The Drosophila Na,K-ATPase alpha-subunit gene: gene structure, promoter function and analysis of a cold-sensitive recessive-lethal mutation

The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelines for sorting of unfixed cells, including known biohazardous samples, using jet-in-air, deflected-droplet cell sorters. There is a risk that personnel operating these instruments could become exposed to droplets and aerosols containing biological agents present in the samples. The following guidelines can aid in the prevention of exposures of laboratory personnel to pathogens contained in the sort samples. The document provides biosafety recommendations for sample handling, operator training and protection, laboratory facility design, and instrument setup and maintenance. In addition, it describes in detail methods for assessment of instrument aerosol containment. Recommendations provided here may also help laboratories to obtain institutional and/or regulatory agency approval for sorting of unfixed and known biohazardous samples.     

Mapping, genomic organization and promoter analysis of the human prostate-specific membrane antigen gene

Prostate-specific membrane antigen (PSMA) is a 100 kDa type II transmembrane protein with folate hydrolase and NAALAdase activity. PSMA is highly expressed in prostate cancer and the vasculature of most solid tumors, and is currently the target of a number of diagnostic and therapeutic strategies. PSMA is also expressed in the brain, and is involved in conversion of the major neurotransmitter NAAG (N-acetyl-aspartyl glutamate) to NAA and free glutamate, the levels of which are disrupted in several neurological disorders including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease and schizophrenia. To facilitate analysis of the role of PSMA in carcinoma we have determined the structural organization of the gene. The gene consists of 19 exons spanning approximately 60 kb of genomic DNA. A 1244 nt portion of the 5' region of the PSMA gene was able to drive the firefly luciferase reporter gene in prostate but not breast-derived cell lines. We have mapped the gene encoding PSMA to 11p11-p12, however a gene homologous, but not identical, to PSMA exists on chromosome 11q14. Analysis of sequence differences between non-coding regions of the two genes suggests duplication and divergence occurred 22 million years ago.     

A new member of the MER2 repeat family is detected in the promoter region of the human X11 gene

AIMS: To evaluate the clinical utility of two new tests for serum trypsinogen 2 and trypsin 2-alpha 1 antitrypsin complex (trypsin 2-AAT) in diagnosing and assessing the severity of acute pancreatitis (AP) induced by endoscopic retrograde cholangiopancreatography (ERCP). PATIENTS: Three hundred and eight consecutive patients undergoing ERCP at Helsinki University Central Hospital in 1994 and 1995. METHODS: Patients were followed prospectively for pancreatitis and clinical outcome. They were tested for serum trypsinogen 2, trypsin 2-AAT, and amylase in samples obtained before and one, six, and 24 hours after ERCP. RESULTS: Pancreatitis developed in 31 patients (10%). Their median serum trypsinogen 2 increased 26-fold to 1401 micrograms/l at six hours after the procedure and trypsin 2-AAT showed an 11-fold increase to 88 micrograms/l at 24 hours. The increase in both markers was stronger in severe than in mild pancreatitis, and in patients without pancreatitis there was no significant increase. Baseline trypsinogen 2 and trypsin 2-AAT concentrations were elevated in 29% and 32% of patients, respectively. The diagnostic accuracy of a threefold elevation over the baseline value was therefore analysed. The sensitivity and specificity of these parameters in the diagnosis of post-ERCP pancreatitis was 93% and 91%, respectively, for serum trypsinogen 2 at six hours after the examination, and 93% and 90%, for trypsin 2-AAT at 24 hours. CONCLUSIONS: Serum trypsinogen 2 and trypsin 2-AAT reflect pancreatic injury after ERCP. High concentrations are associated with severe pancreatic damage. The delayed increase in trypsin 2-AAT compared with trypsinogen 2 appears to reflect the pathophysiology of AP. A greater than threefold increase in trypsinogen 2 six hours after ERCP is an accurate indicator of pancreatitis.     

