A DNA damage checkpoint in Caulobacter crescentus inhibits cell division through a direct interaction with FtsW

Following DNA damage, cells typically delay cell cycle progression and inhibit cell division until their chromosomes have been repaired. The bacterial checkpoint systems responsible for these DNA damage responses are incompletely understood. Here, we show that Caulobacter crescentus responds to DNA damage by coordinately inducing an SOS regulon and inhibiting the master regulator CtrA. Included in the SOS regulon is sidA (SOS-induced inhibitor of cell division A), a membrane protein of only 29 amino acids that helps to delay cell division following DNA damage, but is dispensable in undamaged cells. SidA is sufficient, when overproduced, to block cell division. However, unlike many other regulators of bacterial cell division, SidA does not directly disrupt the assembly or stability of the cytokinetic ring protein FtsZ, nor does it affect the recruitment of other components of the cell division machinery. Instead, we provide evidence that SidA inhibits division by binding directly to FtsW to prevent the final constriction of the cytokinetic ring.     

Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression

Second messengers control a wide range of important cellular functions in eukaryotes and prokaryotes. Here we show that cyclic di-GMP, a global bacterial second messenger, promotes cell cycle progression in Caulobacter crescentus by mediating the specific degradation of the replication initiation inhibitor CtrA. During the G1-to-S-phase transition, both CtrA and its cognate protease ClpXP dynamically localize to the old cell pole, where CtrA is rapidly degraded. Sequestration of CtrA to the cell pole depends on PopA, a newly identified cyclic di-GMP effector protein. PopA itself localizes to the cell pole and directs CtrA to this subcellular site via the direct interaction with a mediator protein, RcdA. We present evidence that c-di-GMP regulates CtrA degradation during the cell cycle by controlling the dynamic sequestration of the PopA recruitment factor to the cell pole. Furthermore, we show that cell cycle timing of CtrA degradation relies on converging pathways responsible for substrate and protease localization to the old cell pole. This is the first report that links cyclic di-GMP to protein dynamics and cell cycle control in bacteria.     

In vivo hypoglycemic effect of methanolic fruit extract of Momordica charantia L

Background

Momordica charantia L. is a medicinal plant commonly used in the management of diabetes mellitus.

Objectives

We investigated the blood glucose lowering effect of the methanolic fruit extract of the Ugandan variety of M. charantia L. in alloxan-induced diabetic albino rats.

Methods

500g of M. charantia powder were macerated in methanol and the extract administered to two groups of alloxan-induced diabetic rats. The first group received 125mg/kg, the second 375mg/kg and a third group 7mg/kg of metformin. A fourth group received 1ml normal saline. Fasting blood glucose (FBG) levels were measured at 0.5,1,2,3,5,8 and 12 hours and compared using one-way ANOVA.

Results

There was an initial rise in FBG for 1 hour after administration of extracts followed by steep reductions. Significant reduction in FBG occurred at 2 hours for 125mg/kg of extract (−3.2%, 313±25.9 to 303±25.0mg/dL, p = 0.049), 375mg/kg of extract (−3.9%, 356±19.7 to 342±20.3mg/dL, p = 0.001), and metformin (−2.6%, 344±21.7 to 335±21.1mg/dL, p = 0.003) when compared to normal saline. The maximum percentage reduction in FBG by both extracts occurred between 3 and 12 hours post dose.

Conclusions

The methanolic fruit extract of M. charantia exhibits dose dependent hypoglycaemic activity in vivo.     

