Biomedical subjects

T Wood

Publications and source records attributed to T Wood.

At least 19 recordsLinked to original sources

PubMed2002

Ultrastaging of early colon cancer using lymphatic mapping and molecular analysis.

Approximately one-third of node-negative colon cancers will recur, possibly due to understaging and inadequate pathological examination of lymph nodes (LNs). We evaluated the sensitivity, accuracy and feasibility of staging based on lymphatic mapping, focused examination, and molecular analysis of the sentinel node (SN) in patients with primary colorectal carcinoma. Between 1996 and 2000, 100 patients with colon carcinoma (CRC) underwent lymphatic mapping immediately after peritumoral injection of 1.0 cc of isosulphan blue dye. All LNs in the CRC specimen were examined by routine haematoxylin and eosin (H&E) staining. Sentinel nodes were examined by step serial sectioning, cytokeratin immunohistochemistry (CK-IHC) and/or reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in an attempt to identify occult micrometastatic disease. Lymphatic mapping was successful in 97% of the cases. There were 5 false-negative cases, predominately associated with T3/T4 tumours. Aberrant lymphatic drainage was identified in 8 patients (8%) altering the operative approach. 26 patients had H&E-positive LNs. In 74 patients who were node-negative by routine H&E, 18 (24%) had occult nodal micrometastases missed on routine H&E examination, but detected by focused analysis of the SN. RT-PCR analysis of the SN was performed in 40 patients, 26 of which were negative by H&E and CK-IHC. In 12/26 (46%) of these patients, there was additional evidence of micrometastatic disease. In this study, focused examination of the SN in conjunction with RT-PCR analysis identified micrometastatic disease in a significant number of node-negative patients. This may have important implications when selecting patients for adjuvant treatment protocols.

Adult↗

PubMed2001

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

Amino Acid Sequence↗

PubMed2001

A framework for sequencing the rice genome.

Rice is an important food crop and a model plant for other cereal genomes. The Clemson University Genomics Institute framework project, begun two years ago in anticipation of the now ongoing international effort to sequence the rice genome, is nearing completion. Two bacterial artificial chromosome (BAC) libraries have been constructed from the Oryza sativa cultivar Nipponbare. Over 100,000 BAC end sequences have been generated from these libraries and, at a current total of 28 Mbp, represent 6.5% of the total rice genome sequence. This sequence information has allowed us to draw first conclusions about unique and redundant rice genomic sequences. In addition, more than 60,000 clones (19 genome equivalents) have been successfully fingerprinted and assembled into contigs using FPC software. Many of these contigs have been anchored to the rice chromosomes using a variety of techniques. Hybridization experiments have shown these contigs to be very robust. Contig assembly and hybridization experiments have revealed some surprising insights into the organization of the rice genome, which will have significant repercussions for the sequencing effort. Integration of BAC end sequence data with anchored contig information has provided unexpected revelations on sequence organization at the chromosomal level.

Chromosome Mapping↗

PubMed2001

Transcriptional regulation of the BCL-X gene by NF-kappaB is an element of hypoxic responses in the rat brain.

Signal transduction pathways that mediate neuronal commitment to apoptosis involve the nuclear factor kappa B (NF-kappaB) transcription factor. The bcl-x gene is a member of the bcl-2 family of genes that regulate apoptosis, and gives rise to two proteins, Bcl-XL and Bcl-XS, via alternative mRNA splicing. BCl-XL protein, like Bcl-2, is a dominant inhibitor of apoptotic cell death, whereas Bcl-XS promotes apoptosis. While there is high expression of Bcl-XL in the developing and adult brain, few transcriptional control elements have been identified in the bcl-x promoter. There are two functional nuclear factor-kappa B (NF-kappaB) DNA binding sites clustered upstream of the brain-specific transcription start site in the upstream promoter region of murine bcl-x. Recombinant NF-kappaB proteins bind to these sites. Also NF-kappaB overexpression, coupled with bcl-x promoter/reporter assays using a series of murine bcl-x promoter and deletion mutants, has identified the downstream 1.1kb of the bcl-x promoter as necessary for basal promoter activity and induction by NF-kappaB in support of the hypothesis that NF-kappaB can act to enhance BCl-XL expression via highly selective interactions with the bcl-x promoter, where NF-kappaB binding and promoter activation are dependent on specific DNA binding site sequences and NF-kappaB protein dimer composition. Hypoxia induces apoptosis in the hippocampus where the NF-kappaB dimers c-Rel/p50 and p50/pS0 bind to the bcl-x promoter NF-kappaB site.

