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Biomedical subjects

S Roberts

Publications and source records attributed to S Roberts.

At least 199 records · Page 11Linked to original sources
PubMed1994

Tamoxifen aziridine, a novel affinity probe for P-glycoprotein in multidrug resistant cells.

In this study for the first time we used an electrophilic analog of tamoxifen, [3H]tamoxifen aziridine, and demonstrated that it covalently and specifically binds to P-glycoprotein in multidrug resistant cells. Tamoxifen and its metabolites, N-desmethyltamoxifen and 4-hydroxytamoxifen, were potent inhibitors of [3H]tamoxifen aziridine binding to P-glycoprotein with 4-hydroxytamoxifen > tamoxifen > N-desmethyltamoxifen. The multidrug resistance-related drugs inhibited [3H]tamoxifen aziridine binding with vinblastine > vincristine > doxorubicin > actinomycin D, while colchicine enhanced the binding. Moreover, the multidrug resistance modulators verapamil, nicardipine, diltiazem, prenylamine, cyclosporin A, FK506, dibucaine, reserpine, monensin and progesterone were all potent inhibitors of [3H]tamoxifen aziridine binding to P-glycoprotein. Our data provide the first evidence that [3H]tamoxifen aziridine directly binds to P-glycoprotein and interacts with the binding sites for multidrug resistance-related drugs and modulators.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

A net +1 frameshift permits synthesis of thymidine kinase from a drug-resistant herpes simplex virus mutant.

Clinical resistance to antiviral drugs requires that a virus evade drug therapy yet retain pathogenicity. Thymidine kinase (TK)-negative mutants of herpes simplex virus are resistant to the drug, acyclovir, but are attenuated for pathogenicity in animal models. However, numerous cases of clinical resistance to acyclovir have been associated with viruses that were reported to express no TK activity. We studied an acyclovir-resistant clinical mutant that contains a single-base insertion in its tk gene, predicting the synthesis of a truncated TK polypeptide with no TK activity. Nevertheless, the mutant retained some TK activity and the ability to reactivate from latent infections of mouse trigeminal ganglia. The mutant expressed both the predicted truncated polypeptide and a low level of a polypeptide that comigrated with full-length TK on polyacrylamide gels and reacted with anti-TK antiserum, providing evidence for a frameshifting mechanism. In vitro transcription and translation of mutant tk genes, including constructs in which reporter epitopes could be expressed only if frameshifting occurred, also gave rise to truncated and full-length polypeptides. Reverse transcriptase-polymerase chain reaction analysis coupled with open reading frame cloning failed to detect alterations in tk transcripts that could account for the synthesis of full-length polypeptide. Thus, synthesis of full-length TK was due to an unusual net +1 frameshift during translation, a phenomenon hitherto confined in eukaryotic cells to certain RNA viruses and retrotransposons. Utilization of cellular frameshifting mechanisms may permit an otherwise TK-negative virus to exhibit clinical acyclovir resistance.

Acyclovir↗
PubMed1994

Transcriptional elongation by RNA polymerase II is stimulated by transactivators.

We report that a variety of transactivators stimulate elongation by RNA polymerase II. Activated transcription complexes have high processivity and are able to read through pausing and termination sites efficiently. In contrast, nonactivated and "squelched" transcription mostly arrests prematurely. Activators differ in the extent to which they stimulate processivity; for example, GAL4-VP16 and GAL4-E1a are more effective than GAL4-AH. The stimulation of elongation can be as important as the stimulation of initiation in activating expression of a reporter gene. We suggest that setting the competence of polymerase II to elongate is an integral part of the initiation step that is controlled by activators cooperating with the general transcription factors.

Animals↗
PubMed1994

Innervation of the spondylolysis "ligament".

SUMMARY OF BACKGROUND DATA: Spondylolysis of the lower lumbar vertebrae is a non-united childhood fracture of the arch of the vertebra, persisting into adult life. Symptoms of disabling low back pain appear in a minority of patients, usually for the first time in adulthood. This pain is considered to arise from several separate sources, one of which may be the spondylolysis ligament. STUDY DESIGN: The innervation of the ligament has been investigated immunohistochemically. METHODS: Specimens from eight patients were divided longitudinally for histology including hematoxylin and eosin, toluidine blue, and elastic van Gieson. Histochemistry involved immunostaining for the neuropeptides: protein gene product, calcitonin gene related peptide, substance P, vasoactive intestinal peptide, and the c-flanking peptide of neuropeptide Y. RESULTS: Immunoreactivity to calcitonin gene-related peptide, the c-peptide of neuropeptide Y, and vasoactive intestinal peptide was identified in the ligament or in the adjacent adipose tissue. CONCLUSION: The movement that the ligament allows at the fracture site may result in stimulation of the nerve endings both in the ligament and in the surrounding soft tissue.

