Subcellular distribution of hydrolase in germinating pea cotyledons

The subcellular distribution of hydrolases in germinating peacotyledons was investigated by differential and sucrose densitygradient centrifugations. Acid phosphatase was present in atleast two kinds of subcellular components. One was slightlyheavier than mitochondria, and the other was so light that itremained on the upper part of the gradient after sucrose densitygradient centrifugation. Protease was localized in the smooth-surfacedmicrosomal fraction which contained antimycin A-insensitiveNADH₂-cytochrome c reductase and no RNA. Aryl esterase existedin a soluble form in the cytoplasm. (Received July 22, 1971; )     

Molecular Structure of Xanthocidin

In order to establish industrial production of 5′-inosinic acid (5′-IMP), a permeability mutant, KY13171, of Brevibacterium ammoniagenes, which accumulated 7 to 8 grams of 5′-IMP per liter and 4 to 6 grams of hypoxanthine (Hx) per liter (calculated as 5′-IMP), was improved by a genetical procedure. Further improved mutants were selected stepwise through repeating mutational work. The finally selected mutant. KY13369, accumulated 20 to 27 grams of 5′-IMP per liter, but not Hx.

Increased productivity of 5′-IMP and decreased productivity of Hx were not caused by the changes in 5′-IMP degrading activity, because these activities were not significantly different among the mutants. These results appear to indicate that the increased accumulation of 5′-IMP may be caused by the improvement in membrane permeability for 5′-IMP. However, the changes in phospholipid and fatty acid compositions were not enough to explain the increased permeability.     

Bioconversion of dethiobiotin to biotin by a cell-free system of a bioYB transformant of Bacillus sphaericus

Abstract Potassium-limited cultures of Candida utilis grown with ammonium chloride as the sole nitrogen source attained a higher dry weight than similar cultures grown with sodium nitrate as the sole nitrogen source. This increase depended on the extracellular ammonia concentration and was not due to accumulation of storage polymers. We conclude that in this yeast ammonium ions can fulfill to some extent the physiological role of potassium ions and that the intracellular concentration of ammonium ions is predominantly determined by the ammonia concentration in the culture medium.     

Activation of Protein Synthesis and Biogenesis of Mitochondrial Particles during Aging of Discs of Sweet Potato Root Tissue

Protein synthesis in mitochondrial and supernatant fractions of sweet potato root tissue was found to be activated up to about 3 and 1.5 times in response to wounding, respectively. The activation seemed to take place within 8 hr after slicing. Sucrose density gradient centrifugation of mitochondrial fraction prepared from the aged sliced tissue showed the presence of new fraction of mitochondrial particles which were not seen in the case of fresh tissue. The particles in the fraction were labeled by radioactive leucine in vivo more rapidly than the other particles. Chloramphenicol treatment of tissue before aging blocked the development of these particles. These results suggest that the particles were newly formed during aging.     

Purification and Properties of Maltose Phosphorylase from Lactobacillus brevis

Maltose phosphorylase (EC 2.4.1.8) from Lactobacillus brevis was purified 29-fold over the crude extract. The final preparation was at least 80% pure and had a specific activity of 18 units/mg protein. The molecular weights of the native enzyme and of the component dissociated in sodium dodecyl sulfate were 150,000 and 80,000, respectively. The enzyme does not contain pyridoxal-5′-phosphate as a cofactor. It can not act on maltitol, malto-triitol, sucrose, lactose and trehalose, and essentially not on isomaltose, maltobionic acid, maltotriose and maltotetraose. Inhibitory effect was observed with CuSO₄, HgCl₂ and p-chloromercuribenzoate. Some other properties were also examined. A possibility of using this enzyme for the analysis of maltose was proposed.     

