Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase

A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD⁺-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg^–1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg^–1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg^–1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 mol g^–1 (cell dry weight) min^–1. In the presence of formate, the intracellular [NADH]/[NAD⁺] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.     

Soluble interleukin-1 receptor type II, IL-18 and caspase-1 in mild cognitive impairment and severe Alzheimer's disease

In the present study, we have determined levels of soluble interleukin-1 (IL-1) receptor type II (sIL-1RII), interleukin-18 (IL-18) and caspase-1 in cerebrospinal fluid and serum from mild cognitive impairment patients that later progressed to Alzheimer's disease (AD) and severe AD patients. Previous studies have shown that a chronic local inflammatory process is a part of AD neuropathology. In this process, activated microglial production of IL-1 seems to play an important role. In a previous study, we have shown increased levels of sIL-1RII in CSF from AD patients in a mild-moderate disease stage. In the present study, we found no significant differences in CSF or serum levels of sIL-1RII in either mild cognitive impairment or advanced AD patients as compared to control subjects. Likewise, there was no significant difference between mild cognitive impairment and severe AD patients. The same was true for caspase-1 and IL-18 serum levels, whereas CSF levels of caspase-1 and IL-18 were below detection limits. Our data indicate that the IL-1 system is relatively intact in the early and late stages of AD.     

The HtrA protease of Campylobacter jejuni is required for heat and oxygen tolerance and for optimal interaction with human epithelial cells

Campylobacter jejuni is a predominant cause of food-borne bacterial gastroenteritis in the developed world. We have investigated the importance of a homologue of the periplasmic HtrA protease in C. jejuni stress tolerance. A C. jejuni htrA mutant was constructed and compared to the parental strain, and we found that growth of the mutant was severely impaired both at 44 degrees C and in the presence of the tRNA analogue puromycin. Under both conditions, the level of misfolded protein is known to increase, and we propose that the heat-sensitive phenotype of the htrA mutant is caused by an accumulation of misfolded protein in the periplasm. Interestingly, we observed that the level of the molecular chaperones DnaK and ClpB was increased in the htrA mutant, suggesting that accumulation of non-native proteins in the periplasm induces the expression of cytoplasmic chaperones. While lack of HtrA reduces the oxygen tolerance of C. jejuni, the htrA mutant was not sensitive to compounds that increase the formation of oxygen radicals, such as paraquat, cumene hydroperoxide, and H2O2. Using tissue cultures of human epithelial cells (INT407), we found that the htrA mutant adhered to and invaded human epithelial cells with a decreased frequency compared to the wild-type strain. This defect may be a consequence of the observed altered morphology of the htrA mutant. Thus, our results suggest that in C. jejuni, HtrA is important for growth during stressful conditions and has an impact on virulence.     

Antiviral reactivities of gammadelta T cells

The complex antiviral immune mechanisms involve both adaptive and innate reactions mediated by gammadelta T lymphocytes, whose unique immunosurveillance contributions are analyzed here in different clinical and experimental settings. It is beyond any doubt that the fast, potent, cytotoxic as well as non-cytolytic antiviral activities of gammadelta T cells are critical in protecting the host against diverse viral pathogens.     

The NF2 tumor suppressor Merlin and the ERM proteins interact with N-WASP and regulate its actin polymerization function

The function of the NF2 tumor suppressor merlin has remained elusive despite increasing evidence for its role in actin cytoskeleton reorganization. The closely related ERM proteins (ezrin, radixin, and moesin) act as linkers between the cell membrane and cytoskeleton, and have also been implicated as active actin reorganizers. We report here that merlin and the ERMs can interact with and regulate N-WASP, a critical regulator of actin dynamics. Merlin and moesin were found to inhibit N-WASP-mediated actin assembly in vitro, a function that appears independent of their ability to bind actin. Furthermore, exogenous expression of a constitutively active ERM inhibits N-WASP-dependent Shigella tail formation, suggesting that the ERMs may function as inhibitors of N-WASP function in vivo. This novel function of merlin and the ERMs illustrates a mechanism by which these proteins directly exert their effects on actin reorganization and also provides new insight into N-WASP regulation.     

Selective membrane protein internalization accompanies movement from the endoplasmic reticulum to the protein storage vacuole pathway in Arabidopsis

In plant cells, certain membrane proteins move by unknown mechanisms directly from the endoplasmic reticulum (ER) to prevacuolar or vacuole-like organelles where membrane is internalized to form a dense, lattice-like structure. Here, we identify a sequence motif, PIEPPPHH, in the cytoplasmic tail of a membrane protein that directs the protein from the ER to vacuoles where it is internalized. A type II membrane protein in Arabidopsis thaliana, (At)SRC2 (for Soybean Gene Regulated by Cold-2), binds specifically to PIEPPPHH and moves from the ER to the same vacuoles where it is internalized. Not all proteins that move in this pathway are internalized because another Arabidopsis type II membrane protein, (At)VAP (for Vesicle-Associated Protein), localizes to the same organelles but remains exposed on the limiting membrane. The identification of (At)SRC2 and its preference for interaction with a targeting motif specific for the ER-to-vacuole pathway may provide tools for future dissection of mechanisms involved in this unique trafficking system.