Highly distinct chromosomal structures in cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>), as revealed by molecular cytogenetic analysis

Cowpea (Vigna unguiculata (L.) Walp) is an important legume, particularly in developing countries. However, little is known about its genome or chromosome structure. We used molecular cytogenetics to characterize the structure of pachytene chromosomes to advance our knowledge of chromosome and genome organization of cowpea. Our data showed that cowpea has highly distinct chromosomal structures that are cytologically visible as brightly DAPI-stained heterochromatic regions. Analysis of the repetitive fraction of the cowpea genome present at centromeric and pericentromeric regions confirmed that two retrotransposons are major components of pericentromeric regions and that a 455-bp tandem repeat is found at seven out of 11 centromere pairs in cowpea. These repeats likely evolved after the divergence of cowpea from common bean and form chromosomal structure unique to cowpea. The integration of cowpea genetic and physical chromosome maps reveals potential regions of suppressed recombination due to condensed heterochromatin and a lack of pairing in a few chromosomal termini. This study provides fundamental knowledge on cowpea chromosome structure and molecular cytogenetics tools for further chromosome studies.     

The Perioperative Lung Transplant Virome: Torque Teno Viruses Are Elevated in Donor Lungs and Show Divergent Dynamics in Primary Graft Dysfunction

Primary graft dysfunction (PGD) is a principal cause of early morbidity and mortality after lung transplantation, but its pathogenic mechanisms are not fully clarified. To date, studies using standard clinical assays have not linked microbial factors to PGD. We previously used comprehensive metagenomic methods to characterize viruses in lung allografts >1 mo after transplant and found that levels of Anellovirus, mainly torque teno viruses (TTVs), were significantly higher than in nontransplanted healthy controls. We used quantitative polymerase chain reaction to analyze TTV and shotgun metagenomics to characterize full viral communities in acellular bronchoalveolar lavage from donor organs and postreperfusion allografts in PGD and non‐PGD lung transplant recipient pairs. Unexpectedly, TTV DNA levels were elevated 100‐fold in donor lungs compared with healthy adults (p = 0.0026). Although absolute TTV levels did not differ by PGD status, PGD cases showed a smaller increase in TTV levels from before to after transplant than did control recipients (p = 0.041). Metagenomic sequencing revealed mainly TTV and bacteriophages of respiratory tract bacteria, but no viral taxa distinguished PGD cases from controls. These findings suggest that conditions associated with brain death promote TTV replication and that greater immune activation or tissue injury associated with PGD may restrict TTV abundance in the lung.     

Optimization of the Manufacturing Process for Oral Formulations Using Multivariate Statistical Methods

Multivariate statistical analysis has and will continue to play an important role in the development of pharmaceutical products. Although many examples have been reported, few have applied multivariate statistical analysis to the overall manufacturing process. In this study, the model drug core tablets were manufactured under different conditions, and the challenge to understand the cause-and-effect relationship between process parameters and response variables was addressed by applying three different multivariate statistical methods. It was confirmed that conventional multivariate statistical methods were able to extract the process parameters (granulation time, drying temperature, blending time, and compression force) that affected both the average and the variance of the response variables (hardness, content uniformity, and dissolution) with a science-based rationale. In order to overcome the multiobjective optimization problem among the response variables, an advanced multivariate statistical method was also applied. It was confirmed that the mathematical models of response variables were determined with sufficiently high accuracy and the optimal levels of both process parameters and response variables were determined with high reliability, which provided a more profound understanding of the process. These methods enable us to develop pharmaceutical products more efficiently and accurately.     

Possible synergic effect of angiotensin-I converting enzyme gene insertion/deletion polymorphism and angiotensin-II type-1 receptor 1166A/C gene polymorphism on ischemic heart disease in patients with Kawasaki disease

ACE I/D and AT1R 1166A/C polymorphisms are considered to comprise individual risk factors for the development of coronary disease. We sought to demonstrate that the ACE I/D and AT1R 1166A/C polymorphisms affect coronary artery stenosis in patients with Kawasaki disease (KD). We examined 147 healthy controls and 281 Japanese children with KD. The patients were further divided into group N (n = 246, no ischemia) and group I (n = 35, severe coronary artery stenosis with myocardial ischemia), and we studied the genotype of ACE I/D and AT1R 1166A/C polymorphisms. We also examined ACE activity in patients with acute KD. We did not detect any prevalent genotypes of the ACE and AT1R polymorphisms between controls and KD patients. However, the prevalence of the D allele in the ACE polymorphism and of the C allele in the AT1R polymorphism tended to be higher in group I than in group N (odds ratios, 2.00 and 2.32, respectively). In addition, the presence of the D and/or C alleles significantly increased the relative risk of developing myocardial ischemia (odds ratio, 2.71; p = 0.038). During the convalescent phase of KD, ACE activity was increased despite significant attenuation during the acute phase. These results suggested that the renin-angiotensin system is associated with the formation of severe coronary artery stenosis and myocardial ischemia.     