Adult↗
PubMed1994

Neuropeptides in the human intervertebral disc.

The innervation of the human intervertebral disc was investigated by immunochemical methods. Immunoreactivity to the general nerve marker protein gene product (PGP 9.5) was found in the outer annulus fibrosus of 11 of 12 discs removed during anterior arthrodesis for back pain. PGP 9.5-immunoreactive fibres ran between and across the collagenous lamellae, both in association with blood vessels and distant from them, and extended at least 3 mm into the disc. No innervation was observed in the nucleus pulposus. Fine fibres (< 1 micron in diameter) immunoreactive to calcitonin gene-related peptide and substance P (neuropeptides located in sensory and possibly nociceptive nerves) were identified in eight and four of the annuli fibrosi, respectively. Nerve fibres immunoreactive to vasoactive intestinal peptide and to the c-flanking peptide of neuropeptide Y were found in the majority of specimens of annulus fibrosus that were examined.

Adult↗
PubMed1994

Site-directed mutagenesis of tryptophan residues to conserved hydrophobic residues inhibits the processing of human KB cell folate receptor.

We are interested in identifying the ligand binding site of the human folate receptor (hFR). Previous reports have suggested a role of tryptophan(W) residues in ligand binding. We used site-directed mutagenesis to change the conserved W residues in positions 86, 116, 142, 143, 156, 160, and 193 of the hFR to either leucine(L) or phenylalanine(F) to examine the role of these W residues in hFR function. Although all W to L changes except W86L produced unstable proteins, W to F changes were tolerated. Based on total folate binding and transport studies, Chinese hamster ovary (CHO) cells transfected with W86L, W116F, and W143F expressed high levels of functional hFR, equivalent to cells transfected with wt hFR. CHO cells transfected with W142F, W156F, W160F, and W193F expressed low or undetectable levels of functional hFR although mRNA was present. Of these four mutants, only W142F expressed easily detectable immunoprecipitable protein but it was not fully glycosylated. Since glycosylation may affect the ability of hFR to bind folate, we expressed W142F in Xenopus oocytes which glycosylate the mutant and wild type proteins to the same apparent extent. In oocytes, the stoichiometry of folate binding was identical between the fully processed mutant protein and the wild type hFR. These results indicate that in CHO cells three of the seven W mutations (W86L, W116F, and W143F) function normally, whereas four of the seven W mutations (W142F, W156F, W160F, and W193F) produce unstable or abnormally processed protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Proteoglycan components of the intervertebral disc and cartilage endplate: an immunolocalization study of animal and human tissues.

Monoclonal antibodies have been used to study the presence and distribution of various components of the proteoglycan molecule in the intervertebral disc and cartilage endplate. Link protein, hyaluronic acid binding region, keratan sulphate and chondroitin 4- and 6-sulphate have been investigated in tissues from humans and other mammals. Exposure of the carbohydrate and protein epitopes was enhanced by chondroitinase and trypsin pretreatment respectively. The degree of immunoreactivity varied with location, being greater in the nucleus pulposus than the annulus fibrosus with least reactivity in the cartilage endplate. In addition, there was increased staining in the pericellular domains, particularly in adult tissues. Areas of ectopic calcification exhibited very different immunoreactivity, depending on the type of calcium salt present. Calcium hydroxyapatite deposits showed greater staining for 8A4 (link protein), while calcium pyrophosphate deposits demonstrated greater staining for 3B3(-), 7D4(-) and 3D5 than the surrounding non-calcified matrix. Staining for chondroitin sulphate isomer epitopes 3B3(-) and 7D4(-), indicative of modified chondroitin sulphate chains, was greater in human tissues of degenerate than non-degenerate appearance. This suggests that expression of these epitopes may be an indicator of disease and subsequent reparative procedures in intervertebral disc and cartilage endplate, similar to that seen in articular cartilage degeneration.

Adolescent↗
PubMed1994

Hypothermia associated with percutaneous nephrolithotomy.

