Arabidopsis thaliana ferrochelatase-I and -II are not imported into Arabidopsis mitochondria

Using in vitro import assays into purified mitochondria and chloroplasts we found that Arabidopsis ferrochelatase-I and ferrochelatase-II were not imported into mitochondria purified from Arabidopsis (or several other plants) but were imported into pea leaf chloroplasts. Other dual targeted proteins could be imported into purified mitochondria from Arabidopsis. As only two ferrochelatase genes are present in the completed Arabidopsis genome, the presence of ferrochelatase activity in plant mitochondria needs to be re-evaluated. Previous reports of Arabidopsis ferrochelatase-I import into pea mitochondria are due to the fact that pea leaf (and root) mitochondria appear to import a variety, but not all chloroplast proteins. Thus pea mitochondria are not a suitable system to either study dual targeting, or to distinguish between isozymes present in mitochondria and chloroplasts.     

Identification of nonsulfated cholecystokinin-58 in canine intestinal extracts and its biological properties

Nonsulfated CCK(58) [CCK(58)(ns)] has not been considered to be of biological importance because CCK(58)(ns) binds poorly to the CCK(A) receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the COOH-terminal region of gastrin and CCK (antibody 5135) was used to monitor the purification of CCK molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange, HPLC step. Characterization of this minor immunoreactive peak demonstrated that it was CCK(58)(ns). CCK(58)(ns) is 14% as immunoreactive as sulfated CCK(8) [CCK(8)(s)]. Amino acid analysis demonstrated that CCK(58)(ns) was present at 50% the amount of CCK(58)(s). In addition, we found that CCK(58)(ns) does not potently displace an (125)I-labeled CCK(10) analog from the CCK(A) receptor in mouse pancreatic membranes and does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK(58)(ns) does bind to CCK(B) receptors and stimulates gastric acid secretion via this receptor. The presence of CCK(58)(ns) and its ability to selectively stimulate the CCK(B) receptor without stimulation of the CCK(A) receptor suggest that CCK(58)(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent.     

Schizosaccharomyces pombe cells deficient in triacylglycerols synthesis undergo apoptosis upon entry into the stationary phase

Triacylglycerols (TAG) are important energy storage molecules for nearly all eukaryotic organisms. In this study, we found that two gene products (Plh1p and Dga1p) are responsible for the terminal step of TAG synthesis in the fission yeast Schizosaccharomyces pombe through two different mechanisms: Plh1p is a phospholipid diacylglycerol acyltransferase, whereas Dga1p is an acyl-CoA:diacylglycerol acyltransferase. Cells with both dga1+ and plh1+ deleted (DKO cells) lost viability upon entry into the stationary phase and demonstrated prominent apoptotic markers. Exponentially growing DKO cells also underwent dramatic apoptosis when briefly treated with diacylglycerols (DAGs) or free fatty acids. We provide strong evidence suggesting that DAG, not sphingolipids, mediates fatty acids-induced lipoapoptosis in yeast. Lastly, we show that generation of reactive oxygen species is essential to lipoapoptosis.     

A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.     

Chlorobium Tepidum: Insights into the Structure,Physiology, and Metabolism of a Green Sulfur Bacterium Derived from the Complete Genome Sequence

Green sulfur bacteria are obligate, anaerobic photolithoautotrophs that synthesize unique bacteriochlorophylls (BChls) and a unique light-harvesting antenna structure, the chlorosome. One organism, Chlorobium tepidum, has emerged as a model for this group of bacteria primarily due to its relative ease of cultivation and natural transformability. This review focuses on insights into the physiology and biochemistry of the green sulfur bacteria that have been derived from the recently completed analysis of the 2.15-Mb genome of Chl. tepidum. About 40 mutants of Chl. tepidum have been generated within the last 3 years, most of which have been made based on analyses of the genome. This has allowed a nearly complete elucidation of the biosynthetic pathways for the carotenoids and BChls in Chl. tepidum, which include several novel enzymes specific for BChl c biosynthesis. Facilitating these analyses, both BChl c and carotenoid biosynthesis can be completely eliminated in Chl. tepidum. Based particularly on analyses of mutants lacking chlorosome proteins and BChl c, progress has also been made in understanding the structure and biogenesis of chlorosomes. In silico analyses of the presence and absence of genes encoding components involved in electron transfer reactions and carbon assimilation have additionally revealed some of the potential physiological capabilities, limitations, and peculiarities of Chl. tepidum. Surprisingly, some structural components and biosynthetic pathways associated with photosynthesis and energy metabolism in Chl. tepidum are more similar to those in cyanobacteria and plants than to those in other groups of photosynthetic bacteria.     

