Antibiosis of Commensal Bacteria Harboring the Gut of Estuarine Water Fish ‘Chelon parsia’

Microbiology - Screening of gut flora of the estuarine water fish ‘Chelon parsia’ for the presence of potential antibiotic producers resulted in finding a new strain Bacillus subtilis...     

Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases

Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.     

Triiodothyronine and melatonin influence antioxidant defense mechanism in a teleost Anabas testudineus (Bloch): in vitro study

The effect of the hormones triiodothyronine (T3) and melatonin on antioxidant defense system was studied in 6-propyl thiouracil (6-PTU)-treated or photoperiod-exposed teleost Anabas testudineus. 6-PTU (2 microg/g) treatment or photoperiod exposure (24 h) increased malondialdehyde (MDA) and conjugated dienes (CD) concentrations, indicating increased lipid peroxidation (LPO) in the experimental conditions. T3 or melatonin (10(-6) M) treatment for 15 min in vitro in PTU-treated fish reversed the activity of superoxide dismutase (SOD), catalase and glutathione content. T3-treated group showed no change in glutathione peroxidase (GPx) activity, whereas melatonin treatment decreased its activity. T3 inhibited glutathione reductase (GR) activity. Photoperiod exposure (physiological pinealotomy) induced a stressful situation in this teleost, as evidenced by LPO products and antioxidant enzyme activities. Melatonin and T3 treatment for 15 min in vitro also reversed the effect of photoperiod on peroxidation products and the SOD and catalase activities. GR activity decreased in photoperiod-exposed group and melatonin and T3 treatment reversed the activities. The antioxidant enzymes responded to the stress situation after 6-PTU treatment and photoperiod exposure by altering their activities. The study suggested an independent effect of T3 and melatonin on antioxidant defence mechanism in different physiological situations in fish.     

Crystal structure of the human dual specificity phosphatase 1 catalytic domain

The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38‐alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.     

Does melatonin have a time-dependent effect on brain and gill ionic metabolism in a teleost, Anabas testudineus (Bloch)?

Exogenous administration of 0.20, 0.40 and 0.60 microg/g body weight melatonin over a 24 hr cycle caused an inhibition of Na+, K+ ATPase activity in both brain and gills of A. testudineus. However, Ca2+ ATPase activity in the brain was significantly inhibited by the highest dose, and that in the gill at all the doses of melatonin. Evening injection of melatonin had an inhibitory effect on both brain and gill Na+ K+ and Ca2+ ATPase activity. Melatonin treatment in the morning for 12 hrs did not have an effect on brain Na+, K+ ATPase, while Ca2+ ATPase was inhibited. Similar treatment stimulated Na+, K+ and Ca2+ ATPase activity in the gills. Sodium, potassium and calcium ions in the gill were significantly reduced in the evening treated group while no change was observed in the morning melatonin injected group. The results suggest that melatonin elicits a time-dependent effect on the enzymes and ionic content in the brain and gills of A. testudineus.     

A comparative study on lipid peroxidation, activities of antioxidant enzymes and viability of cattle and buffalo bull spermatozoa during storage at refrigeration temperature

A comparative study was conducted to monitor the activities of some antioxidant enzymes, lipid peroxidation and viability of cattle and buffalo bull spermatozoa during storage of semen at refrigeration temperature over a period of 72 h. Semen samples, collected from six cross bred cattle bulls (group I) and six Murrah buffalo bulls (group II), were diluted in egg-yolk-citrate and the spermatozoa were separated from seminal plasma by centrifugation at 4 degrees C in a refrigerated centrifuge. The malondialdehyde (MDA) production in group I increased from 1.17+/-0.29 at 0 h to 7.50+/-0.52 nmol/10(8)spermatozoa after 72 h of storage while in group II it increased from 1.99+/-0.26 to 8.70+/-0.10 nmol/10(8)spermatozoa in the same period. However, buffalo bull spermatozoa had a significantly higher (p<0.05) lipid peroxidation at 0 h as well as at 12, 24 and 48 h (p<0.01) periods. The activities of antioxidant enzymes viz. SOD, GPx and G6PD in both the groups showed a similar pattern of change i.e. the activities declined successively in spermatozoa and increased in the seminal plasma. However, the activities of these three enzymes remained significantly higher in the cattle bull spermatozoa than that in buffalo bull spermatozoa. Amount of MDA produced in spermatozoa of both the groups was negatively correlated while SOD, GPx and G6PD activities in spermatozoa were positively correlated to the motility and viability of spermatozoa. Sperm motility as well as viability was significantly less (p<0.05) in group II than that in group I. SOD, GPx and G6PD activities in spermatozoa of both the groups were negatively correlated to lipid peroxidation of spermatozoa cell membrane. The results showed that the less activities of antioxidant enzymes in buffalo bull spermatozoa was due to higher lipid peroxidation that indicated that they were more prone to oxidative stress as compared to cattle bull spermatozoa when stored at refrigeration temperature.     

Dissociation of I domain and global conformational changes in LFA-1: refinement of small molecule-I domain structure-activity relationships

LFA-1 (alphalbeta2) is constitutively expressed on leukocytes, but its activity is rapidly regulated. This rapid activation has been proposed to be associated with conformation changes in the inserted ("I") domain within the headpiece of LFA-1 as well as conversion of the molecules from bent to extended forms. To study these molecular changes as they relate to affinity regulation of LFA-1, we developed and synthesized a fluorescent derivative of BIRT-377 [Kelly et al. (2001) J. Immunol.] to examine changes in LFA-1 affinity in a flow cytometer with live cells. BIRT-377 binds to the ligand-binding or "I" domain of LFA-1. Structure-activity relationships studies indicated that an aminoalkyl group could be added to the central hydantoin group without significantly affecting binding. Using this modified derivative [1-(N-fluoresceinylthioureidobutyl)-[5R]-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-5-methyl-imidazolidine-2,4-dione (FBABIRT)], we analyzed the affinity of FBABIRT binding to LFA-1 on live cells. The binding affinity increases, and the dissociation rate decreases with divalent cation (Mn(2+)) stimulation. We then used FBABIRT with fluorescent resonance energy transfer (FRET) to show that LFA-1 changes its height relative to the cell surface when cells were treated with dithiothreitol (DTT) but not Mn(2+). Competition assays among FBABIRT and BIRT derivatives defined structure-affinity relationships that refine the current model of BIRT-377 binding to the I domain. Our data supports the model in which BIRT-377 binds to the I domain and stabilizes the bent structure of LFA-1, while divalent cation activation results in a small conformational change in the I domain without significant extension of LFA-1. DTT, in contrast, induces a conversion to the extended form of LFA-1 in the presence of BIRT-377 on live cells. The structure-activity studies suggest that BIRT-377 is a fully optimized inhibitor.