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J Baker

Publications and source records attributed to J Baker.

At least 109 records · Page 6Linked to original sources
PubMed1997

Oligomeric forms of the 148 kDa cartilage matrix protein.

The 148 kDa cartilage matrix protein (CMP), composed of three disulphide-bonded subunits, is a cartilage-specific glycoprotein found in association with fibrils of type II collagen and possibly with aggrecan. It is probable that CMP serves a structural role. As cartilage ages, an increasing proportion of the CMP becomes insoluble and resistant to extraction. In the present study, the isolation of CMP has been improved by inclusion of a hydrophobic chromatography step, thereby removing the remaining traces of collagen and proteoglycan. Evidence of self-association of CMP is presented. Higher-molecular-mass forms of CMP, ranging in apparent molecular mass from 270 to 510 kDa and separated by SDS/PAGE, were located using a specific anti-CMP monoclonal antibody. Both CMP and its oligomeric forms are reducible to 52 kDa subunits, and only trace amounts of other proteins. The formation of oligomers, which may constitute 23% of the total cartilage matrix protein, could occur as a byproduct of the normal biosynthetic trimerization of subunits. Alternatively, the oligomers may represent a step toward the age-related cross-linking and insolubilization of CMP.

Animals↗
PubMed1997

Antiviral potency of drug-gene therapy combinations against human immunodeficiency virus type 1.

Gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infection using intracellular immunization strategies is currently being tested in clinical trials. With the continuing development of potent antiretroviral drugs (e.g., reverse transcriptase [RT] and protease [PR] inhibitors), it is likely that HIV-1 gene therapy will be applied to humans concurrently receiving such antiretroviral medication. In this study, we assessed the in vitro antiviral efficacy of two gene therapy strategies (trans-dominant RevM10, Gag antisense RNA) in combination with clinically relevant RT (AZT, ddC) or PR (indinavir) inhibitors. Retrovirally transduced, human T cell lines expressing antiviral gene constructs were inoculated with high doses of HIV-1HXB3 in the presence or absence of inhibitors. The combination of RevM10 or Gag antisense RNA with antiviral drugs inhibited HIV-1 replication 10-fold more effectively than the single antiviral drug regimen alone. More importantly, we also addressed whether gene therapy strategies are effective against drug-resistant HIV-1 isolates. Both the RevM10 and Gag antisense RNA strategies showed antiviral efficacy against several RT inhibitor-resistant HIV-1 isolates equivalent to their inhibition of HIV-1HXB3 replication. In summary, our data demonstrate the greater than additive antiviral efficacy of gene therapy strategies and RT or PR inhibitors, and that gene therapy approaches are effective against drug-resistant HIV-1 viral isolates.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Plasma clearance of cholyl-lysyl-fluorescein: a pilot study in humans.

BACKGROUND/AIMS: Cholyl-lysyl-fluorescein is a fluorescent analogue of the natural bile acid, cholyl glycine. In vivo and in vitro studies showed that this analogue has many biological characteristics similar to cholyl glycine. In this study we analysed cholyl-lysyl-fluorescein plasma clearance in six healthy volunteers as a potential quantitative liver function test. METHODS: The compound in water for injection was administered as an i.v. bolus in the dose of 0.02 mg/kg b.w. RESULTS: The plasma elimination curve showed rapid, intermediate and slow phases of clearance. Half-life (T1/2 time) for the first (t1/2 1st phase), second (t1/2 2nd phase) and third (t1/2 3rd phase) phases of elimination was 1.7+/-0.9 min, 6.7+/-1.6 min and 68+/-17 min, respectively. Ninety-minute plasma retention (% dose/l plasma) was 2.2%. Cholyl-lysyl-fluorescein volume of distribution and residual fluorescence after 60 min were similar to the data obtained by others for natural or radiolabelled bile acids. In five out of six healthy volunteers a 25-fold higher dose of cholyl-lysyl-fluorescein (0.5 mg/kg b.w.) was injected to estimate the safety margins of the compound. This dose was eliminated at a disappearance rate similar to that of the dose of 0.02 mg/kg b.w. and did not cause any adverse reactions. Serum liver tests measured before and after injection did not change significantly. CONCLUSIONS: This study showed that cholyl-lysyl-fluorescein clearance is similar to the clearance of endogenous natural bile acids and may potentially offer a new, dynamic test of liver function.

