Pα-Methyl Deoxynucleoside Triphosphates as Substrates for E. COLI DNA Polymerase I in a Template-Directed Synthesis of DNA

Abstract

Pα-methyl deoxynucleoside triphosphates are used as substrates for E. coli DNA polymerase I in template-directed polymerase reactions. It is shown that the modified compounds are incorporated together with the unmodified deoxynucleoside triphosphates into DNA under both nick-translation and Klenow reaction conditions.     

Histochimie du Pancreas du Chat Avec Discussion du Tissu Langerhansien Endocrine

Résumé Des recherches histologiques et histochimiques sont faites sur le pancréas du chat avec discussion surtout au sujet du tissu endocrine. Quelques particularités sont signalées: la richesse en îlots ganglionnaires, en corpuscules de Pacini et en complexes neuro-insulaires, aussi bien que l'existence dans la partie apicale de l'épithélium des canaux excréteurs de granulations paraldéhyde positives. En principe l'histoenzymologie du pancréas du chat est semblable à celle des autres mammifères mais ne lui est pas toujours identique. Certaines considérations sont faites sur le rôle probable des enzymes étudiées et du Zn dans la cytophysiologie insulaire.

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Summary Morphological and histochemical investigations on the pancreas of cat have been carried out and discussion on the question of the endocrine tissue. Some particularities are marked down: abundance of ganglion islets, of corpuscules of Pacini and neuro-insular complexes, as well as existence of positive paraldehyde granulations in the top portion of the epithelium of the excretory ducts. In general the histoenzymology of the pancreas of the cat is similar to other mammals, but is not always identical. Some considerations on the probable role of the enzymes investigated and on the zinc in the insular cytophysiology are made.

     

PRDM9 Drives Evolutionary Erosion of Hotspots in Mus musculus through Haplotype-Specific Initiation of Meiotic Recombination

Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9 ^Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9^Cst arose, and M.m. domesticus, into which Prdm9^Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9^Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parent''s Prdm9 allele and the opposite parent''s chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.     

Escherichia coli O157:H7 Infection in Dutch Belted and New Zealand White Rabbits

Enterohemorrhagic Escherichia coli (EHEC) produce one or more types of Shiga toxins and are foodborne causes of bloody diarrhea. The prototype EHEC strain, Escherichia coli O157:H7, is responsible for both sporadic cases and serious outbreaks worldwide. Infection with E. coli that produce Shiga toxins may lead to diarrhea, hemorrhagic colitis, or (less frequently) hemolytic uremic syndrome, which can cause acute kidney failure. The exact mechanism by which EHEC evokes intestinal and renal disease has not yet been determined. The development of a readily reproducible animal oral-infection model with which to evaluate the full pathogenic potential of E. coli O157:H7 and assess the efficacy of therapeutics and vaccines remains a research priority. Dutch belted (DB) rabbits are reported to be susceptible to both natural and experimental EHEC-induced disease, and New Zealand white (NZW) rabbits are a model for the intestinal manifestations of EHEC infection. In the current study, we compared the pathology caused by E. coli O157:H7 infection in DB and NZW rabbits. Both breeds of rabbits developed clinical signs of disease and intestinal lesions after experimental infection. In addition, one of the infected DB rabbits developed renal lesions. Our findings provide evidence that both breeds are susceptible to E. coli O157:H7 infection and that both may be useful models for investigating EHEC infections of humans.Abbreviations: EHEC, enterohemorrhagic E. coli; HUS, hemolytic uremic syndrome; DB, Dutch belted; STEC, Shiga-toxin– producing E. coli; NZW, New Zealand whiteEscherichia coli O157:H7 is the prototype enterohemorrhagic strain of Shiga-toxin–producing E. coli (STEC), which cause food and waterborne outbreaks and sporadic cases of serious intestinal disease that manifest as diarrhea or hemorrhagic colitis (or both).^12,13,31 Enterohemorrhagic E. coli (EHEC) are a subset of STEC that, in addition to elaborating Shiga toxins, encode the locus of enterocyte effacement, whose products allow intimate attachment of the bacteria to the epithelium.^16,19 Children infected with STEC are more susceptible than adults and may subsequently develop hemolytic uremic syndrome (HUS) that is characterized by hemolytic anemia, thrombocytopenia, and kidney dysfunction or failure.²⁹ Shiga toxins are considered to be major determinants involved in the pathogenesis of these E. coli-induced infections. Indeed, the capacity of STEC to cause bloody diarrhea and HUS derives from the activity of the Stx.^8,25,30,40 The 2 types of Shiga toxins, Stx1 and Stx2, are quite similar in sequence and structure, although their polyclonal antisera do not crossreact.^7,38,39,42A vaccine is currently not available to protect humans from infection or disease caused by STEC. There is a need to define the pathogenic mechanisms by which STEC cause disease and to develop strategies for the prevention and treatment of STEC-mediated HUS. Achieving this goal would benefit from a small animal model that displays gastroenteritis or signs of HUS similar to those occurring in humans. Naturally infected DB rabbits mimic the clinical and pathologic signs (including diarrhea, hemorrhagic colitis, and HUS) produced by STEC in humans.¹¹ In addition, infant NZW rabbits become infected with EHEC and subsequently exhibit diarrhea and hemorrhagic colitis.^20,28,34,36 The current study compared DB and NZW rabbits for breed-specific differences in response to E. coli O157:H7 infection.     

