Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity

The extracellular poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis T1, which hydrolyzes both hydrophobic poly(3-hydroxybutyrate) and water-soluble oligomers of D(-)-3-hydroxybutyrate, lost its hydrolyzing activity toward the hydrophobic substrate on mile trypsin treatment, but retained its activity toward water-soluble oligomers. The molecular mass of the trypsin-treated enzyme was 44 kDa, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, which was 6 kDa smaller than that of the native enzyme (50 kDa). The trypsin-treated enzyme seemed to be less hydrophobic than the native one, because it was rather weakly adsorbed to a hydrophobic butyl-Toyopearl column compared with the native enzyme, and showed no ability to bind to poly(3-hydroxybutyrate), to which the native enzyme tightly bound. These results suggest that, in addition to a catalytic site, the enzyme has a hydrophobic site, which is not essential for the hydrolysis of water-soluble oligomers, but is necessary for the hydrolysis of hydrophobic substrates, and this hydrophobic site is removed from the enzyme by the action of trypsin.     

Anthocyanin production in callus cultures of roselle (Hibiscus sabdariffa L.)

Callus tissues derived from seedlings of roselle (Hibiscus sabdariffa L.) were shown to produce two cyanidin glycosides as major anthocyanin pigments. Both callus growth and anthocyanin synthesis were remarkably stimulated by 2,4-dichlorophenoxyacetic acid. The highest anthocyanin yield was observed when 1 M 2,4-D in combination with 0.1–1 M kinetin was supplemented to the culture medium. In contrast, gibberellic acid showed inhibitory effect on anthocyanin production.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - GA gibberellic acid     

L-proline is an essential amino acid for hepatocyte growth in culture

For improvement of the culture conditions of adult rat hepatocytes in primary culture in collagen coated dishes, effects of various commercial culture media on the induction of replicative DNA synthesis of the cells stimulated by insulin plus epidermal growth factor were studied. Proline-deficient media, such as Leibovitz's L-15, Eagle's minimal essential medium and Dulbecco's modified minimal essential medium, did not induce DNA synthesis in hepatocytes, whereas proline-rich media, such as Williams medium E, McCoy's 5A and Ham's F-12, induced markedly hepatocyte proliferation. Moreover, when the proline-deficient media were supplemented with L-proline, the cells synthesized DNA in response to the two hormones. Cis-4-hydroxyl-L-proline strongly inhibited the induction of DNA synthesis, without affecting protein synthesis of the cells or showing any cytotoxicity. This inhibition was recovered completely by adding excess proline to the medium. Addition of other amino acids not present in the medium did not promote DNA synthesis. These findings indicate that L-proline is essential for induction of hepatocyte proliferation in culture, through its affect on synthesis of intracellular collagen.     

Chinese hamster cell mutants with defective endocytosis of low-density lipoprotein contain altered fatty acid composition in the membrane

Chinese hamster V79 cell mutants resistant to compactin (ML236B), a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, are defective in endocytosis of low-density lipoprotein (1). Two resistant clones, MF-2 and MF-3, differ in lipid composition from the parental V79 strain. In both the total cells and membrane fraction, the ratio of palmitoleic acid (16:1)/palmitic acid (16:0) is 0.4-0.5 in MF-2 and 1.7-1.8 in MF-3 while that in V79 is 0.2-0.3. By contrast, a hybrid clone between V79 and MF-3 shows a ratio of palmitoleic acid to palmitic acid very similar to that of V79. The synthesis of palmitoleic acid from acetate in the resistant clone is higher than in V79.     

Phylogenetic Position and Molecular Chronology of a Colonial Green Flagellate,Stephanosphaera pluvialis (Volvocales,Chlorophyceae), among Unicellular Algae

The genus Balticola comprises a group of unicellular green flagellate algae and is composed of four species formerly classified in the genus Haematococcus. Balticola is closely related to a colonial green flagellate, Stephanosphaera pluvialis. Although the phylogeny among these genera was previously investigated based on two nuclear gene sequences, the phylogenetic sister of S. pluvialis has yet to be determined. In the present study, the species diversity of Balticola and Stephanosphaera was investigated using 18S rRNA gene sequences, and phylogenetic analyses of combined nuclear and chloroplast gene sequences were performed to understand the evolutionary origin of coloniality in Stephanosphaera. The divergence times of four colonial volvocalean flagellates from their respective unicellular sisters were also estimated. Six Balticola genotypes and a single Stephanosphaera genotype were recognized, and one Balticola genotype was resolved as the sister of S. pluvialis, showing that Balticola is a nonmonophyletic genus. The divergence time of Stephanosphaera from its nearest Balticola relative was estimated to be 4–63 million years ago, and these genera represent the most recently diverged pair of unicellular and colonial flagellates among the Volvocales.     

