Chloroplast division in cultured cells of the hornwortAnthoceros punctatus

Using cultured cells of the hornwortAnthoceros punctatus, the change in the relative chloroplast DNA content in each stage of chloroplast division was investigated to clarify the relationship between the division cycle of a chloroplast and a cell nucleus. Samples of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then observed with an epifluorescence microscope and a chromosome image analyzing system (CHIAS). A chloropiast in cultured cells duplicated DNA with an increase in size. When a chloroplast began to divide, it was constricted in the middle, taking a dumbbell shape, and then divided into two daughter chloroplasts. In cultured cells of this species, the pattern of quantitative change of chloroplast DNA, that is, the DNA replication pattern of chloroplasts, corresponded to that of cell nuclear DNA in mitosis.     

Molecular structure of the Japanese hepatitis C viral genome

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et. al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524–9528)indicated that this virus is a member of a new class of positive-stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin-like serine proteinase. NTPase and RNA-dependent RNA polymerase     

Metabolomic Derangements Are Associated with Mortality in Critically Ill Adult Patients

Objective

To identify metabolomic biomarkers predictive of Intensive Care Unit (ICU) mortality in adults.

Rationale

Comprehensive metabolomic profiling of plasma at ICU admission to identify biomarkers associated with mortality has recently become feasible.

Methods

We performed metabolomic profiling of plasma from 90 ICU subjects enrolled in the BWH Registry of Critical Illness (RoCI). We tested individual metabolites and a Bayesian Network of metabolites for association with 28-day mortality, using logistic regression in R, and the CGBayesNets Package in MATLAB. Both individual metabolites and the network were tested for replication in an independent cohort of 149 adults enrolled in the Community Acquired Pneumonia and Sepsis Outcome Diagnostics (CAPSOD) study.

Results

We tested variable metabolites for association with 28-day mortality. In RoCI, nearly one third of metabolites differed among ICU survivors versus those who died by day 28 (N = 57 metabolites, p<.05). Associations with 28-day mortality replicated for 31 of these metabolites (with p<.05) in the CAPSOD population. Replicating metabolites included lipids (N = 14), amino acids or amino acid breakdown products (N = 12), carbohydrates (N = 1), nucleotides (N = 3), and 1 peptide. Among 31 replicated metabolites, 25 were higher in subjects who progressed to die; all 6 metabolites that are lower in those who die are lipids. We used Bayesian modeling to form a metabolomic network of 7 metabolites associated with death (gamma-glutamylphenylalanine, gamma-glutamyltyrosine, 1-arachidonoylGPC(20:4), taurochenodeoxycholate, 3-(4-hydroxyphenyl) lactate, sucrose, kynurenine). This network achieved a 91% AUC predicting 28-day mortality in RoCI, and 74% of the AUC in CAPSOD (p<.001 in both populations).

Conclusion

Both individual metabolites and a metabolomic network were associated with 28-day mortality in two independent cohorts. Metabolomic profiling represents a valuable new approach for identifying novel biomarkers in critically ill patients.     

Catalytic properties of domain-exchanged chimeric proteins between firefly luciferase and Drosophila fatty Acyl-CoA synthetase CG6178

Firefly luciferase and fatty acyl-CoA synthetase are members of the acyl-CoA synthetase super family, which consists of a large N-terminal domain and a small C-terminal domain. Previously we found that firefly luciferase has fatty acyl-CoA synthetic activity, and also identified that the homolog of firefly luciferase in Drosophila melanogaster (CG6178) is a fatty acyl-CoA synthetase and is not a luciferase. In this study, we constructed chimeric proteins by exchanging the domain between Photinus pyralis luciferase (PpLase) and Drosophila CG6178, and determined luminescence and fatty acyl-CoA synthetic activities. A chimeric protein with the N-terminal domain of PpLase and the C-terminal domain of CG6178 (Pp/Dm) had luminescence activity, showing approximately 4% of the activity of wild-type luciferase. The Pp/Dm protein also had fatty acyl-CoA synthetic activity and the substrate specificity was similar to PpLase. In contrast, a chimeric protein with the N-terminal domain of CG6178 and the C-terminal of PpLase (Dm/Pp) had only fatty acyl-CoA synthetase activity, and the substrate specificity was similar to CG6178. These results suggest that the N-terminal domain of firefly luciferase is essential for substrate recognition, and that the C-terminal domain is indispensable but not specialized for the luminescence reaction.     

Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.     

