Effect of γ Irradiation on the Microflora of Freshwater Fish: II. Generic Identification of Aerobic Bacteria from Yellow Perch Fillets1

Studies on the generic identification of bacteria isolated from nonirradiated and irradiated (0.3 and 0.6 Mrad) yellow perch fillets during the course of microbial spoilage have been conducted. After the enumeration and tabulation of macrocolonies on petri dish cultures obtained from fillets, isolates were examined and keyed out essentially according to modified morphological and biochemical protocols of Shewan. Identification was further confirmed through reference to Bergey''s Manual. Data obtained from each isolate were coded and recorded on IBM cards to facilitate identification. Total aerobic microbial plate counts obtained from nonirradiated perch before storage ranged from 10⁵ to 10⁶ microorganisms per gram of fish. Organisms isolated from these fillets, in order of decreasing number, consisted of Achromobacter, Alcaligenes, Pseudomonas, Brevibacterium, Micrococcus, Flavobacterium, Bacillus, Sarcina, Microbacterium, Corynebacterium, yeasts, Lactobacillus, Vibrio, Aeromonas, and a few Proteus and Escherichia cells. During storage and as spoilage progressed, the flora shifted and the pseudomonads became predominant. Irradiation of fillets to 0.3 and 0.6 Mrad reduced the aforementioned flora to the Achromobacter-Alcaligenes group, which constituted the residual flora throughout fillet storage.     

Effect of gamma Irradiation on the Microflora of Freshwater Fish: II. Generic Identification of Aerobic Bacteria from Yellow Perch Fillets

Studies on the generic identification of bacteria isolated from nonirradiated and irradiated (0.3 and 0.6 Mrad) yellow perch fillets during the course of microbial spoilage have been conducted. After the enumeration and tabulation of macrocolonies on petri dish cultures obtained from fillets, isolates were examined and keyed out essentially according to modified morphological and biochemical protocols of Shewan. Identification was further confirmed through reference to Bergey's Manual. Data obtained from each isolate were coded and recorded on IBM cards to facilitate identification. Total aerobic microbial plate counts obtained from nonirradiated perch before storage ranged from 10⁵ to 10⁶ microorganisms per gram of fish. Organisms isolated from these fillets, in order of decreasing number, consisted of Achromobacter, Alcaligenes, Pseudomonas, Brevibacterium, Micrococcus, Flavobacterium, Bacillus, Sarcina, Microbacterium, Corynebacterium, yeasts, Lactobacillus, Vibrio, Aeromonas, and a few Proteus and Escherichia cells. During storage and as spoilage progressed, the flora shifted and the pseudomonads became predominant. Irradiation of fillets to 0.3 and 0.6 Mrad reduced the aforementioned flora to the Achromobacter-Alcaligenes group, which constituted the residual flora throughout fillet storage.     

Effect of γ-Irradiation on the Microflora of Freshwater Fish: I. Microbial Load, Lag Period, and Rate of Growth on Yellow Perch (Perca flavescens) Fillets

Microbial flora were compared in irradiated and nonirradiated yellow perch fillets. These studies included effects of irradiation on the total microbial population, the lag phase, and rate of growth in this freshwater fishery product. The work was conducted concurrently with sensory and chemical evaluation, and constituted part of an investigation designed to evaluate the effect of substerilization doses (0.3 and 0.6 Mrad) of Co⁶⁰ γ rays on the storage life of yellow perch fillets at 1.0 or 6.0 C. In five storage tests, total plate counts prior to irradiation did not exceed 8.7 × 10⁵ per gram of sample; this count was reduced nearly 100% by irradiation with either 0.3 or 0.6 Mrad. Progressively lower maximal bacterial populations and lengthened lag phases were obtained as more radiation was used. The growth rate of the population did not appear to decrease significantly. Microbial data obtained in these studies confirmed the sensory and chemical studies, by indicating that irradiation can significantly extend the refrigerated shelf life of freshwater fish.     