Adult↗
PubMed1997

High transdominant RevM10 protein levels are required to inhibit HIV-1 replication in cell lines and primary T cells: implication for gene therapy of AIDS.

Expression of antiviral genes in CD4+ T cells has been proposed as a strategy for gene therapy of AIDS. Over the past years, we and others have developed retroviral vectors encoding the RevM10 protein, a dominant-negative mutant of the HIV-1 Rev trans-activator protein. We could demonstrate gene transfer and inhibition of HIV-1 replication in cultured T cell lines and primary T cells. However, little is known about the levels of the antiviral protein required to achieve a therapeutic effect, particularly in primary cells. In this report, we compare different vector designs with regard to expression of the antiviral gene to develop an optimal vector for clinical applications. Our results demonstrate that intracellular steady-state RevM10 protein levels expressed from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV) or mouse embryonic stem cell virus (MESV) promoters located in the long terminal repeat (LTR) were uniformly higher than from internal promoters (eg CMV, PGK). Analysis of selected vectors in acutely and chronically HIV-infected cell lines suggested that threshold levels of RevM10 expression are required to achieve inhibition of HIV replication. LTR-driven RevM10 expression also yielded high steady-state protein levels in activated primary T cells resulting in inhibition of HIV replication, and there was no apparent difference between the MoMLV, MPSV and MESV-LTR vectors. However, RevM10 expression was down-regulated in resting primary cells and consequently anti-HIV efficacy was significantly reduced. Taken together, the data suggest that relatively high steady-state levels of RevM10 protein are required to achieve inhibition of HIV replication and that the MPSV- and MESV-derived retroviral vectors show no advantage over the MoMLV-based vectors for expression of anti-HIV genes in human T cells.

CD4-Positive T-Lymphocytes↗
PubMed1997

Genotypic selection of mutated DNA sequences using mismatch cleavage analysis, a possible basis for novel mutation assays.

A novel technique for the selection of mutated DNA sequences, termed mismatch cleavage-polymerase chain reaction (MC-PCR), is proposed. The method is based on hybridizing genomic DNA with a suitable probe, several 100 bp long. Mutated DNA sequences will form mismatched heteroduplexes which are cleaved by using resolvases. Cleaved heteroduplexes are detected by ligation to an oligonucleotide adaptor and then amplified by using PCR. If practical, this technique would have considerable advantages over the restriction site mutation (RSM) method. Failure to achieve cleavage efficiencies of close to 100% will not compromise success. This is because positive signals (PCR amplification) arise from cleaved mutated sites and not, as in RSM, from DNA sequences resistant to cleavage by restriction endonucleases. Furthermore, the mutational target is much larger than in RSM. It would be possible to screen stretches of DNA several 100 bp in length for mutations. Any mutation, independent of its location, could be identified. The usefulness of MC-PCR for the genotypic selection of mutants will depend on the effectiveness with which a small number of mismatched heteroduplexes can be recognized, cleaved and ligated.

Animals↗
PubMed1997

Mechanism of decreased in vitro murine macrophage cytokine release after exposure to carbon dioxide: relevance to laparoscopic surgery.

