The correlation between microscopical examination and erythrocyte band 3 (AE1) gene deletion in South-east Asian ovalocytosis

Seventy-five patients with brain metastases from solid tumours were treated with whole-brain irradiation at our institution between 1990 and 1993. The primary cancers included 35 cases of lung cancer, 19 cases of breast cancer, nine cases of renal-cell cancer, six cases of melanoma and six cases of other primary sites. In each case the total dose to the whole brain was at least 25 gray (Gy). The primary site, age, performance status, number of brain metastases and the presence of extracranial disease were studied as prognostic factors for survival. The median survival for the whole population was 4 months (range 1-62 months). The patients with the brain as the only metastatic site had significantly better survival (P = 0.019) than those with both intracranial and extracranial metastatic sites. Poor performance status at the time of diagnosis of brain metastases was also related to short survival (P = 0.001). None of the other studied variables had prognostic significance. Four of the 75 patients with primary tumour sites in the breast (two patients) and the kidney (two patients) survived for more than 2 years. In general, patients with breast cancer had better survival than patients with other primary cancers. Our study confirms the generally poor prognosis of cancer with brain metastases, although individual patients may survive several years after whole-brain irradiation. Approximately two-thirds of the patients experienced a relief in symptoms allowing a reduction in the dose of corticosteroid medication, which clearly supports the use of whole-brain radiotherapy as a palliative treatment.     

Novel mutations identified in exon 7 of phenylalanine hydroxylase gene in Chinese

Exon 7 of the phenylalanine hydroxylase (PAH) gene was analyzed in 45 children affected with classic phenylketonuria (PKU) from northern China by using PCR-single strand conformation polymorphism (PCR-SSCP) technique and DNA direct sequencing. Six missense mutations (i.e. R243Q, R241H, G247V, L249H, P254I and G257V) and one silent mutation (V245V) were identified. The latter three missense mutations were demonstrated as novel mutations in comparison with the PAH mutation Database. One missense mutation (R241H) was first documented in Chinese. Our results showed population and regional differences in the PAH mutation distribution and suggest that there is more than one founding population for PKU in China. The finding of novel mutations will enhance our capability in molecular diagnosis of PKU.     

Structural studies on the effects of the deletion in the red cell anion exchanger (band 3, AE1) associated with South East Asian ovalocytosis

We have carried out a solution-state NMR study of synthetic peptides patterned on the first membrane span of normal human band 3, and the same region of the mutant band 3 present in Southeast Asian ovalocytosis (SAO) which has a nine amino acid deletion. In 1:1 (v/v) chloroform/methanol, the 42 residue normal peptide (R389-K430) consisted of three helical regions. The slow solvent exchange of backbone amide protons revealed the helix from P403 to A416 was more stable than the "cytoplasmic" N-terminal helix from P391 to A400. These helices were separated by a sharp bend at P403, which is probably located at the boundary between the cytoplasmic domain and the first transmembrane span. The SAO deletion (A400-A408) removed the bend at P403, to leave a stable helix from P391 to A416 containing the residuum of the normal first transmembrane helix and with a hydrophobic turn replaced by a polar turn in the SAO peptide. Insertion of fragments of normal band 3 and band 3 SAO into microsomal membranes was investigated using a cell free translation system. A fragment composed of the cytoplasmic domain and the putative first membrane domain of normal band 3 (B3(1)) inserted stably into the membrane. However, the corresponding fragment of band 3 SAO [SAO(1)] did not integrate stably into membranes. Our results suggest that in SAO band 3, the region of the first membrane span of normal band 3 does not integrate properly into the membrane because it lacks a sufficiently long hydrophobic segment, and the deletion also disrupts a conserved structural subdomain at the membrane surface.     

Secondary structures of synthetic peptides corresponding to the first membrane-contact portion of normal band 3 and its deletion mutant (Southeast Asian ovalocytosis)

The heat shock response (HSR) was characterized in the gills of two lamprey species that differ with respect to their adult life history. In vivo labelling with [35S]methionine revealed an enhanced synthesis of heat shock proteins (HSPs) having approximate molecular weights of 70 kDa (HSP70) and 90 kDa (HSP90) following heat treatment. Induction of the HSR occurred in larval lampreys (ammocoetes) following temperature elevations of 13-16 degrees C for the parasitic species, the sea lamprey (Petromyzon marinus) and 16-20 degrees C for the nonparasitic species, the brook lamprey (Lampetra appendix). The case in L. appendix represents the greatest increase in temperature required to induce the HSR in gill tissue among aquatic poikilotherms studied to data and induction occurs within a temperature range (25-29 degrees C) not normally experienced by these animals. Western blotting detected the presence of 70 and 90 kDa HSPs and HSP70 levels were greater in post-metamorphic L. appendix than in ammocoetes both before and after heat shock. The HSR of lampreys appears to be induced during times of emergency when large, rapid temperature increases are experienced. The high set-point temperature for induction of the response may be a consequence of both the environments they presently inhabit and their experiences during evolution.     

Regional localization of human M-BCR gene to chromosome 23 band q11 in the great apes

Cassettes based on a hisG-URA3-hisG insert have been modified by the addition of an KmR-encoding gene and flanking polylinker sites, greatly simplifying construction of gene disruption vectors in Escherichia coli. After gene disruption in yeast, URA3 can then be excised by recombination between the hisG repeats flanking the gene, permitting reuse of the URA3 marker.     

Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3

We screened 75 primary hepatocellular carcinomas for somatic mutations in the entire coding region of the beta-catenin gene. We detected somatic mutations in 14 tumors; 12 were considered to cause amino acid substitutions and 2 were interstitial deletions of 51 or 195 nucleotides of genomic DNA, corresponding to exon 3. Among the 12 point mutations, 6 occurred at potential serine/threonine phosphorylation residues of codons 33, 41, or 45. The remaining six tumors contained a mutation at codon 32 (aspartic acid) or 34 (glycine), flanking to the serine residue at codon 33. By Western blot analysis, we confirmed accumulation of beta-catenin in five tumors for which frozen tissues were available; the five included tumors in which amino acid alterations had occurred at codons 32, 34, or 45, and one with a 17-amino acid deletion. Our results suggested that accumulation of beta-catenin due to amino acid substitutions at potential serine/threonine phosphorylation residues or at their neighboring codons or interstitial deletions involving exon 3 could contribute to hepatocellular carcinogenesis.     

Psilocybin and psilocin levels in twenty species from seven genera of wild mushrooms in the Pacific Northwest, U.S.A

The analysis of twenty species from seven genera of Pacific Northwest mushrooms revealed psilocybin (and in some cases psilocin as well) in seven species from three genera. The species found to contain psilocybin (and psilocin) varied from one collection to another by more than a factor of seven in amount present. Total psilocybin and psilocin levels in species known to be in use for recreational and entheogenic purposes varied from 0.1% by dry weight up to a high of nearly 2% by dry weight.     

Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae.     

Cloning and sequencing of nm23-H3b, a new gene identified to nm23

Nm23 is a kind of an effective tumor metastasis suppressor gene which included two types in human: nm23-H1 and nm23-H2. Amino acid identity between nm23-H1 and nm23-H2 was 88%. In this study, we used a pair of primers to flank the part of coding sequence of nm23. The 5'-translated sequence was amplified by PCR from human normal liver genomic DNA. A 375bp clone was characterized to designate pnm 23-H3b. The nm23-H3b nucleotide sequence between 40bp and 70bp was different from nm23-H1 and nm23-H2, and other sequences had 86% and 90% identical to nm23-H1 and nm23-H2, respectively. Southern blot containing Bg1II-digested human liver genomic DNA hybridized to the entire nm23-H3b DNA and showed three bands at 10.5, 7.9 and 4.0 kb. These data demonstrate that nm23-H3b is a new type of gene, which has high homology with human nm23-H1 and nm23-H2. Nm23 is possibly considered a family of closely related genes.     

Linkage disequilibrium mapping of the gene for Margarita Island ectodermal dysplasia (ED4) to 11q23

Margarita Island ectodermal dysplasia (ED4) is an autosomal recessive disorder characterized by unusual facies, dental anomalies, hypotrichosis, palmoplantar hyperkeratosis and onychodysplasia, syndactyly, and cleft lip/cleft palate. We have used an affected-only DNA-pooling strategy to carry out linkage disequilibrium mapping of the ED4 gene to 11q23. Haplotype analysis of four complex Margarita Island ED4 families localized the ED4 gene to an approximately 1-2-Mb interval spanned by just two YACs.     

Characterization of mutations in the myotubularin gene in twenty six patients with X-linked myotubular myopathy

A candidate gene, myotubularin, involved in the pathogenesis of X-linked myotubular myopathy (MTM1) was isolated recently. Mutations originally were identified in 12% of patients examined for 40% of the coding sequence, raising the possibility that additional genes could be responsible for a proportion of X-linked cases. We report here the identification of mutations in 26 of 41 independent male patients with muscle biopsy-proven MTM, by direct genomic sequencing of 92% of the known coding sequence of the myotubularin gene. Eighteen patients had point mutations, including one A/G transition found in four patients which alters a splice acceptor site in exon 12 and leads to a three amino acid insertion. Six patients had small deletions involving <6 bp, while two larger deletions encompassed two or six exons, respectively. No differences were noted among the types of mutations between familial and sporadic cases. However, all of the five patients with a mild phenotype had missense mutations. While 50% of the mutations were found in exons 4 and 12, and three distinct mutations were found in more than one patient, no single mutation accounted for more than 10% of the cases. The low frequency of large deletions and the varied mutations identified suggest that direct mutation screening for molecular diagnosis may require gene sequencing.     

Confirmation of an association between a polymorphism in exon 3 of the low-density lipoprotein receptor-related protein gene and Alzheimer's disease

C766T, a polymorphism in exon 3 of the gene for the low-density lipoprotein receptor-related protein (LRP), was found to be associated with late-onset Alzheimer's disease (AD). We developed a PCR-restriction enzyme-based assay to analyze this allele in 234 AD patients and 103 controls. We confirmed that the LRP C766T polymorphism was in disequilibrium with AD--the C/C genotype was present in 76% of AD patients and 60% of controls (p < 0.01); however, the LRP polymorphism did not influence age at onset of AD.     

A polymorphism in exon b2 of the major breakpoint cluster region (M-bcr) identified in chronic myeloid leukaemia patients

The BCR/ABL junctional region and the b3 exon from chronic myeloid leukaemia (CML) patients were sequenced. In all 21 samples analysed the junctional region, as well as the b3 exon of 8 b3a2 mRNA molecules, presented no differences to the already described sequences. However, we identified a polymorphic base in the b2 exon in two out of seven b3a2 samples, four out of 10 b2a2 samples and all four b3a2/b2a2 samples analysed. In the eighth position before the junctional region of BCR/ABL cDNA, a cytosine replaces thymine in these cases. The polymorphism described here could be a useful marker for the differentiation of normal and rearranged BCR alleles in heterozygotes.