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J Latham

Publications and source records attributed to J Latham.

At least 19 recordsLinked to original sources
PubMed1999

smg-7 is required for mRNA surveillance in Caenorhabditis elegans.

Eukaryotic mRNAs that contain premature stop codons are degraded more rapidly than their wild-type counterparts, a phenomenon termed "nonsense-mediated mRNA decay" (NMD) or "mRNA surveillance." Functions of six previously described Caenorhabditis elegans genes, smg-1 through smg-6, are required for NMD. Whereas nonsense mutant mRNAs are unstable in smg(+) genetic backgrounds, such mRNAs have normal stability in smg(-) backgrounds. Previous screens for smg mutations have likely not identified all genes involved in NMD, but efforts to identify additional smg genes are limited by the fact that almost 90% of smg mutations identified in genome-wide screens are alleles of smg-1, smg-2, or smg-5. We describe a modified screen for smg mutations that precludes isolating alleles of smg-1, smg-2, and smg-5. Using this screen, we have identified and cloned smg-7, a previously uncharacterized gene that we show is required for NMD. smg-7 is predicted to encode a novel protein that contains an acidic carboxyl terminus and two probable tetratricopeptide repeats. We provide evidence that smg-7 is cotranscribed with the previously characterized gene lin-45 and show that null alleles of smg-7 confer a temperature-sensitive defect in NMD.

Amino Acid Sequence↗
PubMed1998

Imaging the problem scaphoid.

Scaphoid fractures can be difficult to diagnose on X-ray, even at 2 weeks. We have compared prospectively bone scanning versus MRI scanning in 22 patients with persistent signs and symptoms suggestive of a scaphoid fracture at 2 weeks and found both methods to be sensitive, but MRI to be more specific, particularly in diagnosing soft tissue injuries like scapho-lunate ligament ruptures and triangular fibrocartilage tears. An early MRI scan at 2 weeks allows the clinician to make the appropriate decision in this group of patients whose diagnoses include; occult scaphoid fractures, other occult carpal and radial fractures, ligamentous injuries and those without apparent injuries. MRI has also been useful in defining the group of injuries which may imitate a clinically occult scaphoid fracture.

Adolescent↗
PubMed1998

Development of a non-high pressure liquid chromatography assay to determine [14C]chlorzoxazone 6-hydroxylase (CYP2E1) activity in human liver microsomes.

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.

Animals↗
PubMed1997

Elevated synthesis of biglycan and decorin in an ovine annular lesion model of experimental disc degeneration.

The aim of this study was to extend our earlier observations on the changes that occur in the proteoglycans (PGs) of discs subjected to experimental injury to the annulus fibrosus (AF). We employed the alginate bead culture method to examine the metabolism of the dermatan sulphate (DS) containing PGs by cells derived from different regions of ovine discs that had been subjected to experimental annular injury. This was compared with the metabolism of the DS-PGs by cells isolated from equivalent regions of normal sham-operated discs. Six months after induction of the annular lesion, AF cells isolated from the lesion produced significantly higher levels of decorin and biglycan in alginate bead culture than did cells from equivalent zones of the controls. Decorin and biglycan were identified in culture media samples by immunoblotting, using specific antibodies (6-B-6, LF-96), and also by positive identification of their de-glycosylated core proteins. The core protein of the DS-PGs has been shown to inhibit type I/II collagen fibrillogenesis, to negatively regulate the action of transforming growth factor-beta (TGF-beta) and to diminish cellular proliferation in vitro; events which may be detrimental to tissue repair. The findings are therefore consistent with our previous observation the annular lesions in the avascular inner annulus have no capacity to heal.

Animals↗
PubMed1997

Topographical variation in the catabolism of aggrecan in an ovine annular lesion model of experimental disc degeneration.

