A facile method to prepare C-terminal fluorescently labelled peptides by an Fmoc strategy

Summary Spectrophotometric peptide probes, derivatized at the C-terminus, are conveniently prepared by means of an Fmoc solid-phase strategy. Using a resin such as Sasrin, the fully protected peptide can be cleaved from the resin with hydrazine, yielding the protected peptide-hydrazide which is subsequently oxidized to the azide. An amino-containing chromophore or fluorophore such as 5-[(2-aminoethyl)-amino]-naphthalene sulfonic acid (EDANS) can be coupled directly to this activated carboxyl group. This allows for specific placement of the fluorophore at the C-terminal carboxyl group in the presence of trifunctional amino acids.     

CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration.

The Nef gene product is a regulatory protein of HIV whose biological function is poorly understood. Nef has been thought to have a negative effect on viral replication in vitro but has been shown in studies with SIV to be necessary in the establishment of viraemia in vivo. In vitro studies in various human cell lines have shown that Nef downregulates the expression of cell surface CD4 and thus could have effects on the immune response. We have generated four transgenic mouse lines, with constructs containing two different Nef alleles under the control of CD2 regulatory elements to examine the interaction of Nef with the host immune system in vivo. In adult transgenic mice we have found marked downregulation in the level of CD4 on the surface of double positive thymocytes and a decrease in the number of CD4+ T cells in the thymus. Functional analyses have revealed a decrease in the total activation of transgenic thymocytes by anti-CD3 epsilon antibody. By specific intracellular staining of T cells in such mice we have found CD4 colocalizing with a Golgi-specific marker. These results strongly suggest a Nef mediated effect on developing CD4 thymocytes resulting from interference of Nef in the intracellular trafficking or post-translational modification of CD4.     

Antitumor response to recombinant murine interferon γ correlates with enhanced immune function of organ-associated,but not recirculating cytolytic T lymphocytes and macrophages

The mechanism of therapeutic activity for recombinant murine interferon-(rMu IFN) in the treatment of metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the tumor-bearing organ) were tested after 1 week and 3 weeks of rMu IFN administration (i.v. three times per week). Natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocyte (CTL) activities against specific and nonspecific targets, and macrophage tumoristatic activity were measured. rMu IFN demonstrated immunomodulatory activity in most assays of immune function. The optimal therapeutic protocol of rMu IFN (2.5×10⁶ U/kg, three times per week) prolonged survival and decreased the number of pulmonary metastatic foci. This therapeutic activity was correlated with specific CTL activity from pulmonary parenchymal mononuclear cells (PPMC), but not from spleen or blood. Macrophage tumoristatic activity in PPMC also correlated with therapeutic activity, but activity in alveolar macrophages did not. However, therapeutic activity did not correlate with NK or LAK activity at any site. These results demonstrate that the optimal therapeutic protocol is the same as the optimal immunomodulatory dose for pulmonary CTL and macrophage activities. Furthermore, while immunological monitoring may help to optimize treatment protocols, current monitoring procedures that use readily accessible sites, particularly peripheral blood, may not accurately predict the therapeutic efficacy of biological response modifiers in clinical trials.By acceptance of this article, the publisher or recipient acknowledges the right of the US. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the articleThis research was sponsored by the DHHS, under contract N01-23910 with Program Resources Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government     

Bacteria associated with crabs from cold waters with emphasis on the occurrence of potential human pathogens.

A diverse array of bacterial species, including several potential human pathogens, was isolated from edible crabs collected in cold waters. Crabs collected near Kodiak Island, Alaska, contained higher levels of bacteria than crabs collected away from regions of human habitation. The bacteria associated with the crabs collected near Kodiak included Yersinia enterocolitica, Klebsiella pneumoniae, and coagulase-negative Staphylococcus species; the pathogenicity of these isolates was demonstrated in mice. Although coliforms were not found, the bacterial species associated with the tissues of crabs collected near Kodiak indicate possible fecal contamination that may have occurred through contact with sewage. Compared with surrounding waters and sediments, the crab tissues contained much higher proportions of gram-positive cocci. As revealed by indirect plate counts and direct scanning electron microscopic observations, muscle and hemolymph tissues contained much lower levels of bacteria than shell and gill tissues. After the death of a crab, however, the numbers of bacteria associated with hemolymph and muscle tissues increased significantly. Microcosm studies showed that certain bacterial populations, e.g., Vibrio cholerae, can be bioaccumulated in crab gill tissues. The results of this study indicate the need for careful review of waste disposal practices where edible crabs may be contaminated with microorganisms that are potential human pathogens and the need for surveillance of shellfish for pathogenic microorganisms that naturally occur in marine ecosystems.     

Volume regulation by flounder red blood cells in anisotonic media

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.     

A high molecular weight peptide hydrolase in erythrocytes

1. A peptide hydrolase has been partially purified from the soluble fraction of erythrocyte lysates. 2. The enzyme has a molecular weight of approximately 600,000 and hydrolyses the chymotrypsin substrate glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin (pH optimum 7.0) and the trypsin substrate CBZ-Gly-Gly-Arg-2-naphthylamide. The two activities could not be separated by the purification procedure used. 3. The activity towards glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin in rat reticulocytes was four times that in mature erythrocytes. 4. Activity was abolished by 10 microM p-hydroxymercuriphenylsulphonic acid.     

Lipogenesis in the liver and adipose tissue of the cardiomyopathic hamster.

The activities of fatty acid synthetase, citrate cleavage enzyme, glucose-6-phosphate dehydrogenase and malic enzyme were measured in the liver and adipose tissue of cardiomyopathic and normal hamsters at age 33, 68 and 108 days. There was no difference in the activity of hepatic fatty acid synthetase between the diseased animals and the controls at any stage in their development. The activity of glucose-6-phosphate dehydrogenase was not different until age 108 days where it was significantly elevated in the BIO 82.62 strain. Citrate cleavage enzyme in the liver was depressed at all stages in the diseased animals as was malic enzyme. In adipose tissue, all enzyme activities were significantly depressed in the cardiomyopathic animals at the three stages. These data suggest that lipogenesis was depressed in the cardiomyopathic hamster.