The human photoreceptor rim protein gene (ABCR): genomic structure and primer set information for mutation analysis

In order to evaluate the effect of thyroid replacement therapy on bone metabolism in congenital hypothyroid children, we studied 23 (10 girls and 13 boys) consecutive patients. Their age ranged from 3 to 8 weeks. One of these patients had familiar dyshormonogenesis, 21 had ectopic glands and one hemiagenesis. As a control group, we studied 46 sex- and age-matched healthy newborns. Before the beginning of therapy, the hypothyroid patients showed higher values of calcium (2.78 +/- 0.04 vs 2.65 +/- 0.07 mmol/l; p < 0.05) and of 1,25-dihydroxy-vitamin D (159.7 +/- 31.6 vs 90.5 +/- 33.1 ng/l; p < 0.01), while they showed lower values of osteocalcin (1.9 +/- 0.8 vs 2.9 +/- 0.9 ng/ml; p < 0.01) than controls. After 3 months of therapy, we found a complete normalization of all these parameters and a progressive increase of osteocalcin. Our data show that in congenital hypothyroid children there are abnormalities in calcium metabolism which seem to be transient and reversible after L-thyroxine replacement therapy.     

Nucleotide sequence of 5''-upstream region and expression of a silkworm gene encoding a new member of the attacin family

The morphometric and morphological changes in the mesothelial cell population were studied in rabbits in peritoneal dialysis with lactate and bicarbonate buffer solution. During dialysis the mesothelial population underwent radical changes in morphology and morphometric analysis showed a significant increase in cell size. Light microscope examination showed two types of changes: hyperplasia of the mesothelial cell with diameters of up to 80 microns, nucleus proportional to the cytoplasm, a large nucleole giving an owl's eye appearance and cytoplasm rich in granular material. The second change was multiple nuclei and arrest of cell division. Nuclear division occurred, but no separation of the cytoplasm. The cells became larger than 200 microns, packed with nuclei and relatively little cytoplasm. Electron microscopy confirmed that the hyperplastic cells had perfect structure whereas the polynucleate cells contained vacuoles and little cytoplasmic reticulum. Immunohistochemistry using monoclonal antibodies SK2-27 and SK 60-61 specific to cytokeratins 14, 16, 17 and 8, 18, respectively, identified the cells as mesothelial. The changes were related to the glucose content of the peritoneal dialysis solution. Glucose is therefore the bioincompatible agent that modifies the mesothelium during peritoneal dialysis, causing it to become hyperplastic or blocking replication.     

Novel mutations in the promoter and coding region of the human 5-HT1A receptor gene and association analysis in schizophrenia

Dysfunction of serotonin systems has been implicated in schizophrenia. In the present study, the human 5-HT1A receptor gene containing the 5' untranslated region was screened in order to detect genetic variations, through which alteration of protein function or level of expression might contribute to schizophrenia. Genomic DNAs were isolated from whole-blood samples of 61 unrelated schizophrenic patients and 100 healthy controls. Genetic variations were screened systematically by single-strand conformational polymorphism (SSCP) analysis, followed by direct sequencing of polymerase chain reaction (PCR) product as well as restriction fragment-length polymorphism (RFLP). The novel mutations (-51T --> C, -152C --> G, -321G --> C, -480delA, and -581C --> A) were found in the 5' untranslated region. Furthermore, we found a novel missense mutation (Gly272Asp) in the coding region in addition to the mutations (Pro16Leu, 294G --> A, and 549C --> T) reported previously. No significant differences in genotype frequencies as well as allele frequencies were found between patients and controls. Our data provided no evidence of association between schizophrenia and the variants in the 5' untranslated region as well as the coding region of the human 5-HT1A receptor gene.     

Identification of mouse CPX-2, a novel member of the metallocarboxypeptidase gene family: cDNA cloning, mRNA distribution, and protein expression and characterization

A novel member of the metallocarboxypeptidase gene family was identified from its homology with carboxypeptidase E and has been designated CPX-2. The cDNA of 2500 nucleotides encodes a protein of 764 amino acids that contains an N-terminal signal peptide-like sequence, a 158-residue discoidin domain, and a 400-residue carboxypeptidase domain. The 400-residue metallocarboxypeptidase domain has 59% amino acid identity with a protein designated AEBP-1; 44% to 46% identity with carboxypeptidases E, N, and Z; and lower homology with other members of the metallocarboxypeptidase gene family. The discoidin domain of CPX-2 has 22% amino acid identity with the carbohydrate-binding domain of discoideum-I, 29% to 34% identity with the phospholipid-binding domain of human factors V and VIII, and 59% identity with the discoidin-like domain on AEBP-1. CPX-2 is missing several of the predicted active-site residues that are conserved in most other members of the metallocarboxypeptidase gene family and which are thought to be required for enzyme activity. Expression of CPX-2 using the baculovirus system produced several forms of protein, from 80 to 105 kDa, but no detectable activity toward a variety of carboxypeptidase substrates. A shorter 50-kDa form of CPX-2, which contains the carboxypeptidase domain but not the discoidin domain, was also inactive when expressed in the baculovirus system. CPX-2 is able to bind to Sepharose-Arg; this binding is blocked by 10 mM Arg. Northern blot analysis showed CPX-2 mRNA in mouse brain, liver, kidney, and lung. In situ hybridization analysis of brain revealed a broad distribution. Areas that are enriched in CPX-2 include the hippocampus, cerebral cortex, median eminence, and choroid plexus. Taken together, these data suggest a widespread function for CPX-2, possibly as a binding protein rather than an active carboxypeptidase.     

