Biomedical subjects

M G Thomas

Publications and source records attributed to M G Thomas.

At least 55 records · Page 3Linked to original sources

PubMed2000

Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy.

Vitamin D3 is believed to reduce the risk of colon cancer, and serum levels inversely correlate with colorectal cancer incidence. The active metabolite, 1alpha,25-dihydroxyvitamin D3, has previously been shown to inhibit growth and promote differentiation of colon cancer cells. The vitamin D analogue, EB1089, is currently under clinical trial in a variety of cancers because of its growth-inhibitory effects in vitro and reduced hypercalcemic effects in vivo. The mechanism of growth inhibition by EB1089, however, remained to be determined. In this study we examined the effects of alpha,25-dihydroxyvitamin D3 and EB1089 on five colorectal tumor cell lines (two adenoma and three carcinoma) to determine the mechanism of growth inhibition and to ascertain whether premalignant adenoma cells were responsive to these agents. 1alpha,25-Dihydroxyvitamin D3 and EB1089 induced p53-independent apoptosis in adenoma and carcinoma cell lines in a dose-dependent manner between 10(-10) and 10(-6) M. EB1089, as well as inducing apoptosis, increased the proportion of cells in the G1 phase, particularly in the adenoma cell lines. In two of the three carcinoma cell lines (SW620 and PC/JW), levels of apoptosis induced by EB1089 were similar or greater than those induced by 1alpha,25-dihydroxyvitamin D3. Although the carcinoma cell line HT29 was relatively resistant to apoptosis induced by EB1089 compared with 1alpha,25-dihydroxyvitamin D3, EB1089 markedly inhibited cell yields. These observations offer promise for the clinical use of EB1089. To determine whether the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 was potentially via a differentiation pathway, alkaline phosphatase activity was measured as a marker of differentiation. Induction of alkaline phosphatase was observed in the floating apoptotic cells as well as in the adherent population. A link between the induction of differentiation and apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 is suggested by the occurrence of apoptosis subsequent to the induction of differentiation. To investigate the molecular pathway to apoptosis induction, members of the Bcl-2 family of proteins were examined (Bcl-2, Bcl-x, Bax, and Bak). Decreased Bcl-2 was observed in some cell lines, particularly in response to EB1089, but was not essential for apoptosis. Levels of the proapoptotic protein Bak, however, were consistently increased in all of the five cell lines in association with apoptosis induced by either agent. The results implicate Bak protein in the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 or its analogue EB1089. The ability of EB1089 to induce apoptosis in colorectal carcinoma cells suggests that this or other vitamin D analogues may prove clinically effective for the treatment of colorectal cancer. Furthermore, the fact that it induces cell cycle arrest and apoptosis in the premalignant adenoma cells may suggest an application in colorectal cancer chemoprevention.

Adenocarcinoma↗

PubMed2000

From a dry bone to a genetic portrait: a case study of sickle cell anemia.

The potential and reliability of DNA analysis for the identification of human remains are demonstrated by the study of a recent bone sample, which represented a documented case of sickle cell anemia. beta-globin gene sequences obtained from the specimen revealed homozygosity for the sickle cell mutation, proving the authenticity of the retrieved residual DNA. Further investigation of mitochondrial and Y chromosome DNA polymorphic markers indicated that this sample came from a male of maternal West African (possibly Yoruban) and paternal Bantu lineages. The medical record, which became available after the DNA analyses had been completed, revealed that it belonged to a Jamaican black male. These findings are consistent with this individual being a descendent of Africans brought to Jamaica during the trans-Atlantic slave trade. This study exemplifies how a "reverse population genetics" approach can be applied to reconstruct a genetic profile from a bone specimen of an unknown individual.

Africa↗

PubMed2000

An assessment of the long-term preservation of the DNA of a bacterial pathogen in ethanol-preserved archival material.

