Differential expression of rat beta-defensins

The innate immunity, utilizes a battery of broad-spectrum antibacterial cationic polypeptides (3-5 kDa) named alpha- and beta-defensins. Several beta-defensins have been isolated and shown to play a role in the defense of various tissues. Herein, we report the expression pattern of two rat beta-defensins, rBD-1 and rBD-2, in liver, kidney, lung, spleen, and brain using RT-PCR. To study polymorphism and verify gene identity, all cDNA products were sequenced. rBD-1 was expressed in the kidney, lung, brain, but not in spleen or liver, whereas rBD-2 was expressed in the lung, but not in the kidney or spleen. In addition, rBD-2 was expressed in the brain and liver. No polymorphism was found in the genes encoding rat beta-defensins. These findings demonstrate a different expression pattern for rBD-2 than what has been reported. We conclude that the rat may be a useful model to investigate the function and contribution of beta-defensins to host defense.     

Exploring the role of the active site cysteine in human muscle creatine kinase

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.     

Diet and diabetic state modify glycogen synthase activity and expression in rat hepatocytes

Glycogen synthase (GS), a key regulatory enzyme in glycogen synthesis, is controlled by multisite phosphorylation and allosteric regulation and is activated by insulin. This study investigated changes in GS activity and expression in hepatocytes isolated from rats under altered nutritional and diabetic conditions. Experiments were carried out in healthy rats fed a chow diet, rats on high simple sugar (60% of energy from fructose and sucrose) or high fat (46% of energy from fat) diet, and in rats with streptozotocin induced diabetes. In the presence of insulin, activated GS activity (GS(I) form) was increased by 89% in hepatocytes isolated from healthy rats. The stimulatory effect of insulin on GS activity and expression was blunted by cycloheximide and actinomycin treatment. In rats fed a high simple sugar or high fat diet, insulin stimulation of GS(I) in isolated hepatocytes was impaired and GS expression was significantly lower in rats fed the high fat diet in comparison to controls. GLUT-2 protein expression was significantly lowered by both the high fat and high simple sugar diets. In hepatocytes isolated from diabetic rats, total GS activity (GS(T)) was lower than in hepatocytes from healthy animals. Insulin added to the incubation medium did not stimulate GS activity, demonstrating impaired sensitivity to insulin in diabetic rats. However, insulin administration significantly increased GS expression indicating that a defect in synthase phosphorylation may be responsible for impaired GS activity in the diabetic state. The results presented in this study further confirm that GS activity is affected by both dietary and hormonal factors which can be measured in a rat hepatocyte model.     

Immune responses to weakly immunogenic virally induced tumors. V. Short in vitro cultivation of YAC changes its antigenic properties.

YAC tumor, a Moloney virus-induced lymphoma of A mice, considerably changes its immunogenic properties following cultivation for 12 to 14 hr in vitro. The cultivated tumor, designated YAC-1, generates anti-YAC and anti-YAC-1 reactive cells, while the original YAC tumor generates suppressor cells with abrogate anti-YAC and anti-YAC-1 cytotoxic responses. In this report, we demonstrate by cold target competition experiments that YAC-1 and YAC tumors have distinctive antigenic structures on their surfaces. We have also found that RBL5, a Rauscher virus-induced lymphoma of C57BL/6 mice, generates anti-YAC, anti-YAC-1, and anti-RBL5 reactive cells in A mice. YAC-1 also generates reactive cells against the allogeneic RBL5 tumor. The total population of antitumor reactive cells was found to contain separate subpopulations directed maximally toward each of the three target tumors. The subpopulation directed toward each tumor was shown to be partially, but not completely, cross-reactive with the other tumors. Further, RBL5 priming and YAC-1 priming were shown to stimulate separate populations of antitumor reactive cells.     

Optical Waveguide Lightmode Spectroscopic Techniques for Investigating Membrane-Bound Ion Channel Activities

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na⁺ and organic cations through gramicidin channels and detecting the Cl^–-channel functions of the (α₅β₂γ₂) GABA_A receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline.