Localization of dolichyl phosphate- and pyrophosphate-dependent glycosyl transfer reactions in Saccharomyces cerevisiae.

Membranes from Saccharomyces cerevisiae protoplasts were fractionated on a continuous sucrose gradient. Six bands were obtained, which contained altogether about 15% of the total cell protein. From their densitites, their behavior in the presence and absence of Mg2+ ions, and the distribution of marker enzymes, it was possible to identify fractions enriched in rough and smooth endoplasmic reticulum and in mitochondria. All glycosyl transfer reactions investigated where dolichyl phosphates served as glycosyl acceptors or where dolichyl phosphate- and pyrophosphate-activated sugars served as glycosyl donors showed the highest specific activity and up to 75% of the total activity in the endoplasmic reticulum. This was the case for the reactions involved in the formation of O-glycosidic as well as N-glycosidic linkages in yeast glycoprotein biosynthesis. Membrane fractions enriched in plasmalemma contained less than 3% of the corresponding activities.     

Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones.

Relaxin is a polypeptide hormone involved in remodeling of the birth canal during parturition. It is synthesized as a preprohormone precursor, which undergoes specific processing to form the mature two-chain disulfide-linked active species that is secreted by the cell. A major part of this processing requires endoproteolytic cleavage at specific pairs of basic amino acid residues, an event necessary for the maturation of a variety of important biologically active proteins, such as insulin and nerve growth factor. Human type 2 preprorelaxin was coexpressed in human kidney 293 cells with the candidate prohormone convertase-processing enzymes mPC1 or mPC2, both cloned from the mouse pituitary tumor AtT-20 cell line, or with the yeast kex2 alpha-mating factor-converting enzyme from Saccharomyces cerevisiae. Prorelaxin expressed alone in 293 cells was secreted into the culture medium unprocessed. Transient coexpression with mPC1 or kex2, but not with mPC2, resulted in the secretion of a low mol wt species with an electrophoretic mobility very similar, if not identical, to that of authentic mature relaxin purified from human placenta. This species was precipitable by monoclonal antibodies specific for relaxin and had a retention time on reverse phase HPLC comparable to that of relaxin. Its analysis by both electrospray and fast atom bombardment mass spectrometry generated mass data that were consistent only with mature relaxin. The basic residues required for mPC1-dependent cleavage of prorelaxin are defined by site-directed mutagenesis.     

Salmonella infection does not increase expression and activity of the high affinity IL-12 receptor

Expression of high affinity IL-12 receptors is required for IL-12-mediated IFN-gamma production. Activation of this pathway has been shown to be critical in generating optimal cell-mediated immunity. Therefore, increased IL-12 receptor expression might be expected in the host response after infection by an intracellular bacterial pathogen. In the present study, we have made the surprising discovery that infection with Salmonella results in an early reduction of high affinity IL-12 receptor expression and activation. After oral inoculation with Salmonella, the level of mRNA expression encoding IL-12 receptor beta2 (IL-12Rbeta2) subunit was diminished 12 h postinfection in the mesenteric lymph nodes and subsequently in the spleen. Furthermore, decreased IL-12Rbeta2 mRNA expression was observed in CD4+ T lymphocytes isolated from the mesenteric lymph nodes and spleens of infected mice. Attenuated IL-12Rbeta2 mRNA expression correlated with reduced receptor signaling, as demonstrated by reduced IL-12-induced STAT4 phosphorylation in enriched T lymphocytes isolated from the mesenteric lymph nodes and spleens of Salmonella-infected mice. These in vivo results were substantiated with an in vitro model system. In this model system, T lymphocytes cocultured with Salmonella-infected macrophages expressed less IL-12Rbeta2 mRNA. The cocultured T cells were also less responsive to IL-12 as assessed by reduced phosphorylation of STAT4 and limited IFN-gamma secretion. Together, these studies suggest that Salmonella can limit an optimal host immune response by reducing the expression and activity of high affinity IL-12 receptors.     

Accelerated G(1) phase progression induced by the human T cell leukemia virus type I (HTLV-I) Tax oncoprotein.

Tax, the human T cell leukemia virus type I oncoprotein, plays a crucial role in viral transformation and the development of the virally associated disease adult T cell leukemia. Because oncogenesis involves alterations in cell growth, it is important to examine the effects of Tax on cell cycle progression. Using a synchronized cell system, we have found that Tax expression accelerates G(1) phase progression and S phase entry with concomitant DNA replication. This accelerated progression is accompanied by an earlier onset of cdk2 kinase activity. In contrast to the shortening of G(1) phase, the length of S phase is unaffected by Tax expression. As a result of a more rapid cell cycle progression, cells expressing Tax exhibit faster growth kinetics and display an altered cell cycle distribution. Additionally, the decreased time allowed for growth in the presence of Tax results in a decreased cell size. Tax-associated acceleration of cell cycle progression may play a role in the ability of this viral oncoprotein to mediate cellular transformation and promote the development of human T cell leukemia virus type I-associated diseases.     

Optic Nerve Diffusion Tensor Imaging after Acute Optic Neuritis Predicts Axonal and Visual Outcomes

Background

Early markers of axonal and clinical outcomes are required for early phase testing of putative neuroprotective therapies for multiple sclerosis (MS).

Objectives

To assess whether early measurement of diffusion tensor imaging (DTI) parameters (axial and radial diffusivity) within the optic nerve during and after acute demyelinating optic neuritis (ON) could predict axonal (retinal nerve fibre layer thinning and multi-focal visual evoked potential amplitude reduction) or clinical (visual acuity and visual field loss) outcomes at 6 or 12 months.

Methods

Thirty-seven patients presenting with acute, unilateral ON were studied at baseline, one, three, six and 12 months using optic nerve DTI, clinical and paraclinical markers of axonal injury and clinical visual dysfunction.

Results

Affected nerve axial diffusivity (AD) was reduced at baseline, 1 and 3 months. Reduced 1-month AD correlated with retinal nerve fibre layer (RNFL) thinning at 6 (R=0.38, p=0.04) and 12 months (R=0.437, p=0.008) and VEP amplitude loss at 6 (R=0.414, p=0.019) and 12 months (R=0.484, p=0.003). AD reduction at three months correlated with high contrast visual acuity at 6 (ρ = -0.519, p = 0.001) and 12 months (ρ = -0.414, p=0.011). The time-course for AD reduction for each patient was modelled using a quadratic regression. AD normalised after a median of 18 weeks and longer normalisation times were associated with more pronounced RNFL thinning and mfVEP amplitude loss at 12 months. Affected nerve radial diffusivity (RD) was unchanged until three months, after which time it remained elevated.

Conclusions

These results demonstrate that AD reduces during acute ON. One month AD reduction correlates with the extent of axonal loss and persistent AD reduction at 3 months predicts poorer visual outcomes. This suggests that acute ON therapies that normalise optic nerve AD by 3 months could also promote axon survival and improve visual outcomes.