Animals↗

PubMed2001

Expression and regulation of ClC-5 chloride channels: effects of antisense and oxidants.

Genetic mutations of the Cl(-) channel ClC-5 cause Dent's disease in humans. We recently cloned an amphibian ortholog of Xenopus ClC-5 (xClC-5) from the A6 cell line. We now compare the properties and regulation of ClC-5 currents expressed in mammalian (COS-7) cells and Xenopus oocytes. Whole cell currents in COS-7 cells transfected with xClC-5 cDNA had strong outward rectification, Cl(-) > I(-) anion sensitivity, and were inhibited at low pH, similar to previous results in oocytes. In oocytes, antisense xClC-5 cRNA injection had no effect on endogenous membrane currents or the heterologous expression of human ClC-5. Activators of cAMP and protein kinase C inhibitors had no significant effects on ClC-5 currents expressed in either COS-7 cells or oocytes, whereas H-89, a cAMP-dependent protein kinase (PKA) inhibitor, and hydrogen peroxide decreased the currents. We conclude that the basic properties of ClC-5 currents were independent of the host cell type used for expression. In addition, ClC-5 channels may be modulated by PKA and reactive oxygen species.

Animals↗

PubMed2000

A cDNA RDA protocol using solid-phase technology suited for analysis in small tissue samples.

cDNA representational difference analysis (cDNA RDA) is a PCR-based subtractive enrichment procedure for the cloning of differentially expressed genes. In this study, we have further developed the procedure to take advantage of solid-phase technology, and to facilitate the use of RDA when starting material is limited. Several parameters of the PCR-based generation of cDNA representations were investigated, and a solid-phase based purification step was introduced to simplify removal of digested adapter-ends and uncleaved fragments. The use of magnetic particles increased the speed of the method, and also eliminated the risk of carry-over contamination between iterative steps of subtraction and PCR amplification. The modified protocol was evaluated in monitoring differences in gene expression in (i) a rat system consisting of livers with and without growth hormone treatment, and in (ii) a human system consisting of normal colon and colon cancer.

Acrylic Resins↗

PubMed2000

Insulin-like growth factor-I cDNA gene transfer in vitro and in vivo.

Our hypothesis is that gene transfer of an IGF-I CMV-cDNA with cholesterol containing cationic liposomes is an efficient tool for transient transfection of growth factors in vitro and in vivo. In vitro, we transiently cotransfected IGF-I cDNA with a CMV construct and a Lac Z beta-galactosidase cDNA/CMV construct using cholesterol containing cationic liposomes and measured beta-galactosidase and IGF-I mRNA and protein. In vivo, we subcutaneously injected 3-month-old male Sprague-Dawley rats with IGF-I cDNA and beta-galactosidase cDNA into rat skin. After IGF-I and beta-galactosidase were cotransfected into PC12 cells, Northern blot analysis showed that the peak time of IGF-I expression was 2 days for mRNA and 5 days for protein. In vivo, a cDNA/liposome ratio of 1:2 was most effective. IGF-I protein expression in IGF-I-transfected skin resulted in significant transfection from day 5 to day 7. In situ determination of beta-galactosidase activity confirmed that transfections resulted in a restricted expression area.

Animals↗

PubMed2000

Identification and characterization of nuclear factor kappaB binding sites in the murine bcl-x promoter.

