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Biomedical subjects

G Clark

Publications and source records attributed to G Clark.

At least 37 records · Page 2Linked to original sources
PubMed1996

Establishment of an in vitro assay to characterize hepatitis C virus NS3-4A protease trans-processing activity.

An in vitro cleavage system was established to measure HCV NS3 protease trans-processing activity. This system utilizes purified NS3-4A protein from baculovirus, purified substrates expressed by in vitro transcription and translation and defined buffer components. The 41-residue substrates, 5A/5B and 4A/4B, were processed efficiently in trans by wild-type NS3 but not by a catalytically inactive mutant protease; radiolabel sequencing confirmed that NS3-mediated cleavage occurred at the correct cysteine/serine sites, thereby authenticating this system. Two striking features of this in vitro assay are: (1) analogous 4B/5A and 3/4A substrates cannot be processed in trans under the same conditions, and (2) in vitro cleavage of the 5A/5B and 4A/4B sites is highly dependent on the presence of NS4A, which we show is not the case in vivo.

Amino Acid Sequence↗
PubMed1996

Within-subject comparison of speech perception benefits for congenitally deaf adolescents with an electrotactile speech processor and a cochlear implant.

This study assessed speech perception benefits for three congenitally deaf adolescents who used an electrotactile speech processor (Tickle Talker(TM)) and subsequently went on to use a Nucleus Minisystem 22 cochlear implant. Both devices provided significant and comparable benefits for all children in the device plus lipreading condition. All children benefited from the additional information provided by either the Tickle Talker(TM) or the cochlear implant, and were able to perceive speech information with these devices that was not available through either aided residual hearing or lipreading. None of the three children were able to understand open-set words or sentences using either hearing aids alone or Tickle Talker(TM) alone, without the aid of lipreading. Two of the children showed significant open-set speech perception benefits while using their cochlear implant alone.

Adolescent↗
PubMed1995

A visual inspection protocol for measuring characteristics of used syringes.

This article reports on the use of a Visual Inspection Protocol (VIP) to measure observable characteristics of syringes deposited at the San Francisco needle exchange program. Syringes received by the program were evaluated by several inspectors using the VIP, and interrater reliability was assessed for each item. In Study I, syringes bearing individual markings made by the user (n = 568) were more likely to be capped at the point end and plunger end, and more often were new in appearance when compared with unmarked syringes (n = 2,820). In Study II, syringes with a short street life (n = 250) were more often new in appearance and were less likely to contain blood than syringes with a long street life (n = 246). Syringes having individual markings also show signs of more careful use, and marking syringes may represent an ad hoc HIV prevention strategy for some injection drug users.

HIV Infections↗
PubMed1994

Isolation of a maize cDNA encoding a protein with extensive similarity to an inhibitor of protein kinase C and a cyanobacterial open reading frame.

A full-length cDNA clone, Mz2-12, with a predicted amino acid sequence showing extensive similarity to the sequence of a protein inhibitor of protein kinase C, purified from bovine brain, has been isolated from maize. The sequence of Mz2-12 is also similar to an open reading frame of unknown function on a cyanobacterial dicistronic message. The extensive similarity of the three protein sequences identifies a novel class of evolutionarily conserved proteins.

Amino Acid Sequence↗
PubMed1994

Somatosensory evoked response source localization using actual cortical surface as the spatial constraint.

We localized right median nerve somatosensory evoked responses in a normal human subject using an equivalent dipole method applied to magnetic field recordings. High resolution, 3-dimensional MRI data were used to confine source locations to the cortical surface. Results localized in Brodmann area 3b corresponding to location of hand somatosensory cortex derived from direct brain stimulation studies. The solution was unique and total computational time for an exhaustive, brute-force search was small and the results realistic due to applied anatomical constraints. This study demonstrates feasibility of accurate, non-invasive, realistic localization of dynamic human cortical function using spatial constraints provided by MRI images.

Adult↗
PubMed1994

Acute haemodynamic and renal effects of cyclosporin and indomethacin in man.