To examine the potential for DNA recovery from spirit-preserved medical material, a set of specimens from the Hunterian Collection of the Royal College of Surgeons was investigated. Using a range of DNA extraction techniques and the PCR, no replicable positive amplifications were made from this material of either human or Helicobacter DNA. Experiments with modern stomach biopsies of H. pylori-positive patients suggest that the bacterial DNA is typically present in a much lower concentration (10(3)-fold) than that of the host. The potential for recovery of this organism from spirit specimens is therefore low. The absence of DNA in this material is probably due to several factors, chiefly the incomplete fixation of the specimen by the ethanol storage fluid. Studies such as this demonstrate the need for a good understanding of specimen history when working with archival material.

Archives↗

PubMed2000

High-resolution Y chromosome haplotypes of Israeli and Palestinian Arabs reveal geographic substructure and substantial overlap with haplotypes of Jews.

High-resolution Y chromosome haplotype analysis was performed in 143 paternally unrelated Israeli and Palestinian Moslem Arabs (I&P Arabs) by screening for 11 binary polymorphisms and six microsatellite loci. Two frequent haplotypes were found among the 83 detected: the modal haplotype of the I&P Arabs (approximately 14%) was spread throughout the region, while its one-step microsatellite neighbor, the modal haplotype of the Galilee sample (approximately 8%), was mainly restricted to the north. Geographic substructuring within the Arabs was observed in the highlands of Samaria and Judea. Y chromosome variation in the I&P Arabs was compared to that of Ashkenazi and Sephardic Jews, and to that of North Welsh individuals. At the haplogroup level, defined by the binary polymorphisms only, the Y chromosome distribution in Arabs and Jews was similar but not identical. At the haplotype level, determined by both binary and microsatellite markers, a more detailed pattern was observed. Single-step microsatellite networks of Arab and Jewish haplotypes revealed a common pool for a large portion of Y chromosomes, suggesting a relatively recent common ancestry. The two modal haplotypes in the I&P Arabs were closely related to the most frequent haplotype of Jews (the Cohen modal haplotype). However, the I&P Arab clade that includes the two Arab modal haplotypes (and makes up 32% of Arab chromosomes) is found at only very low frequency among Jews, reflecting divergence and/or admixture from other populations.

Arabs↗

PubMed2000

Transcriptional regulation of pituitary synthesis and secretion of growth hormone in growing wethers and the influence of zeranol on these mechanisms.

This experiment evaluated relationships between pituitary messenger RNA levels of the transcription factor Pit-1, the growth hormone releasing-hormone receptor (GHRHR), and synthesis and secretion of GH in growing wethers. The experiment also evaluated the influence of the estrogenic compound, zeranol, on these relationships. Seventy wethers that were 9.5 +/- 1 day of age were randomly assigned to a control group or to one of three zeranol treatment groups that were implanted (12 mg, Ralgro) at 0, 45, and (or) 90 days of age. Twenty-eight days after implantation (i.e., Days 28, 73, 118) and on Day 135, sera were collected serially from wethers (n > or = 5) from each group and then their pituitary was collected. As wethers gained weight with age, the pituitary increased in size and so did the relative message levels of Pit-1 and GH (effect of time, P < 0.01). However, as wethers reached 135 days of age, serum concentrations of GH had declined while concentrations of IGF-I had increased (linear contrast, P < 0.01). Additionally, zeranol increased serum concentrations of GH and IGF-I and this effect on GH appeared to be a consequence of increased pulse amplitude, particularly at 73 and 118 days of age (treatment x time, P </= 0.074). This result could have been due to increased pituitary expression of GH (treatment, P < 0.08). Zeranol appeared to modulate pituitary expression of transcripts containing exon 3 of Pit-1 (treatment, P < 0.01) and GHRHR (treatment x time, P = 0.056), however, pituitary message levels of Pit-1 and GHRHR were not positively correlated to pituitary expression of GH mRNA or serum concentrations of GH.

Animals↗

PubMed2000

Effect of cerebroventricular infusion of insulin and (or) glucose on hypothalamic expression of leptin receptor and pituitary secretion of LH in diet-restricted ewes.

