Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N- acetylgalactosaminyltransferase family

A homologous family of UDP- N -acetylgalactosamine: polypeptide N - acetylgalactosaminyltransferases (GalNAc-transferases) initiate O- glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc- transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.      

Oxidative stress in skin fibroblasts cultures from patients with Parkinson's disease

Background  

In the substantia nigra of Parkinson's disease (PD) patients, increased lipid peroxidation, decreased activities of the mitochondrial complex I of the respiratory chain, catalase and glutathione-peroxidase, and decreased levels of reduced glutathione have been reported. These observations suggest that oxidative stress and mitochondrial dysfunction play a role in the neurodegeneration in PD. We assessed enzymatic activities of respiratory chain and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with PD.     

Effect of dietary supplementation on the frequency of spontaneous lacZ mutations in the developing colon

Epidemiological studies have demonstrated that dietary modifications can reduce the incidence of cancer. Specifically, diets high in vegetables and fruits are associated with lower rates of cancer at many sites. Somatic mutations have a critical role in carcinogenesis suggesting the use of in vivo mutation assays as an alternative approach to studying the relationship between diet and cancer. Since the rate of accumulation of spontaneous mutations is highest during growth and development early in life, we tested whether certain foods as dietary supplements could reduce the rate of mutation during this period using lacZ transgenic mice. Pregnant female mice were placed on a control diet or a diet supplemented to 20% final dry weight with broccoli, cabbage, carrots, flaxseed, green peas, green peppers, oranges or strawberries for the entire duration of their pregnancy and lactation. Mutation frequencies were subsequently measured at the lacZ transgene in colonic epithelial cells of the offspring at 3 weeks of age. A small number of measurements were also made on siblings at 8 weeks of age. While the control AIN-96G diet on its own resulted in lower mutant frequencies than had been observed in earlier experiments with lab chow, no significant reduction in mutant frequencies was detected for any of the foods tested as compared to the AIN-93G diet alone. Significantly more mutations were found at 3 weeks of age in mice fed diets supplemented with broccoli or oranges, but the result with oranges may be the result of jackpot mutations.     

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte

By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.     

Prospects for estimating nucleotide divergence with RAPDs

The technique of random amplification of polymorphic DNA (RAPD), which is simply polymerase chain reaction (PCR) amplification of genomic DNA by a single short oligonucleotide primer, produces complex patterns of anonymous polymorphic DNA fragments. The information provided by these banding patterns has proved to be of great utility for mapping and for verification of identity of bacterial strains. Here we consider whether the degree of similarity of the banding patterns can be used to estimate nucleotide diversity and nucleotide divergence. With haploid data, fragments generated by RAPD-PCR can be treated in a fashion very similar to that for restriction-fragment data. Amplification of diploid samples, on the other hand, requires consideration of the fact that presence of a band is dominant to absence of the band. After describing a method for estimating nucleotide divergence on the basis of diploid samples, we summarize the restrictions and criteria that must be met when RAPD data are used for estimating population genetic parameters.      

Prolongation of cardiac allograft survival by pretreatment of recipient mice with donor blood or spleen cells plus cyclophosphamide.

Using six mouse strain combinations, we attempted to prolong cardiac allograft survival by pretreatment of recipients with a single iv injection of donor-specific whole blood or spleen cells plus a single ip injection of cyclophosphamide (Cy). Significant prolongation of cardiac allograft survival occurred in a small proportion of pretreated mice of some strain combinations, with some grafts surviving for periods longer than 6–9 months. Cy injected alone did not influence the normal cardiac allograft rejection time of between 1 and 2 weeks. Depending upon the strain combination, accelerated rejection of all or some of the grafts occurred in mice pretreated with blood or spleen cells or myocardial cells alone.     

A mouse homologue of the Drosophila tumor suppressor l(2)tid gene defines a novel Ras GTPase-activating protein (RasGAP)-binding protein

p120 GTPase-activating protein (GAP) down-regulates Ras by stimulating GTP hydrolysis of active Ras. In addition to its association with Ras, GAP has been shown to bind to several tyrosine-phosphorylated proteins in cells stimulated by growth factors or expressing transforming tyrosine kinase variants. Here we report the cloning and characterization of a novel GAP-binding protein, mTid-1, a DnaJ chaperone protein that represents the murine homolog of the Drosophila tumor suppressor l(2)tid gene. Three alternatively spliced variants of mTid-1 were isolated, two of which correspond to the recently identified hTid-1(L) and hTid-1(S) forms of the human TID1 gene that exhibit opposing effects on apoptosis. We demonstrate that both cytoplasmic precursor and mitochondrial mature forms of mTid-1 associate with GAP in vivo. Interestingly, although mTid-1 is found tyrosine-phosphorylated in v-src-transformed fibroblast cells, GAP selectively binds to the unphosphorylated form of mTid-1. In immunofluorescence experiments, GAP and Tid-1 were shown to colocalize at perinuclear mitochondrial membranes in response to epidermal growth factor stimulation. These findings raise the possibility that Tid chaperone proteins may play a role in governing the conformation, activity, and/or subcellular distribution of GAP, thereby influencing its biochemical and biological activity within cells.