Conversion of gastric mucosa to intestinal metaplasia in Cdx2-expressing transgenic mice

Gastric intestinal metaplasia occurs as a pathological condition in the gastric mucosa. To clarify how an intestine-specific homeobox gene, Cdx2, affects the morphogenesis of gastric mucosa, we generated transgenic mice expressing Cdx2 in parietal cells. Until Day 18 after birth, the number of parietal cells inthegastric mucosa of transgenic mice was the same as for their normal littermates. However, at Day 19, we detected several glands in which parietal cells disappeared and the proliferating zone moved from the isthmus to the base of the glands. Thereafter, parietal cells decreased gradually and disappeared at Day 37. All of the gastric mucosal cells, except for enterochromaffin-like (ECL) cells, were completely replaced by intestinal metaplasia, consisting of goblet cells, enteroendocrine cells, and absorptive cells expressing alkaline phosphatase. Pseudopyloric gland metaplasia was also formed. The transgenic mouse is a very useful model for clarifying physiological differentiation of gastric and intestinal cell lineages and analyzing the molecular events from intestinal metaplasia to adenocarcinoma.     

Adenosine A2A Receptors Measured with [11C]TMSX PET in the Striata of Parkinson's Disease Patients

Adenosine A_2A receptors (A2ARs) are thought to interact negatively with the dopamine D₂ receptor (D2R), so selective A2AR antagonists have attracted attention as novel treatments for Parkinson''s disease (PD). However, no information about the receptor in living patients with PD is available. The purpose of this study was to investigate the relationship between A2ARs and the dopaminergic system in the striata of drug-naïve PD patients and PD patients with dyskinesia, and alteration of these receptors after antiparkinsonian therapy. We measured binding ability of striatal A2ARs using positron emission tomography (PET) with [7-methyl-¹¹C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([¹¹C]TMSX) in nine drug-naïve patients with PD, seven PD patients with mild dyskinesia and six elderly control subjects using PET. The patients and eight normal control subjects were also examined for binding ability of dopamine transporters and D2Rs. Seven of the drug-naïve patients underwent a second series of PET scans following therapy. We found that the distribution volume ratio of A2ARs in the putamen were larger in the dyskinesic patients than in the control subjects (p<0.05, Tukey-Kramer post hoc test). In the drug-naïve patients, the binding ability of the A2ARs in the putamen, but not in the head of caudate nucleus, was significantly lower on the more affected side than on the less affected side (p<0.05, paired t-test). In addition, the A2ARs were significantly increased after antiparkinsonian therapy in the bilateral putamen of the drug-naïve patients (p<0.05, paired t-test) but not in the bilateral head of caudate nucleus. Our study demonstrated that the A2ARs in the putamen were increased in the PD patients with dyskinesia, and also suggest that the A2ARs in the putamen compensate for the asymmetrical decrease of dopamine in drug-naïve PD patients and that antiparkinsonian therapy increases the A2ARs in the putamen. The A2ARs may play an important role in regulation of parkinsonism in PD.     

Mother–young distance in Japanese Black cattle at pasture

Distance between dam and offspring (1–121 days old) in a herd of Japanese Black cattle (Bos taurus) grazing a tropical grass (Paspalum notatum) pasture (1.5 ha) was investigated during 7-h grazing periods over grazing seasons from May (spring) to October (autumn). The mother–young distance was not constant throughout the grazing period, repeatedly increasing and decreasing. Although significant periodicity was always detected in the mother–young distance, there was no consistent dominant cycle, indicating the complexity of the within-day pattern of mother–young distance. The mean mother–young distance over the grazing period increased as a calf aged, reaching a plateau at an age of about 33 days. The mean distance of a calf from its mother was usually shorter than that from a non-mother cow, with the difference between the mean distances decreasing sharply until a calf became about 35 days old. The results and literature show that mutual independence of mother and young rapidly develops in the first 30–50 days after parturition. Electronic Publication     

Changes of aldolase A and B messenger RNA levels in rat liver during azo-dye-induced hepatocarcinogenesis

The expression of aldolase A and B mRNAs during azo-dye-induced carcinogenesis in rat liver was examined. After feeding the dye for 18 weeks, the level of aldolase A mRNA increased to about 11 times that in a normal liver, with the concomitant decrease of aldolase B mRNA level to about 25% of that in a normal liver. These changes did not occur progressively during the carcinogenesis, but occurred as an additional phase after 4 week-feeding of the azo-dye. At this stage, the levels of aldolase A and B mRNAs were about 7 times and 45% of that in a normal liver, respectively. This biphasic pattern in the aldolase isozyme expression in the azo-dye-fed rat liver is discussed together with the kinetic data of the enzyme activity.