Effect of γ-Irradiation on the Microflora of Freshwater Fish: I. Microbial Load,Lag Period,and Rate of Growth on Yellow Perch (Perca flavescens) Fillets

Microbial flora were compared in irradiated and nonirradiated yellow perch fillets. These studies included effects of irradiation on the total microbial population, the lag phase, and rate of growth in this freshwater fishery product. The work was conducted concurrently with sensory and chemical evaluation, and constituted part of an investigation designed to evaluate the effect of substerilization doses (0.3 and 0.6 Mrad) of Co⁶⁰ γ rays on the storage life of yellow perch fillets at 1.0 or 6.0 C. In five storage tests, total plate counts prior to irradiation did not exceed 8.7 × 10⁵ per gram of sample; this count was reduced nearly 100% by irradiation with either 0.3 or 0.6 Mrad. Progressively lower maximal bacterial populations and lengthened lag phases were obtained as more radiation was used. The growth rate of the population did not appear to decrease significantly. Microbial data obtained in these studies confirmed the sensory and chemical studies, by indicating that irradiation can significantly extend the refrigerated shelf life of freshwater fish.     

Differential Subcellular Localization of Protein Phosphatase-1 α, γ1, and δ Isoforms during Both Interphase and Mitosis in Mammalian Cells

Protein phosphatase-1 (PP-1) is involved in the regulation of numerous metabolic processes in mammalian cells. The major isoforms of PP-1, α, γ1, and δ, have nearly identical catalytic domains, but they vary in sequence at their extreme NH₂ and COOH termini. With specific antibodies raised against the unique COOH-terminal sequence of each isoform, we find that the three PP-1 isoforms are each expressed in all mammalian cells tested, but that they localize within these cells in a strikingly distinct and characteristic manner. Each isoform is present both within the cytoplasm and in the nucleus during interphase. Within the nucleus, PP-1 α associates with the nuclear matrix, PP-1 γ1 concentrates in nucleoli in association with RNA, and PP-1 δ localizes to nonnucleolar whole chromatin. During mitosis, PP-1 α is localized to the centrosome, PP-1 γ1 is associated with microtubules of the mitotic spindle, and PP-1 δ strongly associates with chromosomes. We conclude that PP-1 isoforms are targeted to strikingly distinct and independent sites in the cell, permitting unique and independent roles for each of the isoforms in regulating discrete cellular processes.     

Comparative Effects of Chlortetracycline, Freezing, and γ Radiation on Microbial Population of Ocean Perch

The microbial populations in chlortetracycline (CTC)-treated (50, 100, 200, and 500 ppm), frozen (-15 C), and irradiated (0.1 Mrad) ocean perch (Sebastodes alutus) were compared. The control sample spoiled at 7 C, primarily because of the growth of Pseudomonas. Irradiation changed this to Achromobacter-dominated spoilage. Freezing or CTC treatment altered the spoilage pattern very little. CTC was particularly effective against ultraviolet fluorescent Pseudomonas species at the higher concentrations. Freezing and CTC were not effective against "coryneforms."     

Patterns of functional and taxonomic organization of stream fishes: inferences based on α, β, and γ diversities

The primary objective of this study was to determine whether total biodiversity (γ) is partitioned into within‐community (α) and among‐community (β) components differently for taxonomic and functional organization. I hypothesized that α diversity will contribute more to the functional organization of γ diversity and that β diversity will contribute more to the taxonomic organization of γ diversity. A secondary objective was to determine whether the relationship between taxonomic and functional diversity is scale dependent. Species abundance data was obtained from fisheries surveys conducted by the Texas Parks and Wildlife Dept that focused on least disturbed streams from 11 different ecoregions of Texas, including 62 localities from 18 drainages. Functional and taxonomic organization of assemblages was quantified with two different measures of biodiversity, including richness and the numbers equivalent of Shannon diversity. Scale‐dependent effects on these indices were assessed by multiplicatively partitioning γ into α and β components. The contribution of α and β components to γ diversity differed between functional and taxonomic organization and among different measures of biodiversity. Among‐community components were more influential in structuring the taxonomic organization of stream‐fish assemblages, whereas within‐community components were more important in structuring the functional organization of assemblages. The relationship between taxonomic and functional diversity differed between α and β components and between spatial scales. Indeed, ecological patterns not only change with spatial scale, but how they change is dependent on which aspect of biodiversity is considered.     

