Peptide mapping analysis of the avian progesterone receptor

Progesterone receptor from the chicken oviduct has been shown to exist as two 8 S forms (I and II). Form I contains a protein of Mr = 75,000 and form II contains a protein of Mr = 110,000. In addition to these hormone-binding proteins, both receptor forms contain a protein with Mr = 90,000 that does not bind steroid. To investigate the possibility that these proteins are structurally related, they were isolated by preparative sodium dodecyl sulfate gel electrophoresis and subjected to peptide mapping analyses after digestion with Staphylococcus aureus V-8 protease, papain, or alpha-chymotrypsin. Receptor proteins labeled with [32P]orthophosphate in tissue minces were also subjected to peptide mapping analysis. The electrophoretic patterns of peptide fragments of the 90-kDa protein from receptor forms I and II were identical but were different from the peptide patterns obtained from the 75- and 110-kDa proteins which generated similar peptide patterns, indicating that these are structurally related. However, some differences were evident, indicating that these latter two proteins are not identical substrates for proteases. A one-dimensional comparison of the phosphopeptide patterns from the 75- and 110-kDa proteins also showed them to be similar, but not identical. Two-dimensional maps of phosphopeptides generated from the 75- and 110-kDa protein after complete tryptic digestion revealed multiple sites of phosphorylation which were identical except for one phosphopeptide that was unique to the 110-kDa protein. These results show the two progesterone-binding proteins to be very similar in structure, but to differ considerably from the 90-kDa protein.     

Identification of the lipid droplet targeting domain of the Cidea protein

Cidea, the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) domain-containing protein, is targeted to lipid droplets in mouse adipocytes, where it inhibits triglyceride hydrolysis and promotes lipid storage. In mice, Cidea may prevent lipolysis by binding and shielding lipid droplets from lipase association. Here we demonstrate that human Cidea localizes with lipid droplets in both adipocyte and nonadipocyte cell lines, and we ascribe specific functions to its protein domains. Expression of full-length Cidea in undifferentiated 3T3-L1 cells or COS-1 cells increases total cellular triglyceride and strikingly alters the morphology of lipid droplets by enhancing their size and reducing their number. Remarkably, both lipid droplet binding and increased triglyceride accumulation are also elicited by expression of only the carboxy-terminal 104 amino acids, indicating this small domain directs lipid droplet targeting and triglyceride shielding. However, unlike the full-length protein, expression of the carboxy-terminus causes clustering of small lipid droplets but not the formation of large droplets, identifying a novel function of the N terminus. Furthermore, human Cidea promotes lipid storage via lipolysis inhibition, as the expression of human Cidea in fully differentiated 3T3-L1 adipocytes causes a significant decrease in basal glycerol release. Taken together, these data indicate that the carboxy-terminal domain of Cidea directs lipid droplet targeting, lipid droplet clustering, and triglyceride accumulation, whereas the amino terminal domain is required for Cidea-mediated development of enlarged lipid droplets.     

Suppression of bioactive luteinizing hormone and testosterone by a progesterone antagonist ZK 98.734 in adult male common marmosets, Callithrix jacchus

The effects of a progesterone antagonist ZK 98.734 on release of bioactive luteinizing hormone (LH) and testosterone were studied in adult male common marmosets by using the following experimental protocols: (1) the blocking of the nocturnal rise in testosterone levels by ZK 98.734, (2) the pharmacodynamic effects of ZK 98.734 on testosterone and LH levels, (3) the reversal of ZK 98.734-induced decrease in testosterone by treatment with human chorionic gonadotropin (hCG), and (4) the blocking of estradiol-induced positive feedback release of LH by ZK 98.734. Sixteen adult male common marmosets were used for different experiments after resting them for at least 4 wk between experiments. Testosterone and bioactive LH levels were measured by specific radioimmunoassay and in vitro bioassay methods, respectively. Treatment (i.m.) of male common marmosets (n = 6/group) with ZK 98.734 (1 mg or 5 mg/day) at 1700 h for 3 consecutive days significantly (p less than 0.05) suppressed the nocturnal (2200 h) rise in testosterone levels. The effects of the two doses were not dose-related; however, the decrease on the first day of treatment was more pronounced with the 5-mg dose than with the 1-mg dose. Diurnal rhythms were restored during the post-treatment period. Similarly, treatment with ZK 98.734 (5 mg, n = 8/group) at 1000 h caused a decrease in testosterone and LH levels. The levels were significantly (p less than 0.05) lower at 3 and 6 h after treatment compared to pretreatment levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spotted in the News: Using Media Reports to Examine Leopard Distribution,Depredation, and Management Practices outside Protected Areas in Southern India