The objective was to determine the effect of central infusion of insulin and (or) glucose on hypothalamic expression of leptin receptor and pituitary secretion of LH in the ewe. Twenty-two ovariectomized ewes (32 wk of age) were fitted with two lateral cerebroventricular (LCV) cannulae and fed 33% of NRC requirements for 8 wk. Ewes (n> or =5/group) were then infused, via LCV cannulae, with artificial cerebrospinal fluid (aCSF) or aCSF containing physiological concentrations of insulin (INS), glucose (GLU), or INS + GLU; the mass of each increasing linearly from Day 0 (mass = 0 units/h) to Day 8 (mass of INS = 80 mIU/hr and GLU = 10 mg/hr). Jugular serum was collected every 12 min for 4 hr on Days 0, 2, and 4. Ewes treated with INS or INS + GLU had greater (P<0.06) mean concentrations of LH than aCSF treated ewes on Day 2 (13.8+/-1.8 and 12.5+/-1.3 > 8.0+/-3.3 ng/ml). Furthermore, on Day 4, concentrations of LH in INS treated ewes exceeded that (P<0.07) of aCSF treated ewes (14.8+/-2.0 > 7.4+/-3.0 ng/ml). Expression of NPY mRNA did not differ between treatments (P = 0.87). Leptin receptor mRNA expression was dramatically reduced (P<0.0002) in INS+GLU versus aCSF treated ewes. These data provide evidence to suggest that insulin may be an important component of hypothalamic mechanisms regulating secretion of LH and expression of leptin receptors in undernourished ruminants.

Animals↗

PubMed2000

Biosynthesis of L-p-hydroxyphenylglycine, a non-proteinogenic amino acid constituent of peptide antibiotics.

BACKGROUND: The non-proteinogenic amino acid p-hydroxyphenylglycine is a crucial component of certain peptidic natural products synthesized by a non-ribosomal peptide synthetase mechanism. In particular, for the vancomycin group of antibiotics p-hydroxyphenylglycine plays a structural role in formation of the rigid conformation of the central heptapeptide aglycone in addition to being the site of glycosylation. Initial labeling studies suggested tyrosine was a precursor of p-hydroxyphenylglycine but the specific steps in p-hydroxyphenylglycine biosynthesis remained unknown. Recently, the sequencing of the chloroeremomycin gene cluster from Amycolatopsis orientalis gave new insights into the biosynthetic pathway and allowed for the prediction of a four enzyme pathway leading to L-p-hydroxyphenylglycine from the common metabolite prephenate. RESULTS: We have characterized three of the four proposed enzymes of the L-p-hydroxyphenylglycine biosynthetic pathway. The three enzymes are encoded by open reading frames (ORFs) 21, 22 and 17 (ORF21: [PCZA361.1, O52791, CAA11761]; ORF22: [PCZA361. 2, O52792, CAA11762]; ORF17: [PCZA361.25, O52815, CAA11790]), of the chloroeremomycin biosynthetic gene cluster and we show they have p-hydroxymandelate synthase, p-hydroxymandelate oxidase and L-p-hydroxyphenylglycine transaminase activities, respectively. CONCLUSIONS: The L-p-hydroxyphenylglycine biosynthetic pathway shown here is proposed to be the paradigm for how this non-proteinogenic amino acid is synthesized by microorganisms incorporating it into peptidic natural products. This conclusion is supported by the finding of homologs for the four L-p-hydroxyphenylpyruvate biosynthetic enzymes in four organisms known to synthesize peptidic natural products that contain p-hydroxyphenylglycine. Three of the enzymes are proposed to function in a cyclic manner in vivo with L-tyrosine being both the amino donor for L-p-hydroxyphenylglycine and a source of p-hydroxyphenylpyruvate, an intermediate in the biosynthetic pathway.

Actinomycetales↗

PubMed2000

Clinical isolates of Staphylococcus aureus exhibit diversity in fnb genes and adhesion to human fibronectin.

