PATJ regulates directional migration of mammalian epithelial cells

Directional migration is important in wound healing by epithelial cells. Recent studies have shown that polarity proteins such as mammalian Partitioning-defective 6 (Par6), atypical protein kinase C (aPKC) and mammalian Discs large 1 (Dlg1) are crucial not only for epithelial apico-basal polarity, but also for directional movement. Here, we show that the protein associated with Lin seven 1 (PALS1)-associated tight junction protein (PATJ), another evolutionarily conserved polarity protein, is also required for directional migration by using a wound-induced migration assay. In addition, we found that aPKC and Par3 localize to the leading edge during migration of epithelia and that PATJ regulates their localization. Furthermore, our results show that microtubule-organizing centre orientation is disrupted in PATJ RNA interference (RNAi) MDCKII (Madin-Darby canine kidney II) cells during migration. Together, our data indicate that PATJ controls directional migration by regulating the localization of aPKC and Par3 to the leading edge. The migration defect in PATJ RNAi cells seems to be due to the disorganization of the microtubule network induced by mislocalization of polarity proteins.     

Effect of Naturally Occurring nif Reiterations on Symbiotic Effectiveness in Rhizobium phaseoli

Most naturally occurring strains of Rhizobium phaseoli possess reiteration of the nif genes. Three regions contain nitrogenase structural genes in strain CFN42. Two of these regions (a and b) have copies of nifH, nifD, and nifK, whereas the third region (c) contains only nifH. Strains containing mutations in either nif region a or nif region b had significantly diminished symbiotic effectiveness compared with the wild-type strain on the basis of nodule mass, total nitrogenase activity per plant, nitrogenase specific activity, total nitrogen in the shoot, and percentage of nitrogen. A strain containing mutations in both nif region a and nif region b was totally ineffective. These data indicate that both nif region a and nif region b are needed for full symbiotic effectiveness in R. phaseoli.     

Influence of Environmental Factors on Interstrain Competition in Rhizobium japonicum

The effect of several biotic and abiotic factors on the pattern of competition between two strains of Rhizobium japonicum was examined. In two Minnesota soils, Waseca and Waukegan, strain USDA 123 occupied 69% (Waseca) and 24% (Waukegan) of the root nodules on Glycine max L. Merrill cv. Chippewa. USDA 110 occupied 2% of the root nodules in the Waseca soil and 12% of the nodules in the Waukegan soil. Under a variety of other growth conditions—vermiculite, vermiculite amended with Waseca soil, and two Hawaiian soils devoid of naturalized Rhizobium japonicum strains—USDA 110 was more competitive than USDA 123. The addition of nitrate to or the presence of antibiotic-producing actinomycetes in the rhizosphere of soybeans did not affect the pattern of competition between the two strains. However, preexposure of young seedings to USDA 110 or USDA 123 before transplantation into soil altered the pattern of competition between the two strains significantly. In the Waseca soil, preexposure of cv. Chippewa to USDA 110 for 72 h increased the percentage of nodules occupied by USDA 110 from 2 to 55%. Similarly, in the Hawaiian soil Waimea, nodule occupancy by USDA 123 increased from 7 to 33% after a 72-h preexposure.     

Biochemical Properties and Atomic Resolution Structure of a Proteolytically Processed β-Mannanase from Cellulolytic Streptomyces sp. SirexAA-E

β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.     

Genetic Diversity and Population Structure of Acacia senegal (L) Willd. in Kenya

The level of genetic diversity and population structure of Acacia senegal variety kerensis in Kenya was examined using seven polymorphic nuclear microsatellite loci and two chloroplast microsatellite loci. In both chloroplast and nuclear datasets, high levels of genetic diversity were found within all populations and genetic differentiation among populations was low, indicating extensive gene flow. Analysis of population structure provided support for the presence of two groups of populations, although all individuals had mixed ancestry. Groups reflected the influence of geography on gene flow, with one representing Rift Valley populations whilst the other represented populations from Eastern Kenya. The similarities between estimates derived from nuclear and chloroplast data suggest highly effective gene dispersal by both pollen and seed in this species, although population structure appears to have been influenced by distributional changes in the past. The few contrasts between the spatial patterns for nuclear and chloroplast data provided additional support for the idea that, having fragmented in the past, groups are now thoroughly mixed as a result of extensive gene flow. For the purposes of conservation and in situ management of genetic resources, sampling could target a few, large populations ideally distributed among the spatial groups identified. This should ensure the majority of extant variation is preserved, and facilitate the investigation of variation in important phenotypic traits and development of breeding populations.     