A single oral dose of cyclosporin (12 mg/kg) was given to healthy volunteers (n = 9) on day 2 with or without indomethacin pretreatment (100 mg day 1 + 100 mg day 2). All parameters reported were analysed after a water-loading protocol on day 2. As a peak drug effect within 4 h of drug ingestion on day 2 cyclosporin on its own increased mean arterial pressure by 13% (P < 0.05) without causing any sodium retention or renal vasoconstriction. Indomethacin pretreatment did not accentuate this hypertensive effect of cyclosporin. The earliest renal effect observed with cyclosporin on day 2 was on water handling with a marked antidiuretic effect. Free-water clearance decreased by a maximum of 48% (P < 0.05) following cyclosporin. Indomethacin pretreatment induced a similar antidiuresis on day 2 but cyclosporin did not augment this antidiuresis any further. This suggests that inhibition of vasodilator prostaglandins within the kidneys may mediate the antidiuretic effect of cyclosporin such that in the presence of indomethacin, cyclosporin had no additional effect. The inhibition of intrarenal prostaglandins by a single dose of indomethacin did not, however, produce any acute cyclosporin-induced change in glomerular filtration rate or renal blood flow.

Administration, Oral↗
PubMed1994

Association between CYP1A1 genotype, mRNA expression and enzymatic activity in humans.

Genetic susceptibility factors may play a role in determining adverse effects of exposure to environmental toxins. As a preliminary step to a molecular epidemiological study in a population exposed to 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD), we investigated 20 healthy Caucasian volunteers with a set of putative susceptibility markers including a CYP1A1 Msp I restriction fragment length genetic polymorphism (RFLP), CYP1A1 mRNA expression, and ethoxyresorufin-O-deethylase (EROD) activity in cultured and mitogen-activated blood lymphocytes. Both basal (p = 0.008) and induced (p = 0.0001) EROD activity was significantly higher among persons with a mutation in one or both alleles of the CYP1A1 gene (variant CYP1A1 genotype). Induction in vitro by TCDD significantly increased EROD activity in both variant and wild-type CYP1A1 subjects; however, the absolute increase was greater in subjects with variant genotypes. An additive interaction between genotype and TCDD induction was suggested. Expression of CYP1A1 mRNA, both basal and induced, did not vary significantly across the genotypes.

Adult↗
PubMed1994

Roles of Triton X-100 in NADPH-diaphorase histochemistry.

Triton X-100 is widely but not universally used in NADPH-diaphorase histochemical staining. We investigated its effect on the staining and examined nitroblue diformazan (NBF) production under the influence of Triton X-100. Exposure of opossum esophagus, intestine, and colon tissues to Triton X-100 before staining enhanced staining of nerve cells and fibers and suppressed staining of non-neural structures. Long exposures and high concentrations nearly abolished the staining of non-neural structures and decreased the staining of nerves. The use of an incubation medium containing Triton X-100 achieved the best staining of nerve cells and fibers. Addition of Triton X-100 to the incubation medium changed its color from yellow to purple; in the presence of tissues, this color change occurred much more quickly. Spectral analysis showed that Triton X-100 increases the rate of NBF formation in the presence of tissue supernatant. Triton X-100 increases it less in the absence of tissue supernatant. Therefore, Triton X-100 improves the histochemical staining, probably by catalyzing the activity of NADPH-diaphorase, by keeping the extracellular NBF in solution and thus suppressing the staining of non-neural structures, and by increasing the permeability of cell membranes.

Animals↗
PubMed1993

Ligand/receptor binding for 2,3,7,8-TCDD: implications for risk assessment.

There is renewed controversy regarding safe exposure levels for dioxin. At the heart of this controversy is the hypothesis that toxic effects of dioxin are receptor-mediated and therefore a "threshold" should exist below which no toxic effects can occur. Our research focuses on the ability of dioxin to alter protein levels in rodent livers. Established effects of exposure to dioxin are the induction of cytochrome P450-1A1 and P450-1A2 and a reduction in the maximal binding of the epidermal growth factor receptor in rat livers. An initiation-promotion protocol was used to study the effects of dioxin in female Sprague-Dawley rats. Animals were administered a single initiating dose of diethylnitrosamine followed by 16 biweekly gavage doses of 2,3,7,8-TCDD. Steady-state pharmacodynamic models were fit to these data assuming a combination of Hill kinetics and Michaelis-Menten kinetics. Two classes of models were developed which postulate two different mechanisms for the constitutive expression and TCDD-induced alterations in the levels of these proteins. The results are consistent with models which follow proportionate response in the low-dose region (no threshold) and with models which allow for a low-dose threshold. In all cases studied, the best fitting model exhibited no "threshold" for the effects of TCDD on the modulation of these proteins. The finding is consistent with the knowledge that for some receptor-mediated responses, there is a proportional relationship between receptor occupancy and biological response, even at low ligand concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Regulation of epidermal growth factor receptor expression and growth by protein kinase C and retinoic acid in LLC-PK1 cells.