OBJECTIVES: The fibronectin-binding proteins (FnBPs) of Staphylococcus aureus are involved in the pathogenesis of infection, but their characteristics in clinical isolates are incompletely defined. The aim of this study was to evaluate phenotypic and genotypic characteristics of the FnBPs of a large collection of recent isolates. METHODS: The adherence of 163 S. aureus isolates to immobilized fibronectin was compared with that of S. aureus 8325-4 using a microtitre assay. The presence of the genes encoding the fibronectin-binding proteins FnBPA and FnBPB was evaluated by Southern dot blot using probes specific for region A of fnbA or fnbB. RESULTS: The adherence of clinical isolates to fibronectin (expressed as a percentage of the mean adherence of S. aureus 8325-4) was 56%-125% for 155 isolates (95%), and less than 20% for eight isolates (5%). Adherence of the bacterial group associated with orthopaedic implant-associated infection was significantly greater than that for isolates associated with nasal carriage, endocarditis, or septic arthritis/osteomyelitis. Southern dot blot demonstrated that 126/163 isolates had two genes (77%) and 37/163 had one detectable gene (23%). There was no difference in adherence between isolates with one or two fnb, but isolates associated with invasive disease (endocarditis or primary septic arthritis and/or osteomyelitis) were more likely to have two genes. CONCLUSIONS: These data demonstrate diversity in the FnBPs of clinical isolates of S. aureus. The findings suggest that the interplay between pathogenesis and a single virulence determinant is unlikely to be a uniform process across a spectrum of infections. This confirms the need to extend the study of staphylococcal pathogenesis from the laboratory to non-uniform populations of clinically relevant isolates.

Adhesins, Bacterial↗

PubMed2000

Streptococcus pneumoniae septic arthritis in adults.

Septic arthritis is a rarely reported manifestation of disease due to Streptococcus pneumoniae. We have reviewed our recent experience of this disease in 14 adult patients. Common features in patients with S. pneumoniae septic arthritis included advanced age (median=63 y), pre-existing joint disease (6/14), large joint disease (14/14), polyarthritis (6/14), and associated meningitis, pneumonia or both (6/14). Two patients with septic arthritis and meningitis, and another with Down's syndrome and sleep apnoea, died during treatment. In the remaining 11 patients, treatment for at least 19 d, predominantly with intravenous benzyl penicillin, plus joint lavage, resulted in cure.

Adult↗

PubMed2000

Y chromosomes traveling south: the cohen modal haplotype and the origins of the Lemba--the "Black Jews of Southern Africa".

The Lemba are a traditionally endogamous group speaking a variety of Bantu languages who live in a number of locations in southern Africa. They claim descent from Jews who came to Africa from "Sena." "Sena" is variously identified by them as Sanaa in Yemen, Judea, Egypt, or Ethiopia. A previous study using Y-chromosome markers suggested both a Bantu and a Semitic contribution to the Lemba gene pool, a suggestion that is not inconsistent with Lemba oral tradition. To provide a more detailed picture of the Lemba paternal genetic heritage, we analyzed 399 Y chromosomes for six microsatellites and six biallelic markers in six populations (Lemba, Bantu, Yemeni-Hadramaut, Yemeni-Sena, Sephardic Jews, and Ashkenazic Jews). The high resolution afforded by the markers shows that Lemba Y chromosomes are clearly divided into Semitic and Bantu clades. Interestingly, one of the Lemba clans carries, at a very high frequency, a particular Y-chromosome type termed the "Cohen modal haplotype," which is known to be characteristic of the paternally inherited Jewish priesthood and is thought, more generally, to be a potential signature haplotype of Judaic origin. The Bantu Y-chromosome samples are predominantly (>80%) YAP+ and include a modal haplotype at high frequency. Assuming a rapid expansion of the eastern Bantu, we used variation in microsatellite alleles in YAP+ sY81-G Bantu Y chromosomes to calculate a rough date, 3,000-5,000 years before the present, for the start of their expansion.

Africa, Southern↗

PubMed2000

Identification of an alternative nucleoside triphosphate: 5'-deoxyadenosylcobinamide phosphate nucleotidyltransferase in Methanobacterium thermoautotrophicum delta H.

Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5'-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the S. enterica cobU gene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domain Archaea. The corresponding ORF open reading frame from Methanobacterium thermoautotrophicum Delta H (MTH1152; referred to as cobY) was amplified and cloned, and the CobY protein was expressed and purified from Escherichia coli as a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme of S. enterica. NTP:adenosylcobinamide kinase activity was not detected in M. thermoautotrophicum Delta H cell extract, suggesting that this organism may not have this activity. The cobY gene complemented a cobU mutant of S. enterica grown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis. cobY, however, failed to restore adenosylcobalamin biosynthesis in cobU mutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide. Therefore, NTP:adenosylcobinamide kinase activity is not required for de novo cobamide biosynthesis.

Amino Acid Sequence↗

PubMed2000

Coordinated localisation of mucins and trefoil peptides in the ulcer associated cell lineage and the gastrointestinal mucosa.

BACKGROUND AND AIMS: Trefoil factor family (TFF) peptides and the chromosome 11p15.5 mucin glycoproteins are expressed and secreted in a site specific fashion along the length of the gastrointestinal tract. Evidence for coexpression of mucins and trefoil peptides has been suggested in numerous gastrointestinal mucosal pathologies. The ulcer associated cell lineage (UACL) occurs at sites of chronic ulceration in Crohn's disease, expresses all three trefoil peptides, and is implicated in mucosal restitution. We tested the hypothesis that individual trefoil peptides are uniquely localised with specific mucins in the UACL and normal gastrointestinal epithelia. METHODS: Expression of mucin genes in the UACL from small bowel tissue of patients with Crohn's disease was detected by in situ hybridisation, and localisation of the products by immunohistochemistry. Colocalisation of mucins and trefoil peptides was demonstrated by immunofluorescent colabelling in UACL and normal gastrointestinal epithelia. RESULTS: MUC5AC and TFF1 were colocalised in distal ductular and surface elements of the UACL and in foveolar cells of the stomach, whereas MUC6 and TFF2 were colocalised to acinar and proximal ductular structures in the UACL, in the fundus and deep antral glands of the stomach, and in Brunner's glands of the duodenum. MUC5B was found sporadically throughout the UACL and gastric body. MUC2 was absent from the UACL, Brunner's glands, and stomach. MUC2 and TFF3 were colocalised throughout the large and small bowel mucosa. CONCLUSIONS: The UACL has a unique profile of mucin gene expression. Coordinated localisation of trefoil peptides and mucins in UACL and normal gastrointestinal epithelia suggests they may assist each others' functions in protection and repair of gastrointestinal mucosa.

Crohn Disease↗

PubMed2000

Lack of mucin MUC5AC field change expression associated with tubulovillous and villous colorectal adenomas.

BACKGROUND: MUC5AC is a secreted mucin aberrantly expressed by polypoid colorectal adenomas. It has been hypothesised that the "normal" surrounding colorectal mucosa expresses MUC5AC as a field change phenomenon that can be used to predict adenoma recurrence following resection. AIM: To determine if there is a field change of de novo MUC5AC expression in histologically normal rectal mucosa adjacent to villous and tubulovillous adenomas, and thus whether MUC5AC expression can be used as a marker of early tumour recurrence. METHODS: In a prospective cohort study paired mucosal biopsies of adenomatous and macroscopically "normal" mucosa were obtained from 11 patients with villous and 11 patients with tubulovillous adenomas who underwent primary resection for purpose of cure. The tissues were studied to determine MUC5AC gene expression by immunohistochemistry and in situ hybridisation. Patients were followed up by flexible sigmoidoscopy to detect the presence of early local recurrence. RESULTS: 10 villous adenomas showed mature MUC5AC glycoprotein and all 11 expressed MUC5AC mRNA. Five tubulovillous adenomas showed mature MUC5AC glycoprotein and 10 expressed MUC5AC mRNA. Neoexpression of the MUC5AC mucin gene was not detected in any of the mucosal biopsies taken adjacent to either villous or tubulovillous adenomas, even in three patients with early, locally recurrent disease. CONCLUSIONS: Aberrant MUC5AC gene expression is not a "field change" in the colorectal mucosa in patients with rectal adenomas and therefore cannot be used to predict local recurrence of villous and tubulovillous adenomas.

Adenoma, Villous↗

PubMed2000

Estrogen agonist (zeranol) treatment in a castrated male lamb model: effects on growth and bone mineral accretion.