β-glucosidase from <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>: a new and efficient purification procedure and use as a suitable marker in immuno-enzymatic assay

The β-glucosidase enzyme β-glu2 isolated from Sclerotinia sclerotiorum was purified and used as tracer in enzyme linked immunosorbent assay. A novel purification procedure of the protein was developed that consists of ammonium sulphate precipitation, gel filtration on Sephacryl S-200-HR column, ion exchange chromatography on DEAE-Toyopearl and polybuffer exchanger PBE 94 TM chromatofocusing. The pI value was 4.45. K _m and V _max values for the enzyme towards p-nitrophenyl-β-D-glucopyranoside were respectively 0.45 mM and 0.2 U/mL. Thermal stability showed that β-glu2 has a half-life of 85 min at 55 °C and of 25 min at 65 °C. β-glu2 was conjugated to goat anti-rabbit antibodies with glutaraldehyde as cross linking agent according to the one-step method. Conjugates were purified by HPLC gel filtration on TSK 2000. Enzymatic and immunological activities of the β-glucosidase conjugate component were tested by the ELISA method.     

Genotypic and Phenotypic Profiles of Enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Associated with Acute Diarrhea in Tunis,Tunisia

No past studies of acute diarrhea in Tunisia have examined the phenotypic and genotypic profiles of enterotoxigenic Escherichia coli (ETEC) isolates. We determined 65 ETEC isolates derived from a total of 327 E. coli isolates collected from a previous study (acute diarrheal and healthy persons, children and adults n = 214) and 32 E. coli isolates derived from an acute diarrheal outbreak in Kabaria-Ennour city, Tunis. All E. coli isolates were screened by polymerase chain reaction (PCR) for ETEC virulence genes: sta (heat-stable toxin gene) and elt (heat-labile toxin gene). Seventy-two percent (47 of 65) of ETEC strains expressed the sta gene only, 21.5% (14 of 65) expressed the elt gene and 6.1% (4 of 65) expressed both genes. For the outbreak isolates, the elt gene was predominant (10 isolates out of 14). Ganylioside GM1 enzyme-linked immunosorbent assay (GM1-ELISA) was used to validate the PCR results and this was confirmed by dot blot assay. The same results were obtained. The most common colonization factors (CFs) were CFA/I (44.6%) and coli surface antigen 6 (CS6) (11%), and 44.6% of the isolates showed no association with either CFAs. Resistance of ETEC isolates to tetracycline (38.5%), streptomycin (26%), and beta-lactam agents (ticarcillin 26%, amoxicillin 24.6%, cephalotin 21.5%) was common. Regarding serotypes, the majority of ETEC isolates serotyped as O86:H(-) (n = 16), O128:H2 (n = 11), and O127:H21 (n = 10). Other serotypes found were O111:H(-) (n = 6) and O126: H(-) (n = 5). DNA macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE) using the XbaI enzyme was conducted to investigate the epidemiological clonal relationship among ETEC isolates. Major patterns were identified among which some of outbreak ETEC isolates belonged. These data suggest that a proportion of acute diarrhea in Tunis represents the confluence of small epidemics by clonality-related ETEC isolates that are transiently introduced or that persist in our community.     

Propagation of centromeric chromatin requires exit from mitosis

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A-containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.     

NMDA-mediated release of glutamate and GABA in the subthalamic nucleus is mediated by dopamine: an in vivo microdialysis study in rats

The present study investigated the effects of N-methyl-D-aspartic acid.H2O (NMDA) on the dopamine, glutamate and GABA release in the subthalamic nucleus (STN) by using in vivo microdialysis in rats. NMDA (100 micromol/L) perfused through the microdialysis probe evoked an increase in extracellular dopamine in the STN of the intact rat of about 170%. This coincided with significant increases in both extracellular glutamate (350%) and GABA (250%). The effect of NMDA perfusion on neurotransmitter release at the level of the STN was completely abolished by co-perfusion of the selective NMDA-receptor antagonist MK-801 (10 micromol/L), whereas subthalamic perfusion of MK-801 alone had no effect on extracellular neurotransmitter concentrations. Furthermore, NMDA induced increases in glutamate were abolished by both SCH23390 (8 micromol/L), a selective D1 antagonist, and remoxipride (4 micromol/L), a selective D2 antagonist. The NMDA induced increase in GABA was abolished by remoxipride but not by SCH23390. Perfusion of the STN with SCH23390 or remoxipride alone had no effect on extracellular neurotransmitter concentrations. The observed effects in intact animals depend on the nigral dopaminergic innervation, as dopamine denervation, by means of 6-hydroxydopamine lesioning of the substantia nigra, clearly abolished the effects of NMDA on neurotransmitter release at the level of the STN. Our work points to a complex interaction between dopamine, glutamate and GABA with a crucial role for dopamine at the level of the STN.