Signal transduction pathways that mediate neuronal commitment to apoptosis involve the nuclear factor kappaB (NF-kappaB) transcription factor. Bcl-X(L) is a potent regulator of apoptosis in the CNS and is highly expressed in the developing and adult brain. We identified three putative NF-kappaB DNA binding sequences clustered upstream of the brain-specific transcription start site in the upstream promoter region. Recombinant p50/p50 and NF-kappaB proteins from nuclear extracts bound to these sites as determined by electrophoretic mobility shift assay and biotin-oligonucleotide/streptavidin affinity assays. NF-kappaB overexpression, coupled with bcl-x promoter/reporter assays using a series of murine bcl-x promoter and deletion mutants, has identified the downstream 1.1 kb of the bcl-x promoter as necessary for basal promoter activity and induction by NF-kappaB. The mutagenic removal of NF-kappaB binding sites individually or in combination revealed altered response patterns to p49/p65 and p50/p65 overexpression. These results support the hypothesis that NF-kappaB can act to enhance Bcl-X(L) expression via highly selective interactions, where NF-kappaB binding and bcl-x promoter activation are dependent on both DNA binding site sequence and NF-kappaB subunit composition. Our data suggest that molecular events associated with NF-kappaB promote regulation of neuronal apoptosis in the developing or injured CNS.

5' Untranslated Regions↗

PubMed2000

Nuclear factor kappaB/p49 is a negative regulatory factor in nerve growth factor-induced choline acetyltransferase promoter activity in PC12 cells.

Anovel nuclear factor kappaB (NF-kappaB) binding site has been identified within the promoter region of the mouse gene encoding choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine and has been implicated in the cognitive deficits associated with aging and Alzheimer's disease. This binding site, which is located within the nerve growth factor (NGF)-responsive enhancer element, was recognized by the NF-kappaB protein p49 but not p65 or p50. p49 from both basal forebrain and PC12 nuclear extracts interacted with this specific sequence in electrophoretic mobility shift assays. Mutation of the NF-kappaB site caused an increase in NGF-induced promoter activation, whereas overexpression of p49 in NGF-differentiated PC12 cells caused a decrease in endogenous ChAT enzyme activity and a decrease in promoter activity that was specifically mediated through this NF-kappaB binding site. Treatment of PC12 cells with NGF resulted in a drastic reduction in nuclear p49 binding to the ChAT NF-kappaB site after 24 h, but nuclear p49 levels were not altered, suggesting that late NGF-mediated events prevent binding of p49 to the ChAT promoter by an unknown mechanism other than nuclear translocation. Decreased ChAT expression and increased NF-kappaB activity in the brain are associated with aging and Alzheimer's disease. These data indicate that p49 is a negative regulator of ChAT expression and suggest a possible mechanism for aging-associated declines in cholinergic function.

Aging↗

PubMed2000

Mouse staufen genes are expressed in germ cells during oogenesis and spermatogenesis.

The Drosophila melanogaster staufen gene encodes an RNA binding protein (Dm Stau) required for the localization and translational repression of mRNAs within the Drosophila oocyte. In mammals translational repression is important for normal spermatogenesis in males and storage of mRNAs in the oocytes of females. In the present study we identified two mouse cDNA expressed sequence tags (ESTs), encoding proteins with significant homology to Dm Stau and used these firstly to screen a mouse kidney cDNA library and secondly to determine whether staufen mRNAs are expressed in the ovaries and testes of mice and rats. Sequence analysis of the cDNAs revealed that they originated from two different genes. Using Northern blots of RNAs from kidneys, ovaries and testes, both cDNAs hybridized to mRNA species of approximately 3 kb in all three tissues. On sections of mouse ovaries, staufen mRNA was localized specifically to oocytes. On sections of mouse testes, staufen mRNA was expressed in spermatocytes found in seminiferous tubules at stages VI-XII of the spermatogenic cycle. In conclusion, we have shown that the mammalian homologues of Dm stau are expressed in germ cells in both male and female mice, consistent with a role for these RNA binding proteins in mammalian gametogenesis.

Amino Acid Sequence↗

PubMed2000

Diagnostic and therapeutic choledochoscopy.