The importance of receptor expression and protein kinase C to epidermal growth factor (EGF)-induced cell proliferation was studied in LLC-PK1 kidney cells. These cells have both high- and low-affinity binding sites for EGF. Neither transforming growth factor-beta nor tumor necrosis factor altered EGF receptor expression. On the other hand, retinoic acid induced a concentration-dependent increase in EGF binding that was maximal at 1 mumol/L. One micromolar of retinoic acid increased EGF binding from 0.38 +/- 0.01 fmol/10(6) cells in controls to 1.10 +/- 0.03 fmol/10(6) in treated cells at 18 hours (n = 8, P < 0.001). The increase in binding was the result of an increase in the Bmax of the high-affinity receptor. The upregulation of the EGF receptor induced by retinoic acid was associated with enhanced EGF-induced growth promotion. A 45-minute incubation of cells with phorbol 12-myristate 13-acetate caused a concentration-dependent decrease in EGF binding that was prevented by a 40-hour, 2 mumol/L pre-exposure to phorbol 12-myristate 13-acetate; 10(-8) mol/L EGF also caused a downregulation of the EGF receptor that was not prevented by phorbol 12-myristate 13-acetate or retinoic acid. Downregulation of protein kinase C did not interfere in the capacity of EGF to induce growth in these cells. These studies demonstrate that EGF receptor upregulation plays an important role in the control of EGF-induced cell growth. Protein kinase C regulates EGF binding in these cells; however, it is not necessary for EGF-induced growth promotion or receptor downregulation.

Cell Division↗
PubMed1993

Detection of antisense transcripts in transgenic plants by RT-PCR.

A reverse transcriptase-polymerase chain reaction (RT-PCR) where one oligonucleotide primer is end-labelled has been used to analyse expression in transgenic plants carrying antisense gene constructs. Specific detection of both sense and antisense RNA transcripts of the spliceosomal protein gene, U2B'', was achieved using the same pair of oligonucleotide primers. To maintain specificity, a reaction step in which reverse transcriptase was inactivated and RNA digested was found to be essential.

Base Sequence↗
PubMed1993

Dose response for TCDD promotion of hepatocarcinogenesis in rats initiated with DEN: histologic, biochemical, and cell proliferation endpoints.

The present study examines the dose-response relationship for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) promotion of histologic and biochemical parameters by using a two-stage model for hepatocarcinogenesis in female Sprague-Dawley rats initiated with a single intraperitoneal dose of 175 mg of diethylnitrosamine (DEN)/kg body weight at 70 days of age. Starting 2 weeks after initiation, treatment groups of 8-10 rats were given TCDD by gavage in corn oil once every 2 weeks for 30 weeks. Doses were 3.5, 10.7, 35.7, and 125 ng TCDD/kg body weight/day. A significant body weight reduction was present in the noninitiated group that received 125 ng TCDD. Relative liver weight was statistically increased in initiated rats treated with > or = 10.7 ng TCDD and in noninitiated rats treated with > or = 35.7 ng TCDD. Histopathologic evidence of cytotoxicity was dose-related in all TCDD-treated groups. There was a statistically significant dose response in the bromodeoxyuridine (BrdU) S-phase labeling index (LI) in the DEN-initiated rats (p < 0.01) and a marginally significant trend in the saline-treated rats (p = 0.10), but proliferating cell nuclear antigen S-phase LI and growth fraction within altered hepatic foci showed no increase. Among the DEN-initiated groups there was a significant increase in glutathione S-transferase altered hepatic foci stereological parameters in the 125 ng TCDD group. This study demonstrates that dose-response relationships for TCDD's effects on cell proliferation growth of altered hepatic foci are different from previously reported effects on P450 gene expression, indicating that different biological or biochemical responses may exhibit different dose-response relationships.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