Culture medium enhances semicarbazide-sensitive amine oxidase activity

Components of fetal calf serum (FCS) are known to contribute to growth and maintenance of cultured cells. Fetal calf serum supplementation of media also may contribute to the cytotoxicity of other substances to cells grown in vitro. Semicarbazide-sensitive amine oxidase (SSAO) enzyme, present in FCS, metabolizes primary amines and contributes to amine cytotoxicity in vascular smooth muscle cells (VSMC). In cell culture experiments, the media used may greatly affect enzymic activities such as SSAO. In these studies, the SSAO activity in FCS, cultured rat aortic VSMC, and rat plasma was determined in the presence and absence of various culture media. Semicarbazide-sensitive amine oxidase activity in FCS (5-20 microl) was significantly enhanced (approximately 1.5- to 2-fold) in the presence of various culture media, with Dulbecco modified Eagle medium (DMEM), causing the greatest enhancement. Dulbecco modified Eagle medium enhanced the SSAO activity of cultured VSMC in two of the four passages but reduced activity in two passages. Activity in rat plasma was reduced by approximately 25% in the presence of DMEM. The concentrations of various media components, such as glucose, sodium pyruvate, pyridoxine.HCl, and L-glutamine, were not correlated with enhancement. This study identifies an important enhancement effect of culture media on the FCS enzyme, SSAO, although the media components responsible for the enhancement are yet to be identified.     

Tiny telomere DNA

We describe the design, synthesis and biophysical characterization of a novel DNA construct in which a folded quadruplex structure is joined to a standard double helix. Circular dichroism, gel electrophoresis, three-dimensional UV melting and differential scanning calorimetry were all used to characterize the structure. Rigorous molecular dynamics simulations were used to build a plausible atomic-level structural model of the DNA construct. This novel DNA construct provides a model for the duplex–quadruplex junction region at the end of chromosomal DNA and offers a system for the study of structure-selective ligand binding.     

The composition,structure and stability of a group II chaperonin are temperature regulated in a hyperthermophilic archaeon

The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86 degrees C) and decreased by cold shock (30 min, 60 degrees C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75-79 degrees C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo-oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero-oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero-oligomeric with an average subunit ratio of 1alpha:1beta:0.1gamma in cultures grown at 75 degrees C, a ratio of 1alpha:3beta:1gamma in cultures grown at 60 degrees C and a ratio of 2alpha:3beta:0gamma after 86 degrees C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7 degrees C, 96.7 degrees C and 80.5 degrees C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between species.     

Linkage of prostate cancer susceptibility loci to chromosome 1

Three prostate cancer susceptibility genes have been reported to be linked to different regions on chromosome 1: HPC1 at 1q24-25, PCAP at 1q42-43, and CAPB at 1p36. Replication studies analyzing each of these regions have yielded inconsistent results. To evaluate linkage across this chromosome systematically, we performed multipoint linkage analyses with 50 microsatellite markers spanning chromosome 1 in 159 hereditary prostate cancer families (HPC), including 79 families analyzed in the original report describing HPC1 linkage. The highest lod scores for the complete dataset of 159 families were observed at 1q24-25 at which the parametric lod score assuming heterogeneity (hlod) was 2.54 (P=0.0006) with an allele sharing lod of 2.34 (P=0.001) at marker D1S413, although only weak evidence was observed in the 80 families not previously analyzed for this region (hlod=0.44, P=0.14, and allele sharing lod=0.67, P=0.08). In the complete data set, the evidence for linkage across this region was very broad, with allele sharing lod scores greater than 0.5 extending approximately 100 cM from 1p13 to 1q32, possibly indicating the presence of multiple susceptibility genes. Elsewhere on chromosome 1, some evidence of linkage was observed at 1q42-43, with a peak allele sharing lod of 0.56 (P=0.11) and hlod of 0.24 (P=0.25) at D1S235. For analysis of the CAPB locus at 1p36, we focused on six HPC families in our collection with a history of primary brain cancer; four of these families had positive linkage results at 1p36, with a peak allele sharing lod of 0.61 (P=0.09) and hlod of 0.39 (P=0.16) at D1S407 in all six families. These results are consistent with the heterogeneous nature of hereditary prostate cancer, and the existence of multiple loci on chromosome 1 for this disease.     

