Efficient production of thermostable Thermus thermophilus xylose isomerase in Escherichia coli and Bacillus brevis

Summary The xylose (glucose) isomerase from the thermophile Thermus thermophilus seems to have potential for the development of new isomerization processes using high temperatures and slightly acidic pH. The isomerase has an optimum temperature at 95° C, and is also very stable at high temperatures. The optimum pH is around 7.0, close to where by-product formation is minimal. Since Thermus produces only a little of this useful isomerase, the production of the cloned gene in Escherichia coli and Bacillus brevis were compared. Especially B. brevis was able to produce the isomerase effeciently, more than 1 g/l, in spite of the high G + content (67%) of the Thermus gene, and the presence of codons not frequently used in E. coli or B. brevis.Offprint requests to: S. Udaka     

T3-hydrocortisone synergism on adult-type erythroblast proliferation and T3-mediated apoptosis of larval-type erythroblasts during erythropoietic conversion in Xenopus laevis

 The conversion of an erythropoietic system from larval to adult type in anuran amphibia may possibly come about through cell replacement. The hormonal regulation of apoptosis of larval-type precursor cells and adult-type cell proliferation has yet to be examined in detail. In amphibians, corticoids synergize T₃ action during metamorphosis. In the present study, examination was made of the process of larval-to-adult conversion in the liver erythropoietic site of Xenopus laevis, with special attention to how these metamorphic hormones, T₃ and corticoid, regulate programmed cell death specific for larval erythroblasts and the proliferation of adult cells. Immunohistochemical analysis of liver sections indicates that the number of larval erythroblasts decreased to less than 50% at the early climax stage (stages 59–60) of metamorphosis. Overall liver morphology greatly changed subsequent to the climax stage from the three-lobe to the two-lobe shape. The addition of T₃ (10^-8 M) to premetamorphic tadpoles induced considerable liver morphological change and a 50% decrease in larval-type erythroblasts. These erythroblast decreases seem to take place through the apoptotic process, since double-staining experiments with in situ DNA nick-end labeling (TUNEL) and hemoglobin immunostaining revealed that DNA breakage of nuclei, a well-known feature of apoptosis, occured specifically in larval erythroblasts during prometamorphosis. Hydrocortisone (HC), which modulates T₃ action during metamorphosis, was found not to be a factor in larval cell decrease. But adult erythroblasts increased by 8 times as much through the action of T₃ and 32 times as much by the action of T₃ plus HC, indicating the important action of T₃–HC synergism. It thus follows that the erythropoietic system is converted during metamorphosis effectively by two distinct hormonal mechanisms, T₃–HC synergism on adult erythroblast proliferation and T₃-mediated programmed death of larval precursor cells. Accepted: 14 January 1999     

Development of a novel neodymium compound for in vivo fluorescence imaging.

We developed a novel fluorescent probe that contains the neodymium(III) complex moiety and fluorescein moiety. This probe can emit long-lived near-infrared luminescence derived from a Nd ion through excitation of the fluorescein moiety with visible light (lambda(ex) = 488 nm, lambda(em) = 880 nm, lifetime = 2.3 micros). These results indicate the possibility of the probe as a candidate for in vivo fluorescence molecular imaging.     

The budding yeast mei5 and sae3 proteins act together with dmc1 during meiotic recombination

Here we provide evidence that the Mei5 and Sae3 proteins of budding yeast act together with Dmc1, a meiosis-specific, RecA-like recombinase. The mei5 and sae3 mutations reduce sporulation, spore viability, and crossing over to the same extent as dmc1. In all three mutants, these defects are largely suppressed by overproduction of Rad51. In addition, mei5 and sae3, like dmc1, suppress the cell-cycle arrest phenotype of the hop2 mutant. The Mei5, Sae3, and Dmc1 proteins colocalize to foci on meiotic chromosomes, and their localization is mutually dependent. The localization of Rad51 to chromosomes is not affected in either mei5 or sae3. Taken together, these observations suggest that the Mei5 and Sae3 proteins are accessory factors specific to Dmc1. We speculate that Mei5 and Sae3 are necessary for efficient formation of Dmc1-containing nucleoprotein filaments in vivo.     