Effect of Behavior Modification on Outcome in Early- to Moderate-Stage Chronic Kidney Disease: A Cluster-Randomized Trial

Objectives

Owing to recent changes in our understanding of the underlying cause of chronic kidney disease (CKD), the importance of lifestyle modification for preventing the progression of kidney dysfunction and complications has become obvious. In addition, effective cooperation between general physicians (GPs) and nephrologists is essential to ensure a better care system for CKD treatment. In this cluster-randomized study, we studied the effect of behavior modification on the outcome of early- to moderate-stage CKD.

Design

Stratified open cluster-randomized trial.

Setting

A total of 489 GPs belonging to 49 local medical associations (clusters) in Japan.

Participants

A total of 2,379 patients (1,195 in group A (standard intervention) and 1,184 in group B (advanced intervention)) aged between 40 and 74 years, who had CKD and were under consultation with GPs.

Intervention

All patients were managed in accordance with the current CKD guidelines. The group B clusters received three additional interventions: patients received both educational intervention for lifestyle modification and a CKD status letter, attempting to prevent their withdrawal from treatment, and the group B GPs received data sheets to facilitate reducing the gap between target and practice.

Main outcome measure

The primary outcome measures were 1) the non-adherence rate of accepting continuous medical follow-up of the patients, 2) the collaboration rate between GPs and nephrologists, and 3) the progression of CKD.

Results

The rate of discontinuous clinical visits was significantly lower in group B (16.2% in group A vs. 11.5% in group B, p = 0.01). Significantly higher referral and co-treatment rates were observed in group B (p<0.01). The average eGFR deterioration rate tended to be lower in group B (group A: 2.6±5.8 ml/min/1.73 m²/year, group B: 2.4±5.1 ml/min/1.73 m²/year, p = 0.07). A significant difference in eGFR deterioration rate was observed in subjects with Stage 3 CKD (group A: 2.4±5.9 ml/min/1.73 m²/year, group B: 1.9±4.4 ml/min/1.73 m²/year, p = 0.03).

Conclusion

Our care system achieved behavior modification of CKD patients, namely, significantly lower discontinuous clinical visits, and behavior modification of both GPs and nephrologists, namely significantly higher referral and co-treatment rates, resulting in the retardation of CKD progression, especially in patients with proteinuric Stage 3 CKD.

Trial registration

The University Hospital Medical Information Network clinical trials registry UMIN000001159     

Changes in Cognitive Functions in the Elderly Living in Temporary Housing after the Great East Japan Earthquake

On March 11, 2011, Japan experienced an earthquake of magnitude 9.0 and subsequent enormous tsunamis. This disaster destroyed many coastal cities and caused nearly 20,000 casualties. In the aftermath of the disaster, many tsunami survivors who lost their homes were forced to live in small temporary apartments. Although all tsunami survivors were at risk of deteriorating health, the elderly people were particularly at a great risk with regard to not only their physical health but also their mental health. In the present study, we performed a longitudinal cohort study to investigate and analyze health conditions and cognitive functions at 28, 32, and 42 months after the disaster in the elderly people who were forced to reside in temporary apartments in Kesennuma, a city severely damaged by the tsunamis. The ratio of people considered to be cognitively impaired significantly increased during the research period. On the other hand, the mean scores of the Kessler Psychological Distress Scale-6 and Athens Insomnia Scale improved based on the comparison between the data at 24 and 42 months. The multiple logistic regression analysis revealed that frequency of “out-of-home activities” and “walking duration” were independently associated with an increase in the ratio of people with cognitive impairment. We concluded that the elderly people living in temporary apartments were at a high risk of cognitive impairment and “out-of-home activities” and “walking” could possibly maintain the stability of cognitive functions.