Candida antarctica lipase-B-catalyzed kinetic resolution of 1,3-dialkyl-3-hydroxymethyl oxindoles

Candida antarctica (CAL-B) lipase-catalyzed resolution of 1,3-dialkyl-3-hydroxymethyl oxindoles has been performed to obtain (R)-1,3-dialkyl-3-acetoxymethyl oxindoles with up to 99% ee and (S)-1,3-dialkyl-3-hydroxymethyl oxindoles with up to 78% ee using vinyl acetate as acylating agent and acetonitrile as solvent transforming (S)-3-allyl-3-hydroxymethyl oxindole to (3S)-1′-benzyl-5-(iodomethyl)-4,5-dihydro-2H-spiro[furan-3,3′-indolin]-2′-one. The optically active 3-substituted-3-hydroxymethyl oxindoles and spiro-oxindoles are among the key synthons in the synthesis of potentially biologically active molecules.     

Cutting edge: diminished T cell TLR expression and function modulates the immune response in human filarial infection

Patent lymphatic filariasis is characterized by profound Ag-specific T cell hyporesponsiveness with impaired IFN-gamma and IL-2 production. Because T cells have been shown to express a number of TLR and to respond to TLR ligands, we hypothesized that diminished T cell TLR function could partially account for the T cell hyporesponsiveness in filariasis. T cells expressed TLR1, TLR2, TLR4, and TLR9, and the baseline expression of TLR1, TLR2, and TLR4, but not TLR9 was significantly lower in T cells of the filarial-infected individuals compared with the uninfected individuals (both endemic and nonendemic). TLR function was significantly diminished in the T cells of filarial-infected individuals based on decreased T cell activation/cytokine production in response to TLR ligands. Thus, diminished expression and function of T cell TLR is a novel mechanism underlying T cell immune tolerance in lymphatic filariasis.     

Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.Vesicular stomatitis virus (VSV), the prototypic rhabdovirus within the family Rhabdoviridae and the order Mononegavirales, is an enveloped virus with a negative-stranded RNA genome of 11,161 nucleotides. The viral genome encodes five proteins, namely, the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the large polymerase protein (L) (35). The genome is present within the virion core as a ribonucleoprotein (RNP) or nucleocapsid (NC) complex tightly encapsidated by the N protein and associated with the viral RNA-dependent RNA polymerase, a multiprotein complex of the viral L and P proteins. The G protein forms spikes on the viral envelope, binds to cell surface receptors, and plays a role in entry of virus into susceptible cells. The M protein is multifunctional; it plays a role in virus assembly and is responsible for cytopathogenesis observed in virus-infected cells (6).Studies on viral protein transport and virus motility in infected cells have been facilitated by imaging of fluorescent proteins fused to viral structural proteins (for reviews, see references 8 and 23). Recent advances in imaging techniques, coupled with the ability to genetically tag viral structural proteins with fluorescent proteins or to label viral membranes with lipophilic dyes, have allowed studies of the dynamic events of virus entry as well as of virus-cell interactions (17, 27-29, 31, 34, 40, 43, 51, 53). For enveloped viruses, the hallmark event of infection is the fusion of the viral envelope and the release of the NC (or RNP) into the cytoplasm. To examine the infection process by fluorescence microscopy and to distinguish between the enveloped virion and the uncoated NC, it is essential to differentially label the viral envelope and the NC core. Dually fluorescent viruses in which the viral core component, such as the NC or the RNP, is labeled with one fluorescent color and the envelope component is labeled with another color are thus powerful reagents for studies of virus entry and NC uncoating during early stages of infection as well as for studies of virus assembly during late stages of infection. Recently, dually fluorescent rabies virus (27) and human immunodeficiency virus (HIV) (9, 30) have been generated successfully. Using the dually fluorescent rabies virus, it was demonstrated that complete virus particles are transported in a retrograde manner (27).Successful recovery of a recombinant VSV encoding the P protein fused in frame with enhanced green fluorescent protein (PeGFP) allowed us to track the intracellular transport of viral NCs by live-cell imaging (14). This study demonstrated that microtubules were involved in viral NC transport toward the cell periphery (14), presumably to the plasma membrane for virus assembly. Whether the M protein interacts with the viral NCs before transport to the plasma membrane or at the plasma membrane prior to virus assembly remains a fundamental question in VSV assembly. Previous studies have shown that the M protein and the NCs do interact in vitro and in vivo (11, 12, 26), although more recent studies suggest that such interactions may occur only at the plasma membrane (18, 54). To examine the events of virus entry, uncoating, and also assembly, we wanted to generate a dually fluorescent VSV encoding PeGFP and monomeric red fluorescent protein fused in frame with the M protein at its carboxy terminus (MmRFP). Although repeated attempts to recover VSV with the fluorescently tagged M protein in place of wild-type (wt) M were unsuccessful, viruses encoding the fluorescently tagged M protein could be recovered when it was inserted as an extra cistron at the G-L gene junction. Further use of this virus for studies of virus entry, uncoating, and egress was limited because the MmRFP fusion protein was not incorporated into the virions.Recently, a new method of genetic tagging of proteins for fluorescence imaging was developed wherein the protein is tagged with a relatively smaller tetracysteine (tc) motif (CCPGCC). This motif can be recognized specifically by membrane-permeable biarsenical dyes that fluoresce when covalently bound to the cysteine pairs in the tc motif (1, 24, 39). Such a small tag can be fused to the protein of interest with minimal disruption of protein function. This is a powerful approach for real-time visualization of nascent protein synthesis and trafficking, as the existing and newly synthesized pools of proteins can be labeled differentially with the two fluorescent biarsenical dyes, FlAsH (green) and ReAsH (red) (38, 48). Using a tc-tagged M protein (Mtc) encoded in place of the wt M protein in the VSV genome, we rescued recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) and demonstrated that the Mtc was incorporated into infectious virions in amounts similar to that observed for M protein in wt VSV. Moreover, dynamic imaging of newly synthesized M protein by sequential labeling with the two biarsenical dyes revealed that the M protein is transported from the site of synthesis inside the cytoplasm to the plasma membrane in less than 30 min. We have also shown that the M protein reaches the plasma membrane independent of NCs and the microtubules. Additionally, our results show that following adsorption, entry and uncoating of VSV in the infected cells occur with a half-life of approximately 28 min.     