Regulation of cytokine gene expression by adjuvants in vivo

Antibody isotype affects biological activity of the antibodies and therefore should be considered in prevention of disease by vaccination. In previous reports, we demonstrated that adjuvants affect the antibody isotype switching process and favour the production of certain isotypes. The present study extends these findings and shows fundamental differences in the cytokine induction pattern according to the adjuvant used. Cytokine mRNA levels were determined by in situ RNA–RNA hybridization performed on splenocytes isolated from mice injected with different adjuvants. The results revealed that Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Al(OH)₃ and QuilA administration results in a type-2 (humoral) response, increasing IL-4, IL-5 and IL-13 gene expression, while poly I:C exhibits a type-1 (cell-mediated) response, increasing the production of interferon-gamma (IFN-γ), IL-2 and IL-6 mRNA. Finally, BeSO₄ and poly A:U augment IL-5 and IL-6 mRNA production, while lipopolysaccharide (LPS) and LiCl augment IL-6 and tumour necrosis factor-alpha (TNF-α) mRNA production. Also, the adjuvants appear capable of overcoming the inherent IL-2/IFN-γ and IL-4 dichotomy of C57Bl/6 and BALB/c mice, respectively, in response to cellular antigens such as Leishmania and herpessimplex virus (HSV). The overall data suggest that adjuvants direct the isotype switching process via induction of certain cytokines, a finding that can be useful in selection of the most efficient isotype of protective antibodies for disease prevention by vaccination.     

The mglA gene and its flanking regions in Brucella: the role of mglA in tolerance to hostile environments, Fe-metabolism and in vivo persistence

We previously demonstrated that a spontaneous smooth small-colony variant of Brucella abortus S19 is characterized by increased in vivo persistence and the differential expression of a gene predicted to encode a galactoside transport ATP binding protein (mglA). In order to further investigate the role of this gene in the context of its flanking regions, we analyzed the respective DNA sequences from the formerly described B. abortus S19 as well as from avirulent B. neotomae 5K33 and compared these with published data from other Brucella species. Deletion mutagenesis of mglA in the large-colony variant of B. abortus S19 resulted in increased tolerance of the deletion mutant to a hyperosmotic (toxic), galactose-containing medium as well as to oxidative stress (H(2)O(2)). Whilst the deletion mutant is characterized by reduced growth on solid Fe(3+)-containing minimal medium (small-colony morphology), in vivo studies in mice demonstrated statistical significant differences in the bacterial load of spleens in the pre-immune, but not in the late phase of the infection.     

UNC-51/ATG1 kinase regulates axonal transport by mediating motor–cargo assembly

Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor–cargo assembly are not well understood. Here we show that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser¹⁴³, and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor−cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser¹⁴³ phosphorylation in axonal transport. Thus, our data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins.     

STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo

Alternative splicing of pre-mRNAs greatly contributes to the spatiotemporal diversity of gene expression in metazoans. However, the molecular basis of developmental regulation and the precise sequence of alternative pre-mRNA processing in vivo are poorly understood. In the present study, we focus on the developmental switching of the mutually exclusive alternative splicing of the let-2 gene of Caenorhabditis elegans from the exon 9 form in embryos to the exon 10 form in adults. By visualizing the usage of the let-2 mutually exclusive exons through differential expression of green fluorescent protein (GFP) and red fluorescent protein (RFP), we isolated several switching-defective mutants and identified the alternative splicing defective-2 (asd-2) gene, encoding a novel member of the evolutionarily conserved STAR (signal transduction activators of RNA) family of RNA-binding proteins. Comparison of the amounts of partially spliced let-2 RNAs in synchronized wild-type and asd-2 mutant worms suggested that either of the introns downstream from the let-2 mutually exclusive exons is removed prior to the removal of the upstream ones, and that asd-2 promotes biased excision of intron 10 in the late larval stages. We propose that the developmental switching between alternative sequences of intron removal determines the ratio between the mature let-2 mRNA isoforms.     

Helicobacter pylori: Bacterial Strategy for Incipient Stage and Persistent Colonization in Human Gastric Niches

Helicobacter pylori (H. pylori) undergoes decades long colonization of the gastric mucosa of half the population in the world to produce acute and chronic gastritis at the beginning of infection, progressing to more severe disorders, including peptic ulcer disease and gastric cancer. Prolonged carriage of H. pylori is the most crucial factor for the pathogenesis of gastric maladies. Bacterial persistence in the gastric mucosa depends on bacterial factors as well as host factors. Herein, the host and bacterial components responsible for the incipient stages of H. pylori infection are reviewed and discussed. Bacterial adhesion and adaptation is presented to explain the persistence of H. pylori colonization in the gastric mucosa, in which bacterial evasion of host defense systems and genomic diversity are included.     

