A novel type of human alpha-amylase produced in lung carcinoid tumor

A novel type of alpha-amylase was detected in a lung carcinoid tissue after surveying the cDNA library constructed from this tumor mRNA. Nucleotide sequence analysis showed that the amylase expressed in this carcinoid tumor has 13 and 6 amino acid substitutions when compared with salivary amylase (Amy1) and pancreatic amylase (Amy2), respectively. The nucleotide sequence homologies of cDNAs between this carcinoid amylase and amy1, amy2 are 97.5% and 98.2%, respectively. The nucleotide sequence comparison strongly suggests that this new amylase is the product of the amy3 gene that has been detected in human genome [Emi et al., Gene 62 (1988) 229-235]     

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

Effects of the Histamine H3-Agonist (R)-α-Methylhistamine and the Antagonist Thioperamide on Histamine Metabolism in the Mouse and Rat Brain

To study the feedback control by histamine (HA) H3-receptors on the synthesis and release of HA at nerve endings in the brain, the effects of a potent and selective H3-agonist, (R)-alpha-methylhistamine, and an H3-antagonist, thioperamide, on the pargyline-induced accumulation of tele-methylhistamine (t-MH) in the brain of mice and rats were examined in vivo. (R)-alpha-Methylhistamine dihydrochloride (6.3 mg free base/kg, i.p.) and thioperamide (2 mg/kg, i.p.), respectively, significantly decreased and increased the steady-state t-MH level in the mouse brain, whereas these compounds produced no significant changes in the HA level. When administered to mice immediately after pargyline (65 mg/kg, i.p.), (R)-alpha-methylhistamine (3.2 mg/kg, i.p.) inhibited the pargyline-induced increase in the t-MH level almost completely during the first 2 h after treatment. Thioperamide (2 mg/kg, i.p.) enhanced the pargyline-induced t-MH accumulation by approximately 70% 1 and 2 h after treatment. Lower doses of (R)-alpha-methylhistamine (1.3 mg/kg) and thioperamide (1 mg/kg) induced significant changes in the pargyline-induced t-MH accumulation in the mouse brain. In the rat, (R)-alpha-methylhistamine (3.2 mg/kg, i.p.) and thioperamide (2 mg/kg, i.p.) also affected the pargyline-induced t-MH accumulation in eight brain regions and the effects were especially marked in the cerebral cortex and amygdala. These results indicate that these compounds have potent effects on HA turnover in vivo in the brain.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.     

Expression and transport into mitochondria of bovine cytochrome P-450(SCC) in insect cells using the baculovirus expression system.

Bovine cytochrome P-450(SCC) introduced with the baculovirus host vector system was found to be expressed in Spodoptera frugiperda cells. Cell fractionation analysis indicated that the P-450(SCC) expressed as the precursor form was transported into mitochondria and converted to a mature form. However, this form did not exhibit definite activity for cholesterol side chain cleavage. These findings suggest that most of the P-450(SCC) expressed by this system is an inactive protein within mitochondria that is not folded to the conformation of the active enzyme and/or does not incorporate heme appropriately.     

Effect of fructose on glycogen synthesis in the perfused rat liver

The effect of fructose on glycogen synthesis was examined in the perfused liver of starved rats. With increasing fructose concentration in the perfusate, glycogen synthesis and the % a form of glycogen synthase increased to a maximum at 2 mM and then decreased, progressively. The glucose 6-P level increased with the increase in fructose concentration. On the other hand, the ATP content was unchanged at a concentration of 2 mM or less and decreased at 3 mM or more. We also showed that the stimulation of glycogen synthesis by fructose at a concentration of 2 mM or less was due to activation of glycogen synthase by accumulated glucose 6-P and that ATP depletion at a concentration of 3 mM or more caused an increase in phosphorylase a and a decrease in glycogen synthase activity even in the presence of a high concentration of glucose 6-P.