OBJECTIVE: The objective of this study was to determine the effect of carbon dioxide (CO2) on the function of peritoneal macrophages. SUMMARY BACKGROUND DATA: Laparoscopic surgery is associated with minimal pain, fever, and low levels of inflammatory cytokines. To understand the mechanisms involved, the authors investigated the effect of different gases on murine peritoneal macrophage intracellular pH and correlated these alterations with alterations in LPS-stimulated inflammatory cytokine release. METHODS: Peritoneal macrophages were incubated for 2 hours in air, helium, or CO2, and the effect of the test gas on immediate or next day lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) and interleukin-1 release compared. Cytosolic pH of macrophages exposed to test gases was measured using single-cell fluorescent imaging. The in vivo effects of test gases were determined in anesthetized rats during abdominal insufflation. RESULTS: Macrophages incubated in CO2 produced significantly less TNF and interleukin-1 in response to LPS compared to incubation in air or helium. Cytokine production returned to normal 24 hours later. Exposure to CO2, but not air or helium, caused a marked cytosolic acidification. Pharmacologic induction of intracellular acidification to similar levels reproduced the inhibitory effect. In vitro studies showed that CO2 insufflation lowered tissue pH and peritoneal macrophage LPS-stimulated TNF production. CONCLUSIONS: The authors propose that cellular acidification induced by peritoneal CO2 insufflation contributes to blunting of the local inflammatory response during laparoscopic surgery.

Animals↗
PubMed1997

Risk factors for osteoarthritis in the tibiofemoral and patellofemoral joints of the knee.

OBJECTIVE: Risk factors for osteoarthritis (OA) in the different compartments of the knee are important in the pathogenesis of knee OA. We examined the risk factors associated with OA of the tibiofemoral and patellofemoral compartments of the knee. METHODS: A population-based study of 325 unrelated, middle aged females was performed. Knee and hand radiographs for each individual were graded for joint space narrowing and osteophytes using a standard atlas. Individuals with knee OA were classified as having tibiofemoral OA only, patellofemoral OA only, or a combination of both tibiofemoral and patellofemoral OA. Information on risk factors was obtained by a structured interview and examination. RESULTS: Obesity was associated with all the categories of knee OA. This relationship was strongest for the combined tibiofemoral and patellofemoral OA [OR (95% CI) = 7.26 (2.36-22.31) for the highest vs lowest third]. There was a strong association between distal interphalangeal OA and isolated tibiofemoral OA [OR = 6.67 (1.94-22.94)], but no association with patellofemoral OA. There was an inverse association between premenopausal status and patellofemoral OA [OR = 0.23 (0.06-0.84)]. CONCLUSION: The pathogenetic mechanisms involved in patellofemoral and tibiofemoral OA may differ; clarification of the etiology of OA for the separate compartments of the knee is needed.

Aged↗
PubMed1996

Metabolism of serotonin to N-acetylserotonin, melatonin, and 5-methoxytryptamine in hamster skin culture.

Biotransformation of [3H]serotonin by cultured hamster skin to 3H-metabolites corresponding to N-acetylserotonin (NAS), melatonin, and 5-methoxytryptamine (5-MT) was demonstrated. This process was time-dependent, with the highest production of radioactive NAS and melatonin metabolites after 3 and 5 h of incubation followed by a decrease in the rate of metabolite release into the media. Conversely, the formation of radioactive metabolite corresponding to 5-MT increased gradually during skin culture, reaching the highest level after 24 h of incubation. The production of 3H-metabolites, corresponding to NAS, melatonin, and 5-MT, was stimulated by forskolin with a maximum effect of forskolin at 10 microM concentration. The gas chromatographic/mass spectroscopy analysis of the fraction eluting at the retention time of NAS standard material showed that it contained NAS, further confirming production and release of NAS into the media by hamster skin. Therefore, we conclude that mammalian skin can acetylate serotonin to NAS and postulate that the NAS is further metabolized by the skin to form melatonin which is subsequently transformed to 5-MT.

5-Methoxytryptamine↗
PubMed1996

Genetic influences on osteoarthritis in women: a twin study.

