Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

In vitro proliferation of saffron (Crocus sativus L.) stigma

Excised young intact stigmas plus ovaries of Crocus sativus L. were cultured on Linsmaier-Skoog media supplemented with either a cytokinin or an auxin alone or in combinations. Benzyladenine and kinetin at concentrations of 0.1, 1, and 5 mgl^-1 supported growth, and crocin was biosynthesized in the stigmas in vitro. Auxins had little effect. Young excised single stigmas or half ovaries were also cultured so as to form stigma-like structures in order to explore a possible new approach to industrial production of the spice, saffron. On Linsmaier-Skoog and Nitsch media supplemented with kinetin at concentration of 1 or 5 mgl^-1 and alpha-naphthalene acetic acid or indole-butyric acid at concentration of 0.1 or 10 mgl^-1 in combinations, stigma-like structures appeared directly and indirectly (through meristematic tissue), grew and matured. The maximum number of structures were 75 per half ovary. Three kinds of yellow pigments including crocin were tentatively identified by TLC in the stigma-like structures as was the case for the in vivo grown natural stigma, although the contents were lower.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-acetic acid - IBA indole-butyric acid - NAA alpha-naphthalene-acetic acid - TLC thin layer chromatography     

Plasma cholecystokinin-octapeptide like immunoreactivity in patients with hepatic cirrhosis

Molecular forms of cholecystokinin (CCK) in the peripheral circulation were studied in normal subjects and cirrhotic patients. Fractionation of plasma extract collected 20 min after intraduodenal infusion of fat revealed four major peaks by Sephadex G-50 column chromatography in normal subjects. Peak I eluted at a position similar to CCK-33, peaks II and III eluted between CCK-33 and CCK-14, and peak IV eluted between CCK-14 and CCK-8. In cirrhotic patients, there was a prominent peak (peak V) eluted at a position similar to CCK-8, in addition to those four peaks. These findings are consistent with the previous observations of hepatic elimination of CCK-8, and suggest that smaller forms of CCK similar in size to CCK-8 are not major forms of CCK in plasma in normal subjects but circulate substantially in cirrhotic patients.     

Cloning and nucleotide sequence of the penicillinase antirepressor gene penJ of Bacillus licheniformis.

The penicillinase antirepressor gene, penJ, of Bacillus licheniformis ATCC 9945a was cloned in Escherichia coli by using pMB9 as a vector plasmid. The penicillinase gene, penP, its repressor gene, penI, and penJ were encoded on the cloned 5.2-kilobase HindIII fragment of the recombinant plasmid pTTE71. The penJ open reading frame was composed of 1,803 bases and 601 amino acid residues (molecular weight, 68,388). A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site. Since this sequence was located in the 3'-terminal region of the penI gene, penJ might be transcribed together with penI as a polycistronic mRNA from the penI promoter. Frameshift mutations of penJ were constructed in vitro from pTTE71, and the penJ mutant gene was introduced into B. licheniformis by chromosomal recombination. The transformant B. licheniformis U173 (penP+ penI+ penJ) turned out to be uninducible for penicillinase production, whereas the wild-type strain (penP+ penI+ penJ+) was inducible. Only when these three genes (penP, penI, and PenJ) were simultaneously subcloned in Bacillus subtilis did the plasmid carrier exhibit inducible penicillinase production, as did wild-type B. licheniformis. It was concluded that penJ is involved in the penicillinase induction. The regulation of penP expression by penI and penJ is discussed.     

5'-Nucleotidase in rat liver lysosomes

5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (Mr = 72,000). 5'-Nucleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.     

Detection of point mutation in the tyrosinase gene of a Japanese albino patient by a direct sequencing of amplified DNA

Summary Enzymatic DNA amplification and direct DNA sequencing were used to detect a mutation in the tyrosinase gene of an albino patient. Single-base change could be detected by direct sequencing. This base change (G to A) is thought to result in an amino acid change (Arg to Gln) in tyrosinase of the patient.     

Effects of water level fluctuation on the growth ofNelumbo nucifera Gaertn. in Lake Kasumigaura,Japan

Investigations were made of the growth ofNelumbo nucifera, an aquatic higher plant, in a natural stand in Lake Kasumigaura. A rise of 1.0 m in the water level after a typhoon in August 1986 caused a subsequent decrease in biomass ofN. nucifera from the maximum of 291 g d.w. m⁻² in July to a minimum of 75 g d.w. m⁻². The biomass recovered thereafter in shallower regions. The underground biomass in October tended to increase toward the shore. The total leaf area index (LAI) is the sum of LAI of floating leaves and emergent leaves. The maximum total LAI was 1.3 and 2.8 m² m⁻² in 1986 and 1987, respectively. LAI of floating leaves did not exceed 1 m² m⁻². The elongation rates of the petiole of floating and emergent leaves just after unrolling were 2.6 and 3.4 cm day⁻¹, respectively. The sudden rise in water level (25 cm day⁻¹) after the typhoon in August 1986 caused drowning and subsequent decomposition of the mature leaves. Only the young leaves were able to elongate, allowing their laminae to reach the water surface. The fluctuation in water level, characterized by the amplitude and duration of flooding and the time of flooding in the life cycle, is an important factor determining the growth and survival ofN. nucifera in Lake Kasumigaura.     

Prostaglandin D2 diminishes transmucosal potential difference in rat colonic mucosa in vitro in contrast to the increasing effect of prostaglandin E1

The effect of Prostaglandin D2 (PGD2) on ion transport was investigated in the rat colon in vitro. Ion transport across the intestinal mucosa was estimated by transmucosal potential difference (PD) and short circuit current (Isc) in the Ussing chamber. PGD2 added to the serosal reservoir induced a sustained reduction in PD and Isc at the concentration of higher than 10(-7)M, producing the maximal decrease at 10(-5)M. PGD2 at 10(-5)M completely blocked the increase in PD elicited by prostaglandin E1 (PGE1), theophylline, dibutyryl cAMP or serotonin. Adenylate cyclase activity was determined in the colonic mucosal homogenates after addition of PGD2 and PGE1. Treatment with PGD2 or PGE1 caused a significant increase in the enzyme activity. Combined treatment with both prostaglandins induced no more increase than that elicited by PGE1 alone. These results suggest that PGD2 has an anti-secretory effect on the rat colon and it may regulate the ion transport process through other mechanism than the modification of cyclic AMP concentration in mucosal cells.     

Purification and characterization of acid phosphatase in rat liver lysosomal contents

Acid phosphatase in rat liver lysosomal contents, C-APase I, was purified about 5,700-fold over the homogenate with 8.0% recovery, to apparent homogeneity as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included; preparation of crude lysosomal contents, DEAE-Sephacel ion exchange chromatography, hydroxylapatite chromatography, and gel filtration with Sephacryl S-300. The enzyme is composed of three identical subunits with an apparent molecular weight of 48K. The enzyme contains about 11% carbohydrate and the carbohydrate moiety was composed of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine in a molar ratio of 20:3:11:1. Sialic acid was not detected in the enzyme. Antisera against the purified C-APase I were raised in goat and the C-APase I was rapidly purified with high yield (10%) by using the specific antibodies coupled to Sepharose 6B.