Hepatic microsomal conversion of pregnenolone to 3β,5,6β-trihydroxy-5α-pregnan-20-one via pregnenolone α- and β-epoxides

In the presence of ferrous ion, ADP, and an NADPH-generating system, [4-¹⁴C]pregnenolone was oxidized by bovine liver microsomes to its α-epoxide (5,6α-epoxy-3β-hydroxy-5α-pregnan-20-one), β-epoxide (5,6β-epoxy-3β-hydroxy-5β-pregnan-20-one), trihydroxypregnanone (3β,5,6β-trihydroxy-5β-pregnan-20-one) which were separated, isolated on an octadecylsilicone column in 70% aq. methanol by high performance liquid chromatography, identified with respective synthetic specimens by gas-liquid chromatography-mass spectrometry. The microsomal Δ⁵-oxidation products of pregnenolone were detected in trace yield either when EDTA was added to the incubation mixture or when ferrous ion was omitted from the mixture. The microsomal oxidation system generated malondialdehyde significantly. It, however, was retarded to a negligible extent either by the addition of EDTA or by the omission of ferrous ion. Therefore, the microsomal formation of the significant yields of Δ⁵-oxygenated pregnenolones was reasonably attributed to a reaction linked to microsomal lipid peroxidation. The ratio of pregnenolone α- to β-epoxides formed was 1:3. A comparable study carried out under the same conditions by using [4-¹⁴C]cholesterol as the substrate resulted in the similar Δ⁵-epoxidation with concomitant formation of cholestane-3β,5α,6β-triol; cholesterol α- and β-epoxides formed were in the ratio 1:4.Both pregnenolone α- and β-epoxides were hydrolyzed by the microsomes to trihydroxypregnanone as the sole metabolite at a relative rate of 0.6:1. A similar relative value was also obtained in the microsomal hydrolysis of cholesterol α- and β-epoxides to the cholestanetriol.     

Hypertension, hypokalemia and hypoaldosteronism with suppressed renin: a clinical study of a patient with Liddle's syndrome

A 24-yr-old woman with hypertension, hypokalemic alkalosis, low plasma renin and hypoaldosteronism was studied. Plasma aldosterone, renin and potassium returned to normal and blood pressure fell after sodium restriction or the administration of triamterene. Thiazide therapy also normalized her blood pressure while dexamethasone, spironolactone and furosemide did not improve her symptoms. Plasma aldosterone levels were low and responded poorly to a short term ACTH injection, but responded well to the maximal adrenal stimulation by ACTH-Z. Plasma levels of cortisol, corticosterone and deoxycorticosterone were within the normal range. Adrenal scintigram with 131I-adosterol and abdominal computed axial tomography did not reveal the presence of a sizeable adrenal tumor. In addition, the urinary kallikrein excretion was low after sodium restriction and showed no response to saline infusion. These findings suggest that the excessive secretion of unusual mineralocorticoids may not exist in this case. From these observations and the results of the therapeutic responses to the diuretic agents, we conclude that the primary cause of the disorder of this patient seems to be a renal defect in the distal tubule in handling sodium and potassium which is similar to that in Liddle's syndrome.     

Purification of a eukaryotic site-specific endonuclease, Endo.Sce I, from Saccharomyces cerevisiae and effectors on its specificity and activity

A site-specific endonuclease (Endo.Sce I) which caused double-strand scission of DNA was highly purified from a eukaryote, Saccharomyces cerevisiae IAM4274. The molecular weight of the active form of Endo.Sce I was estimated to be 120,000 and 110,000 by sedimentation analysis on a glycerol density gradient and gel filtration on Ultrogel AcA34, respectively. Analysis of the fractions from the last column chromatography by polyacrylamide gel-electrophoresis in the presence of sodium dodecyl sulfate and by an assay of the endonucleolytic activities suggested that Endo.Sce I consists of two non-identical subunits with molecular weights of 75,000 and 50,000. Unlike restriction endonucleases, Endo.Sce I was active on chromosomal DNA of the cells which produced Endo.Sce I. Single-stranded DNA was not cleaved by Endo.Sce I, but inhibited the endonucleolytic activity of the enzyme on double-stranded DNA. The endonucleolytic activity of Endo.Sce I required the magnesium ions (Mg2+) as a sole cofactor; Mg2+ could not be replaced by Ca2+ or Zn2+. When Mg2+ was replaced by manganese ions (Mn2+), extensively purified Endo.Sce I cleaved double-stranded DNA at many other sites in addition to the sites at which DNA was cleaved in the presence of Mg2+. Experiments indicated that this is not the activation of contaminating endonuclease in the preparation of Endo.Sce I, but the result of relaxation in the site-specificity of cleavage.     

A 3' to 5' exonuclease activity is associated with phage 029 DNA polymerase

Bacteriophage 029 produces its own DNA polymerase which is encoded by gene 2 [Watabe, K. and Ito, J. (1983) Nucleic Acid Res. 11, 8333]. This 029 DNA polymerase has been purified by phospho-cellulose, DEAE-cellulose, double-stranded DNA cellulose chromatography and glycerol gradient centrifugation. An exonuclease activity associated with the DNA polymerase was found through all the steps of the purification. This nuclease preferably degrades single-stranded DNA from the 3' to the 5' terminus direction, suggesting that the enzyme plays a role for proofreading during DNA replication. While DNA polymerase activity isolated from cells infected with temperature sensitive mutant of gene 2 is thermolabile, the nuclease activity is not significantly reduced at the restrictive temperature.     