Pulmonary blastoma, carcinosarcoma and spindle-cell carcinoma: an immunohistochemical study of keratin intermediate filaments

We have examined 18 primary malignant lung tumours categorized as either carcinosarcoma, blastoma or spindle-cell carcinoma according to accepted criteria. Two monoclonal antibodies to keratins, CAM 5.2 and LP 34, were used to determine whether the non-epithelial or spindle-cell components of each tumour showed evidence of keratin expression. By this means the epithelial nature of the five tumours classified as spindle-cell carcinomas was confirmed. In all four pulmonary blastomas and in five of nine carcinosarcomas, the sarcomatous elements failed to stain for keratin but in the remaining four carcinosarcomas there was focal staining. The histogenesis of these tumours is discussed and it is suggested that the sarcomatous component of a carcinosarcoma may be derived from malignant epithelial cells by a process of mesenchymal metaplasia with a switch in intermediate filament type. It remains uncertain whether blastomas are derived from both endoderm and mesoderm, or from either one of these tissues, with one component representing complete metaplastic transformation.     

Dietary Flaxseed Mitigates Impaired Skeletal Muscle Regeneration: in Vivo,in Vitro and in Silico Studies

Background: Diets enriched with n-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to exert a positive impact on muscle diseases. Flaxseed is one of the richest sources of n-3 PUFA acid α-linolenic acid (ALA). The aim of this study was to assess the effects of flaxseed and ALA in models of skeletal muscle degeneration characterized by high levels of Tumor Necrosis Factor-α (TNF).Methods: The in vivo studies were carried out on dystrophic hamsters affected by muscle damage associated with high TNF plasma levels and fed with a long-term 30% flaxseed-supplemented diet. Differentiating C2C12 myoblasts treated with TNF and challenged with ALA represented the in vitro model. Skeletal muscle morphology was scrutinized by applying the Principal Component Analysis statistical method. Apoptosis, inflammation and myogenesis were analyzed by immunofluorescence. Finally, an in silico analysis was carried out to predict the possible pathways underlying the effects of n-3 PUFAs.Results: The flaxseed-enriched diet protected the dystrophic muscle from apoptosis and preserved muscle myogenesis by increasing the myogenin and alpha myosin heavy chain. Moreover, it restored the normal expression pattern of caveolin-3 thereby allowing protein retention at the sarcolemma. ALA reduced TNF-induced apoptosis in differentiating myoblasts and prevented the TNF-induced inhibition of myogenesis, as demonstrated by the increased expression of myogenin, myosin heavy chain and caveolin-3, while promoting myotube fusion. The in silico investigation revealed that FAK pathways may play a central role in the protective effects of ALA on myogenesis.Conclusions: These findings indicate that flaxseed may exert potent beneficial effects by preserving skeletal muscle regeneration and homeostasis partly through an ALA-mediated action. Thus, dietary flaxseed and ALA may serve as a useful strategy for treating patients with muscle dystrophies.     

Immunohistochemical distribution of serum proteins in living mouse heart with In vivo cryotechnique

In vivo cryotechnique (IVCT), which immediately cryofixes target organs in situ, was used to clarify the morphological features of beating heart tissue of living mice. IVCT was performed for diastolic heart tissue under the condition of monitoring with electrocardiogram (ECG). Other mouse hearts were prepared with conventional perfusion-fixation (PF-DH) or immersion-fixation followed by dehydration (IM-DH), and quick-freezing of resected heart tissues (FQF). Immunolocalizations of albumin, immunoglobulin G1 (IgG1), intravenously injected bovine serum albumin (BSA), and connexin 43 were examined after different intervals of BSA injection. In the case of IVCT, the exact stop time of beating mouse hearts was recorded by ECG, and open blood vessels with flowing erythrocytes were observed with less artificial tissue shrinkage than with conventional preparation methods. Both albumin and BSA were well preserved in intercalated discs and t-tubules of cardiomyocytes in addition to blood vessels and interstitial matrices. IgG1 was immunolocalized in interstitial matrices of heart tissues in addition to their blood vessels. At 4 hr after BSA injection, it was immunolocalized in the intercalated discs of cardiomyocytes and lost later at 8 hr. IVCT should prove to be more useful for the morphofunctional examination of dynamically changing heart tissue than conventional preparation methods.     

