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Biomedical subjects

P Carter

Publications and source records attributed to P Carter.

At least 55 records · Page 3Linked to original sources
PubMed1998

Glutamate transport asymmetry in renal glutamine metabolism.

D-Glutamate (Glu) was previously shown to block L-Glu uptake and accelerate glutaminase flux in cultured kidney cells [Welbourne, T. C., and D. Chevalier. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E367-E370, 1997]. To test whether D-Glu would be taken up by the intact functioning kidney and effect the same response in vivo, male Sprague-Dawley rats were infused with D-Glu (2.6 mumol/min), and renal uptake of D- and L-Glu was determined from chemical and radiolabeled arteriovenous Glu concentration differences times renal plasma flow. The amount removed was then compared with that amount filtered to obtain the antiluminal contribution. In the controls, L-Glu uptake measured as net removal was 33% of the arterial L-Glu load and not different from that filtered, 27%; however, the unidirectional uptake was actually 58% of the arterial load, indicating that antiluminal uptake contributes at least half to the overall Glu consumption. Surprisingly, the kidneys showed a more avid removal of D-Glu, removing 73% of the arterial load, indicating uptake predominantly across the antiluminal cell surface. Furthermore, uptake of D-Glu was associated with a 55% reduction in L-Glu uptake, with the residual amount taken up equivalent to that filtered; D-Glu did not increase the excretion of the L-isomer. However, elevating plasma L-Glu concentration reduced uptake of the D-isomer, suggesting a shared antiluminal transporter. Thus there is an apparent asymmetrical distribution of the D-Glu transporter. Under these conditions, kidney cortex L-Glu content decreased 44%, whereas net glutamine (Gln) uptake increased sevenfold (170 +/- 89 to 1,311 +/- 219 nmol/min, P < 0.01) and unidirectional uptake nearly threefold (393 +/- 121 to 1,168 +/- 161 nmol/min, P < 0.05); this large Gln consumption was paralleled by an increase in ammonium production so that the ratio of production to consumption approaches 2, consistent with accelerated Gln deamidation and subsequent Glu deamination. These results point to a functional asymmetry (antiluminal vs. luminal) for Glu transporter activity, which potentially plays an important role in modulating Gln metabolism and renal function.

Animals↗
PubMed1997

Mutations in the glutathione-gated KefC K+ efflux system of Escherichia coli that cause constitutive activation.

The kefC gene of Escherichia coli encodes a potassium efflux system that is gated by glutathione (GSH) and by GSH adducts. Independently isolated kefC mutations that result in spontaneous activation of the efflux system have been analyzed. Three mutations affect residues located adjacent to the conserved Rossman fold in the carboxyl-terminal domain. Two mutations lie in a sequence predicted to form a cytoplasmically located loop in the membrane domain of KefC. All of the mutants retain normal regulation by the YabF protein and by GSH adducts. A mutation in the Rossman fold, R416S, alters the normal regulation of KefC by GSH. In contrast to the wild-type protein, which is inactive in the presence of GSH, the R416S mutant is only active in the presence of GSH or its analogue, ophthalmic acid. Other mutations in this region or elsewhere in the protein have their spontaneous activity augmented by depletion of the GSH pool. These data identify a specific role for the carboxyl-terminal domain of KefC in regulating KefC activity and are discussed in the light of recent data that suggest that GSH adducts can bind within a Rossman fold.

Bacterial Proteins↗
PubMed1997

Elevated expression of eIF4E and FGF-2 isoforms during vascularization of breast carcinomas.

The translation initiation factor eIF4E is a novel protooncogene found over expressed in most breast carcinomas (Kerekatte et al., 1995), but the pathology where this elevation is initially manifested and its possible role in cancer progression are unknown. We report that eIF4E is markedly increased in vascularized malignant ductules of invasive carcinomas, whereas necrotic and avascular ductal carcinomas in situ display significantly lower levels. eIF4E facilitates the synthesis of FGF-2, a powerful tumor angiogenic factor. Conversely, reducing eIF4E with antisense RNA in MDA-435 cells suppresses their tumorigenic and angiogenic properties, consistent with loss of FGF-2 synthesis. These findings suggest a causal role for eIF4E in tumor vascularization.

