Two-dimensional kinetics regulation of alphaLbeta2-ICAM-1 interaction by conformational changes of the alphaL-inserted domain

The leukocyte integrin alphaLbeta2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of alphaLbeta2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with alphaLbeta2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by approximately 8000- and approximately 30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type alphaLbeta2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the alphaLbeta2, is required for affinity and on-rate up-regulation.     

Keratinophilic fungi of poultry farm and feather dumping soil in Tamil Nadu,India

Soils of 10 poultry farms from Namakkal and 12 feather dumping sites from Chennai were studied for the presence of keratinophilic fungi. A total of 34 species belonging to 19 genera and one non-sporulating fungus were recovered. Sixteen species of fungi and one non-sporulating fungi were common to both sites, eight species were specific to Namakkal and nine species were specific to Chennai. Dermatophytes and closely related fungi were represented by six species belonging to five genera. Fungal species commonly found in the soil samples included Chrysosporium keratinophilum (73%), Trichophyton mentagrophytes (68.2%), Microsporum gypseum (64%), Myceliopthora vellerea (32%), Chrysosporium state of Arthroderma tuberculatum (27.3%) and Geomyces pannorum (23%). Non-dermatophyte fungi were represented by 28 species belonging to 14 genera and one non-sporulating fungus.     

Signal-specific activation and regulation of human neutrophil Fc gamma receptors

FcgammaRs with the ITIM domain have been shown to regulate the inflammatory signal delivered by the ITAM-containing FcgammaRs. In this study, we demonstrate that the function of human neutrophil FcgammaR type IIA (CD32A) is regulated in a distinct manner by different cell activation signals at the ligand-binding stage. Activation of neutrophils with fMLP up-regulated the ligand-binding function of CD32A, whereas PMA-mediated activation completely abolished ligand binding without altering CD32A expression. Furthermore, PMA treatment also abolished CD16B-dependent ligand binding irrespective of the level of expression. The effect of PMA was cell type specific, because the ligand-binding function of CD32A expressed on cultured cells such as K562 and CHO-CD32A transfectants was not affected by PMA. Interestingly, phorbol 12,13-dibutyrate, another phorbol ester, and IL-8 up-regulated CD32A-dependent ligand-binding function. These results demonstrate that regulation of CD32A-dependent ligand binding in human neutrophils is not only cell type specific but also activation signal specific. Moreover, these results suggest the possibility that signals delivered to neutrophils by various inflammatory stimuli can exert opposing effects on the function of human FcgammaRs, representing a novel inside-out regulatory mechanism of FcgammaR ligand binding.     

An enzymatic method to determine receptor-mediated endocytosis

Many studies have measured receptor-mediated endocytosis using radiolabeled ligands or antibodies. Upon ligation and cross-linking, the labeled ligand or antibody is endocytosed and the internalization of the radioisotope is assayed after stripping the uninternalized ligand from the cell membrane. This study reports on an enzymatic assay to measure receptor-mediated endocytosis and compares it with the radioactive method. The results show that receptor-mediated endocytosis measured using the peroxidase conjugated antibody is two fold higher than that measured with a radiolabeled antibody. Thus, approximately 38% endocytosis of CD3 is measured using an ¹²⁵I-labeled antibody, whereas approximately 79% endocytosis is detected by peroxidase conjugated antibody method. Similar increases are also found with CD2 receptor-mediated endocytosis. Our study has demonstrated that the enzymatic method could be employed in determining receptor-mediated endocytosis. In addition to increased sensitivity, the enzymatic assay eliminates the use of radioactive materials.     

Isolation of hydroquinone (benzene-1,4-diol) metabolite from halotolerant <Emphasis Type="Italic">Bacillus methylotrophicus</Emphasis> MHC10 and its inhibitory activity towards bacterial pathogens

A halotolerant bacterial isolate-MHC10 with broad spectrum antibacterial activity against clinical pathogens was isolated from saltpans located in Tuticorin and Chennai (India). 16S rRNA gene analysis of MHC10 revealed close similarity to that of Bacillus methylotrophicus. The culture conditions of B. methylotrophicus MHC10 strain were optimized for antibacterial production using different carbon and nitrogen sources, as well as varying temperature, pH, sodium chloride (NaCl) concentrations and incubation periods. The maximum antibacterial activity of B. methylotrophicus MHC10 was attained when ZMB was optimized with 1 % (w/v) glucose, 0.1 % (w/v) soybean meal which corresponded to a C/N ratio of 38.83, temperature at 37 °C, pH 7.0 and 8 % NaCl. The activity remained stable between 72 and 96 h and then drastically decreased after 96 h. Solvent extraction followed by chromatographic purification steps led to the isolation of hydroquinone (benzene-1,4-diol). The structure of the purified compound was elucidated based on FTIR, ¹H NMR, and ¹³C NMR spectroscopy. The compound exhibited efficient antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens. The minimum inhibitory concentration (MIC) for Gram-positive pathogens ranged from 15.625 to 62.5 µg/mL^?1, while it was between 7.81 and 250 µg/mL^?1 for Gram-negative bacterial pathogens. This is the first report of hydroquinone produced by halotolerant B. methylotrophicus exhibiting promising antibacterial activity.     