OBJECTIVES: To characterize the perioperative fall in core body temperature associated with percutaneous nephrolithotomy, and to identify patients at greater risk of hypothermia. METHODS: A retrospective review was undertaken of core body temperatures in 77 patients who underwent 95 percutaneous nephrolithotomy (PNL) procedures using room temperature irrigants over a 3 year period. Intraoperative temperature measurements were obtained via an esophageal thermistor probe. RESULTS: Mean core body temperature fell from 36.7 degrees C preoperatively to 35.8 degrees C after retrograde manipulations and subsequent patient positioning before commencing the renal puncture. The mean core temperature fell an additional mean 1.0 degrees C during the percutaneous nephrolithotomy. This additional temperature drop was proportional to the length of the procedure. Female patients had a significantly greater decline in intraoperative core body temperature than male patients (p < 0.01). Obese patients preserved their preoperative temperature more efficiently. Blood loss and advanced age had no impact on the occurrence of hypothermia. Mean preoperative temperature was regained on average within 3 hours postoperatively. CONCLUSIONS: Hypothermia occurs in the majority of patients undergoing PNL. Nearly an equivalent fall in temperature is seen during presurgical preparation, induction of anesthesia, and patient positioning as is seen during the surgical procedure itself. Efforts to preserve core body temperature both before and during the percutaneous procedure may reduce the degree of hypothermia and its potential complications.

Body Temperature↗
PubMed1994

Induction of nitric oxide synthase in rat C6 glioma cells.

We have examined the induction of nitric oxide synthase (NOS) activity in the rat astrocyte-derived C6 glioma cell line. In contrast to the previous results with primary astrocyte cultures, incubation of C6 cells with bacterial endotoxin lipopolysaccharide (LPS; 1 microgram/ml for 24 h) did not stimulate NO2 production. However, addition of either tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose-dependent manner (EC50 values of 39 ng/ml of TNF-alpha and 9.4 U/ml of IFN-gamma), and the effect of TNF-alpha could be further potentiated (twofold) by the presence of interleukin-1 beta. The simultaneous presence of TNF-alpha and IFN-gamma yielded a greater response than either cytokine alone; however, the respective EC50 values were not affected. A cytoplasmic extract from induced C6 cells catalyzed the Ca(2+)-independent conversion of L-arginine to L-citrulline, with an apparent Km of 51.2 microM, and this activity could be blocked by L-arginine analogues in the potency order amino > methyl > nitroarginine. Immunoblot analysis revealed an apparent molecular mass of 125 kDa for the NOS protein induced in C6 cells. These results indicate that the combination of LPS plus cytokines can induce NOS activity in C6 glioma cells with properties similar to those of the enzyme expressed in primary astrocyte cultures.

Amino Acid Oxidoreductases↗
PubMed1994

Lethal mutations within the conserved stem-loop of African cassava mosaic virus DNA are rapidly corrected by genomic recombination.

The nonanucleotide motif TAATATTAC occurs in the intergenic region of all geminiviruses that have been examined to date. The motif is invariably located within the loop of a potential stem-loop structure that has been implicated in viral DNA replication. To investigate the contribution of these sequences to virus proliferation, African cassava mosaic virus (ACMV) DNA B mutants have been screened for their ability to infect Nicotiana benthamiana when co-inoculated with DNA A. Mutants in which the putative stem structure was altered by the introduction of single nucleotide mismatches remained as infectious as the wild-type virus and the mutations were retained in the progeny. Mutants containing nucleotide substitutions within the loop sequences were similarly infectious but analysis of progeny showed that in most cases wild-type sequences were restored by recombination with DNA A. Stem-loop deletion mutants of both genomic components were not infectious when co-inoculated, although they were once again efficiently rescued by recombination when inoculated with the wild-type components. Co-inoculation of genomic components containing the motif TAGTATTAC did not result in a systemic infection while mutants containing the motif TAATACTAC were infectious and the mutation was stable. The results demonstrate that ACMV will tolerate some modification to this highly conserved region of the genome that might allow more precise mapping of the position at which the viral DNA is nicked during replication.

Base Sequence↗
PubMed1994

Retinopathy of prematurity in infants less than 29 weeks' gestation at birth.

