Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

Purification and characterization of α-glucosidases produced by Saccharomyces in response to three distinct maltose genes

α-Glucosidases or maltases (EC 3.2.1.20) were purified to electrophoretic homogeneity from a respective strain of Sacchromyces cerevisiae which carries a single MAL gene, either MALα, MALβ or MALγ, using gluconate-Sepharose affinity chromography and isoelectrofocusing. Of these maltases, two types of maltase were obtained from the MALγ strain, the pI values of which were 5.6 and 5.9. From the MALα and MALβ strain was obtained only one type of maltase with the pI at 5.6 which was identical to one of the maltases from the MALγ strain. These four maltases possessed the same properties, except for pI. They were monomers with molecular weights of between 66 000 and 67 000. With regard to the substrate specificity, they hydrolyzed maltose and sucrose exclusively but not α-methulglucoside nor maltooligosaccharide. They did not differ in immunological properties.     

Successful pregnancy in a woman with ovarian failure associated with mutation in the beta-subunit of luteinizing hormone.

BACKGROUND: We report a successful pregnancy in a woman with severe ovarian dysfunction and infertility associated with a variant beta-subunit of luteinizing hormone (LH). METHOD/OUTCOME: A 35-year-old woman consulted our unit for infertility. Laparoscopy and ultrasonography showed obstruction of the right tube and ovulation from the right ovary only. Human menopausal gonadotrophin (hMG) therapy was used for six subsequent cycles, but did not result in conception. Subsequently, marked elevation of follicle-stimulating hormone (FSH) and testosterone, together with polycystic ovary (PCO) were noted. The patient failed to respond to ovarian stimulation by hMG. Severe ovarian dysfunction such as premature ovarian failure (POF) was strongly suspected. Sequence analysis of the LH beta-subunit gene indicated heterozygosity for point mutations Trp(8) to Arg(8) and Ile(15) to Thr(15) in the coding sequence. LH hypersecretion resembling that seen in PCO syndrome was observed. Induction of ovulation by hMG was successful in the first cycle in which the basal LH and FSH were well controlled with gonadotrophin-releasing hormone analog following estrogen-progesterone replacement. She conceived and delivered a healthy male infant at term. CONCLUSION: Clinicians should be clinically aware of patients with immunologically anomalous LH variant who might be at risk of developing ovarian failure within a relatively short time span. Pertinent treatment should be applied without delay in such cases.     

Single amino acid replacements affecting the thermostability of kanamycin nucleotidyltransferase

Summary Amino acid residues of the carboxyl-terminal region of kanamycin nucleotidyltransferase were modified using segment-directed mutagenesis. Six different mutant enzymes with single amino acid replacements were selected out of 59 clones by DNA sequence analyses. The mutant enzymes were purified and it was found that the thermostability of one mutant enzyme was identical to the wild type, whereas the other five were less thermostable at varying degrees. The data suggested that changes in the enzyme thermostability depend not only on the position but also on the species of amino acid residue replaced.     

Diazo-reaction positive substance observed in the cortex of Chattonella antiqua

In order to investigate the causative factors responsible for removal of mucous coat from the gill lamellae of young yellowtail, Seriola quinqueradiata by red tide, diazo-reactions were employed for planktons and their media. The concentration of NO2- in the medium containing the raphidophyceae, Chatonella antiqua (ca 2000 cells/ml), was 0.70 +/- 0.05 (mu g/ml +/- SEM). In addition, diazo-reaction positive substances (NOx) which may degenerate the mucous, was highly concentrated in the cortex (perikaryon) of Chattonella antiqua. Morphologically, mucocysts, and chloroplats were likewise present in the cortex. Mucocysts were packed with fine fibrous content. Histochemically, the mucocysts were stained with PAS and had an abundance of nitrogen oxides (NOx). We observed discharge of the fibrous material from the mucocysts. These results suggest that when Chattonella antiqua is passing between the gill lamellae, NOx discharged from the mucocysts may act on the mucous, leading to the degeneration and concomitant removal of the mucous coat from gill lamellae.     

Molecular cloning and nucleotide sequence of a gene for alkaline cellulase from Bacillus sp. KSM-635

A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a sigma 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.     

Kinetic studies on CO binding to reconstituted myoglobins with four synthetic hemes; structural control in ligand binding to myoglobin.

Examination was made of CO binding reactions to four kinds of modified sperm whale myoglobin (Mb), whose heme was reconstituted by iron complexes of synthetic porphyrins such as porphine (Por), meso-tetramethylporphyrin (TMeP), meso-tetraethylporphyrin (TEtP) and meso-tetra(n-propyl)porphyrin (TnPrP), using flash photolysis and stopped-flow methods. The CO association rate was found to be 5- to 20-times and dissociation rate 10- to 36-times accelerated by replacement with synthetic hemes. These features could be explained based on characteristic structures of modified Mbs indicated by X-ray crystallography. The side chain of Arg-45 protruded from the heme vicinity into the solvent region and heme was tilted by interactions of meso-alkyl side chains with surrounding peptides, resulting in the formation of widely opened channels and pockets for ligand passage. These structural features indicate the CO ligand to more easily enter or exit from heme pockets of reconstituted myoglobins, compared to native Mb.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.