Changes in Flavor Components of Onion by γ-Irradiation

The effect of γ-irradiation on the flavor of Onions (Senshu Yellow) associated with sprout-inhibition has been investigated. The relative amounts of propionaldehyde, n-propyl mercaptan and di-n-propyl disulfide in onions stored for 0, 5, 10, 20 and 30 days, respectively, after irradiation were estimated by measurement of peak areas of gas chromatograms. It was observed that the lachrymatory character and the pungent flavor had been decreased by γ-irradiation (remarkably at 70 and 700 Krads). In this connection, the amounts of propionaldehyde and di-n-propyl disulfide were decreased with increasing radiation dose and storage period at room temperature (20 to 25°C) and at low temperature (4°C). Moreover, it was observed that the sweetness had been decreased by γ-irradiation, but the amount of n-propyl mercaptan was increased with radiation dose and storage period. Therefore it seems that there is no correlation between the sweetness of onion and the amount of n-propyl mercaptan.     

Effect of Cycloheximide on Development of Mitochondria in Germinating Pea Cotyledons

Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50% PEG 4,000, 15 µg protamine sulfate/ml, 0.15 m sucrose, and 10 m m MgSO₄ in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 × 10⁵ PFU/µg DNA. The eclipse period of growth of progeny phages in the transfectants was 3 hr and the average burst size was 200.     

Skin Autofluorescence Is Associated with the Progression of Chronic Kidney Disease: A Prospective Observational Study

Background

Advanced glycation end product (AGE) accumulation is thought to be a measure of cumulative metabolic stress that has been reported to independently predict cardiovascular disease in diabetes and renal failure. The aim of this study was to evaluate the association between AGE accumulation, measured as skin autofluorescence, and the progression of renal disease in pre-dialysis patients with chronic kidney disease (CKD).

Methods

Skin autofluorescence was measured noninvasively with an autofluorescence reader at baseline in 449 pre-dialysis patients with CKD. The primary end point was defined as a doubling of serum creatinine and/or need for dialysis.

Results

Thirty-three patients were lost to follow-up. Forty six patients reached the primary end point during the follow-up period (Median 39 months). Kaplan-Meier analysis showed a significantly higher risk of development of the primary end points in patients with skin autofluorescence levels above the optimal cut-off level of 2.31 arbitrary units, derived by receiver operator curve analysis. Cox regression analysis revealed that skin autofluorescence was an independent predictor of the primary end point, even after adjustment for age, gender, smoking history, diabetes, estimated glomerular filtration rate and proteinuria (adjusted hazard ratio 2.58, P = 0.004).

Conclusions

Tissue accumulation of AGEs, measured as skin autofluorescence, is a strong and independent predictor of progression of CKD. Skin autofluorescence may be useful for risk stratification in this group of patients; further studies should clarify whether AGE accumulation could be one of the therapeutic targets to improve the prognosis of CKD.     

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.     

Diverse effects of poly-basic amino acids, heparin and ionic strength on the phosphorylation of various substrates by cytosolic protein-tyrosine kinase from porcine spleen

1. Effects of poly-basic amino acids, heparin and ionic strength on the activity of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) have been studied. 2. Both polylysine and polyarginine stimulated the phosphorylation of [Val5]angiotensin II and E11 G1 (synthetic peptide of EDAEYAARRRG), but could neither stimulate nor inhibit the phosphorylation of random copolymers; poly(EY)4:1 and poly(EAY)6:3:1. 3. Heparin stimulated the phosphorylation of poly(EY)4:1 by 2.5-fold, however, it inhibited those of E11G1, poly(EAY)6:3:1, casein and H2B histone. 4. Elevation of ionic strength of either NaCl, KCl or (NH4)2SO4 stimulated the phosphorylation of poly(EY)4:1 by greater than 5-fold, but inhibited those of casein, tubulin, H2B histone, E11G1 and poly(EAY)6:3:1. 5. These effectors did not change the Km for substrates but increased the Vmax. 6. These results suggest that the effects of poly-basic amino acids, heparin and ionic strength on the activity of CPTK-40 are mainly on the substrates employed rather than on the enzyme itself.