Immune Complex-Induced,Nitric Oxide-Mediated Vascular Endothelial Cell Death by Phagocytes Is Prevented with Decoy FcγReceptors

Autoimmune vasculitis is an endothelial inflammatory disease that results from the deposition of immune-complexes (ICs) in blood vessels. The interaction between Fcgamma receptors (FcγRs) expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is essential to prevent the IC-mediated blood vessel damage. Thus we tested if uncoupling the interaction of FcγRs and ICs prevents endothelium damage. Herein, we demonstrate that dimeric FcγR-Igs prevented nitric oxide (NO) mediated apoptosis of human umbilical vein endothelial cells (HUVECs) in an in vitro vasculitis model. Dimeric FcγR-Igs significantly inhibited the IC-induced upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release by murine monocytic cell line. However, FcγR-Igs did not affect the exogenously added NO-induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in HUVECs. In conclusion, these data suggest that IC-induced NO could be one of the major inflammatory mediator promoting blood vessel inflammation and endothelial cell death during IC-mediated vasculitis which can be effectively blocked by dimeric decoy FcγRs.     

Interspecific diversity reduces and functionally substitutes for intraspecific variation in biofilm communities

Diversity has a key role in the dynamics and resilience of communities and both interspecific (species) and intraspecific (genotypic) diversity can have important effects on community structure and function. However, a critical and unresolved question for understanding the ecology of a community is to what extent these two levels of diversity are functionally substitutable? Here we show, for a mixed-species biofilm community composed of Pseudomonas aeruginosa, P. protegens and Klebsiella pneumoniae, that increased interspecific diversity reduces and functionally substitutes for intraspecific diversity in mediating tolerance to stress. Biofilm populations generated high percentages of genotypic variants, which were largely absent in biofilm communities. Biofilms with either high intra- or interspecific diversity were more tolerant to SDS stress than biofilms with no or low diversity. Unexpectedly, genotypic variants decreased the tolerance of biofilm communities when experimentally introduced into the communities. For example, substituting P. protegens wild type with its genotypic variant within biofilm communities decreased SDS tolerance by twofold, apparently due to perturbation of interspecific interactions. A decrease in variant frequency was also observed when biofilm populations were exposed to cell-free effluents from another species, suggesting that extracellular factors have a role in selection against the appearance of intraspecific variants. This work demonstrates the functional substitution of inter- and intraspecific diversity for an emergent property of biofilms. It also provides a potential explanation for a long-standing paradox in microbiology, in which morphotypic variants are common in laboratory grown biofilm populations, but are rare in diverse, environmental biofilm communities.     

Surfactant-cobalt(III) complexes: synthesis, critical micelle concentration (CMC) determination, DNA binding, antimicrobial and cytotoxicity studies

A new class of surfactant-cobalt(III) complexes, cis-[Co(bpy)₂(C₁₁H₂₃NH₂)Cl]²⁺ (1) and cis-[Co(phen)₂(C₁₁H₂₃NH₂)Cl]²⁺ (2) (bpy = 2,2′-bipyridyl, phen = 1,10-phenanthroline), have been synthesized and characterized. The critical micelle concentration (CMC) values of these complexes in aqueous solution were obtained from conductance measurements. The specific conductivity data (at 298, 308, 318 and 328 K) served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization . The interaction between these complexes and calf thymus DNA in aqueous solution was investigated adopting electronic absorption spectroscopy, emission spectroscopy and viscosity measurements. Results suggest that the two complexes can bind to DNA via groove binding, van der Waals interactions and/or electrostatic interactions. The complexes showed moderate antibacterial and antifungal activities against certain selected microorganisms. The cytotoxic activity of the complexes on HBL-100 human breast cancer cells was determined adopting MTT assay and specific staining techniques, which revealed that the viability of the cells thus treated was significantly decreased and the cells succumbed to apoptosis as seen in the changes in the nuclear morphology and cytoplasmic features. Furthermore, the influence of complexes on normal cell lines from green monkey kidney was also determined and the results indicate that the effect is small on inhibition of viability.