromonas was isolated in pure culture from five infections. Twenty-five infections were related to trauma and were community acquired, while two were probably nosocomial. Of the 25 community acquired infections, 16 were associated with contamination by soil and one with water. Twenty-six cases were surgically debrided or a limb or digit amputated, but in only one case was amputation performed because of uncontrolled infection. Fifteen patients received antibiotics inappropriate for the infection, yet the outcome was uniformly good. This series is discussed with a review of the English language literature. Aeromonas is most likely to be isolated when there is contamination of a wound by soil or water; adequate surgical debridement is probably of primary importance.

Adult↗
PubMed1988

Long-term cytotoxicity of orthodontic direct-bonding adhesives.

The purpose of this study was to examine the potential toxic effects of several orthodontic adhesives immediately after polymerization and at various time intervals up to 2 years postopolymerization by means of an in vitro overlay assay. Adhesive samples were incubated on tissue cultures containing an agar overlay with a vital dye (neutral red). Viable cells were stained red; nonviable cells were unstained. The circular pattern of nonviable cells demonstrated a zone of inhibition that was measured and compared. All materials tested showed cytotoxic effects immediately after polymerization and the toxic effect decreased with time postpolymerization. However, 2 years after initial polymerization, significant zones of inhibition indicating continued in vitro toxicity were still evident in all but one of the adhesives.

Adhesives↗
PubMed1987

Diabetic patient discharges from Middlemore Hospital in 1983.

Diabetes mellitus is a significant health problem in south Auckland. A retrospective case note review of all diabetic admissions to Middlemore Hospital in 1983 was performed to establish a database to identify current problem areas and permit ongoing analysis. Three hundred and seventeen admissions from 225 patients using 6086 hospital days were reviewed: 204 diabetes-related admissions occupied 4040 days. One hundred and nine patients were male. One hundred and fifty-seven patients were European, 45 were Maori and 21 were Polynesian. European patients were older (mean age 64 years) and lighter (mean weight 69kg) than the non-European patients (mean age 56 years, mean weight 83kg). One hundred and eighty patients had noninsulin dependent diabetes mellitus: European patients were discharged on insulin treatment in 38% of admissions compared with 17% for nonEuropean patients. The patterns of admission were similar for all racial groups except for admissions for chronic renal failure where 21 of 22 admissions were by nonEuropean patients. A comparative cost analysis was performed: admissions for peripheral vascular disease contributed 34% to the calculated total cost of diabetes-related admissions despite only accounting for 17% of admissions. Further research, especially in the nutritional field, is necessary before realistic cost-benefit interventions can be devised and implemented.

Adult↗
PubMed1987

Cystic hydatid disease in Auckland, New Zealand, 1967-1982.

The development of anthelmintic chemotherapy has provoked re-evaluation of the management of hydatid disease. We reviewed the case records of 74 patients with 93 episodes admitted to hospitals in Auckland, New Zealand, between 1967 and 1982. Median stay was 29 d (range 1-172). 46 (62%) of the patients were male: 34 (45%) were European and 38 (51%) Maori. The median age was 39 years (range 14-85). The majority of patients presented with symptoms and signs related to local effects of the cyst. 57 (77%) patients had at least one operation and while 34 (60%) had a major surgical complication, only 3 died. At follow-up in 1984, 38 (67%) of the surgical patients and 10 of the 17 (65%) who were not operated on were alive. 9 mebendazole recipients were evaluated: 5 (55%) responded symptomatically, but only one was cured by mebendazole alone. One stopped mebendazole because of side-effects. 2 patients took albendazole: one was cured and one had a symptomatic response. Thus surgery is not always needed. The role of chemotherapy requires further evaluation.

Adolescent↗
PubMed1986

Idiopathic giant cell myocarditis: case report.

Idiopathic giant cell myocarditis has not been previously reported from Australasia; a typical case is presented, and the clinical and pathological findings discussed. Possible aetiologies are discussed in the light of positive serology to coxsackie B viruses.

Adult↗