Identification of ENV determinants in V3 that influence the molecular anatomy of CCR5 utilization

The V3 loop of the ENV glycoprotein exerts a dominant influence on the interaction of gp120 with coreceptors. Primary env genes cloned from sequential isolates from two seroconverters revealed Pro-->Ala conversion in the conserved GPG motif of the V3 crown in seven of 17 R5 ENV. ENV containing the GPG motif in the V3 crown had fusogenic activity with chimeric receptors containing either the N terminus or loops of CCR5, whereas those with the GAG variant utilized only the former. Site-directed mutagenesis of multiple primary and prototypic R5 env genes demonstrated that the GPG motif was necessary for dual utilization of the N terminus and body of CCR5 in both gain and loss-of-function experiments. All ENV containing the GPG V3 crown showed CCR5 binding in the presence of soluble CD4, whereas it was not detected with the GAG variants. Molecular dynamic simulations of a V3 peptide predicts that the Pro-->Ala substitution results in a conformational change with loss of the crown structure. These studies demonstrate that sequences in the third hypervariable region determine the specificity of coreceptor utilization for fusion, and that a conserved motif in the crown directly influences the molecular anatomy of the interaction between gp120 and CCR5.     

Analysis of HPC1, HPCX, and PCaP in Icelandic hereditary prostate cancer

Putative prostate cancer susceptibility loci have recently been identified by genetic linkage analysis on chromosomes 1q24-25 (HPC1). 1q44.243 (PCaP), and Xq27-28 (HPCX). In order to estimate the genetic linkage in Icelandic prostate cancer families, we genotyped 241 samples from 87 families with eleven markers in the HPC1 region, six markers at PCaP, and eight at HPCX. Concurrently, we assessed allelic imbalance at the HPC1 and PCaP loci in selected tumors from the patients. For each of the candidate regions, the combined parametric and non-parametric LOD scores were strongly negative. Evidence for linkage allowing for genetic heterogeneity was also insignificant for all the regions. The results were negative irrespective of whether calculations were performed for the whole material or for a selected set of early age at onset families. The prevalence of allelic imbalance was relatively low in both the HPC1 (0%-9%) and PCaP (5%-20%) regions and was not elevated in tumors from positively linked families. Our studies indicate that the putative cancer susceptibility genes at chromosomes 1q24-25, 1q44.2-43, and Xq27-28 are unlikely to contribute significantly to hereditary prostate cancer in Iceland and that selective loss of the HPC1 and PCaP loci is a relatively rare somatic event in prostate cancers.     

Characterization of the active site of group B streptococcal hyaluronan lyase

Xin Li was inadvertently dropped from the list of authors for this article. The authors regret this error.     

Cloning and tissue expression of the mouse ortholog of AIM1, a βγ-crystallin superfamily member

We report the isolation of the murine ortholog of AIM1, a human gene whose expression is associated with the reversal of tumorigenicity in an experimental model of melanoma. Mouse and human AIM1 are more than 90% identical in amino acid sequence in the βγ-crystallin repeats and the C-terminal domain, and more than 75% identical in the extended N-terminal domain. Consistent with the isolated cDNA representing the authentic AIM1 ortholog, linkage analysis localized mouse Aim1 to proximal mouse Chromosome (Chr) 10 in a conserved linkage group with genes localized to human Chr band 6q21. Searches of EST databases identified a second AIM1-like gene in both mouse and human, suggesting the existence of a gene family. Northern analysis demonstrates Aim1 is expressed most abundantly in adult skin, lung, heart, liver, and kidney and is temporally regulated during embryogenesis. Aim1 is expressed highly in the shaft region of the hair follicles and the presumptive ectoderm, but not at detectable levels in melanocytes or melanocyte precursor cells. Received: 18 February 1998 / Accepted: 8 May 1998