17β-Oestradiol inhibits doxorubicin-induced apoptosis via block of the volume-sensitive Cl(-) current in rabbit articular chondrocytes

BACKGROUND AND PURPOSE

Chondrocyte apoptosis contributes to disruption of cartilage integrity in osteoarthritis. Recent evidence suggested that the volume-sensitive organic osmolyte/anion channel [volume-sensitive (outwardly rectifying) Cl⁻ current (I_Cl,vol)] plays a functional role in the development of cell shrinkage associated with apoptosis (apoptotic volume decrease) in several cell types. In this study, we investigated the cellular effects of 17β-oestradiol on doxorubicin-induced apoptotic responses in rabbit articular chondrocytes.

EXPERIMENTAL APPROACH

Whole-cell membrane currents and cross-sectional area were measured from chondrocytes using a patch-clamp method and microscopic cell imaging, respectively. Caspase-3/7 activity was assayed as an index of apoptosis.

KEY RESULTS

Addition of doxorubicin (1 µM) to isosmotic bath solution rapidly activated the Cl⁻ current with properties similar to those of I_Cl,vol in chondrocytes. Doxorubicin also gradually decreased the cross-sectional area of chondrocytes, followed by enhanced caspase-3/7 activity; both of these responses were totally abolished by the I_Cl,vol blocker DCPIB (20 µM). Pretreatment of chondrocytes with 17β-oestradiol (1 nM) for short (approximately 10 min) and long (24 h) periods almost completely prevented the doxorubicin-induced activation of I_Cl,vol and subsequent elevation of caspase-3/7 activity. These effects of 17β-oestradiol were significantly attenuated by the oestrogen receptor blocker ICI 182780 (10 µM), as well as the phosphatidyl inositol-3-kinase (PI3K) inhibitors wortmannin (100 nM) and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM). Testosterone (10 nM) had no effect on the doxorubicin-induced Cl⁻ current.

CONCLUSIONS AND IMPLICATIONS

17β-Oestradiol prevents the doxorubicin-induced cell shrinkage mediated through activation of I_Cl,vol and subsequent induction of apoptosis signals, through a membrane receptor-dependent PI3K pathway in rabbit articular chondrocytes.     

Evaluation of hepatic glutathione transferase Mu 1 and Theta 1 activities in humans and mice using genotype information

We investigated the impact of glutathione transferases Mu 1 (GSTM1)- and glutathione transferase Theta 1 (GSTT1)-null genotypes on hepatic GST activities in humans and compared the results with those of Gstm1- and Gstt1-null mice. In liver with GSTM1/Gstm1-null genotype, GST activity toward p-nitrobenzyl chloride (NBC) was significantly decreased in both humans and mice. In addition, in liver with GSTT1/Gstt1-null genotype, GST activity toward dichloromethane (DCM) was significantly decreased in both humans and mice. Therefore, null genotypes of GSTM1/Gstm1 and GSTT1/Gstt1 are considered to decrease hepatic GST activities toward NBC and DCM, respectively, in both humans and mice. This observation shows the functional similarity between humans and mice for GSTM1 and GSTT1 toward some substrates. In the case of NBC and DCM, Gst-null mice would be relevant models for humans with GST-null genotype. In addition, decreases in GST activities toward 1,2-dichloro-4-nitrobenzene, trans-4-phenyl-3-buten-2-one, and 1-chloro-2,4,-dinitrobenzene were observed in Gstm1-null mice, and a decrease in GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane was observed in Gstt1-null mice. However, an impact of GST-null genotypes on GST activities toward these substrates was not observed in humans. In the case of these mouse-specific substrates, Gst-null mice may be relevant models for humans regardless of GST genotype, because GST activities, which are higher in wild-type mice than in humans, were eliminated in Gst-null mice. This study shows that comparison of hepatic GST activities between humans and mice using genotype information would be valuable in using Gst-null mice as human models.     

Pharmacokinetics and metabolism of NO-1886, a lipoprotein lipase-promoting agent,in cynomolgus monkey

1. The study was conducted to investigate the pharmacokinetics and metabolism of NO-1886 (diethyl 4-[(4-bromo-2-cyanophenyl) carbamoyl] benzylphosphonate) in cynomolgus monkeys.

2. After single intravenous administration of NO-1886 at a dose of 3?mg?kg^?1, the total clearance (CL_tot), area under the plasma concentration–time curve (AUC_0–t), half-life (t_1/2), and volume of distribution (V_d) in cynomolgus monkeys were 531?ml?h^?1?kg^?1, 5.63?µg?h?ml^?1, 0.96?h and 679?ml?kg^?1, respectively. The AUC_0–t for oral administration of NO-1886 (3?mg?kg^?1) was 4.23?µg?h?ml^?1 and the bioavailability was 75%.

3. M-2 (ethyl 4-[(4-bromo-2-cyanophenyl) carbamoyl] benzylphosphonate) and M-3 (4-[(diethoxy-phosphoryl) methyl)] benzoic acid) were present as metabolites in plasma and urine. In faeces, M-2 was present but M-3 was not.

4. The major metabolite of NO-1886 in liver S9 or microsomes was M-2 in the presence of NADPH. On the other hand, M-3 was formed in the absence of NADPH in liver S9 or microsomes and its formation was inhibited by bis-(?p-nitrophenyl) phosphate (BNPP) in liver S9, suggesting that the formation of M-3 was catalysed by carboxylesterase.

5. The findings suggest that the main metabolic pathway of NO-1886 in cynomolgus monkeys is the O-deethylation of NO-1886 to M-2, as in rats and humans, and that the hydrolysis of the amide bond is a minor metabolic pathway.