OBJECTIVES: To assess the relative contribution of genetic and environmental factors to common forms of osteoarthritis of the hands and knees. DESIGN: Classic twin study with unselected twins who were screened radiologically for osteoarthritis. SUBJECTS: 130 identical and 120 non-identical female twins aged 48-70 recruited from a London based twin register and through a national media campaign. MAIN OUTCOME MEASURES: Similarity in identical compared with non-identical twin pairs for radiographic changes at the interphalangeal and first carpometacarpal joints of the hands and the tibiofemoral joint and patellofemoral joint of the knee expressed as intraclass correlations. RESULTS: The intraclass correlations of radiographic osteophytes and narrowing at most sites and the presence of Heberden's nodes and knee pain were higher in the identical pairs. The intraclass correlation of the total radiographic osteoarthritis score in identical pairs (rMZ) was 0.64 (SE 0.05) compared with 0.38 (0.08) in non-identical pairs. The proportion of genetic variance of total osteoarthritis score (osteophytes and narrowing) with modelling techniques was estimated at 0.54 (95% confidence interval 0.43 to 0.65) and ranged from 0.39 to 0.65 for different sites and features (p < 0.001) after adjustment for age and weight. CONCLUSIONS: These results demonstrate for the first time a clear genetic effect for radiographic osteoarthritis of the hand and knee in women, with a genetic influence ranging from 39-65%, independent of known environmental or demographic confounders. The results of this study should lead to further work on isolating the gene or genes involved in the pathogenesis of the common disabling disease.

Aged↗
PubMed1996

Shared record keeping in the multidisciplinary team.

This article describes an attempt to improve discharge planning procedures by introducing multidisciplinary patient records and a joint approach to patient care. The one-year project involved collecting information about the record keeping system in use, before introducing a comprehensive shared package of documents kept at the patient's bedside. Evaluation showed dramatic overall changes in record keeping, although some duplication of documents still occurred. Positive benefits were also noted in team communication and discharge planning.

Aged↗
PubMed1996

Humanized anti-Lewis Y antibodies: in vitro properties and pharmacokinetics in rhesus monkeys.

ABL 364 is a murine monoclonal IgG3 antibody directed against the Lewis Y carbohydrate antigen (Le(y)) expressed on the surface of many epithelial cell tumors. The antibody mediates cytotoxicity via activation of human complement or human effector cells, and has been evaluated in several clinical trials including two Phase I/II trials in relapsed small cell lung cancer and metastatic breast cancer. To improve the effector functions of the antibody, increase its half-life in circulation, and avoid the human antimouse antibody response, two chimeric and several humanized antibodies were constructed for evaluation. The chimeric IgG1 is more potent than the murine IgG3 in tumor cell lysis via activation of human peripheral mononuclear cells (10-fold), but somewhat less effective in complement-dependent lysis (2-3 fold). The chimeric IgG3 is slightly less potent than the IgG1. A humanized IgG1 was constructed by combining the complementarity-determining regions of the ABL 364 antibody with human framework and constant regions. Several additional variants were subsequently constructed to improve the binding affinity and increase expression of the antibody. Two of the variants, designated I and K, differ by a single amino acid at position 75 of the heavy chain. Both variants have affinity within 2-fold of the chimeric IgG1 antibody and retain the cytolytic activities toward tumor cell lines. However, it was possible to express variant K at a significantly higher level (5- 10-fold) than variant I. Pharmacokinetics of the humanized ABL 364 antibody variant K was compared with that of the parent murine antibody in rhesus monkeys. It was shown that the terminal half-life of the humanized antibody in rhesus monkeys is 14-20 days, with a mean of 16.3 days, while that of the parent murine antibody is only 1.9 days.

Amino Acid Sequence↗
PubMed1996

The heritability of bone mineral density, ultrasound of the calcaneus and hip axis length: a study of postmenopausal twins.

