Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities

Serine palmitoyltransferase (SPT) catalyzes the first committed step in sphingolipid biosynthesis. In yeast, SPT is composed of a heterodimer of 2 highly-related subunits, Lcb1p and Lcb2p, and a third subunit, Tsc3p, which increases enzyme activity markedly and is required for growth at elevated temperatures. Higher eukaryotic orthologs of Lcb1p and Lcb2p have been identified, but SPT activity is not highly correlated with coexpression of these subunits and no ortholog of Tsc3p has been identified. Here, we report the discovery of 2 proteins, ssSPTa and ssSPTb, which despite sharing no homology with Tsc3p, each substantially enhance the activity of mammalian SPT expressed in either yeast or mammalian cells and therefore define an evolutionarily conserved family of low molecular weight proteins that confer full enzyme activity. The 2 ssSPT isoforms share a conserved hydrophobic central domain predicted to reside in the membrane, and each interacts with both hLCB1 and hLCB2 as assessed by positive split ubiquitin 2-hybrid analysis. The presence of these small subunits, along with 2 hLCB2 isofoms, suggests that there are 4 distinct human SPT isozymes. When each SPT isozyme was expressed in either yeast or CHO LyB cells lacking endogenous SPT activity, characterization of their in vitro enzymatic activities, and long-chain base (LCB) profiling revealed differences in acyl-CoA preference that offer a potential explanation for the observed diversity of LCB seen in mammalian cells.     

Mechanisms regulating the development and function of natural regulatory T cells

The key of the immune system is to protect the host from foreign threat posed by pathogens and from the internal threat posed by self-attacking lymphocytes. The ability to discriminate self versus non-self ensures that only “non-self” pathogens, but not the self antigens, are attacked. Such tolerance to “self” arises from the central tolerance mechanisms that include the deletion of thymocytes with high reactivity to self antigens and also the induction of unresponsiveness of autoreactive T cells in the periphery. Natural regulatory T cells (nTregs) directly inhibit effector T cells, and keep their proliferation in control. Apart from preventing autoimmune reactions, Tregs also contribute to peripheral immune homeostasis as evidenced by the excessive lymphocyte accumulation in peripheral lymphoid organs and intestinal inflammation in the absence of nTregs. Here we discuss the molecular aspects of the development and suppressive function of naturally occurring Tregs. Accumulating evidence shows the importance of these Tregs in autoimmunity, tumor immunity, organ transplantation, allergy, and microbial immunity.     

Increased release of interleukin-1 from mouse peritoneal macrophages in vitro after cisplatin treatment

The supernatants collected from cis-platin (2 micrograms, 5 micrograms, 10 micrograms, treated macrophage monolayers enhance the proliferation of thymocytes by a submitogenic concentration of con A (1 microgram/ml). The supernatants collected from the untreated macrophage monolayers show a gradual ten fold increase in the interleukin-1 (IL-1) activity during 30 min to 48 h incubation at 37 degrees C. Supernatants collected from macrophage monolayers treated with 2 micrograms/ml of cis-platin show only a marginal increase in IL-1 activity as compared to untreated monolayers. However, compared to controls, 30 to 40 fold increases in IL-1 activity were measured in supernatants collected from the macrophage monolayers incubated with 5, 10 and 20 micrograms/ml cis-platin at 37 degrees C. The IL-1 activity in supernatants collected from macrophage monolayers treated with cis platin and LPS are also compared.     