The Laminin α Chains: Expression, Developmental Transitions, and Chromosomal Locations of α1-5, Identification of Heterotrimeric Laminins 8–11, and Cloning of a Novel α3 Isoform

Laminin trimers composed of α, β, and γ chains are major components of basal laminae (BLs) throughout the body. To date, three α chains (α1–3) have been shown to assemble into at least seven heterotrimers (called laminins 1–7). Genes encoding two additional α chains (α4 and α5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant α4 and α5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that α4 and α5 assemble into four novel laminin heterotrimers (laminins 8–11: α4β1γ1, α4β2γ1, α5β1γ1, and α5β2γ1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of α1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, α4 and α5 exhibited the broadest patterns of expression, while expression of α1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the α chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one α chain, all α chains were present in multiple BLs, and some BLs contained two or three α chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different α chains and two different β chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five α chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length α3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

Isolation and Characterization of Novel Psychrophilic, Neutrophilic, Fe-Oxidizing, Chemolithoautotrophic α- and γ-Proteobacteria from the Deep Sea

We report the isolation and physiological characterization of novel, psychrophilic, iron-oxidizing bacteria (FeOB) from low-temperature weathering habitats in the vicinity of the Juan de Fuca deep-sea hydrothermal area. The FeOB were cultured from the surfaces of weathered rock and metalliferous sediments. They are capable of growth on a variety of natural and synthetic solid rock and mineral substrates, such as pyrite (FeS₂), basalt glass (~10 wt% FeO), and siderite (FeCO₃), as their sole energy source, as well as numerous aqueous Fe substrates. Growth temperature characteristics correspond to the in situ environmental conditions of sample origin; the FeOB grow optimally at 3 to 10°C and at generation times ranging from 57 to 74 h. They are obligate chemolithoautotrophs and grow optimally under microaerobic conditions in the presence of an oxygen gradient or anaerobically in the presence of nitrate. None of the strains are capable of using any organic or alternate inorganic substrates tested. The bacteria are phylogenetically diverse and have no close Fe-oxidizing or autotrophic relatives represented in pure culture. One group of isolates are γ-Proteobacteria most closely related to the heterotrophic bacterium Marinobacter aquaeolei (87 to 94% sequence similarity). A second group of isolates are α-Proteobacteria most closely related to the deep-sea heterotrophic bacterium Hyphomonas jannaschiana (81 to 89% sequence similarity). This study provides further evidence for the evolutionarily widespread capacity for Fe oxidation among bacteria and suggests that FeOB may play an unrecognized geomicrobiological role in rock weathering in the deep sea.     

Complementary Roles for Receptor Clustering and Conformational Change in the Adhesive and Signaling Functions of Integrin αIIbβ3

Integrin α_IIbβ₃ mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to α_IIbβ₃ function, α_IIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified α_IIb subunits were expressed with β₃ in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when α_IIbβ₃ affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of α_IIbβ₃ also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72^Syk and fibrinogen-dependent phosphorylation of pp125^FAK, even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in α_IIbβ₃ function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of α_IIbβ₃ in platelets.     

Inhibition of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae with aromatic sulphonamides and clinically licenced drugs – a joint docking/molecular dynamics study

The binding mode of aromatic sulphonamides and clinically licenced drugs to the three carbonic anhydrase (CA, EC 4.2.1.1) isoforms from the human pathogen V. cholerae was here thouroghly characterised by a joint docking and molecular dynamics in silico protocol. In fact, VchCA, VchCAβ, and VchCAγ are crucial in the pathogen life cycle and growth and represent innovative targets to fight V. cholerae proliferation overcoming the spreading chemoresistance to the available drugs. A set of 40 sulphonamides/sulfamates VchCAs inhibitors was studied using the proteins homology built 3 D models unveiling the key and stable interactions responsible for a potent CA inhibition. This study has the aim to offer insights and guidelines for the future rational design of potent and selective inhibitors targeting CA isoforms from V. cholerae or other human pathogens.     

Regulation of Endothelial Cell Motility by Complexes of Tetraspan Molecules CD81/TAPA-1 and CD151/PETA-3 with α3β1 Integrin Localized at Endothelial Lateral Junctions

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell–cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell–cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with α3β1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for α3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.     

Effect of thyroid status on α- and β-catecholamine responsiveness of hamster adipocytes

It has been suggested that part of the increased β-catecholamine responsiveness in hyperthyroid animals is due to a decrease in α-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the α₂-adrenergic inhibition of adenylate cyclase or the α₁-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamster. No effect of hyperthyroidism was found on the K_d of [³H]dihydroegocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to β-catecholamines was enhanced in adipocytes from hyperthyroid hamster, as was lipolysis. However, in adipocytes from hyperthyroid hamster the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on α-catecholamine action but enhances the activation of lipolysis by β-catecholamine agonists.