Population based studies have demonstrated that having a first degree relative with a hip fracture is predictive of future hip fractures. Postmenopausal bone mineral density (BMD), ultrasound of calcaneus and hip axis length are associated with hip fracture, with the association for ultrasound and hip axis length being independent of BMD. The aim of this study was to determine the genetic component of these three important risk factors. We performed a classical twin study using 500 normal female twins, 128 identical and 122 non-identical pairs, aged 50 to 70 years. We measured bone mineral density at multiple sites, hip axis length (distance from the inner rim of the acetabulum to the greater trochanter), broadband ultrasound attenuation and velocity of sound of the calcaneus. Bone density had a strong genetic component at all sites with estimates of heritability ranging from 0.46 to 0.84. Hip axis length and velocity of sound had major genetic components with estimates of 0.62 and 0.61 respectively, which remained virtually unchanged after adjustment for bone mineral density. Broadband ultrasound attenuation had a moderate genetic component with an estimate of 0.53, which was reduced further to 0.45 after adjustment for BMD. In summary, all three bone measurements, which are independently associated with hip fracture, are independently heritable. This study suggests that a combination of different genetic factors acting on the structure, dimensions and density of bone may explain the importance of family history as a risk factor for hip fracture.

Aged↗
PubMed1996

Discordant reprogramming of LPS-stimulated cytokine gene transcription and secretion by macrophages after LPS pretreatment.

Dysregulated macrophage (Mphi) cytokine release occurs during systemic inflammation and may predispose to organ failure. We showed that Mphis pretreated (PreRx) in vitro with low-dose LPSp are "reprogrammed" to release less TNF and more IL-1 in response to subsequent LPS activation (LPSa). The effects of this LPSp "reprogramming" on Mphi cytokine gene transcription were investigated in the present study. Murine peritoneal exudate Mphis were cultured in vitro 48 hr, then PreRx 24 hr +/- 100 ng/ml of LPSp. Cultures were stimulated with 0-1000 ng/ml LPSa and 6-hr supernatant TNF and IL-1 were measured using specific bioassays. Cytokine gene transcription was estimated 6 hr after LPSa using RT-PCR. PreRx with LPSp inhibited TNF and augmented IL-1 release by LPSa. PreRx with LPSp significantly inhibited cytokine gene transcription; however, messages for both TNF and IL-1 were detectable after high-dose LPSa. Despite LPSp inhibition of IL-1 transcription by most LPSa concentrations, IL-1 protein was augmented by PreRx. High-dose LPSa can override LPSp reprogrammed inhibition of cytokine gene transcription, but altered TNF and IL-1 protein release after LPSp may be regulated posttranscriptionally.

Animals↗
PubMed1996

Kinetics of decreased LPS-stimulated cytokine release by macrophages exposed to CO2.

The mechanisms responsible for the lack of inflammation after laparoscopic surgery remain unknown. Peritoneal macrophages (M phi) incubated in carbon dioxide (CO2) but not air or helium (He), had significant, reversible inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) and interleukin-1 (IL-1) release. In these experiments the kinetics of these C02-induced alterations in cytokine secretion were examined. Murine peritoneal Mphi were stimulated with LPS for 4 hr and incubated in different test gases (95% air/5% CO2,80%CO2/20%O2,80% He/20% O2) for intervals between 0.25 and 4 hr. Time between gas incubation and LPS stimulation was varied to determine the persistence of CO2 inhibition. Parallel M phi groups received LPS stimulation 24 hr later. Supernatant TNF and IL-1 were measured by bioassay and polymerase chain reaction was used to examine cytokine mRNA. Significant reversible inhibition of TNF and IL-1 was seen with CO2, but not He or air. Inhibition of IL-1 occurred 15 min after CO2 exposure, was associated with decreased IL-1 mRNA, and was rapidly lost following incubation in the control atmosphere. TNF inhibition was seen despite normal levels of TNF message, required more than 30 min of CO2 exposure, and persisted after CO2 removal. CO2 produced profound, reversible, inhibition of LPS-stimulated cytokine release by peritoneal Mphi. The transient inability to secrete inflammatory cytokines after CO2 exposure may explain the lack of systemic inflammation after laparoscopic surgery with CO2.

Animals↗