Animals↗
PubMed1997

Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library.

Structure-guided phage display was used to select for combinations of interface residues for antibody C(H)3 domains that promote the formation of stable heterodimers. A C(H)3 "knob" mutant was made by replacement of a small residue, threonine, with a larger one, tryptophan: T366W. A library of C(H)3 "hole" mutants was then created by randomizing residues 366, 368 and 407, which are in proximity to the knob on the partner C(H)3 domain. The C(H)3 knob mutant was fused to a peptide flag and the C(H)3 hole library was fused to M13 gene III. Phage displaying stable C(H)3 heterodimers were recovered by panning using an anti-flag antibody. Phage-selected C(H)3 heterodimers differed in sequence from the previously designed heterodimer T366W-Y407'A, and most clones tested were more stable to guanidine hydrochloride denaturation. The thermal stability of individual C(H)3 domains secreted from Escherichia coli was analyzed by differential scanning calorimetry. One heterodimer, T366W-T366'S:L368'A:Y407'V, had a t(m) of 69.4 degrees C, which is 4.0 deg.C higher than that for the designed heterodimer and 11.0 deg.C lower than that for the wild-type homodimer. The phage-selected C(H)3 mutant maintained the preference for forming heterodimers over homodimers as judged by near-quantitative formation of an antibody/immunoadhesin hybrid in a cotransfection assay. Phage optimization provides a complementary and more comprehensive strategy to rational design for engineering homodimers for heterodimerization.

Amino Acid Sequence↗
PubMed1997

Sterically stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro.

Liposomes (70-100 nm) of 1-palmitoyl-2-oleoylphosphatidylcholine, cholesterol, and poly(ethylene glycol) (PEG)-modified phosphatidylethanolamine (PEG-DSPE) were conjugated to Fab' fragments of a humanized recombinant MAb against the extracellular domain of HER2/neu to create sterically stabilized immunoliposomes (anti-HER2 SL) as a drug carrier targeting HER2-overexpressing cancers. Conjugation employed maleimide-terminated membrane-anchored spacers of two kinds: a short spacer, providing attachment of Fab' close to the liposome bilayer, or a long spacer, with Fab' attachment at the distal terminus of the PEG chain. Confocal microscopy and spectrofluorometry of HER2-overexpressing breast cancer cells incubated with fluorescently labeled anti-HER2 SL prepared with either spacer showed binding of liposomes (8000-23000 vesicles/cell) followed by endocytosis (rate constant ke = 0.012-0.033 min-1) via the coated-pit pathway, evidenced by intracellular acidification and colocalization with transferrin. Uptake of anti-HER2 immunoliposomes by breast cancer cells with low HER2 expression, or after preincubation of cells with free anti-HER2 Fab', was less than 0.2% and 4.3%, respectively, of the uptake by HER2-overexpressing cells. Increasing PEG-DSPE content (up to 5.7 mol %) in anti-HER2-SL prepared with the short spacer decreased liposome-cell binding affinity 60-100-fold, while ke decreased only 2-fold; however, when Fab' fragments were conjugated via a PEG spacer, both binding affinity and ke were unaffected by PEG-DSPE content. Cell binding and internalization of anti-HER2 immunoliposomes increased at higher surface density of conjugated Fab' fragments, reaching plateaus at approximately 40 Fab'/liposome for binding and approximately 10-15 Fab'/liposome for internalization. Uptake of anti-HER2 immunoliposomes correlated with the cell surface density of HER2 and significantly (p < 0.005) correlated with the antiproliferative effect of the targeting antibody but not with the total level of cellular HER2 expression. The results obtained were used to optimize in vivo preclinical studies of anti-HER2 SL loaded with antineoplastic drugs.

