Mutational analysis of regulation of MHC and anti-viral genes.

CTL-mediated selection for loss of expression of Mta by H-2-heterozygous SV40-transformed mouse fibroblasts (line 24SV) produced an unusual phenotypic class of maternally transmitted Ag negative mutants defective in both MHC expression and in anti-viral activity. Severely reduced surface expression of class I MHC Ag from multiple loci of both haplotypes correlated with low levels of MHC H chain and beta 2-microglobulin mRNA. Inasmuch as IFN can up-regulate class I expression and some fibroblasts elaborate autocrine IFN-beta, we examined whether IFN could restore wild-type expression of class I MHC Ag. However, IFN could not restore wild-type expression. Moreover, the fold-increases in class I Ag and mRNA expression were significantly reduced in mutant cells compared to wild-type cells. These results suggested that the mutants might have generalized defects in IFN response. Inasmuch as the induction of an anti-viral state is a hallmark of IFN responses, we exposed cells to IFN-alpha, -beta, or -gamma and challenged with virus. 24SV cells, exposed to any of the three IFNs, were completely protected from destruction by vesicular stomatitis, mengovirus or respiratory syncytial viruses. In contrast, MHC and anti-viral defective mutants could not be protected from virus-induced lysis by any IFN. Somatic cell hybridization analyses indicated that both basal MHC and IFN-inducible phenotypes were recessive to wild-type, and that a trans-acting regulatory factor required for basal MHC expression is defectively expressed in the mutants. Such a factor may integrate the organismal response to virus infection, encompassing both immune and nonimmune anti-viral responses.     

Obesity-inducing lesions of the central nervous system alter leptin uptake by the blood-brain barrier.

Leptin regulates body adiposity by decreasing feeding and increasing thermogenesis. Obese humans and some obese rodents are resistant to peripherally administered leptin, suggesting a defect in the transport of leptin across the blood-brain barrier (BBB). Defective transport of exogenous leptin occurs in some models of obesity, but in other models transport is normal. This shows that factors other than obesity are associated with impairment of leptin transport across the BBB. In order to further investigate these factors, we determined leptin transport in rats made obese by lesioning of the ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), or posterodorsal amygdala (PDA). These regions all contain leptin receptors and lesions there induce obesity and hyperleptinemia and alter the levels of many feeding hormones which might participate in leptin transporter regulation. We measured the uptake of radioactively labeled leptin by the BBB by multiple-time regression analysis which divides uptake into a reversible phase (Vi, e.g., receptor/transporter binding to the brain endothelial cell) and an irreversible phase (Ki, complete transport across the BBB). Leptin uptake was not affected in rats with VMH lesions. No significant change occurred in the entry rate (Ki) for any group, although Ki declined by over 35% in rats with PVN lesions. Decreased uptake was observed in rats with PVN lesions and with PDA lesions. This was primarily due to a reduced Vi (about 21% for the PDA). This decreased uptake is most likely explained by decreased binding of leptin to the brain endothelial cell, which could be because of decreased binding by either receptors or transporters. This suggests that some of the feeding hormones controlled by the PVN and PDA may participate in regulating leptin uptake by the BBB.     

Characterization of the Phosphorylation Site of PP59, a Substrate for Cyclic AMP-Dependent Protein Kinase That Is Enriched in the Synaptic Membrane Fraction of Rat Cerebellum

Abstract: We have identified previously a synaptic membrane-associated protein, PP59, that serves as a substrate for cyclic AMP-dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCl. A 290-fold purification of PP59 was achieved by selective solubilization, followed by continuous-elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP-dependent protein kinase phosphorylation site within PP59, the partially purified ³²P-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed-phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala-Arg-Glu-Arg-Ser-Asp-Ser(P)-Thr-Gly-Ser-Ser-Ser-Val-Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14-amino acid sequence was used to develop polyclonal anti-peptide antibodies that were affinity-purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP-dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.     

Vasculature of the head and respiratory organs in an obligate air-breathing fish,the swamp eel Monopterus (=Amphipnous) cuchia

Methyl methacrylate vascular corrosion replicas were used to examine the macrocirculation in the head region and the microcirculation of respiratory vessels in the air-breathing swamp eel Monopterus cuchia. Fixed respiratory tissue was also examined by SEM to verify capillary orientation. The respiratory and systemic circulations are only partially separated, presumably resulting in supply of mixed oxygenated and venous blood to the tissues. A long ventral aorta gives rise directly to the coronary and hypobranchial arteries. Two large shunt vessels connect the ventral aorta to the dorsal aorta, whereas the remaining ventral aortic flow goes to the respiratory islets and gills. Only two pairs of vestigial gill arches remain, equivalent to the second and third arches, yet five pairs of aortic arches were identified. Most aortic arches supply the respiratory islets. Respiratory islet capillaries are tightly coiled spirals with only a fraction of their total length in contact with the respiratory epithelium. Valve-like endothelial cells delimit the capillary spirals and are unlike endothelial cells in other vertebrates. The gills are highly modified in that the lamellae are reduced to a single-channel capillary with a characteristic three-dimensional zig-zag pathway. There are no arterio-arterial lamellar shunts, although the afferent branchial artery supplying the gill arches also supplies respiratory islets distally. A modified interlamellar filamental vasculature is present in gill tissue but absent or greatly reduced in the respiratory islets. The macro- and micro-circulatory systems of M. cuchia have been considerably modified presumably to accommodate aerial respiration. Some of these modifications involve retention of primitive vessel types, whereas others, especially in the microcirculation, incorporate new architectural designs some of whose functions are not readily apparent.     

Messenger RNA orientation on the ribosome. Placement by electron microscopy of antibody-complementary oligodeoxynucleotide complexes

Messenger RNA orients on the small ribosomal subunit by base pairing with a complementary sequence in ribosomal RNA. We have positioned this ribosomal RNA segment and thus oriented the mRNA using a new technique--localization of an antibody-recognizable modified complementary oligodeoxynucleotide by electron microscopy. A synthetic oligodeoxynucleotide complementary to the message-positioning ribosomal RNA sequence was modified at either or both ends with different antigenic markers. Electron microscopy of subunit-oligodeoxynucleotide-antibody complexes allowed separate placement of each terminal marker of the oligodeoxynucleotide probe. The 5'-end of the complementary sequence contacts the subunit at the platform tip (rRNA nucleotide 1542). The message then extends along the interior side of the platform to the level of the fork of the cleft separating the platform from the subunit body, and displaced slightly to the convex side of the platform (rRNA nucleotide 1531). Based on our results and data from other laboratories, we propose a model for the positioning of messenger RNA on the 30 S subunit.     

FBXO44 promotes DNA replication-coupled repetitive element silencing in cancer cells

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Foraging Efficacy of a Larval Parasitoid in a Cotton Patch: Influence of Chemical Cues and Learning

Plant-herbivore chemical signals and behavioral plasticity may enhance parasitoid host-foraging efficacy in the field; however, no studies have quantified the potential benefits from these factors under field-type conditions. The effect of plant-herbivore signals and learning on the foraging efficacy of Microplitis croceipes was quantified by directly observing and recording total and sequential duration of various foraging behaviors relative to 5 randomly placed herbivore-damaged and host-infested cotton plants and 20 undamaged and non-host-infested plants. Microplitis croceipes spent significantly more time searching (flying and antennation) on host infested versus uninfested plants. Antennation time was significantly and negatively correlated with successive host stings. Contrary to expectations of increased duration, flight time remained constant throughout the foraging bout, which may indicate that there was some learning associated with flight. These results suggest that plant-herbivore chemical signals and learning enhances the foraging efficacy of M. croceipes.     

Lipase-catalyzed transamidation of non-activated amides in organic solvent

A novel biocatalytic reaction of transamidation of non-activated amides with amines is reported. Among 45 different lipolytic and proteolytic enzymes tested, only the lipase from Candida antarcticawas able to catalyze this reaction. The reaction proceeded with up to ca. 80% conversion in anhydrous methyl tert-butyl ether and worked with both N-substituted and unsubstituted amides. The biocatalytic transamidation is an equilibrium process and, therefore, higher conversions to the desired amide were achieved by using increased concentrations of the amine nucleophile.