High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.     

Effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infections in young children in Brazil

A beneficial effect of periodic vitamin A supplementation on childhood mortality has been demonstrated, but the effect on morbidity is less clear. We investigated the effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infections (ALRI) in children from northeastern Brazil in a randomised, double-blind, placebo-controlled community trial. 1240 children aged 6-48 months were assigned vitamin A or placebo every 4 months for 1 year. They were followed up at home three times a week, and data about the occurrence and severity of diarrhoea and ALRI were collected. Any child with cough and respiratory rate above 40 breaths per min was visited by a paediatrician. The overall incidence of diarrhoea episodes was significantly lower in the vitamin-A-supplemented group than in the placebo group (18.42 vs 19.58 x 10(-3) child-days; rate ratio 0.94 [95% Cl 0.90-0.98]). The benefit of supplementation was greater as regards severe episodes of diarrhoea; the incidence was 20% lower in the vitamin A group than in the placebo group (rate ratio 0.80 [0.65-0.98]). With the standard definition of diarrhoea (> or = 3 liquid or semi-liquid stools in 24 h) the effect of vitamin A on mean daily prevalence did not reach significance, but as the definition of diarrhoea was made more stringent (increasing number of stools per day), a significant benefit became apparent, reaching for diarrhoea with 6 or more liquid or semi-liquid stools in 24 h a 23% lower prevalence. We found no effect of vitamin A supplementation on the incidence of ALRI. The reduction in severity of diarrhoea may be the most important factor in the lowering of mortality by vitamin A supplementation.     

Unusual chromosomal organization of telomeric sequences and expeditious karyotypic differentiation in the recently evolved Mus terricolor complex

The Mus terricolor complex displays a stable homozygous arrangement of autosomal heterochromatin variations in the form of accretion of definitive autosomal short arms among three nonoverlapping populations, in concert with an expeditious evolutionary differentiation into three chromosomal species: M. terricolor I, II, and III. In contrast to the highly conservative M. musculus-like chromosomes in the coexisting sibling species, M. booduga, reshuffling and differentiation of centric heterochromatin has occurred in harmony with a revision of centric configurations, resulting in acrocentric and submetacentric autosomes. The chromosomal distribution of the prevalent vertebrate telomeric sequence (TTAGGG)n was examined by fluorescence in situ hybridization to metaphase cells of M. terricolor I, II, and III. An unusual centric organization of internal telomeric sequences was detected in all the submetacentric and acrocentric autosomes. An auxiliary role of these presumably fragile, recombinogenic telomeric sequences in the evolutionary revision of centric configurations in the terricolor complex is hypothesized.     

Effects of hypertonia on voltage-gated ion currents in freshly isolated hippocampal neurons, and on synaptic currents in neurons in hippocampal slices

A NAD-dependent mannitol dehydrogenase (MtlD) was purified to homogeneity from P. fluorescens DSM50106 and the N-terminal amino acid sequence was determined. An oligonucleotide deduced from this peptide sequence was used as a probe to isolate the mannitol dehydrogenase gene (mtlD) from a genomic library of P. fluorescens. Nucleotide sequence analysis of a 1.8 kb NruI fragment containing the entire mtlD gene revealed an open reading frame of 1482 bp encoding a protein with a calculated molecular weight of 54.49 kDa. The enzyme shared a high similarity with a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall identity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-1-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activity of the mannitol dehydrogenase synthesized in E. coli was increased from 0.02 U (mg protein)(-1) to 10 U (mg protein)(-1). After fusion of six histidine codons to the 3' end of mtlD gene and expression in E. coli active mannitol dehydrogenase could be purified in a two-step procedure by affinity chromatography using a Ni2+ matrix column. The purified enzyme exhibited a specific activity of 46 U (mg protein)(-1) and was shown to be a polyol dehydrogenase with a broad substrate spectrum oxidizing efficiently mannitol, sorbitol and arabitol.     

A prospective study of the effect of fundoplication on primary and secondary peristalsis in the esophagus

BACKGROUND: Delay of prenatal care is an important risk for poor birth outcome, yet its association with maternal knowledge and beliefs remains insufficiently studied. This research examined the relationship of unintended childbearing and beliefs about the importance of prenatal care with initiation after the first trimester, adjusting for key sociodemographic determinants. METHODS: One hundred fifty-four Texas hospitals accounting for 80 percent of state births were asked to collect surveys from all women delivering infants during a one-week interval in 1986. Seventy-four percent of hospitals and 70 percent of women participated (n = 2032). No differences occurred between the sample and the population on rates of delayed care and low birthweight or maternal demographics. RESULTS: Since delayed prenatal care is more frequent among low-income women, analyses were limited to those below the 200 percent poverty level. After adjustment for maternal age, marital status, education, parity, race, and health insurance status employing logistic regression, unintended births were 1.6 times more likely to involve delayed care. Mothers who believed prenatal care was unimportant were 2.1 times more likely to delay care. These coefficients exceeded or about equaled those for the covariates. CONCLUSIONS: Preconception education about the value of prenatal care and family planning programs to prevent unintended pregnancies should be conducted together with efforts to overcome financial and structural barriers if progress toward national prenatal care objectives is to be achieved.     