An estradiol matrix transdermal system for the prevention of postmenopausal bone loss

OBJECTIVE: The aim of this study was to evaluate the efficacy, safety, and tolerability of 2 years' application of an estradiol matrix transdermal system for the prevention of postmenopausal bone loss. METHODS: In this multicenter, randomized, placebo-controlled, parallel-group study, 261 surgically or naturally postmenopausal women were randomized to apply the estradiol matrix transdermal system (0.025, 0.0375, 0.05, or 0.1 mg/d) or matching placebo twice a week for 2 years. The study was double blind with respect to treatment (active vs placebo) but not to the dose levels of active treatment (because of the differing sizes and shapes of the patches). In addition to receiving the assigned treatment, the 100 nonhysterectomized women received 2.5 mg medroxyprogesterone acetate daily throughout the study. RESULTS: The evaluable group (n = 259) had a mean age of 52 years and a mean duration of menopause of 32 months. Following 2 years of treatment, there were significant differences in favor of estradiol between all doses of the estradiol matrix transdermal system and placebo in terms of the percentage change from baseline in the bone mineral density (BMD) of the L1-L4 anteroposterior lumbar spine (0.1 and 0.05 mg/d, P < 0.001; 0.0375 mg/d, P = 0.024; 0.025 mg/d, P = 0.002). Percentage changes from baseline in the BMD of the femoral neck after 2 years of treatment also consistently demonstrated the efficacy of the estradiol matrix transdermal system compared with placebo (all, P < or = 0.044). The estradiol matrix transdermal system was well tolerated. CONCLUSION: The estradiol matrix transdermal system was effective in preventing postmenopausal bone loss at dosages of 0.025 to 0.1 mg/d, and had a safety profile consistent with the known effects of estrogen/progestin.     

Utility of B-cell epitopes based peptides of RD1 and RD2 antigens for immunodiagnosis of pulmonary tuberculosis

Tuberculosis (TB) continues to be a major health problem due to lack of accurate, rapid, and cost-effective diagnostic tests. Serodiagnostic tests incorporating highly specific region of difference (RD) antigens (early secretory antigenic target 6 [ESAT-6], culture filtrate protein 10 [CFP-10], culture filtrate protein 21 [CFP-21], and mycobacterial protein from species tuberculosis 64 [MPT-64]) have recently been shown to be promising for specific diagnosis of TB in our lab. However, only few studies have reported the use of synthetic peptides of RD antigens, and none has used them to differentiate TB from sarcoidosis, a close mimic of smear-negative pulmonary TB (PTB) with entirely different management. The present study was conducted with an aim to study the utility of B-cell epitopes based peptides of RD1 (ESAT-6, CFP-10) and RD2 (CFP-21, MPT-64) antigens for immunodiagnosis of PTB for which sputum smear-positive PTB patients, sputum smear-negative PTB patients, sarcoidosis patients, and healthy controls (n = 24/group) were recruited. Bioinformatic software Bcepred was used to predict linear B-cell epitopes, using physico-chemical properties on a non-redundant dataset. Seven peptides as representative B-cell epitopes of ESAT-6, CFP-10, CFP-21, and MPT-64 were evaluated as targets of the antibody responses in TB patients and controls by enzyme-linked immunosorbent assay (ELISA). The current study showed sensitivity with individual peptides ranging from 37.5% to 83.3% for smear positive, 25% to 58.3% for smear negative as compared to 4.16% to 20.8% for sarcoidosis. Four out of 7 peptides that showed higher reactivity with TB patients and better discrimination from sarcoidosis patients representing ESAT-6, CFP-10, CFP-21, and MPT-64 were selected for multiepitope ELISA. The combination of peptides yielded 83.3% sensitivity for smear positive, 62.5% for smear negative, and only 4.16% for sarcoidosis. The specificity, however, for all the peptides/combination was 100%. Combination of peptides has proven to be better than individual peptides as per the latest criteria of the World Health Organization according to which a test that can replace smear microscopy with sensitivity of >90% for smear-positive patients and >65% for smear-negative TB patients with a specificity >95%, and thus, the present study suggests that a test based on combination of peptides selected from mycobacterial RD1 and RD2 antigens could be important for promoting an early diagnosis and management of otherwise difficult to diagnose smear-negative PTB patients. Moreover, it can also be used to discriminate sarcoidosis from PTB, thus preventing the misdiagnosis and mismanagement.