Laparoscopic cholecystectomy and common bile duct exploration are common surgical procedures for the general surgeon. To successfully remove common bile duct stones, choledochoscopy is an important technique for efficient and effective management. The choledochoscope can be introduced intraoperatively through the cystic duct or directly into the common bile duct and it also can be inserted transhepatically through a T-tube tract and per orally using a therapeutic duodenoscope. All of these methods allow for many options for the surgeon to consider, and one must be familiar with each of these techniques to maximize care. Successful stone extraction with the choledochoscope can be achieved in 75% to 95% of the cases with a morbidity less than 5%. Using this technique, the surgeon can improve the cost benefit of bile duct exploration.

Choledochostomy↗

PubMed1999

Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans.

Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.

Animals↗

PubMed1999

Comparison of amphibian and human ClC-5: similarity of functional properties and inhibition by external pH.

Loss of function mutations of the renal chloride channel, ClC-5, have been implicated in Dent's disease, a genetic disorder characterized by low weight proteinuria, hypercalciuria, nephrolithasis and, in some cases, eventual renal failure. Recently, our laboratory used an RT-PCR/RACE cloning strategy to isolate an amphibian cDNA from the renal epithelial cell line A6 that had high homology to human ClC-5. We now report a full-length native ClC-5 clone (xClC-5, containing 5' and 3' untranslated regions) isolated by screening a cDNA library from A6 cells that was successfully expressed in Xenopus oocytes. In addition, we compared the properties of xClC-5 and hClC-5 using isogenic constructs of xClC-5 and hClC-5 consisting of the open reading frame subcloned into an optimized Xenopus expression vector. Expression of the full-length "native" xClC-5 clone resulted in large, strongly rectifying, outward currents that were not significantly affected by the chloride channel blockers DIDS, DPC, and 9AC. The anion conductivity sequence was NO-3 > Cl- = I- > HCO-3 >> glutamate for xClC-5 and NO-3 > Cl- > HCO-3 > I- >> glutamate for hClC-5. Reduction of the extracellular pH (pHo) from 7.5 to 5.7 inhibited outward ClC-5 currents by 27 +/- 9% for xClC-5 and 39 +/- 7% for hClC-5. The results indicate that amphibian and mammalian ClC-5 have highly similar functional properties. Unlike hClC-5 and most other ClC channels, expression of xClC-5 in oocytes does not require the removal of its untranslated 5' and 3' regions. Acidic solutions inhibited both amphibian and human ClC-5 currents, opposite to the stimulatory effects of low external pH on other ClC channels, suggesting a possibly distinct regulatory mechanism for ClC-5 channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Sex-specific transcripts of the Dstpk61 serine/threonine kinase gene in Drosophila melanogaster.

We describe the characterization of several transcripts of the Drosophila serine/threonine protein kinase 61 (Dstpk61) gene. Dstpk61 produces at least four transcripts, including a 3.0-kb testis-specific transcript, a 4.5-kb female-specific carcass transcript, a 3.5-kb ovary-specific transcript, and a 4.7-kb non-sex-specific transcript. Two cDNAs, a 4.5-kb cDNA (cDNAB) and a 3.0-kb cDNA (cDNAA), likely to correspond to either the non-specific or the female-specific carcass and the testis-specific transcript, respectively, were fully sequenced and found to encode a novel OPA-repeat-containing serine/threonine-specific protein kinase. cDNAA and cDNAB both contain the entire ORF that encodes this predicted protein, but differ in the untranslated regions. The cDNAs contain translational control elements which are found in transcripts under male germline-specific translational control, and doublesex-like 13-nucleotide repeat elements, which are required for transformer/transformer-2-mediated splicing of the female doublesex transcript. The complex tissue and sex-specific transcripts, differing in the untranslated regions which are likely to be crucial in translational control, suggest that this kinase may have both general and sex-specific functions. The protein is homologous to human 3-phosphoinositide dependent protein kinase, which is involved in transduction of insulin signalling.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1998

Cloning, sequencing and expression of the flagellin core protein and other genes encoding structural proteins of the Vibrio cholerae flagellum.