Antibodies, Monoclonal↗
PubMed1997

Remodeling domain interfaces to enhance heterodimer formation.

An anti-p185HER2/anti-CD3 humanized bispecific diabody was previously constructed from two cross-over single-chain Fv in which YH and VL domains of the parent antibodies are present on different polypeptides. Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers. A disulfide-stabilized diabody was obtained by introducing two cysteine mutations, VL L46C and VH D101C, at the anti-p185HER2.VL/VH interface. The fraction of recovered diabody that was functional following expression in Escherichia coli was improved for the disulfide-stabilized compared to the parent diabody (> 96% versus 72%), whereas the overall yield was > 60-fold lower. Eleven "knob-into-hole" diabodies were designed by molecular modeling of sterically complementary mutations at the two VL/VH interfaces. Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinity for both antigens close to that of the parent diabody. For example, diabody variant v5 containing the mutations VL Y87A:F98M and VH V37F:L45W at the anti-p185HER2 VL/VH interface was recovered as 92% functional heterodimer while maintaining overall recovered yield within twofold of the parent diabody. The binding affinity of v5 for p185HER2 extracellular domain and T cells is eightfold weaker and twofold stronger than for the parent diabody, respectively. Domain interface remodeling based upon either sterically complementary mutations or interchain disulfide bonds can facilitate the production of a functional diabody heterodimer. This study expands the scope of domain interface engineering by demonstrating the enhanced assembly of proteins interacting via two domain interfaces.

Antibodies, Bispecific↗
PubMed1997

Expression of the SU glycoprotein of maedi visna virus in baculovirus.

The envelope glycoprotein, gp 135, of the ovine lentivirus maedi visna virus is the main target for a specific antibody response in vivo, however, little is known about the specific regions of gp 135 which elicit this response. Research on the function of gp 135 has been hampered by the lack of reagents to study such structure/function relationships. We have used a baculovirus expression system to express gp 135 lacking the viral signal sequence. This recombinant protein is glycosylated and recognised by immune sera from clinically affected animals.

Animals↗
PubMed1997

The role of growth hormone in substrate utilization.

Substrate fluxes in response to growth hormone administration depend on both the calorie as well as acid-base balance. Growth hormone's acidogenic action as a consequence of promoting fatty acid utilization yields protons required for driving hepatic glutamate efflux; effective uncoupling of nitrogenous precursors from ureagenesis and recycling as glutamate bound for the periphery appears dependent upon this mechanism. Subsequent peripheral retrieval of the salvaged glutamate requires insulin-like growth factor-1 (IGF-1) activated uptake and acid-base homoeostasis. In addition to this nitrogen sparing acidogenic effect, growth hormone is also basogenic in combination with IGF-1 and acting on the kidney as a target organ. Therefore acid-base and nitrogen homoeostasis are normally attuned to one another through the co-ordinated action of growth hormone/IGF-1 on substrate fluxes. However during starvation ketoacid production as the consequence of incomplete fatty acid oxidation and ketone excretion swamps the basogenic limb and full-blown metabolic acidosis prevails; under this condition growth hormone's effectiveness in sparing nitrogen for anabolic processes is curtailed as glutamate (emanating from the liver) and glutamine (derived from muscle proteolysis) are directed to the kidneys, supporting ammoniogenesis: nitrogen balance is now sacrificed for acid-base homoeostasis. Underlying this state is an intracellular acidosis that may contribute to insulin resistance and developing hyperglycaemia in response to growth hormone. In acute injury, an additional acid load contributed from muscle proteolysis and cytokines reinforces an intracellular acidosis that further blunts growth hormone responsiveness and suppresses coupled IGF-1 production. From this perspective growth hormone's acidogenic and basogenic actions should balance for an effective anabolic response during hypermetabolic catabolic illnesses.

Animals↗
PubMed1997

Engineering antibodies for imaging and therapy.