Caspase-3 is transiently activated without cell death during early antigen driven expansion of CD8(+) T cells in vivo

Background

CD8⁺ T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8⁺ T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8⁺ T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8⁺ T cell responses has yet to be examined.

Methods and Findings

We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8⁺ T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8⁺ T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3^hi and caspase-3^low CD8⁺ T cells. The expression of active caspase-3 peaked before effector phenotype (CD62L^low) CD8⁺ T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8⁺ cells remained active caspase-3^low throughout the contraction phase.

Conclusions

Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8⁺ T cells. Furthermore, the contraction of CD8⁺ T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3.     

Crystal structure of the MarR family regulatory protein, ST1710, from Sulfolobus tokodaii strain 7

The emergence of bacterial resistance to multiple drugs poses a serious and growing health concern. Understanding and deciphering the mechanisms of these multiple drug resistance regulatory proteins through structural or biochemical means is an important endeavor. Here, we present the crystal structure of ST1710 from Sulfolobus tokodaii strain 7 in two different crystal forms, at 1.80 and 2.0A, respectively. The overall structure of the ST1710 dimer shares the topology of the MarR family of proteins, with each subunit containing a winged helix-turn-helix DNA-binding motif. We also show the protein-DNA interactions by biochemical methods. Our molecular modeling analysis suggested that Asp88 and Arg90 are the key residues in ST1710 involved in the protein-DNA interactions.     

Non-invasive assessment of oxidative capacity in young Indian men and women: a 31P magnetic resonance spectroscopy study

It is generally assumed that men display greater strength and muscle capacity than women. However, previous biochemical and histological studies have shown that men have greater capacity for anaerobic metabolism and women have higher or similar oxidative metabolism. Therefore, in the present study, we estimated oxidative capacity of gastrocnemius muscle and compared in Indian men and women using non-invasive in vivo 31P magnetic resonance spectroscopy (MRS). Healthy subjects (8 young males and 9 females, age-matched) performed plantar flexion exercise within a magnet and MRS measurements of inorganic phosphate (Pi), phosphocreatine (PCr), ADP, and pH of the calf muscles were carried out using a 1.5 T whole-body MRI system. PCr values during recovery were fitted to an exponential curve, and oxidative capacity was calculated using rate constant (k(PCr)), as an index of oxidative phosphorylation. When men and women were compared for different metabolic ratios, ADP, pH, k(PCr) and oxidative capacity, all parameters turned out to be statistically insignificant. The results showed no gender effect on skeletal muscle oxidative metabolism. The study demonstrated the usefulness of such non-invasive method to indirectly measure the oxidative capacity of the muscle based on PCr recovery.     

Mutation in the Fas pathway impairs CD8+ T cell memory

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-gamma and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections.     

Dynamic change of neural cell adhesion molecule polysialylation on human neuroblastoma (IMR-32) and rat pheochromocytoma (PC-12) cells during growth and differentiation

Polysialic acid (PSA) is a regulatory epitope of neural cell adhesion molecule (NCAM) in homophilic adhesion of neural cells mediated by NCAM, is also known to be re-expressed in several human tumors, thus serves as an oncodevelopmental antigen. In this study, using a recently developed ultrasensitive chemical method in addition to immunochemical methods, growth stage-dependent and retinoic acid (RA)-induced differentiation-dependent changes of PSA expression in human neuroblastoma (IMR-32) and rat pheochromocytoma (PC-12) cells were analyzed both qualitatively and quantitatively. Both IMR-32 and PC-12 cells expressed PSA on NCAM, and the level of PSA expressed per unit weight of cells increased with post-inoculation incubation time. The most prominent feature was seen at the full confluence stage. RA induced neuronal differentiation in both IMR-32 and CP-12 cells that paralleled the change in the PSA level. Chemical analysis revealed the presence of NCAM glycoforms differing in the degree of polymerization (DP) of oligo/polysialyl chains, whose DP was smaller than 40. DP distribution of PSA was different between the cell lines and was changed by the growth stage and the RA treatment. Thus DP analysis of PSA is important in understanding both mechanism and biological significance of its regulated expression.     

Aspartate modulates the circadian patterns of a few biochemical variables in Wistar rats

D-aspartate was used to demonstrate possible sources of excitatory input to the suprachiasmatic nucleus (SCN) in rats. Aspartate (50 mg/kg bodyweight) was orally administrated chronically for 60 days to Wistar rats and 24 h rhythmic patterns of glucose, cholesterol, total protein and aspartate transaminase (AST) were studied under light - dark (LD 12:12 h) cycle. Our results showed acrophase advances in glucose and delays in cholesterol and AST rhythms. Increased mesor and altered amplitude values were found in all rhythms; aspartate levels in the brain were found to be significantly increased in aspartate treated animals. We hypothesised that the altered biochemical rhythms in aspartate treated rats could be due to (1) modulation of neurotransmission in SCN, (2) behavioural rhythms and (3) feeding rhythms.