Molecular cloning of an ice nucleation gene from Erwinia ananas and its expression in Escherichia coli

Abstract An approximately 7 kbp genomic DNA fragment was cloned from an ice nucleation-active (ina) strain of Erwinia ananas and defined as to its restriction enzyme site. When the DNA fragment was introduced into E. coli MM294, a potent ice nucleation activity was expressed. Both 0.7 kbp truncation from the 5'-end and 1.7 kbp truncation from the 3'-end were also effective in expressing the ice nucleation activity in E. coli . Therefore, the resulting DNA fragment of approximately 5 kbp was considered to be an ina gene and named ina A. It existed as a unique gene in this strain of E. ananas . No corresponding ina gene existed in an ice nucleation-inactive strain of E. milletiae .     

Proregion of Bombyx mori cysteine proteinase functions as an intramolecular chaperone to promote proper folding of the mature enzyme.

A cDNA encoding the proform of Bombyx cysteine proteinase (BCP) was expressed at a high level in Escherichia coli using the T7 polymerase expression system. The insoluble recombinant zymogen was solubilized and renatured by modifying a method applied to human pro-cathepsin L. Like the natural BCP precursor, the recombinant proenzyme was spontaneously converted to an active proteinase at pH 3.75. A deletion in the central region of the propeptide resulted in much loss of the activity, suggesting that the propeptide is essential for proper folding during renaturation. In contrast, the renatured mature form of recombinant BCP was not active but regained activity by including the propeptide in the renaturing buffer, suggesting that the propeptide, acting as an intramolecular chaperone, promotes refolding of the associated proteinase domain into an active conformation. The mature form of natural BCP rapidly lost its activity at neutral pH, whereas its proform was stable. The mature enzyme retained some activity in the presence of the propeptide. Arch.     

Biochemical genetic evidence for a hybrid origin of spined loach, Cobitis taenia taenia, in Japan

Starch gel electrophoresis and isoelectric focusing were performed on sarcoplasmic proteins from three taxa of cobitid fish in Japan, Cobitis biwae , C. taenia striata and C. l. taenia . These taxa are hardly distinguishable from each other by external appearance or morphological characters. Electrophoretic patterns of sarcoplasmic proteins from white muscle suggested fixed allelic differences between C. biwae and C. t. striata at two protein-coding loci (parvalbumin and creatine kinase). The third taxon, C. t. taenia , showed composite banding patterns with respect to the above two proteins, suggesting C. t, taenia originated from hybridization between C. biwae and C, t. striata . Previously, C. t, taenia and C. t. striata were believed to have shared a common ancestor subsequent to their divergence from C. biwae .     

Relationship between Trusting Behaviors and Psychometrics Associated with Social Network and Depression among Young Generation: A Pilot Study

Maladaptive social interaction and its related psychopathology have been highlighted in psychiatry especially among younger generations. In Japan, novel expressive forms of psychiatric phenomena such as “modern-type depression” and “hikikomori” (a syndrome of severe social withdrawal lasting for at least six months) have been reported especially among young people. Economic games such as the trust game have been utilized to evaluate real-world interpersonal relationships as a novel candidate for psychiatric evaluations. To investigate the relationship between trusting behaviors and various psychometric scales, we conducted a trust game experiment with eighty-one Japanese university students as a pilot study. Participants made a risky financial decision about whether to trust each of 40 photographed partners. Participants then answered a set of questionnaires with seven scales including the Lubben Social Network Scale (LSNS)-6 and the Patient Health Questionnaire (PHQ)-9. Consistent with previous research, male participants trusted partners more than female participants. Regression analysis revealed that LSNS-family (perceived support from family) for male participants, and item 8 of PHQ-9 (subjective agitation and/or retardation) for female participants were associated with participants’ trusting behaviors. Consistent with claims by social scientists, our data suggest that, for males, support from family was negatively associated with cooperative behavior toward non-family members. Females with higher subjective agitation (and/or retardation) gave less money toward males and high attractive females, but not toward low attractive females in interpersonal relationships. We believe that our data indicate the possible impact of economic games in psychiatric research and clinical practice, and validation in clinical samples including modern-type depression and hikikomori should be investigated.     

DNA polymerase action blocked by adenine adducts induced by 5-hydroxymethylchrysene sulfate.

Modification of M13mp10 single-stranded DNA with 5-hydroxymethylchrysene (5HCR) sulfate, the ultimate carcinogenic metabolite of 5-methylchrysene, resulted in formation of N6[(chrysen-5-yl)methyl]adenine and N2[(chrysen-5-yl)methyl]-guanine at the ratio of 2.7:1. Measurement of DNA synthesis using this modified template and E.coli DNA polymerase I (Klenow fragment) demonstrated that increasing levels of adducts caused a progressive decline in replication. Analysis of reaction products on DNA-sequence gels revealed DNA elongation to be arrested exclusively at adenine adducts in -AAAGGA- and -AACA- sequences.