SOCS3 is a physiological negative regulator for granulopoiesis and granulocyte colony-stimulating factor receptor signaling

The suppressor of cytokine signaling-3 (SOCS3/CIS3) has been shown to be an important negative regulator of cytokines, especially cytokines that activate STAT3. To examine the role of SOCS3 in neutrophils and the granulocyte colony-stimulating factor (G-CSF) signaling in vivo, we compared neutrophils from two types of conditional knockout mice, LysM-Cre:SOCS3(fl/fl) mice and Tie2-Cre:SOCS3(fl/fl) mice, in which the Socs3 gene had been deleted in mature neutrophils and hematopoietic stem cells, respectively. The size of the G-CSF-dependent colonies from Tie2-Cre:SOCS3(fl/fl) mouse bone marrow was much larger than that of colonies from control wild-type mice, while the size of interleukin-3-dependent colonies was similar. Moreover, LysM-Cre:SOCS3(fl/fl) mice had more neutrophils than SOCS3(fl/fl) mice, suggesting that SOCS3 is a negative regulator of G-CSF signaling in neutrophils. Consistent with this notion, G-CSF-induced STAT3 as well as mitogen-activated protein kinase activation was much stronger and prolonged in SOCS3-deficient mature neutrophils than in wild-type neutrophils. The preventive effect of G-CSF on apoptosis was more prominent in SOCS3-deficient mature neutrophils than in control neutrophils. These data indicate that SOCS3 negatively regulates granulopoiesis and G-CSF signaling in neutrophils and may contribute to neutrophilia or neutropenia.     

Abnormal lipid metabolism in cystathionine beta-synthase-deficient mice, an animal model for hyperhomocysteinemia

Hyperhomocysteinemia (HHCY) is a consequence of impaired methionine/cysteine metabolism and is caused by deficiency of vitamins and/or enzymes such as cystathionine beta-synthase (CBS). Although HHCY is an important and independent risk factor for cardiovascular diseases that are commonly associated with hepatic steatosis, the mechanism by which homocysteine promotes the development of fatty liver is poorly understood. CBS-deficient (CBS(-/-)) mice were previously generated by targeted deletion of the Cbs gene and exhibit pathological features similar to HHCY patients, including endothelial dysfunction and hepatic steatosis. Here we show abnormal lipid metabolism in CBS(-/-) mice. Triglyceride and nonesterified fatty acid levels were markedly elevated in CBS(-/-) mouse liver and serum. The activity of thiolase, a key enzyme in beta-oxidation of fatty acids, was significantly impaired in CBS(-/-) mouse liver. Hepatic apolipoprotein B100 levels were decreased, whereas serum apolipoprotein B100 and very low density lipoprotein levels were elevated in CBS(-/-) mice. Serum levels of cholesterol/phospholipid in high density lipoprotein fractions but not of total cholesterol/phospholipid were decreased, and the activity of lecithin-cholesterol acyltransferase was severely impaired in CBS(-/-) mice. Abnormal high density lipoprotein particles with higher mobility in polyacrylamide gel electrophoresis were observed in serum obtained from CBS(-/-) mice. Moreover, serum cholesterol/triglyceride distribution in lipoprotein fractions was altered in CBS(-/-) mice. These results suggest that hepatic steatosis in CBS(-/-) mice is caused by or associated with abnormal lipid metabolism.     

Chronological characterization of diabetes development in male Spontaneously Diabetic Torii rats

To characterize the underlying mechanisms of diabetes development in males of the Spontaneously Diabetic Torii (SDT) rat, a novel spontaneous model for diabetes, we chronologically examined them, focusing on their diabetic features and the pathological changes in the pancreatic islets. Male SDT rats exhibited glucose intolerance with impaired insulin secretion after 14 weeks and developed diabetes with remarkable hyperglycemia and marked hypoinsulinemia after 20 weeks. At prediabetic stage (10-20 weeks), they were normoglycemic, but had significantly lower insulin levels of plasma and pancreas than the normal rats. Their beta-cell volume was already smaller significantly at 10 weeks than that of normal rats. The primary changes of the pancreatic islets were microvascular events such as congestion and hemorrhage at 8-10 weeks. Thereafter, the SDT rat islets were affected with inflammation and progressive fibrosis (at 10-20 weeks), and eventually atrophied with a loss of beta-cells (at 38 weeks). These results indicate that the male SDT rats develop spontaneous diabetes with an absolute decrease in the insulin secretory capacity of the islets.     

Identification and characterization of a novel component of the cornified envelope, cornifelin

Psoriasis is recognized as a chronic inflammatory disease characterized by epidermal hyperproliferation. To identify psoriasis-related genes, we compared the mRNA populations of normal and psoriatic skin. We identified one gene, designated as cornifelin, which showed increased expression in psoriatic skin. Human cornifelin contains 112 amino acids and is expressed in the uterus, cervix, and skin. In situ hybridization analysis demonstrated the presence of human cornifelin in the granular cell layer of the epidermis. To investigate the function of cornifelin, we established a transgenic mouse line overexpressing human cornifelin. Using these mice, we have shown that cornifelin is directly or indirectly cross-linked to at least two other cornified envelope proteins, loricrin and involucrin, in vivo. Overexpression of human cornifelin correlated with decreased loricrin expression and increased involucrin expression in the transgenic mouse. However, abnormality of epidermal differentiation was not observed in the transgenic mouse.