The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly

We report here the cloning and initial characterization of PAS4, a gene required for peroxisome assembly in the yeast Pichia pastoris. The PAS4 gene encodes a 24-kDa protein (Pas4p) that is located on the cytoplasmic surface of peroxisomes and is induced during peroxisome proliferation. Analysis of the Pas4p sequence revealed a high degree of similarity to ubiquitin-conjugating enzymes, particularly in the region surrounding the putative active-site cysteine residue with which ubiquitin forms a thioester bond. As expected for a ubiquitin-conjugating enzyme, substitution of alanine or serine for the conserved active-site cysteine residue abolished PAS4 function. In addition, a small amount of a 32 kDa form of Pas4p (the predicted size of a Pas4p-ubiquitin conjugate) was detected both in vivo and in vitro. This species was eliminated by reducing agents and was not detected in the cysteine to alanine substitution mutant, suggesting that it is a Pas4p-ubiquitin conjugate. Using a yeast strain that overexpresses a Myc-ubiquitin fusion protein, we demonstrate directly that this conjugate contains ubiquitin. We conclude from these observations that PAS4 is a member of the ubiquitin-conjugating enzyme gene family and that one or more ubiquitination reactions are required for peroxisome assembly.     

Post-translational processing of the insulin-like growth factor-2 precursor. Analysis of O-glycosylation and endoproteolysis

Insulin-like growth factor-2 (IGF-2) is expressed in most embryonic tissues and is required for normal development during gestation. After birth IGF-2 expression is extinguished in most tissues, but the gene is often reactivated during tumorigenesis. Tumors secrete high molecular weight forms of IGF-2 that result from aberrant post-translational processing of pro-IGF-2. As a first step toward understanding how high molecular weight IGF-2 peptides might contribute to tumor progression, we have characterized the biosynthesis of IGF-2 in a human embryonic cell line. We have found that pro-IGF-2 can initially form two disulfide isomers that undergo rearrangement to a single conformation in vivo. The addition of N-acetylgalactosamine to Ser71, Thr72, Thr75, and Thr139 likely occurs in the cis- Golgi apparatus. Sialic acid addition begins in the trans- Golgi apparatus, but IGF-2 peptides must reach the trans-Golgi network for oligosaccharide maturation to be completed. Endoproteolysis occurs concomitant to or slightly after oligosaccharide maturation. Cleavage was observed only at Arg104, resulting in the secretion of IGF-2-(1-104) and free E-peptide. Proteolysis required basic residues in the P1 (Arg104) and P4 (Arg101) positions, was completely blocked by a furin inhibitor, and was enhanced by coexpression with furin, PACE4, PC6A, PC6B, and LPC. These data suggest that members of the subtilisin-related proprotein convertase family mediate processing of pro-IGF-2 at Arg104. We did not detect the IGF-2 peptides that are most abundant in normal serum, mature IGF-2, and IGF-2-(1-87), in this expression system, which indicates that novel endoproteases are responsible for generating these products.     

Allelotype analysis of oesophageal adenocarcinoma: loss of heterozygosity occurs at multiple sites

Deletions of tumour-suppressor genes can be detected by loss of heterozygosity (LOH) studies, which were performed on 23 cases of adenocarcinoma of the oesophagus, using 120 microsatellite primers covering all non-acrocentric autosomal chromosome arms. The chromosomal arms most frequently demonstrating LOH were 3p (64% of tumours), 5q (45%), 9p (52%), 11p (61%), 13q (50%), 17p (96%), 17q (55%) and 18q (70%). LOH on 3p, 9p, 13q, 17p and 18q occurred mainly within the loci of the VHL, CDKN2, Rb, TP53 and DCC tumour-suppressor genes respectively. LOH on 5q occurred at the sites of the MSH3 mismatch repair gene and the APC tumour-suppressor gene. 11p15.5 and 17q25-qter represented areas of greatest LOH on chromosomes 11p and 17q, and are putative sites of novel tumour-suppressor genes. LOH on 9p was significantly associated with LOH on 5q, and tumours demonstrating LOH at both the CDKN2 (9p21) and MSH3 (5q11-q12) genes had a significantly higher fractional allele loss than those retaining heterozygosity at these sites. Six of nine carcinomas displaying microsatellite alterations also demonstrated LOH at CDKN2, which may be associated with widespread genomic instability. Overall, there are nine sites of LOH associated with oesophageal adenocarcinoma.     

Lovastatin disrupts early events in insulin signaling: a potential mechanism of lovastatin's anti-mitogenic activity

The mechanism by which lovastatin lowers cholesterol levels is well characterized but little is known about its anti-mitogenic and anti-tumorigenic mechanism. Here we demonstrate that lovastatin disrupts early events in the mitogenic signaling pathways of insulin. Insulin treatment (200 mM) of quiescent HIR rat-1 fibroblasts results in an 8-fold stimulation of phosphatidylinositol-3-kinase (PI-3-K) activity. Overnight pretreatment of cells with lovastatin (20 microM) inhibits insulin stimulation of PI-3-K activity by 75%. Immunoprecipitation and immunoblotting experiments using antibodies against the regulatory subunit of PI-3-K (p85), phosphotyrosine, and insulin receptor alpha and beta subunits demonstrate that lovastatin inhibits the association of p85 with tyrosine phosphorylated insulin receptor substrate-1 and the beta subunit of the insulin receptor. Furthermore, lovastatin dramatically reduces (70-100%) the level of tyrosine phosphorylated insulin receptor beta subunit following insulin stimulation. These results clearly demonstrate that lovastatin disrupts early events of insulin mitogenic signaling by reducing the levels of tyrosine phosphorylated beta subunit and suggest that this disruption is a potential mechanism for the anti-mitogenic effect of lovastatin.     

Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis

BACKGROUND & AIMS: Membrane lymphotoxin (LT) alpha/beta, a member of the tumor necrosis factor (TNF) family of immune regulatory molecules, is involved both in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in the adult. Defects observed in the mucosal immune system in animals with a genetically disrupted LTalpha/beta pathway coupled with the expression of LTalpha/beta in activated T cells motivated an examination of the importance of this pathway in experimental colitis. METHODS: Soluble LTbeta receptor (LTbetaR) immunoglobulin fusion protein was used to inhibit the LTalpha/beta/light axis in two independent rodent models of colitis: CD45RBhi CD4(+)-reconstituted SCID mice and bone marrow-transplanted tg26 mice (BM --> tg26). RESULTS: Treatment with LTbetaR immunoglobulin attenuated the development of both the clinical and histological manifestations of the disease in these two murine models of colitis. Given the success of TNF inhibitors in the treatment of human Crohn's disease, the effects of LTbetaR immunoglobulin have been compared with antibody to TNF in the BM --> tg26 model, and both treatments were equally efficacious. CONCLUSIONS: The LT pathway plays a role in the development of colitis as important as that of the TNF system and, therefore, represents a potential novel intervention point for the treatment of inflammatory bowel disease.     

Testosterone worsens endothelial dysfunction associated with hypercholesterolemia and environmental tobacco smoke exposure in male rabbit aorta

OBJECTIVES: To assess the effects of interaction of sex hormones, hypercholesterolemia (HC) and environmental tobacco smoke (ETS) exposure on endothelium-dependent relaxation, we examined vascular reactivity in vitro in an animal model of atherogenesis. BACKGROUND: Animal and human studies indicate the presence of interactions between classic coronary artery disease risk factors and endothelium-dependent relaxation. Sex hormones have also been shown to influence release of endothelium-derived relaxing factor. METHODS: New Zealand White rabbits were randomized to receive either an HC diet (n = 8) or ETS exposure plus HC diet (n = 8). Eight rabbits receiving a normal diet, without exposure to ETS, served as the control group. The HC diet consisted of 3% soybean oil and 0.3% cholesterol by weight over 13 weeks. The source of ETS was sidestream smoke of 4 cigarettes/15 min, 6 h/day, 5 days/week over 10 weeks in a smoking chamber. Rabbits were killed, and fresh aortic rings were harvested and maintained in oxygenated Krebs solution in an organ bath at 37 degrees C. Rings were precontracted with norepinephrine and exposed to acetylcholine in increasing doses, and isometric tension was recorded. Rings were also exposed to physiologic concentrations (1 nmol/liter) of either 17-beta-estradiol, testosterone or progesterone before pre-contraction with norepinephrine and relaxation with acetylcholine. Endothelium-independent relaxation was studied using nitroglycerin. The surface area of the ring covered by lipids was measured by Sudan IV staining. RESULTS: HC and ETS significantly reduced endothelium-dependent relaxation (p = 0.01 and p < 0.0005, respectively) and caused atherogenesis (p < 0.0005 and p = 0.047, respectively) but did not affect endothelium-independent relaxation. Incubation with estradiol and estradiol plus progesterone did not influence endothelium-dependent relaxation. Testosterone reduced endothelium-dependent relaxation (p = 0.049) and augmented the endothelial dysfunction associated with ETS exposure and HC (p = 0.03). CONCLUSIONS: Both HC and ETS are atherogenic and impair endothelial function but do not affect endothelium-independent relaxation. Physiologic levels of estradiol and estradiol plus progesterone do not affect endothelium-dependent relaxation. Physiologic levels of testosterone impair relaxation and augment the endothelial dysfunction associated with ETS exposure and HC.     

Shoulder joint kinetics during the push phase of wheelchair propulsion

The purpose of this investigation was to quantify the forces and moments at the shoulder joint during free, level wheelchair propulsion and to document changes imposed by increased speed, inclined terrain, and 15 minutes of continuous propulsion. Data were collected using a six-camera VICON motion analysis system, a strain gauge instrumented wheel, and a wheelchair ergometer. Seventeen men with low level paraplegia participated in this study. Shoulder joint forces and moments were calculated using a three-dimensional model applying the inverse dynamics approach. During free propulsion, peak shoulder joint forces were in the posterior (46 N) and superior directions (14 N), producing a peak resultant force of 51 N at an angle of 185 degrees (180 degrees = posterior). Peak shoulder joint moments were greatest in extension (14 Newton-meters [Nm]), followed by abduction (10 Nm), and internal rotation (6 Nm). With fast and inclined propulsion, peak vertical force increased by greater than 360%, and the increase in posterior force and shoulder moments ranged from 107% to 167%. At the end of 15 minutes of continuous free propulsion, there were no significant changes compared with short duration free propulsion. The increased joint loads documented during fast and inclined propulsion could lead to compression of subacromial structures against the overlying acromion.     

Mu and kappa opioid receptors in periaqueductal gray and rostral ventromedial medulla

The periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) are important brain stem pain modulating regions. Recent evidence suggests that kappa opioids antagonize the effects of mu opioids in the RVM. However, the anatomical relationship between mu and kappa opioid receptors in PAG and RVM is not well characterized. This study examined relationships between mu and kappa opioid receptor immunoreactivity (IR) and mRNA in PAG and RVM. Brain slices were processed for either immunocytochemistry or in situ hybridization. We found considerable anatomical overlap of mu and kappa opioid IR and mRNA in the RVM and PAG. These results provide an anatomical basis for recent behavioral and electrophysiological findings in RVM, and suggest modulatory interactions between mu and kappa opioids in PAG.