The purification and properties of NADPH-dependent carbonyl reductases from rat ovary

Two carbonyl reductases have been highly purified from rat ovary to apparent homogeneity. Though they have similarities in terms of molecular weight (33,000), substrate specificities, inhibitor sensitivities, amino acid composition, and immunological properties, they differed in pI values (6.0 and 5.9). Both enzymes reduced aromatic aldehydes, ketones, and quinones at higher rates, compared to prostaglandins and 3-ketosteroids, whereas they showed higher affinity for prostaglandins and 3-ketosteroids. The enzymes also catalyzed oxidation of the 9-hydroxy group of prostaglandin F2 alpha. Moreover, they showed the remarkable characteristic of catalyzing the reduction of not only the 9-keto group of prostaglandin E2 but also the 15-keto group of 13,14-dihydro-15-keto-prostaglandin F2 alpha. Both enzymes were inhibited by SH-reagents, quercitrin, indomethacin, furosemide, and disulfiram. The results of immunoinhibition, using antibody against the purified enzymes, indicated that the enzymes were solely responsible for the overall catalytic activities of prostaglandin E series reduction, as well as 13,14-dihydro-15-keto-prostaglandin F2 alpha reduction and prostaglandin F2 alpha oxidation in rat ovarian cytosol. Western-blot analysis revealed that immunoreactive proteins were present in adrenal gland and various reproductive tissues except uterus of rats.     

Cytologic appearance of carcinosarcoma (malignant ameloblastoma and fibrosarcoma) of the maxilla. A case report.

The cytologic appearance of a carcinosarcoma (malignant ameloblastoma and fibrosarcoma) of the maxilla in a 63-year-old man is described. On his first admission the diagnosis of malignant ameloblastoma was made on biopsy. After five surgical excisions, radiotherapy and chemotherapy, the diagnosis was changed to carcinosarcoma because the stromal cells of the tumor had become malignant. Aspiration biopsy cytology of the tumor, with a cystic lesion found at the left suborbital area, revealed malignant epithelial cells, indicating malignant ameloblastoma. Imprint smears of both surgically resected and autopsy material showed two types of malignant neoplastic cells, of epithelial and mesenchymal origin.     

Hormonal stimulation of tyrosinase activity in human foreskin organ cultures

Summary A human foreskin organ culture system has been developed to study the response of human skin to hormonal stimulation. Foreskins are maintained in culture on floating plastic supports which allows the epidermal surface to be exposed to air while the dermis is bathed in nutrient medium. Both black and white human foreskins can be maintained in organ culture for at least 1 wk with no change in the tissue structure or cell viability as determined by histochemical staining and by dopa reaction staining. Tyrosinase activity in both black and white human foreskin cultures decays markedly during the first 2 d of culture to a new steady state level which remains stable throughout the culture period. Both black and white foreskin cultures consistently demonstrate 2- to 10-fold increases in tyrosinase activity when treated with theophylline (1 mM). Approximately 90% of all skin cultures examined showed an increase in enzyme activity when treated with this phosphodiesterase inhibitor. Dibutyryl cAMP (0.1 mM) and [Nle⁴, D-phe⁷]-alpha MSH (10⁻⁸ M), were also found to markedly stimulate tyrosinase activity in some skin cultures, whereas alpha-MSH and prostaglandin E₁ produced only an inconsistent and small increase in the activity of the enzyme. Histamine (1 μM), vitamin D₃ (1 μM), and retinoic acid (1μM) failed to stimulate tyrosinase activity in either white or black foreskin cultures. This hormone-responsive organ culture system can be utilized to characterize the molecular processes responsible for the regulation of tyrosinase and pigmentation in human skin. This work was supported by a research contract from the Oklahoma Center for the Advancement of Science and Technology (OCAST) and by a research grant from the Presbyterian Health Foundation.     

Effects of 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on carnation flower longevity

The effects of a novel preservative for cut carnation flowers, 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS), were investigated. DPSS extended the vase life of cut carnation flowers not only by continuous treatment but pulse treatment as well. This inhibition of senescence by DPSS appeared to depend on that of ethylene production in carnation flowers. DPSS provided no protection from the action of ethylene nor did it inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. It did inhibit ACC-dependent ethylene production in carnation petal discs, suggesting possible potential for inhibiting ACC oxidase.     

BTKbase, mutation database for X-linked agammaglobulinemia (XLA).

