Chitinolytic properties of Bacillus pabuli K1

The chitinolytic properties of Bacillus pabuli K1 isolated from mouldy grain was studied. Chitinase activity was measured as the release of p -nitrophenol from p -nitrophenyl-N, N'-diacetylchitobiose. Influences of substrate concentration and different environmental variables on growth and chitinase activity were determined. The optimum environmental conditions for chitinase production were: 30°C, initial pH 8, initial oxygen 10% and a_w > 0.99. Chitinase production was induced when B. pabuli K1 was grown on colloidal chitin. The smallest chito-oligosaccharide able to induce chitinase production was N, N'-diacetylchitobiose, (GlcNAc)₂. Production was also induced by (GlcNAc)₃ and (GlcNAc)₄. When the bacterium was grown on glucose or N -acetylglucosamine, no chitinases were formed. The highest chitinase production observed was obtained with colloidal chitin as substrate. The production of chitinases by B. pabuli K1 growing on chitin was repressed by high levels (0.6%) of glucose. The production was also repressed by 0.6% starch, laminarin and β-glucan from barley and by glycerol. The addition of pectin and carboxymethyl cellulose increased chitinase production.     

Trophic level and overlap of sea lions (Zalophus californianus) in the Gulf of California, Mexico

Stable isotope and scat analyses were used in concert to determine trophic level and dietary overlap among California sea lions from different rookeries in the Gulf of California. Isotopic analysis of the fur of sea lion pups revealed differences in δ¹⁵N and δ¹³C values among rookeries during the breeding season. Mean δ¹⁵N and δ¹³C values varied from 20.2‰ to 22.4‰ and from −15.4‰ to −14.0‰, respectively. The pattern of differences among rookeries was similar between years in most cases. Isotopic variations among rookeries were associated with differences in prey consumption. There was a significant correlation between δ¹⁵N value and trophic level, as determined by scat analysis. Joint application of isotopic and scat analyses allowed us to identify how the feeding habits of sea lions vary with location. Our results suggest the presence of spatial structure in available prey as well as the localized use of prey by sea lions across the Gulf of California.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil

Aims:  Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent.
Methods and Results:  About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx ₁, stx ₂, eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)–PCR was performed to investigate the variants of stx ₁ and stx ₂, and the flagellar antigen ( fli C) genes in nonmotile isolates. Five isolates were eae ⁺ and stx ⁻, and belonged to serotypes O128:H2/β-intimin (2), O145:H2/γ, O153:H7/β and O178:H7/ε. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx _1c stx _2d-O118 (46·9%), stx _1c (27·2%), stx _2d-O118 (23·4%), and stx _1c stx _2dOX3a (2·5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates.
Conclusions:  This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC.
Significance and Impact of the Study:  As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.     

Borrelia burgdorferi BBB07 interaction with integrin alpha3beta1 stimulates production of pro-inflammatory mediators in primary human chondrocytes

Borrelia burgdorferi , the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin α₃β₁ on human chondrocytes activates signalling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin α₃β₁ and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg–Gly–Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin α₃β₁ and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro-inflammatory mediators in primary human chondrocyte cells by interaction with integrin α₃β₁. This interaction is specific, as P66, another integrin ligand of B. burgdorferi , does not activate signalling through α₃β₁. In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin α₃β₁ and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells.     

Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABA_A) receptor-gated Cl⁻ channel response is a common action of structurally diverse anesthetics, suggesting that the GABA_A receptor plays an important role in anesthesia. To determine if GABA_A receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl⁻ currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABA_A receptor protein subunits; the order of sensitivity was α₁β₁ > α₁β₁γ_2s=α₁β₁γ_2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABA_A receptor protein complex to modulate the Cl⁻ channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.     

Developmental Characterization of Thymosin β4 and β10 Expression in Enriched Neuronal Cultures from Rat Cerebella

Abstract: The β₄ and β₁₀ thymosins are G-actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin β₄ is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin β₁₀ is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the β-thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both β-thymosins were initially expressed in granule cells, although expression, especially of thymosin β₄, was truncated compared with the in vivo time course. As in vivo, thymosin β₄ was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin β₁₀. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the β-thymosins show that modulation of tissue culture conditions could be used to identify factors regulating β-thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.     