An established model of experimental disc degeneration (Osti et al., Spine 15:762, 1990; Melrose et al., J Orthop Res 10:655, 1992) was used in this study. Four 2-year-old sheep received anterolateral incisions (4 x 10 mm) in the outer one-third of the annulus fibrosus of their L2-L3 and L4-L5 discs (lesion group). The annulus was not incised in another four sham-operated animals. After 6 months the sheep were killed, lumbar discs were dissected into lateral halves of the annulus fibrosus and the nucleus pulposus. Cells were isolated from disc tissues enzymatically and were grown in alginate bead culture to examine the proteoglycan metabolism of cells from lesion and control zones. The media of lesion zone cultures contained relatively high levels (compared with sham cultures) of catabolic fragments of the large, high-buoyant-density proteoglycans as demonstrated by Western blotting using monoclonal antibodies (5-D-4, 3-B-3, 1-C-6) and biotinylated hyaluronan and also by gel chromatography. Furthermore, cells from the vicinity of the lesion site also synthesized significantly lower levels (compared with sham cultures) of aggrecan that was retained within the alginate beads. Collectively, these data indicated that focal depletion of large, high-buoyant-density proteoglycans was evident within lesion sites in this model of experimental disc degeneration. The introduction of an annular lesion therefore significantly affected the proteoglycan metabolism of endogenous disc cell populations. The unique hydrodynamic and viscoelastic properties of the intervertebral disc are dependent to a large degree on the tissue levels of aggrecan. The focal depletion of aggrecan by annular lesions therefore may represent an important predisposing factor to the subsequent degeneration of these intervertebral discs.

Aggrecans↗
PubMed1996

Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity.

The stability of cytochrome P450 enzymes, cytochrome b5, and NADPH-cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at -80 degrees C, (B) human liver microsomes suspended in 250 mM sucrose and stored at -80 degrees C, and (C) human liver microsomes subjected to as many as 10 cycles of thawing and freezing. In study A, microsomes from five human livers were prepared from fresh (unfrozen) tissue and from tissue that was stored frozen at -80 degrees C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b5 and the activity of NADPH-cytochrome c reductase decreased 20-40% as a result of freezing the liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases were observed in the activities of cytochrome P450 enzymes belonging to several gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4'- hydroxylation), CYP2D6 (dextromethorphan O-de-methylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4solidus5 (testosterone 6beta-hydroxylation), and CYP4A9solidus11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, but it increased the contamination of liver microsomes with hemoglobin or other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cytochromes P450 and b5 and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and b5, the activity of NADPH-cytochrome c reductase, and the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at -80 degrees C for approximately 0, 1, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome b5, and NADPH-cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at -80 degrees C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b5 concentration, or NADPH-cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at -80 degrees C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activity. Overall, these results suggest that our current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity.

Adult↗
PubMed1995

Cytokine treatment of human bone marrow activates anti-leukaemia effector cells: monitoring of purging by polymerase chain reaction and DNA analysis.

The aims of this study were to investigate the role of cytokines (tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) and interleukin-2 (IL-2) in augmenting graft-versus-leukaemia (GVL). We have investigated the effector cells involved in GVL, by studying the role of these cells in purging of the cell line K562 in short-term bone marrow cultures. The effect of the addition in vitro of rGCSF was also studied. Monitoring of purging was achieved by cytotoxicity assays, DNA analysis and the use of the polymerase chain reaction for the detection of bcr/abl transcripts in the Philadelphia positive (Ph+) K562 cell line. Supernatants from IL-2-treated and non-treated bone marrow were tested for cytokine production (TNF alpha and IFN gamma). The results have shown that the main cytotoxic effector cells in the bone marrow generated by IL-2 have the CD56+ CD8+ phenotype. Overnight incubation of bone marrow was sufficient to generate cytotoxic cells as measured by Chromium51 (Cr51) release assays. Measurable levels of TNF alpha but not IFN gamma were also detected in supernatants. Addition of TNF alpha and IFN gamma to the IL-2 in the bone marrow cultures augmented the cytotoxicity but tended to inhibit progenitor cell growth as measured by granulocyte-macrophage colony-forming unit (GM-CFU) and erythroid blast-forming unit (BFU-e) assays. An estimate of the purging of the marrow could also be achieved by DNA analysis of K562 DNA in bone marrow. The bcr/abl transcript could still be detected by PCR analysis in marrow containing 1% K562 and treated with IL-2 for 24 h, but by 6 days of incubation the bcr/abl transcript was weak or undectable. The results suggest that although reduction in the proportion of leukaemia in contaminated marrow can be detected after incubation with IL-2 for 24 h, complete elimination of minimal residual disease requires longer incubation times.