Vibrio cholerae is a Gram-negative bacterium with a single polar flagellum. Motility is an important virulence factor for this non-invasive pathogen. We cloned and sequenced a locus in V. cholerae V86 (El Tor, Inaba) that contained five different structural genes of the flagellum. The cloned genes and their products were assigned names and functions based on homology with sequences of similar genes and their products from other related bacteria. All of these genes of V. cholerae V86, namely, flgI, J, M, L and flaA, were transcribed in the same direction. These genes respectively encoded the P- and L-ring proteins, the hook-associated proteins 1 and 3 and the flagellin core protein of the flagellum. Our data indicated the presence of more than one flagellar locus in V. cholerae which could provide a means of immunoavoidance during infection. When compared with homologs in other bacteria, the flagellin core protein of V. cholerae exhibited conservation in the N- and C-termini, but had diverged in the central region.

Amino Acid Sequence↗

PubMed1998

Comparison of injuries in elite senior and junior Australian football.

Three thousand and thirty one AFL and 1034 injuries in the VSFL U/18 competition were recorded by club doctors over the 1992, 1993 and 1994 seasons. Hamstring strains had the highest incidence (86.4 per 10,000 player hours) and prevalence (30.2 hours missed per 1000 hours) of any injury in the AFL, but were significantly less common in the U/18 competition. Other injuries which were common in both competitions were ankle sprains, thigh haematomas, concussion, groin strains and head lacerations. Injury prevalence was higher overall in the AFL, with lower limb muscle strains (hamstring, calf, quadriceps) being significantly more prevalent than in the U/18 competition. Injuries which were significantly more prevalent in the U/18 competition included stress fractures and concussion. Subsequent to this study, coaches and medical staff in the U/18 competition were made aware of the high risk of stress fractures in young footballers with heavy training loads. The AFL injury survey is ongoing and in the process of being computerised; risk factors for specific injuries with high rates are being studied further.

Adolescent↗

PubMed1998

Signal transduction in neuronal death.

Apoptosis in the nervous system is a necessary event during the development of the nervous system and is also present after genotoxic events, be they chronic as in aging or more acute after trauma and ischemia. Apoptotic events reflect an interplay between intrinsic signaling events that rely on cytokines, neurotransmitters, and growth factors and responses to extrinsic events that increase levels of radical oxygen species. Both intrinsically and extrinsically driven signal-transduction pathways act via transcription factors that regulate the coordinated timely expression of stress-response genes as part of a decision-making process that can commit cells to apoptosis or survival. Here we discuss the role of two transcription factors that participate in apoptosis in the nervous system: the activator protein AP-1 and nuclear factor kappaB.

Animals↗

PubMed1997

Comparison of DNA sequences with protein sequences.

The FASTA package of sequence comparison programs has been expanded to include FASTX and FASTY, which compare a DNA sequence to a protein sequence database, translating the DNA sequence in three frames and aligning the translated DNA sequence to each sequence in the protein database, allowing gaps and frameshifts. Also new are TFASTX and TFASTY, which compare a protein sequence to a DNA sequence database, translating each sequence in the DNA database in six frames and scoring alignments with gaps and frameshifts. FASTX and TFASTX allow only frameshifts between codons, while FASTY and TFASTY allow substitutions or frameshifts within a codon. We examined the performance of FASTX and FASTY using different gap-opening, gap-extension, frameshift, and nucleotide substitution penalties. In general, FASTX and FASTY perform equivalently when query sequences contain 0-10% errors. We also evaluated the statistical estimates reported by FASTX and FASTY. These estimates are quite accurate, except when an out-of-frame translation produces a low-complexity protein sequence. We used FASTX to scan the Mycoplasma genitalium, Haemophilus influenzae, and Methanococcus jannaschii genomes for unidentified or misidentified protein-coding genes. We found at least 9 new protein-coding genes in the three genomes and at least 35 genes with potentially incorrect boundaries.

Amino Acid Sequence↗