Several advances made during the past year will probably facilitate the development of therapeutic antibodies. Most notably, significant progress has been made in the rapid isolation of high affinity human antibodies from phage display libraries and by immunization of transgenic mice. The therapeutic potential of bispecific antibody fragments and Fc-containing proteins has been greatly enhanced by improved production methods. The utility of radiolabeled antibody fragments has been improved by the development of site-specific labeling methods and by the advent of the 'minibody', an engineered fragment that has proved to be highly successful for tumor imaging in mice.

Animals↗
PubMed1997

Glutamate transport regulation of renal glutaminase flux in vivo.

We proposed that glutamate transport into cultured kidney cells represses cellular glutaminase activity and hence regulates glutamine utilization. To test this putative regulatory mechanism in vivo, glutamine uptake and conversion to glutamate as well as ammonium production were measured in the intact functioning rat kidney. Glutamine uptake was determined as net removal, arteriovenous concentration difference times renal plasma flow, and also as unidirectional uptake from the fractional extraction of tracer L-[14C]glutamine. Ammonium production was measured as that released into the renal vein plus that excreted, and intracellular glutamine conversion to glutamate was assessed from the rise in cortical glutamate radiolabel specific activity. Cellular glutamate content was reduced 50-60% by infusing D-aspartate (a high-affinity glutamate transporter inhibitor) over 30 min, consistent with interdiction of glutamate uptake. This reduction in the glutaminase repressor was associated with a three- to fivefold increase in glutamine uptake and intracellular conversion to glutamate and ammonium. These results are consistent with and predictable from our previous in vitro model and point to an important role for this regulatory mechanism in the intact functioning organ.

Acid-Base Equilibrium↗
PubMed1997

Prevalence of sexual dysfunction in heterosexual patients attending a central London genitourinary medicine clinic.

Our objective was to determine the prevalence of sexual dysfunction among new heterosexual attendees at a central London genitourinary medicine (GUM) clinic. We carried out a cross-sectional study in which patients completed a self-administered questionnaire-the Golombok-Rust Inventory of Sexual Satisfaction (GRISS) and participated in a brief interview during which additional information was sought regarding the patient's sexual history. An overall transformed score of >5 on the GRISS was defined as indicative of overall sexual dysfunction and a score of >5 on any of the subscales as indicative of a specific sexual dysfunction. Twenty-five (24%) men and 10 (9%) women had a GRISS score in keeping with overall sexual dysfunction, the prevalence being significantly lower in women (P=0.01, chi2=6.56, 1df). Sixty-three men (59%) and 63 (60%) women produced scores indicative of significant abnormality on at least one subscale, including, in men: erectile dysfunction 20 (19%), premature ejaculation 23 (22%), and in women: vaginismus 26 (25%) and anorgasmia 23 (22%). Neither an abnormal overall or subscale score on the GRISS was associated with a current STD on KC60 diagnosis or a history of sexual assault for either men or women. There is a substantial prevalence of sexual dysfunction in new heterosexual attendees at our clinic, the service implications of which need to be addressed.

Adolescent↗
PubMed1997

The provision of psychosexual services by genitourinary medicine physicians in the United Kingdom.

A postal questionnaire survey, enquiring about the provision of psychosexual services, was sent to each GUM clinic in the UK. Of the 246 questionnaires distributed, replies were received from 166 directors responsible for 171 (69.5%) clinics. Of the 140 (84%) who supported the provision of a sexual dysfunction service, 59 (42%) currently provided such a service. Doctors and psychologists were the health care professionals most commonly involved in sexual dysfunction services for patients who were referred both internally and from external sources such as general practice and diabetic clinics. Patients with a variety of dysfunctions were being treated with a broad range of therapies, a reflection probably of the multidisciplinary nature of the team providing the service. However, it appears that junior doctors are not being trained in this field at present.

Female Urogenital Diseases↗
PubMed1997

The most frequently prescribed medications in the elderly and their impact on dental treatment.