There is increasing evidence of large carnivore presence outside protected areas, globally. Although this spells conservation success through population recoveries, it makes carnivore persistence in human-use landscapes tenuous. The widespread distribution of leopards in certain regions of India typifies this problem. We obtained information on leopard-human interactions at a regional scale in Karnataka State, India, based on systematic surveys of local media reports. We applied an innovative occupancy modelling approach to map their distribution patterns and identify hotspots of livestock/human depredation. We also evaluated management responses like removals of ‘problem’ leopards through capture and translocations. Leopards occupied around 84,000 km² or 47% of the State’s geographic area, outside designated national parks and wildlife sanctuaries. Their presence was facilitated by extent of vegetative cover- including irrigated croplands, rocky escarpments, and prey base in the form of feral and free-ranging dogs. Higher probabilities of livestock/human attacks by leopards were associated with similar ecological features as well as with capture/removals of leopards. Of the 56 cases of leopard removals reported, 91% did not involve human attacks, but followed livestock predation or only leopard sightings. The lack of knowledge on leopard ecology in human-use areas has resulted in unscientific interventions, which could aggravate the problem rather than mitigating it. Our results establish the presence of resident, breeding leopards in human-use areas. We therefore propose a shift in management focus, from current reactive practices like removal and translocation of leopards, to proactive measures that ensure safety of human lives and livelihoods.     

Peripartum Cardiomyopathy Presenting With Ventricular Tachycardia: A Rare Presentation

A 25-year-old previously asymptomatic pregnant woman at 36 weeks'' gestation was noticed to have repetitive monomorphic ventricular tachycardia. A dilated left ventricle with moderately reduced systolic function was found on echocardiographic examination. This is a very rare presentation of peripartum cardiomyopathy (PPCMP) presenting with repetitive monomorphic ventricular tachycardia.     

Association between Gender,Process of Care Measures,and Outcomes in ACS in India: Results from the Detection and Management of Coronary Heart Disease (DEMAT) Registry

Background

Studies from high-income countries have shown that women receive less aggressive diagnostics and treatment than men in acute coronary syndromes (ACS), though their short-term mortality does not appear to differ from men. Data on gender differences in ACS presentation, management, and outcomes are sparse in India.

Methods and Results

The Detection and Management of Coronary Heart Disease (DEMAT) Registry collected data from 1,565 suspected ACS patients (334 women; 1,231 men) from ten tertiary care centers throughout India between 2007–2008. We evaluated gender differences in presentation, in-hospital and discharge management, and 30-day death and major adverse cardiovascular event (MACE; death, re-hospitalization, and cardiac arrest) rates. Women were less likely to present with STEMI than men (38% vs. 55%, p<0.001). Overall inpatient diagnostics and treatment patterns were similar between men and women after adjustment for potential confounders. Optimal discharge management with aspirin, clopidogrel, beta-blockers, and statin therapy was lower for women than men, (58% vs. 65%, p = 0.03), but these differences were attenuated after adjustment (OR = 0.86 (0.62, 1.19)). Neither the outcome of 30-day mortality (OR = 1.40 (0.62, 3.16)) nor MACE (OR = 1.00 (0.67, 1.48)) differed significantly between men and women after adjustment.

Conclusions

ACS in-hospital management, discharge management, and 30-day outcomes did not significantly differ between genders in the DEMAT registry, though consistently higher treatment rates and lower event rates in men compared to women were seen. These findings underscore the importance of further investigation of gender differences in cardiovascular care in India.     

IL-13 receptor-targeted cytotoxin cancer therapy leads to complete eradication of tumors with the aid of phagocytic cells in nude mice model of human cancer

Tumor-directed therapeutic approaches require unique or overexpressed specific Ag or receptor as a target to achieve selective tumor killing. However, heterogeneous expression of these targets on tumor cells limits the efficacy of this form of therapy. In this study, we forced abundant expression of IL-13Ralpha2 chain by plasmid-mediated gene transfer in head and neck, as well as prostate tumors to provide a potential target. This was followed by successfully treating xenograft tumor-bearing nude mice with IL-13R-directed cytotoxin (IL13-PE38QQR). Although we did not observe an indirect cytotoxic bystander effect conveyed to nontransduced tumor cells in vitro, our approach in vivo led to a complete regression of established tumors transfected with IL-13Ralpha2 chain in most animals. We found that the tumor eradication was achieved in part by infiltration of macrophages and NK cells, assessed by immunohistochemistry. Moreover, head and neck tumors xenografted in macrophage-depleted nude mice were less sensitive to the antitumor effect of IL-13 cytotoxin. Because we did not observe vector-related toxicity in any vital organs, our novel combination strategy of gene transfer of IL-13Ralpha2 chain and receptor-directed cytotoxin therapy may be a useful approach for the treatment of localized cancer.     

Enhanced expressions of endometrial tumour necrosis factor alpha and its receptors during early pregnancy in bonnet monkeys

Tumour necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine may play an active role in stimulating inflammatory reactions during pregnancy. However, the expression of endometrial TNF-alpha has not been investigated especially during early pregnancy, a phenomenon invariably accompanied by inflammatory reaction. In the present study, the endometrial expressions of TNF-alpha and its receptors (TNFR1 and TNFR2) during early pregnancy, when the embryo lies free in the zona hatched state in the uterine lumen, were analyzed by immunohistochemistry. The endometrial expressions of TNF-alpha, TNFR1 and TNFR2 were found to be significantly up-regulated (p < 0.05) in the glandular epithelium on day 6 post-ovulation in pregnant animals. The alteration in the expression of these molecules may contribute to the induction of local inflammatory reactions during implantation.     

The last exon of SNAP-23 regulates granule exocytosis from mast cells

SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.