Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.     

Purification and properties of glutathione transferase from Issatchenkia orientalis.

Glutathione transferase (GST) (EC 2.5.1.18) was purified from a cell extract of Issatchenkia orientalis, and two GST isoenzymes were isolated. They had molecular weights of 37,500 and 40,000 and were designated GST Y-1 and GST Y-2, respectively. GST Y-1 and GST Y-2 gave single bands with molecular weights of 22,000 and 23,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GST Y-1 and GST Y-2 were immunologically distinguished from each other. GST Y-1 showed specific activity 10.4-times and 6.0-times higher when 1-chloro-2,4-dinitrobenzene and o-dinitrobenzene were used as substrates, respectively, than GST Y-2. GST activity was not detected for either isoenzyme when other substrates such as bromosulfophthalein and trans-4-phenyl-3-buten-2-one were used. GST Y-1 and GST Y-2 had Km values of 0.51 and 0.75 mM for glutathione, respectively, and of 0.16 and 4.01 mM for 1-chloro-2,4-dinitrobenzene. GST Y-1 was significantly inhibited by Cibacron blue 3G-A, and GST Y-2 was significantly inhibited by bromosulfophthalein.     

The inheritance of β-amylase null in storage roots of sweet potato,Ipomoea batatas (L.) Lam.

Summary Several sweet potato genotypes were found to lack completely or to have only traces of-amylase in their storage roots. Such genotypes do not increase in sweetness during cooking because, without a sufficient amount of-amylase, the normal hydrolysis of starch to maltose does not occur in the cooking process. In order to study the inheritance of this biochemical variant in the genotype, 41 families were generated. The following conclusions were drawn from analyzing these families. (1) This trait is controlled by one recessive allele (designated-amy) (2) It is inherited in a hexasomic or tetradisomic manner, but not disomically or tetrasomically. This conclusion supports previous cytological data that sweet potato is an autohexaploid or has two identical genomes plus one genome which is somewhat different. (3) The-amy allele appears to exist at a high frequency in cultivated germplasm. (4) Breeding sweet potato for low-amylase activity is relatively easy. New types of sweet potato without normal-amylase activity have great potential for processing and as a staple food.     

A 1H-NMR study of the solution conformation of cyclo(GRGDSPA): conformational effects on the physiological activity.

Solution conformations of cyclo(GRGDSPA) have been analyzed by the use of two-dimensional proton nuclear magnetic resonance spectroscopy and the dynamical simulated annealing calculation. It has been shown that the RGDS segment in cyclo(GRGDSPA) takes a beta-turn conformation. We have concluded that this beta-turn conformation is essential for the physiological activity of cyclo(GRGDSPA).     

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan,Europe and North America

Sixty-one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA-DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340-350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.     

Transfer of conjugative plasmid pAMβ1 from Lactococcus lactis to mouse intestinal bacteria

Conjugal transfer of plasmid pAMβ1 from Lactococcus lactis to intestinal bacteria of BALB/c mice was studied. Plasmid transfer was observed to Enterococcus faecalis in vitro by a filter mating method with transfer frequencies of 2.3 × 10⁻³ and with lower frequencies to other species. In vivo , using gastric intubation with the pAMβ1-bearing Lactococcus lactis as donor and Ent. faecalis as recipient, a few transconjugants were detected from faecal Ent. faecalis . However, when these mice were given erythromycin through drinking water, a large number of conjugated Ent. faecalis were detected in faeces. Plasmid transfer to Ent. faecalis occurred at high frequency, 1.2 × 10⁻³, in mice whose anus was artificially closed after gastric intubation with pAMβ1-bearing Lactococcus lactis . These results demonstrate clearly that pAMβ1 transfer occurs between Gram-positive bacteria in the gut of mice harbouring many species of bacteria.     

Bilateral giant myelolipoma in the adrenal of a cotton-top tamarin (Saguinus oedipus)

Abstract: A rare case of bilateral adrenal myelolipomas in a female cotton-top tamarin is reported. Large bilateral masses in the adrenal glands were composed of mature adipose cells containing varying amounts of hematopoietic cells of the myeloid, erythroid, and megakaryocyte series. The gross and histologic features of this case closely resemble human “giant” adrenal myelolipomas.