Alterations in beta 1- and beta 2-adrenergic receptor density in the cerebellum of aging rats

Abstract: The densities of β¹, and β₂-adrenergic receptors were determined in homogenates of cerebral cortex and cerebellum of rats between 3 and 14 mo of age. No change in either receptor population occurred in the cortex during this period. In the cerebellum, a 20–25% decrease in the density of β₂, receptors and a 3509% increase in the density of β₁, receptors occurred. The increase in β₁ receptors in the cerebellum may be the result of a decrease in the function of the noradrenergic projections from the locus coeruleus which synapse on cerebellar Purkinje cells.     

Platelet activation by a relapsing fever spirochaete results in enhanced bacterium–platelet interaction via integrin αIIbβ3 activation

Borrelia hermsii , a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro . Platelet binding was promoted by the platelet integrin α_IIbβ₃: the bacterium bound to purified integrin α_IIbβ₃, and bacterial binding to platelets was diminished by α_IIbβ₃ antagonists or by a genetic defect in this integrin. Integrin α_IIbβ₃ undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of α_IIbβ₃ that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin α_IIbβ₃ activation, as indicated by the expression of the platelet activation marker P-selectin and integrin α_IIbβ₃ in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I₂, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high-level bacterial attachment.     

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.     

Differential Effects of 4'-Chlorodiazepam on Expressed Human GABAA Receptors

Abstract: The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABA_A receptor cDNAs were determined. Cotransfection of human α₂, β₁, and γ₂ subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA-activated chloride channel. This site is found on GABA_A receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α₁ or α₃ for the α₂ subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [³⁵S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α₂ and β₁ alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [³⁵S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides.     

Alteration of medial-edge epithelium cell adhesion in two Tgf-beta3 null mouse strains

Abstract Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf -β₃ null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf -β₃ null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α₅- and β₁-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β₃ or neutralizing antibodies against fibronectin or the α₅-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf -β₃ null mutants; the importance of TGF-β₃ to restore their normal pattern of expression; and the crucial role of fibronectin and the α₅-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf -β₃ null mutant mice.     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

Salmonellae in feral pinnipeds off the Southern California coast

Rectal swabs were collected from 90 Northern fur seal (Callorhinus ursinus) and 50 California sea lion (Zalophus californianus) pups on San Miguel Island for Salmonella screening. Three serotypes (newport, heidelberg, and oranienburg) were recovered from 33% of the fur seals and 40% of the sea lions.     

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence

The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin α₂β₁. Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin α₂β₁ and affects the biological outcome of host–pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the α₂β₁ integrin, because (i) both adherence and internalization of the scl1- inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-α₂ integrin-subunit antibody and type I collagen, (iii) recombinant α₂-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the α₂β₁ integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.     

In Situ Hybridization Evidence of Differential Modulation by Pentobarbital of GABAA Receptor α1- and β3-Subunit mRNAs

Abstract: Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABA_A receptor α₁- and β₃-subunit mRNA was conducted using synthetic 3'- end ³⁵S-dATP-labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of α₁-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of β₃-subunit mRNA were not affected. Dramatically increased levels of GABA_A receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the α₁ subunit and in cerebral cortex only for the β₃-subunit. These data provide further support to the structural and pharmacological GABA_A receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up- and down-regulation observed in receptor ligand binding studies.     

Phylogenetic relationships within the eared seals (Otariidae: Carnivora): implications for the historical biogeography of the family.

Phylogenetic relationships within the family Otariidae were investigated using two regions of the mitochondrial genome. A 360-bp region of the cytochrome b gene was employed for the primary phylogenetic analysis, while a 356-bp segment of the control region was used to enhance resolution of the terminal nodes. Traditional classification of the family into the subfamilies Arctocephalinae (fur seals) and Otariinae (sea lions) is not supported, with the fur seal Callorhinus ursinus having a basal relationship relative to the rest of the family. This is consistent with the fossil record which suggests that this genus diverged from the line leading to the remaining fur seals and sea lions about 6 million years ago (mya). There is also little evidence to support or refute the monophyly of sea lions. Four sea lion clades and five fur seal clades were observed, but relationships among these clades are unclear. Similar genetic divergences between the sea lion clades (D(a) = 0.054-0.078), as well as between the major Arctocephalus fur seal clades (D(a) = 0.040-0.069) suggest that these groups underwent periods of rapid radiation at about the time they diverged from each other. Rapid radiations of this type make the resolution of relationships between the resulting species difficult and indicate the requirement for additional molecular data from both nuclear and mitochondrial genes. The phylogenetic relationships within the family and the genetic distances among some taxa highlight inconsistencies in the current taxonomic classification of the family.