Collateral blood supply through the ophthalmic artery: a steal phenomenon analyzed by color Doppler imaging

OBJECTIVE: This study aimed to evaluate the retrobulbar circulatory effects of reversed ophthalmic artery flow (ROAF) on the ophthalmic artery branches by means of color Doppler imaging. DESIGN: The design was a case-controlled study. PARTICIPANTS: Among 56 consecutive patients with severe (>70% stenosis) occlusive carotid artery disease, 15 patients (26.8%) with ROAF were identified. The control group consisted of 15 patients with similar degrees of carotid artery stenosis and forward ophthalmic artery flow. INTERVENTION: Arteriography and measurement of the retrobulbar hemodynamic parameters with color Doppler imaging were performed. MAIN OUTCOME MEASURES: Blood flow velocities and resistive index in the ophthalmic, central retinal, and temporal short posterior ciliary arteries were measured. RESULTS: Arteriography confirmed the diagnosis of ROAF in all 15 patients. There was no patient with ROAF diagnosed by arteriography and not diagnosed by color Doppler imaging. The frequency of bilateral severe occlusive carotid artery disease was significantly higher in the ROAF group (40%) compared to the control group (6.6%) (P = 0.04). Patients with ROAF showed significantly reduced vascular resistance in the ophthalmic artery (P = 0.03), higher vascular resistance, and lower blood flow velocities in the central retinal and temporal short posterior ciliary arteries (P < 0.05). CONCLUSION: This study suggests that patients with ROAF show a steal phenomenon, characterized by a shunt to the low-resistance intracranial circuit and reduction of retrobulbar blood flow.     

The role of human papillomavirus DNAs in cervical carcinoma and risk of lymph node metastasis: association with 72-kilodalton metalloproteinase immunostaining

BACKGROUND: The role of human papillomavirus (HPV) as a prognostic factor in cervical carcinoma is not understood completely and little is known regarding the intrinsic mechanisms involved in the metastatic process of HPV positive carcinoma. The authors evaluated HPV status with respect to clinical features in early stage cervical carcinoma, with special emphasis on lymph node spread. The authors also analyzed the relation between HPV, lymph node involvement, and 72-kilodalton (kDa) metalloproteinase immunostaining, an enzyme that cleaves Type IV collagen and may play a role in tumor metastasis. METHODS: Thirty-two patients with International Federation of Gynecology and Obstetrics Stage I and IIA squamous cell cervical carcinoma treated by primary radical surgery were reviewed. Histologic grade of differentiation, tumor size, fractional depth of invasion, and lymph node spread were evaluated with respect to HPV status and 72-kDa metalloproteinase immunostaining. HPV DNA was detected by polymerase chain reaction and the primers potentially recognized at least the following HPV subtypes: 6, 11, 16, 18, 31, 33, 34, 35, 42, 51, 56, and 58. Immunohistochemical staining was performed using the avidin-biotin complex technique. Affinity-purified rabbit anti-72-kDa metalloproteinase antibody was used. RESULTS: HPV DNA was detected in a total of 69% of cases, and HPV-16 was the most frequent type detected. HPV positive carcinomas showed a significantly higher rate of lymph node metastases than HPV negative carcinomas (45% vs. 10%; P = 0.03); similarly, 72-kDa metalloproteinase index was significantly higher (P = 0.001). CONCLUSIONS: These findings suggest a relation between HPV and risk of lymph node metastasis, which may be mediated by an increased production of 72-kDa metalloproteinase.     

Transport and turnover of microtubules in frog neurons depend on the pattern of axonal growth

Empirical analysis of the profitability of moderate grazing are presented based on sample dairy farms in Pennsylvania and New York. Net income per cow was higher for dairy farms that employed moderate intensive grazing than for dairy farms that employed extensive grazing. Income appeared to be adequate for family living expenses, but moderate intensive grazing could not be considered a high profit system. Detailed analyses of dairy farms that employed moderate and extensive grazing in northern Pennsylvania indicated that returns to management and owner equity were higher for pasture enterprises than for corn silage or hay enterprises. Positive dairy profits were related to lower feed costs. Milk production was lower on farms that employed moderate grazing than on farms that employed extensive grazing. Logit regression analysis characterized farms that employed moderate intensive grazing as oriented toward dairy rather than toward crop production; these farms had lower culling rates and a greater dependence on milk sales as a share of total sales. The reduced use of fertilizers and chemicals suggests that moderate grazing had environmentally sustainable features.     

Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria

Microbial Resistance to antibiotics is on the rise, in part because of inappropriate use of antibiotics in human medicine but also because of practices in the agricultural industry. Intensive animal production involves giving livestock animals large quantities of antibiotics to promote growth and prevent infection. These uses promote the selection of antibiotic resistance in bacterial populations. The resistant bacteria from agricultural environments may be transmitted to humans, in whom they cause disease that cannot be treated by conventional antibiotics. The author reviews trends in antibiotic use in animal husbandry and agriculture in general. The development of resistance is described, along with the genetic mechanisms that create resistance and facilitate its spread among bacterial species. Particular aspects of resistance in bacterial species common to both the human population and the agrifood industry are emphasized. Control measures that might reverse the current trends are highlighted.     

Laparoscopic esophagomyotomy and antireflux procedure with intraoperative manometry

We have been routinely performing laparoscopic cholecystectomy and antireflux procedures. Having this experience, we decided to assess the feasibility and safety of performing a laparoscopic esophagomyotomy and antireflux procedure. Here we present a case of a 37-year-old man with a history of progressive dysphagia and a diagnosis of achalasia, made on the basis of clinical, endoscopic, and manometric studies. Preoperative manometry reported a pressure of 52 mm Hg (normal, 15-25 mm Hg) for 4.5 cm (normal, > 3 cm). Laparoscopic esophagomyotomy and anterior fundoplication were performed. The esophagomyotomy included a 6-cm segment of distal esophagus and 2 cm of stomach; postoperative manometry was 18 mm Hg for 3 cm. Eight months postoperatively, a barium swallow demonstrated no reflux. Laparoscopic esophagomyotomy and antireflux procedure can be performed with efficacy and safety, with the advantage of a shorter hospitalization and an early recovery compared with the traditional procedure. Also, we emphasize the importance of the intraoperative manometry in the relevance of a concomitant antireflux procedure.