The mechanism of estrogen's action on bone mineralization in children has received little attention. Our objective was to determine the effect of time (developmentally) and duration of exposure to an estrogen agonist (zeranol) on bone growth and mineralization using a castrated male lamb model. At birth, 40 male lambs were castrated and within 14 days of birth (day = 0) they were assigned (n = 10 per group) to age-matched control lambs (C-AGE) or to receive a 12.5-mg zeranol implant as follows: E-0, implanted on days 0, 45, 90, and 135; E-90, implanted on days 90 and 135; and E-0, 90, implanted on days 0, 90, and 135. Lambs were studied for 163 days. Serum was collected on days 28, 73, 118, 135, and 163 and analyzed for minerals (Ca, P, and Mg), markers of bone remodeling (bone alkaline phosphatase [ALP] and tartrate resistant acid phosphatase [TRAP]), 1,25-dihydroxyvitamin D [1,25(OH)2D], growth hormone (GH), and insulin-like growth factor I (IGF-I). Whole-body bone mineral content (BMC), bone mineral density (BMD), fat mass, and lean mass were determined by dual energy X-ray absorptiometry (DEXA) on days 28, 73, 118, and 163. There was a linear increase in growth at all time points. Whole-body BMC, weight, and lean mass of C-AGE and E-90 lambs were less than E-0, and E-0, 90 lambs at all time points. Whole-body BMD of C-AGE and E-90 lambs was less than E-0 and E-0, 90 lambs at 28 days and 73 days; however, after implantation at day 90 whole-body BMD of E-90 lambs was similar to E-0 and E-0, 90 lambs at day 118 and day 163 and all three were greater than C-AGE lambs. There was no effect of treatment on calcium absorption, serum minerals, hormones, or markers of bone remodeling. We conclude from these data that treatment of growing castrated lambs with an estrogen agonist from birth augments growth, whereas delaying estrogen agonist treatment does not facilitate growth but appears to augment bone mineral accretion. We suggest these observations may have clinical relevance, and deserve consideration when treating children with delays in growth and bone mineral accretion.

Animals↗

PubMed2000

Effect of dietary energy on milk production and metabolic hormones in thin, primiparous beef heifers.

Thirty-six primiparous heifers were used to determine the effect of dietary energy on postpartum interval, milk production, serum concentrations of insulin, insulin-like growth factor I (IGF-I), growth hormone, and cerebrospinal fluid (CSF) concentrations of neuropeptide-Y (NPY). Low-quality hay was fed during the last trimester of pregnancy to achieve suboptimal calving weight (370 +/- 5 kg) and condition score (4.0 +/- .1). After calving, cows were allotted to one of four dietary treatments that differed in metabolizable energy. Experimental diets were fed at 2.5% of shrunk body weight and formulated to provide 1.8 (low), 2.1 (maintenance), 2.4 (maintenance high), or 2.7 (high) Mcal of ME/kg DM. Daily milk production was estimated at approximately 30, 60, and 90 d postpartum. Condition score change and weight change were defined as change from calving to d 90 postpartum. As energy intake increased, condition score change (P < .001), IGF-I (P < .001) and insulin (P < .01) increased and postpartum interval decreased (P = .04). No relationship existed between postpartum interval and CSF concentration of NPY (P > .1). Condition score change was positively associated with IGF-I and insulin (r = .71, P < .001; r = .38, P = .02, respectively) and negatively associated with GH (r = -.67, P < .001). Weight change and serum concentrations of GH did not differ (P > .10) among treatments. Increasing dietary energy intake was associated with a curvilinear increase in milk yield (P = .04) and percentage milk fat (P = .03) and a linear increase (P = .04) in energy available for milk production. Greater milk yield at d 30 was associated with a longer postpartum interval (r = .34, P < .05). In conclusion, a greater proportion of net energy is partitioned to maternal tissue realimentation when cows receive high compared to low-, maintenance-, or moderate-energy diets.

Animal Feed↗

PubMed1999

Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) complexed with GMP: evidence for a substrate-induced transferase active site.