From January 1986 to December 1991 we examined the eyes of 206 infants born at Westmead Hospital, Neonatal Intensive Care Unit who were less than 29 weeks' gestation at birth to determine the incidence of retinopathy of prematurity. Eighty-five infants (41.3%) had no retinopathy of prematurity (ROP) in either eye, 82 infants had stages 1 or 2 ROP (39.8%), 29 had stage 3 ROP (14.1%) and 11 had stage 4 ROP (5.3%). Of these, cryotherapy was performed in 18; six now have bilateral retinal detachment and are blind. The more severe stages of ROP were significantly associated with an increase in the number of days of oxygen supplementation, an increase in the number of days of mechanical ventilation and the presence of patent ductus arteriosus. Infants receiving steroids for mechanical ventilator dependence had a significantly greater chance of requiring cryotherapy (11 or 22 receiving steroids versus seven of 43 without steroids; P < 0.01).

Blindness↗
PubMed1994

The quality of routinely collected maternity data.

OBJECTIVE: To assess the validity of clinical information held on a regional maternity database, the St Mary's Maternity Information System (SMMIS). DESIGN: A retrospective review of 892 maternity case notes and matched SMMIS records, by a midwife trained in clinical coding techniques. SETTING: Three maternity units in the North West Thames Region. MAIN OUTCOME MEASURES: Percentage agreement for 17 directly recorded SMMIS data items and equivalent data abstracted from the notes. Frequencies of diagnosis codes abstracted from case notes, as compared with those generated by SMMIS on the basis of directly recorded data. RESULTS: A generally high level of agreement was observed between the abstracts of the notes and the SMMIS records. Of the 17 data items examined, 10 showed 95% agreement or better, and all but two exceeded 80% agreement. Little difference was found between the levels of agreement observed at the three sites. A greater number and range of diagnosis codes were abstracted from the notes than were generated by SMMIS. CONCLUSIONS: The directly recorded clinical data held on the SMMIS regional database is largely accurate and consistently recorded across a variety of units. The database can therefore be considered a valuable resource for the comparative audit of maternity practice. The SMMIS technique for deriving, on a semi-automatic basis, diagnosis codes from the directly recorded fields, appears to work moderately well. We suggest that the direct method of data collection used in SMMIS could provide a model for other specialties in the National Health Service.

Data Collection↗
PubMed1994

Mutational analysis of human papillomavirus E4 proteins: identification of structural features important in the formation of cytoplasmic E4/cytokeratin networks in epithelial cells.

We have previously demonstrated that human papillomavirus type 1 (HPV 1) and 16 (HPV 16) E4 proteins form cytoplasmic filamentous networks which specifically colocalize with cytokeratin intermediate-filament (IF) networks when expressed in simian virus 40-transformed keratinocytes. The HPV 16 (but not the HPV 1) E4 protein induced the collapse of the cytokeratin networks. (S. Roberts, I. Ashmole, G. D. Johnson, J. W. Kreider, and P. H. Gallimore, Virology 197:176-187, 1993). The mode of interaction of E4 with the cytokeratin IFs is unknown. To identify E4 sequences important in mediating this interaction, we have constructed a large panel of mutant HPV (primarily HPV 1) E4 proteins and expressed them by using the same simian virus 40-epithelial expression system. Mutation of HPV 1 E4 residues 10 to 14 (LLGLL) abrogated the formation of cytoplasmic filamentous networks. This sequence corresponds to a conserved motif, LLXLL, found at the N terminus of other E4 proteins, and similar results were obtained on deletion of the HPV 16 motif, LLKLL (residues 12 to 16). Our findings indicate that this conserved motif is likely to play a central role in the association between E4 and the cytokeratins. An HPV 1 E4 mutant protein containing a deletion of residues 110 to 115 induced the collapse of the cytokeratin IFs in a manner analogous to the HPV 16 E4 protein. The sequence deleted, DLDDFC, is highly conserved between cutaneous E4 proteins. HPV 1 E4 residues 42 to 80, which are rich in charged amino acids, appeared to be important in the cytoplasmic localization of E4. In addition, we have mapped the N-terminal residues of HPV 1 E4 16-kDa and 10/11-kDa polypeptides expressed by using the baculovirus system and shown that they begin at tyrosine 16 and alanine 59, respectively. Similar-sized E4 proteins are also found in vivo. N-terminal deletion proteins, which closely resemble the 16-kDa and 10/11-kDa species, expressed in keratinocytes were both cytoplasmic and nuclear but did not form cytoplasmic filamentous networks. These findings support the postulate that N-terminal proteolytic processing of the E1-- E4 protein may modulate its function in vivo.

Adult↗