The high prevalence of multiple chronic medical conditions in the elderly and the likelihood of multiple drug therapies dramatically increase the chance of drug-specific adverse effects and drug-drug interactions. This article reviews the age-associated alterations in pharmacokinetics and pharmacodynamics along with the most frequently prescribed medications for the elderly. Adverse drug reactions, drug-drug interactions and oral side effects are reviewed along with the impact these medications may have on dental treatment planning and management.

Aged↗
PubMed1996

Overexpression of the proto-oncogene/translation factor 4E in breast-carcinoma cell lines.

The expression of the proto-oncogene, translation factor elF-4E, was examined in breast-cell lines: 5 carcinomas and 2 normal. At the protein level, elF-4E was 10 times higher in the carcinoma lines than in normal cells, which is comparable to the level found in breast-cancer biopsies. The elevation appears to be due to increased transcription, since the elF-4E mRNA was correspondingly increased. These results demonstrate that cells isolated from naturally occurring breast carcinomas maintain an elevated expression of the factor. Turnover rates for elF-4E (mRNA and protein) were determined for normal and cancer cells. We found that elF-4e mRNA is relatively stable during an 8-hr incubation with Actinomycin D, but the half-life of the protein is fairly short (approximately 4.5 hr). This suggests that, in proliferating cells, elF-4E may be turning over rapidly, possibly to fine-tune the changes in translation rates which occur during the cell cycle.

Base Sequence↗
PubMed1996

Stereotactic breast biopsy is accurate, minimally invasive, and cost effective.

BACKGROUND: We reviewed our experience with stereotactic core needle breast biopsy (SCNBB) for accuracy, complication rate, and staging profile of malignancies diagnosed. METHODS: Since March 1993, 530 stereotactic biopsies were performed. Of these, 25 cases underwent stereotactic core needle biopsy with subsequent wire-guided biopsy. RESULTS: In 25 patients with stereotactic and open biopsy, there was an accuracy for SCNBB of 96%. The number of biopsies rose from 100 to 250 biopsies annually, with an equivalent pre-test positive predictive value for mammography (17% to 19% historical versus 20% with SCNBB). The total number of de novo cancer diagnoses have increased from a mean of 57 to a mean of 71 annually. The percentage of tumors in situ, stage I or stage II, has increased from 60% to 69%. CONCLUSIONS: Stereotactic core needle biopsy combines a high accuracy with a low complication rate. Its aggressive application for tissue diagnosis in suspicious nonpalpable mammographic lesions has increased the proportion of early (in situ and T1 or T2) tumors discovered, and increased the total number of breast cancers diagnosed.

Biopsy, Needle↗
PubMed1996

High level secretion of a humanized bispecific diabody from Escherichia coli.

Clinical development of bispecific antibodies (BsAb) has been effectively stymied by the lack of efficient production methods. We therefore attempted to produce a humanized BsAb fragment using an expression system that has proved very successful for secretion of monospecific Ab fragments from E. coli. An anti-p185HER2/anti-CD3 BsF(ab')2 was first recast into the diabody format and then periplasmically secreted from E. coli grown to high cell density in a fermentor. The diabody was recovered in very high yield (up to 935 mg/l) after protein A purification and predominantly (> or = 80%) as a dimer as judged by size exclusion chromatography. Diabody dimers were found to be mainly functional heterodimers (approximately 75%) by titration with p185HER2 extracellular domain. The diabody binds p185HER2 extracellular domain and human T lymphocytes with affinities close to those of the parent BsF(ab')2. Furthermore, the diabody is capable of simultaneous binding to tumor cells overexpressing p185HER2 and CD3 on T cells as shown by cellular rosetting. The diabody is equally potent as the parent BsF(ab')2 in retargeting IL-2 activated T-enriched peripheral blood lymphocytes to lyse tumor cells overexpressing p185HER2.

Amino Acid Sequence↗