Biomarkers, Tumor↗
PubMed1994

Assessment and measurement of pain.

This paper provides an overview of issues relating to pain assessment and management. Areas to be covered include the problems involved in assessing pain as a subjective phenomenon, the constraints of assessment within the clinical field, factors that affect pain, and pain assessment tools and questionnaires. The aim of the reference list is to provide back-up reading to assist in exploring the options available when considering the possibility of compiling a pain assessment protocol for individual clinical areas.

Humans↗
PubMed1990

Effect of a new computed tomographic image transfer system on management of referrals to a regional neurosurgical service.

A computed tomographic image transfer system ('Image Link') was used to link scanners within the Oxford region, UK, to the regional neurosurgical service. 100 consecutive neurosurgical referrals were examined by this system; 43% of the emergency referrals did not require transfer, 31% were transferred electively, and 26% urgently. Most of the patients defined as potentially hazardous for transfer (10/11) did not require neurosurgical intervention. The delay by the use of the system was negligible and early detection of intracranial haematoma contributed to good outcome in some head injured patients. Overall, 3,170 miles of ambulance journey were avoided. Use of image link has led to substantial improvements in management of neurosurgical emergency referrals, cost-effectiveness of neurosurgical and ambulance facilities, and interhospital communication between doctors.

Adolescent↗
PubMed1990

Effects of phenytoin sodium on doubling time, deoxyuridine suppression, 3H-methotrexate uptake and 57Co-cyanocobalamin uptake in HL60 cells.

Phenytoin sodium (5-50 micrograms/ml) caused a dose-dependent prolongation of the doubling time of the human promyelocytic leukaemia cell line, HL60. This effect was unassociated with any alteration in cell viability. HL60 cells which were pre-incubated with 15 micrograms/ml phenytoin sodium for 1 or 48 h and then incubated with the same concentration of the drug plus either 3H-methotrexate (3H-MTX) or 57Co-cyanocobalamin for 90 min, showed an altered accumulation of both radioactive compounds when compared with control cells. Control cells were not pre-incubated with the drug and were subsequently studied in the absence of the drug. Pre-incubation with the drug for 1 h resulted in a 34% increase, and pre-incubation for 48 h in a 19% reduction in the accumulation of 3H-MTX. Pre-incubation for 1 or 48 h caused a 29% reduction in the accumulation of 57Co-cyanocobalamin. Cells cultured in the presence of 15 micrograms/ml phenytoin sodium for 48 h also gave a slightly increased deoxyuridine-suppressed value; this abnormality was partially corrected by the addition of 50 micrograms/ml folinic acid to the test system but was unaffected by the addition of 1 microgram/ml cyanocobalamin. The data indicate that the effects of phenytoin sodium on the proliferation of HL60 cells may have been slightly mediated via a reduced uptake of folate and possibly also of vitamin B12. They also suggest that one of the mechanisms underlying some of the undesirable effects of long-term therapy with phenytoin may be a drug-related impairment of both folate and vitamin B12 uptake by certain cells, including haemopoietic and neural cells.

Cell Division↗
PubMed1979

Control of Christmas holiday census and staffing.

Most hospitals experience decreased census during certain holiday periods. The costs related to keeping units open during these periods are major problems for nursing administrators. This article describes a holiday staffing plan that addresses the personal needs of staff and the need to control operations. The same principles may apply at other low census periods such as during summer months, vacation periods, or strikes.

Holidays↗