Static and Dynamic Factors Limit Chromosomal Replication Complexity in Escherichia coli,Avoiding Dangers of Runaway Overreplication

We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC∼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the “natural” CRC limit of ∼8 (cells divide more slowly); the “functional” CRC limit of ∼22 (cells divide extremely slowly); and the “tolerance” CRC limit of ∼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter.     

Paradoxical effects of maternal stress on fetal steroids and postnatal reproductive traits in female mice from different intrauterine positions

We examined effects of maternal stress on prenatal serum concentrations of testosterone and estradiol and on postnatal reproductive traits in female mice from different intrauterine positions. On Day 18 of fetal life, control females positioned in utero between two male fetuses (2M females) had higher concentrations of testosterone and lower concentrations of estradiol in serum than control female fetuses located between two females (0M females). Control females positioned between a male and a female fetus (1M females) had intermediate levels of both hormones. Prior intrauterine position in control females accounted for differences in genital morphology (length of the anogenital separation) at birth and length of estrous cycles during adulthood. Maternal stress eliminated these postnatal differences due to prior intrauterine position: all 0M, 1M, and 2M female offspring of stressed mothers exhibited postnatal traits that were indistinguishable from those of control 2M females. Maternal stress resulted in an increase of over 1 ng/ml in serum testosterone in all female fetuses; the magnitude of the increase was similar for 0M, 1M, and 2M females. There was no effect of maternal stress on serum concentrations of estradiol in 0M and 2M female fetuses. Maternal stress resulted in a dramatic change in the postnatal traits of 0M females, whereas 2M females showed no change. Since the effect of maternal stress on sex steroids was similar among fetuses from different intrauterine positions but postnatal response to maternal stress varied by intrauterine position, other components of the endocrine system may mediate effects of maternal stress on these postnatal characteristics.     

Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup.     

A simple device for collecting root exudates chromatographic studies

Plant and Soil - A simple apparatus is described for the collection of root exudates at different intervals without disturbing the root system of the seedling.     

Inactivation of Kupffer Cells by Selenizing Astragalus Polysaccharides Prevents CCl<Subscript>4</Subscript>-Induced Hepatocellular Necrosis in the Male Wistar Rat

Selenizing astragalus polysaccharides-3 (sAPS₃) was prepared by nitric acid–sodium selenite method. The effects of sAPS₃ on carbon tetrachloride (CCl₄) induced hepatocellular necrosis, and its underlying mechanisms were studied in male Wistar rats. Hepatic damage was induced by intraperitoneal injection of CCl₄ twice a week, for 3 weeks. Meanwhile, the rats in addition to CCl₄ were also exposed to sodium selenite (SS), astragalus polysaccharides (APS), SS + APS or sAPS₃, in parallel by oral gavage once a day for 3 weeks. At the end of 3 weeks, blood and liver tissue were taken. Serum was collected to test the levels of alanine aminotransferase, aspartate aminotransferase and antioxidant status parameters. Liver tissue was collected for histopathological examination and determination of messenger RNA (mRNA) expression levels of CD68, TNF-α, IL-1β and ATG7 followed by the measurements of CD68, IL-1β and LC3II by immunohistochemistry assay (IHC), or TNF-α by immunofluorescence assay (IFA). The results showed that sAPS₃ effectively ameliorated CCl₄ induced hepatocellular necrosis and inflammation and significantly decreased the levels of aspartate aminotransferase, alanine aminotransferase, malondialdehyde and the expression levels of Kupffer cells (KCs)-specific biomarker CD68 and proinflammatory cytokines produced by activated KCs such as IL-1β and TNF-α (P < 0.01). While increasing the levels of total antioxidant capacity, glutathione, glutathione peroxidase and superoxide dismutase (P < 0.05) and reduced the expression levels of a key regulator of autophagy in KCs ATG7 or LC3II (P < 0.05). These findings indicate that sAPS₃ could ameliorate CCl₄-induced hepatocellular necrosis by inactivation of Kupffer cells and its activity may be superior to the application of selenium, APS or combination of selenium with APS.     

First reported chloroplast genome sequence of <Emphasis Type="Italic">Punica granatum</Emphasis> (cultivar Helow) from Jabal Al-Akhdar,Oman: phylogenetic comparative assortment with <Emphasis Type="Italic">Lagerstroemia</Emphasis>

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It has grown in popularity and is a profitable fruit crop due to its attractive features including a bright red appearance and its biological activities. Scientific exploration of the genetics and evolution of these beneficial traits has been hampered by limited genomic information. In this study, we sequenced the complete chloroplast (cp) genome of the native P. granatum (cultivar Helow) cultivated in the mountains of Jabal Al-Akhdar, Oman. The results revealed a P. granatum cp genome length of 158,630 bp, characterized by a relatively conserved structure containing 2 inverted repeat regions of 25,466 bp, an 18,686 bp small single copy regions, and an 89,015 bp large single copy region. The 86 protein-coding genes included 37 transfer RNA genes and 8 ribosomal RNA genes. Comparison of the P. granatum whole cp genome with seven Lagerstroemia species revealed an overall high degree of sequence similarity with divergence among intergenic spacers. The location, distribution, and divergence of repeat sequences and shared genes of the Punica and Lagerstroemia species were highly similar. Analyses of nucleotide substitution, insertion/deletions, and highly variable regions in these cp genomes identified potential plastid markers for taxonomic and phylogenetic studies in Myrtales. A phylogenetic study of the cp genomes and 76 shared coding regions generated similar cladograms. The complete cp genome of P. granatum will aid in taxonomical studies of the family Lythraceae.     

Neurosecretion of the mediodorsal cells of the central nervous system of the snail Helisoma duryi

Summary The neurosecretory mediodorsal cells that produce a putative growth hormone of the snail Helisoma duryi were studied in fast-growing virgin snails and in slow-growing reproducing snails. There are about 60 mediodorsal cells in clusters on each side of the cerebral commissure of the central nervous system, and they contain dense-cored granules which are 100–200 nm in diameter. The cells of virgin snails have dense Golgi bodies, scattered ER cisternae, and few granules, while those of reproducing snails have pale Golgi bodies, stacked ER cisternae, and numerous granules. Thus the mediodorsal cells of the virgin snails appear to be more active synthetically than those of the reproducing snails. The cells near the endocrine dorsal bodies contain many dorsal body precesses in their membrane interdigitations. There are junction-like interactions between some of the interdigitations. Gap junction-like contacts are seen between mediodorsal cells and glial cells. The axon endings of the mediodorsal cells at the neurohemal area in the labial nerve show more release profiles in virgin snails than in reproducing snails. A daily pattern of release has been observed in reproducing snails, and rates of release are higher in the evening than in the morning.     

Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost

In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.