Alkaline environmental pH has no effect on ammonia excretion in the mudskipper Periophthalmodon schlosseri but inhibits ammonia excretion in the related species Boleophthalmus boddaerti

Experiments were performed to evaluate the effects of alkaline environmental pH on urea and ammonia excretion rates and on tissue urea, ammonia, and free amino acid concentrations in two mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti. Periophthalomodon schlosseri is known to be capable of actively excreting ammonia. The rate of ammonia excretion in B. boddaerti exposed to 50% seawater (brackish water, BW) at pH 9 decreased significantly during the first 2 d of exposure when compared with that of specimens exposed to pH 7 or 8. This suggested that B. boddaerti was dependent on NH(3) diffusion for ammonia excretion, as in most fishes. It was incapable of detoxifying the accumulating endogenous ammonia to urea but could store and tolerate high concentrations of ammonia in the muscle, liver, and plasma. It did not undergo reductions in proteolysis and/or amino acid catabolism in alkaline water, probably because the buildup of endogenous ammonia was essential for the recovery of the normal rate of ammonia excretion by the third day of exposure to a pH 9 medium. Unlike B. boddaerti, P. schlosseri did not accumulate ammonia in the body at an alkaline pH (i.e., pH 9) because it was capable of actively excreting ammonia. Periophthalmodon schlosseri did not undergo partial amino acid catabolism (no accumulation of alanine) either, although there might be a slight reduction in amino acid catabolism in general. The significant decrease in blood pCO(2) in B. boddaerti at pH 9 might lead to respiratory alkalosis in the blood. In contrast, P. schlosseri was able to maintain its blood pH in BW at pH 9 despite a decrease in pCO(2) in the blood. With 8 mM NH(4)Cl in BW at pH 7, both mudskippers could actively excrete ammonia, although not to the same extent. Only P. schlosseri could sustain ammonia excretion against 8 mM NH(4)Cl in BW at pH 8. In BW containing 8 mM NH(4)Cl at pH 9, both mudskippers died within a short period of time. Boleophthalmus boddaerti consistently died faster than did P. schlosseri. This indicates that the body surfaces of these mudskippers were permeable to NH(3), but the skin of P. schlosseri might be less permeable to NH(3) than that of B. boddaerti. Both mudskippers excreted acid (H(+)) to alter the pH of the alkaline external medium. Such a capability, together with modifications in gill morphology and morphometry as in P. schlosseri, might be essential to the development of an effective mechanism for the active excretion of NH+4.     

CCK-58 is the only detectable endocrine form of cholecystokinin in rat

CCK-58 differs from CCK-8 in patterns of expression of pancreatic secretion of fluid and amylase and gallbladder contraction. These differences have physiological relevance only if CCK-58 release is stimulated by nutrients entering the intestine and if CCK-58 circulates in sizeable amounts. In this study, we report that when radiolabeled CCK-58 is added to rat blood and plasma is formed, there is extensive loss and degradation of the radioactive peptide. Therefore, a new method was developed to minimize loss and degradation of this label. This method recovered >85% of the label with no detectable degradation. Furthermore, the optimized method recovered all unlabeled exogenous cholecystokinin molecular forms in >80% yields. Blood from fasted rats and rats in which cholecystokinin release was stimulated by the trypsin inhibitor camostat contained only CCK-58 (3.5 +/- 0.5 and 17 +/- 1.5 fmol/ml, respectively). Because CCK-58 predominates in the blood, this molecular form should be used in studies on the physiology and pathophysiology of cholecystokinin.     

The sleeper Bostrichthys sinensis (family Eleotridae) stores glutamine and reduces ammonia production during aerial exposure

Bostrichthys sinensis inhabits brackish water, living in the crevices of the river mouths of Shang Xi and Guangdong, China. In its natural habitat, it may encounter aerial exposure frequently during low tides, and it usually remains quiescent in the absence of water. Upon aerial exposure in the laboratory, the ammonia excretion rate decreased to one-fourth that of the submerged control. Although all the enzymes of the ornithine-urea cycle were detected in the liver of this fish, the activity of hepatic carbamoyl phosphate synthetase was too low for the cycle to be functioning. Indeed, ammonia accumulated in the tissues and was not converted to urea. Results indicate that ammonia produced through amino acid catabolism was detoxified to glutamine during the first 24 h of aerial exposure. The excess amount of glutamine stored in the muscle during this period couldaccount approximately for the reduction in ammonia equivalent excreted. There was indeed a significant increase in the activity of glutamine synthetase from the liver of specimens exposed to terrestrial conditions. In contrast to the production of alanine, formation of glutamine is energetically expensive. Since B. sinensis remained relatively inactive on land, the reduction in energy demand for muscular activity might provide it with the opportunity to exploit glutamine formation as a means to detoxify ammonia. After 72 h of aerial exposure, B. sinensis reduced internal ammonia production, possibly through reductions in proteolysis and amino acid catabolism, to avoid excessive accumulation of ammonia.