The X-ray crystal structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) from Salmonella typhimurium bound to GMP has been determined by molecular replacement to 2.2 A resolution. CobU is a bifunctional enzyme, which catalyzes the phosphorylation of the 1-amino-O-2-propanol side chain of the adenosylcobinamide ring and subsequently functions as a guanylyltransferase to form adenosylcobinamide.GDP. The transferase activity involves a covalent enzyme-guanylyl intermediate that is most likely a phosphoramidate linkage to His(46). Previous studies have shown that the enzyme is a homotrimer and adopts a pinwheel shape. Each subunit consists of a single domain of six parallel beta-strands and one antiparallel strand flanked on either side by a total of five alpha-helices and one helical turn. Interestingly, His(46) in the apoenzyme is located a considerable distance from the kinase active site or P-loop motif and is solvent-exposed [Thompson, T. B., et al. (1998) Biochemistry 37, 7686-7695]. To examine the structural relationship of the two active sites, CobU was cocrystallized with GTP and pyrophosphate. Crystals belong to space group P2(1)2(1)2(1) with the following unit cell dimensions: a = 58. 4 A, b = 87.8 A, and c = 101.6 A. The structure shows electron density for the hydrolysis product GMP rather than the expected covalent guanylyl intermediate which appears to have been hydrolyzed in the crystal lattice. Even so, CobU exhibits a substantial conformational rearrangement. The helix axis containing His(46), the site of guanylylation, rotates 30 degrees and translates 11 A relative to the apo structure and is accompanied by compensatory unwinding and rewinding at the helix ends to allow the induction of a guanosine binding pocket between beta-strand 2 and alpha-helix 2. This conformational change brings the C(alpha) of His(46) approximately 10 A closer to the P-loop motif such that a phosphate ion located in the P-loop is only 6 A from the alpha-phosphate of GMP. This suggests that the P-loop motif may be used to coordinate the terminal phosphates in both the transferase and kinase reactions and implies that the active sites for both reactions overlap.

Binding Sites↗

PubMed1999

High throughput analysis of 10 microsatellite and 11 diallelic polymorphisms on the human Y-chromosome.

We describe an integrated approach to the determination of complex Y chromosome haplotypes that is both fast and relatively inexpensive. The method employs GeneScan technology to enable a researcher to assay repeat number variation at ten microsatellite loci and determine the status of 11 diallelic polymorphisms. The method requires only four PCRs and four GeneScan runs per sample and is relatively insensitive to sample DNA concentration.

Genotype↗

PubMed1999

Injection of neuropeptide Y into the third cerebroventricle differentially influences pituitary secretion of luteinizing hormone and growth hormone in ovariectomized cows.

Hypothalamic neurons that control the luteinizing hormone (LH) and growth hormone (GH) axes are localized in regions that also express neuropeptide Y (NPY). Increased hypothalamic expression of NPY due to diet restriction has been associated with suppressed secretion of LH and enhanced secretion of GH in numerous species. However, these physiological relationships have not been described in cattle. Thus, two studies were conducted to characterize these relationships using ovariectomized (Experiment 1) or ovariectomized estrogen-implanted (Experiment 2) cows. In Experiment 1, four well-nourished, ovariectomized cows received third cerebroventricular (TCV) injections of 50 and 500 micrograms of NPY in a split-plot design. Venous blood was collected at 10-min intervals from -4 hr (pre-injection control period) to +4 hr (postinjection treatment period) relative to TCV injection. NPY suppressed (P < or = 0.04) tonic secretion of LH irrespective of dose and tended to stimulate (P < or = 0.10) an increase in tonic secretion of GH. In Experiment 2, six ovariectomized cows that were well nourished and implanted with estradiol received TCV injections of 0, 50, or 500 micrograms of NPY in a replicated 3 x 3 Latin Square. Both doses of NPY suppressed (P < 0.06) mean concentration of LH relative to the 0-microgram dose. The 50-microgram dose of NPY tended (P < 0.10) to increase the amplitude of GH pulses. In conclusion, TCV injection of NPY suppressed pituitary secretion of LH and simultaneously tended to increase pituitary secretion of GH.

Animals↗