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 225 entries from 189 unrelated families showing 148 unique molecular events. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites forming arginine residues. A decreased frequency of missense mutations was found in the TH, SH3 and upper lobe of the kinase domain. The putative structural implications of all the missense mutations are given in the database.     

Molecular basis for subtypic differences of the “a” subunit of coagulation factor XIII with description of the genesis of the subtypes

The “a” subunit of human coagulation factor XIII (F13A) exhibits genetic polymorphism defined by four common alleles, F13A*1A, *1B, *2A, and *2B. We have previously suggested on the basis of the isoelectric focusing patterns of the four allele products that point mutations at two separate sites and one intragenic crossing over might be involved in the genes of F13A polymorphism. Here, we report nucleotide substitutions associated with F13A polymorphism. A C/T transition of the second nucelotide of codon 564 in exon 12 is responsible for the difference between F13A*1A and *1B and that between F13A*2A and *2B, and a set of two base changes in codons 650 and 651 in exon 14 leads to the differences between F13A*1A and *2A and those between F13A*1B and *2B. The four combinations of the point mutations at the two exons thus correspond to the four alleles, two of which were generated by the point mutations from ancestral monomorphic gene. The results suggest strongly that intragenic crossing over must be involved in the genesis of the fourth allele. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods discriminating these base changes in exons 12 and 14 are also presented.     

The SV2 Protein of Synaptic Vesicles Is a Keratan Sulfate Proteoglycan

Abstract: We have determined that synaptic vesicles contain a vesicle-specific keratan sulfate integral membrane proteoglycan. This is a major proteoglycan in electric organ synaptic vesicles. It exists in two forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., the L form, which migrates like a protein with an M_r of 100, 000, and the H form, with a lower mobility that migrates with an M_r of ∼250, 000. Both forms contain SV2, an epitope located on the cytoplasmic side of the vesicle membrane. In addition to electric organ, we have analyzed the SV2 proteoglycan in vesicle fractions from two other sources, electric fish brain and rat brain. Both the H and L forms of SV2 are present in these vesicles and all are keratan sulfate proteoglycans. Unlike previously studied synaptic vesicle proteins, this proteoglycan contains a marker specific for a single group of neurons. This marker is an antigenically unique keratan sulfate side chain that is specific for the cells innervating the electric organ; it is not found on the synaptic vesicle keratan sulfate proteoglycan in other neurons of the electric fish brain.     

Additive Induction of Egr-1 (zif/268) mRNA Expression in Neuroblastoma × Glioma Hybrid NG108-15 Cells via Cholinergic Muscarinic, α2-Adrenergic, and Bradykinin Receptors

Abstract: Administration of carbachol, noradrenaline, and bradykinin induced Egr-1 mRNA expression within 1 h in mouse neuroblastoma × rat gliomahybrid NG108–15 cells. With specific receptor antagonists, the Egr-1 inductions by carbachol and noradrenaline were shown to be mediated via cholinergic muscarinic and α₂-adrenergic receptors, respectively. At their saturation levels for Egr-1 induction, the two agonists had additive effects when added together, but no prolongation of the effect on Egr-1 induction was observed. Addition of carbachol or noradrenaline 6 h after primary stimulation with carbachol or noradrenaline did not result in secondary Egr-1 induction, probably because of receptor desensitization. On the other hand, bradykinin consistently had an additive effect on Egr-1 induction, irrespective of the time of its addition, suggesting that the signal pathways for Egr-1 induction by carbachol or noradrenaline and by bradykinin are different. Treatment of cells with pertussis toxin or cholera toxin strongly inhibited Egr-1 induction by carbachol or noradrenaline but only partially inhibited the induction by bradykinin. Thus, the signals transduced in NG108–15 cells by different neurotransmitter receptors appear to have different effects on Egr-1 induction, depending on the times of stimulation and the combinations of receptors stimulated.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Regulation of the orientation of cortical microtubules inSpirogyra cells

The orientation of cortical microtubules (MTs) was synchronously regulated inSpirogyra cells. While the reorganized MTs in distilled water for 1.5 hr, after 1 hr treatment with amiprophos-methyl (APM) and complete depolymerization of the MTs, were all transverse, those reorganized in 0.30 M mannitol were all oblique or longitudinal. After the MTs had reorganized in 0.30 M mannitol, these cells were then incubated in distilled water for 6 hr, and the orientation of the MTs, in the cells in which MTs could be observed, all became transverse.