The Red clover necrotic mosaic virus origin of assembly is delimited to the RNA-2 trans-activator

The bipartite RNA genome of Red clover necrotic mosaic virus (RCNMV) is encapsidated into icosahedral virions that exist as two populations: i) virions that co-package both genomic RNAs and ii) virions packaging multiple copies of RNA-2. To elucidate the packaging mechanism, we sought to identify the RCNMV origin of assembly sequence (OAS). RCNMV RNA-1 cannot package in the absence of RNA-2 suggesting that it does not contain an independent packaging signal. A 209 nt RNA-2 element expressed from the Tomato bushy stunt virus CP subgenomic promoter is co-assembled with genomic RNA-1 into virions. Deletion mutagenesis delimited the previously characterized 34 nt trans-activator (TA) as the minimal RCNMV OAS. From this study we hypothesize that RNA-1 must be base-paired with RNA-2 at the TA to initiate co-packaging. The addition of viral assembly illustrates the critical importance of the multifunctional TA element as a key regulatory switch in the RCNMV life cycle.     

Psidin, a conserved protein that regulates protrusion dynamics and cell migration

Dynamic assembly and disassembly of actin filaments is a major driving force for cell movements. Border cells in the Drosophila ovary provide a simple and genetically tractable model to study the mechanisms regulating cell migration. To identify new genes that regulate cell movement in vivo, we screened lethal mutations on chromosome 3R for defects in border cell migration and identified two alleles of the gene psidin (psid). In vitro, purified Psid protein bound F-actin and inhibited the interaction of tropomyosin with F-actin. In vivo, psid mutations exhibited genetic interactions with the genes encoding tropomyosin and cofilin. Border cells overexpressing Psid together with GFP-actin exhibited altered protrusion/retraction dynamics. Psid knockdown in cultured S2 cells reduced, and Psid overexpression enhanced, lamellipodial dynamics. Knockdown of the human homolog of Psid reduced the speed and directionality of migration in wounded MCF10A breast epithelial monolayers, whereas overexpression of the protein increased migration speed and altered protrusion dynamics in EGF-stimulated cells. These results indicate that Psid is an actin regulatory protein that plays a conserved role in protrusion dynamics and cell migration.     

Evaluation of Phytochemical and Antibacterial Potential of Helicteres Isora L. Fruits Against Enteric Bacterial Pathogens

Antibacterial activities of aqueous, acetone, ethanol and methanol extracts of fruits of Helicteres isora (Mororphali) were studied. The fruit aqueous extracts of H. isora showed prominent antibacterial activities against E.coli, Staphylococcus epidermidis, Salmonella typhimurium and Proteus vulgaris; moderate activity against Enterobacter aerogenes, Staphylococcus aureus, Salmonella typhi and least activity against Pseudomonas aeruginosa. The aqueous extract showed maximal, the ethanol and methanol extract moderate and acetone extracts least antibacterial activities. Phytochemical screening revealed the presence of carbohydrates, anthraquinon glycosides, proteins, tannin and phenolic compounds and steroids These antibacterial properties supports its traditional use of fruits of H. isora in the treatment of enteric or diarrhoeal infections.     

Identification of the determinants of tRNA function and susceptibility to rapid tRNA decay by high-throughput in vivo analysis

Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4_oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an ochre mutation, fluorescence-activated cell sorting of a library of SUP4_oc mutant yeast strains, and deep sequencing, we scored 25,491 variants. Unexpectedly, SUP4_oc tolerates numerous sequence variations, accommodates slippage in tertiary and secondary interactions, and exhibits genetic interactions that suggest an alternative functional tRNA conformation. Furthermore, we used this methodology to define tRNA variants subject to rapid tRNA decay (RTD). Even though RTD normally degrades tRNAs with exposed 5′ ends, mutations that sensitize SUP4_oc to RTD were found to be located throughout the sequence, including the anti-codon stem. Thus, the integrity of the entire tRNA molecule is under surveillance by cellular quality control machinery. This approach to assess activity at high throughput is widely applicable to many problems in tRNA biology.