Patterns of recombination activity on mouse chromosome 11 revealed by high resolution mapping

The success of high resolution genetic mapping of disease predisposition and quantitative trait loci in humans and experimental animals depends on the positions of key crossover events around the gene of interest. In mammals, the majority of recombination occurs at highly delimited 1-2 kb long sites known as recombination hotspots, whose locations and activities are distributed unevenly along the chromosomes and are tightly regulated in a sex specific manner. The factors determining the location of hotspots started to emerge with the finding of PRDM9 as a major hotspot regulator in mammals, however, additional factors modulating hotspot activity and sex specificity are yet to be defined. To address this limitation, we have collected and mapped the locations of 4829 crossover events occurring on mouse chromosome 11 in 5858 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. This chromosome was chosen for its medium size and high gene density and provided a comparison with our previous analysis of recombination on the longest mouse chromosome 1. Crossovers were mapped to an average resolution of 127 kb, and thirteen hotspots were mapped to <8 kb. Most crossovers occurred in a small number of the most active hotspots. Females had higher recombination rate than males as a consequence of differences in crossover interference and regional variation of sex specific rates along the chromosome. Comparison with chromosome 1 showed that recombination events tend to be positioned in similar fashion along the centromere-telomere axis but independently of the local gene density. It appears that mammalian recombination is regulated on at least three levels, chromosome-wide, regional, and at individual hotspots, and these regulation levels are influenced by sex and genetic background but not by gene content.     

Validated methods for the quantification of biologically active constituents of poplar-type propolis

The validation of rapid, low-cost spectrophotometric procedures for the quantification of the three main groups of bioactive substances (flavones and flavonols, flavanones and dihydroflavonols, and total phenolics) in poplar-type propolis has been performed. A spectrophotometric assay based on the formation of an aluminium chloride complex was applied for the quantification of total flavones and flavonols using galangin as standard. Because of the high amount of flavanones and dihydroflavonols in "poplar type" propolis, the introduction of a distinct procedure for their quantification was considered of special significance and the DAB9 colorimetric method was applied for the purpose. Total phenolic content was measured by the Folin-Ciocalteu procedure using a mixture of pinocembrin and galangin as a reference. The procedures were validated using a model mixture of compounds representing the poplar-type propolis composition as found in previous studies. The accuracy (recovery) varied in the range 84-109%, and the relative standard deviation was 0.5-6.2%. The developed spectrophotometric procedures were applied to six poplar type propolis samples. The results were verified independently by a HPLC procedure. The two sets of results agreed satisfactory, as proven by Student's t-test.     

Computational model of dot-pattern selective cells

A computational model of a dot-pattern selective neuron is proposed. This type of neuron is found in the inferotemporal cortex of monkeys. It responds strongly to groups of dots and spots of light intensity variation but very weakly or not at all to single dots and spots that are not part of a pattern. This non-linear behaviour is quite different from the spatial frequency filtering behaviour exhibited by other neurons that react to spot-shaped stimuli, such as neurons with centre-surround receptive field profiles found in the lateral geniculate nuclei and layer 4Cβ of V1. It is implemented in the proposed computational model by using an AND-type non-linearity to combine the responses of centre-surround cells. The proposed model is capable of explaining the results of neurophysiological experiments as well as certain psychophysical observations. Received: 20 December 1999 / Accepted in revised form: 3 March 2000     

Identification of differentially expressed genes in epithelial stem/progenitor cells of fetal rat liver

Differentially expressed cDNA clones from fetal rat liver were isolated using suppression subtractive hybridization, combined with an efficient screening strategy. Approximately 30,000 clones were screened, yielding 643 genes whose expression was induced, of which 201 clones were distinct and 68 represented ESTs or newly discovered genes of unknown function. Based on their expression patterns in different organs, fetal liver, liver regeneration models, and gut epithelial progenitor cell lines, the subtracted clones presented in this work were placed into four categories: (1) hepatoblast-specific genes; (2) hematopoietic cell-specific genes; (3) genes expressed in hepatoblasts, in hematopoietic cells, and at varying levels in other tissues; and (4) genes overexpressed in fetal liver, in models of activation of liver progenitor cells, and in epithelial progenitor cell lines. Hepatoblast-specific clones and those representing genes induced during liver regeneration are under further study to define their specific function(s) in liver cell growth control and/or differentiation.