Hexokinase II gene: structure, regulation and promoter organization

In order to optimize a method for quantitative assessment of bradykinesia, we evaluated the three-dimensional sources of a movement signal of the wrist and influence of tremor on the reliability of bradykinesia measurements. A total of 33 patients with Parkinson's disease, three patients with Multiple System Atrophy and 29 healthy controls performed a test procedure to measure slowness of movement, consisting of a tap rate (TR) test and a movement time (MT) test. Simultaneously, accelerometers were mounted on the wrist and mean bi- and tri-axial vectors were calculated. Thus the acquired means of acceleration were correlated with the commonly used measures of bradykinesia. i.e. tap rate and movement time. Our results show that bradykinesia is reliably measured by the evaluation of the mean acceleration of movements, and support the use of any of the three bi-axial vectors. Compared to the bi-axial vectors, the tri-axial vector provided no relevant additional information. Additionally, the presence of a moderate to severe resting tremor did not influence the assessment of bradykinesia. Because of the possibility of continuous assessment of bradykinesia this new monitor may prove to be of great value in pharmacodynamic studies and the longitudinal follow-up of patients in drug trials.     

Rapid isolation of genomic clones for individual members of human multigene families: identification and localisation of UBE2L4, a novel member of a ubiquitin conjugating enzyme dispersed gene family

We describe a rapid, PCR-based, screening procedure for the isolation of human genomic clones in lambda bacteriophage, containing sequences coding for individual homologous members of a multigene family. The approach is based upon the identification, by dilution, of sub-pools of the genomic library that contain members of the gene family, prior to phage isolation. The presence of specific genes is established by PCR of aliquots of individually amplified library pools, using consensus primers and subsequent sequencing. We have used the approach to isolate a fourth member of the UBE2L gene family, UBE2L4, and located it on chromosome 19q13.1-->q13.2. This PCR-based approach to library screening has wider applicability in that it could be used to isolate alternate-spliced products from cDNA libraries.     

The human LARGE gene from 22q12.3-q13.1 is a new, distinct member of the glycosyltransferase gene family

Meningioma, a tumor of the meninges covering the central nervous system, shows frequent loss of material from human chromosome 22. Homozygous and heterozygous deletions in meningiomas defined a candidate region of >1 Mbp in 22q12.3-q13.1 and directed us to gene cloning in this segment. We characterized a new member of the N-acetylglucosaminyltransferase gene family, the LARGE gene. It occupies >664 kilobases and is one of the largest human genes. The predicted 756-aa N-acetylglucosaminyltransferase encoded by LARGE displays features that are absent in other glycosyltransferases. The human like-acetylglucosaminyltransferase polypeptide is much longer and contains putative coiled-coil domains. We characterized the mouse LARGE ortholog, which encodes a protein 97.75% identical with the human counterpart. Both genes reveal ubiquitous expression as assessed by Northern blot analysis and in situ histochemistry. Chromosomal mapping of the mouse gene reveals that mouse chromosome 8C1 corresponds to human 22q12.3-q13.1. Abnormal glycosylation of proteins and glycosphingolipids has been shown as a mechanism behind an increased potential of tumor formation and/or progression. Human tumors overexpress ganglioside GD3 (NeuAcalpha2,8NeuAcalpha2, 3Galbeta1,4Glc-Cer), which in meningiomas correlates with deletions on chromosome 22. It is the first time that a glycosyltransferase gene is involved in tumor-specific genomic rearrangements. An abnormal function of the human like-acetylglucosaminyltransferase protein may be linked to the development/progression of meningioma by altering the composition of gangliosides and/or by effect(s) on other glycosylated molecules in tumor cells.