Regulation of mitochondrial contact sites in neonatal,juvenile and diabetic hearts

Mitochondrial contact sites (MiCS) are dynamic structures involved in high capacity transport of energy from mitochondria into the cytosole. Previous studies revealed that in normal conditions the actual number of MiCS is in correlation with the energy requirements of the heart, particularly with those for its contractile work. Although the detailed mechanisms of signalling between the processes of energy utilisation and MiCS formation in the heart are not yet elucidated, it is known that intracellular Ca²⁺ transients are intimately involved in this crosstalk. The present study is devoted to investigation of Ca²⁺-linked MiCS formation in healthy adult hearts and in hearts with modified Ca²⁺-handling such as in developing, in juvenile and diabetic myocardium. Experiments were performed on hearts of healthy rats on the 22nd embryonal day, 1st, 4th, 7th and 14th postnatal days as well as on adult hearts. Diabetic hearts were investigated on the 8th day after streptozotocin injection (45 mg.kg^–1 i.v.) to adult rats. Intracellular Ca²⁺ movements were affected by modulation of Ca²⁺ concentration in perfusion solution (1.6 or 2.2 mmol.l^–1) in isolated, Langendorff-perfused hearts, by calcium paradox (CaP) or by replacing of Ca²⁺ by Cd²⁺ ions. Elevation of extracellular Ca²⁺ was reflected by 30.1, 10.4 and 24.1% increase in intracellular free Ca²⁺ concentration in healthy adult, diabetic and 14-day old hearts respectively. In developing hearts the amount of MiCS was culminating on the 4th postnatal day. In adult hearts, elevated calcium in the perfusion solution, CaP as well as diabetes led to a significant increase in the amounts of MiCS formed (58.1, 77.2 and 86.5% respectively; p < 0.05). Diabetic and 14-day old hearts naturally exhibited amounts of MiCS comparable to those obtained by Ca²⁺-stimulation of MiCS formation in adult healthy hearts. In contrast to healthy controls, perfusion of diabetic and 14-day old hearts with elevated Ca²⁺ as well as induction of CaP exerted little influence on MiCS formation (4.4 and 8.2% for elevated Ca²⁺; 2.9 and 10.7% for CaP; p > 0.05). A replacement of Ca²⁺ by Cd²⁺ ions lowered the amount of MiCS in healthy adult and diabetic hearts (61 and 52.2%; p < 0.05). In conclusion, during development, the formation of MiCS may be influenced by both, permanent stimulation by Ca²⁺-signalling and the availability of mCPK. In healthy adult hearts the amount of MiCS is modulated by intracellular Ca²⁺ transients in response to changes in extracellular Ca²⁺ concentration. In diabetic hearts the modulation of MiCS formation is naturally attenuated, apparently as a consequence of persisting alterations in Ca²⁺-handling.     

Polyketide-nonribosomal peptide epothilone antitumor agents: the EpoA,B, C subunits

The epothilones are a family of macrolactone natural products from the myxobacterial species Sorangium cellulosum. Similar to taxol, they are of current clinical interest as anticancer agents. Sequence analysis of the epothilone gene cluster allowed the identification of polyketide synthase and nonribosomal peptide synthetase modules involved in catalyzing epothilone biosynthesis. Given this information, it has been possible to test the predicted functions of several modules to date. EpoA ACP, EpoB, and EpoC have been overproduced in Escherichia coli, allowing in vitro reconstitution of the EpoA/B/C interface and production of the expected epothilone precursor. Further experiments probed the tolerance of EpoB and EpoC for unnatural substrates. These studies of the first three modules of the epothilone biosynthetic cluster suggest that combinatorial biosynthesis may lead to the production of a variety of epothilone analogs that incorporate diversity into the heterocycle starter unit. Additional efforts with the remaining modules, coupled with increased understanding of the macrocyclizing thioesterase domain, may lead to the production of epothilone variants with improved clinical properties.     

Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-coenzyme a thiolase into glyoxysomes

Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.     

Small intestinal submucosa in abdominal wall repair after TRAM flap harvesting in a rat model

The strength of porcine small intestinal submucosa in abdominal wall repair after transverse rectus abdominis myocutaneous flap harvesting was examined in a rat model. Changes in the levels of selected molecular markers of inflammation after small intestinal submucosa implantation were also studied. Eighty-three rats were divided into three groups. In experimental group I, an abdominal wall defect created by removal of the rectus abdominis muscle was repaired with placement of a 1.5 x 5-cm2 patch of small intestinal submucosa. In experimental group II, the muscle defect was repaired with a combination of small intestinal submucosa patch placement and fascial closure. In the control group, the defect was repaired with direct fascial closure. At postoperative times of 3 days, 2 weeks, 1 month, and 2 months, the muscle tissues adjacent to the abdominal wall repair site were subjected to biopsies for assessment of inflammation markers. Full-thickness sections of the abdominal wall from the repair site in each animal were removed for tensile strength testing and histological examinations. The results demonstrated that interleukin-6 and interferon-gamma levels were increased in the two experimental, small intestinal submucosa-treated groups at 3 days and 2 weeks postoperatively. The results of mechanical testing demonstrated that the average tensile strength of the repaired abdominal wall in the repair model with combined small intestinal submucosa placement and fascial repair was significantly greater than the values for repairs with fascial closure or small intestinal submucosa placement alone. The use of small intestinal submucosa placement in combination with fascial repair can significantly improve the strength of the repaired abdominal wall after transverse rectus abdominis myocutaneous flap harvesting.     

Enamel thickness,microstructure and development in Afropithecus turkanensis

Afropithecus turkanensis, a 17-17.5 million year old large-bodied hominoid from Kenya, has previously been reported to be the oldest known thick-enamelled Miocene ape. Most investigations of enamel thickness in Miocene apes have been limited to opportunistic or destructive studies of small samples. Recently, more comprehensive studies of enamel thickness and microstructure in Proconsul, Lufengpithecus, and Dryopithecus, as well as extant apes and fossil humans, have provided information on rates and patterns of dental development, including crown formation time, and have begun to provide a comparative context for interpretation of the evolution of these characters throughout the past 20 million years of hominoid evolution. In this study, enamel thickness and aspects of the enamel microstructure in two A. turkanensis second molars were quantified and provide insight into rates of enamel apposition, numbers of cells actively secreting enamel, and the time required to form regions of the crown. The average value for relative enamel thickness in the two molars is 21.4, which is a lower value than a previous analysis of this species, but which is still relatively thick compared to extant apes. This value is similar to those of several Miocene hominoids, a fossil hominid, and modern humans. Certain aspects of the enamel microstructure are similar to Proconsul nyanzae, Dryopithecus laietanus, Lufengpithecus lufengensis, Graecopithecus freybergi and Pongo pygmaeus, while other features differ from extant and fossil hominoids. Crown formation times for the two teeth are 2.4-2.6 years and 2.9-3.1 years respectively. These times are similar to a number of extant and fossil hominoids, some of which appear to show additional developmental similarities, including thick enamel. Although thick enamel may be formed through several developmental pathways, most Miocene hominoids and fossil hominids with relatively thick enamel are characterized by a relatively long period of cuspal enamel formation and a rapid rate of enamel secretion throughout the whole cusp, but a shorter total crown formation time than thinner-enamelled extant apes.     

The Quality of Mental Health Care for African Americans

In response to the Surgeon General's request for more research on racial disparities in mental health care, especially research that includes high-need populations (e.g., the homeless, incarcerated, children in foster care, and substance abusers), we examined racial disparities in the provision of mental health counseling, psychotherapy, and pharmacotherapy in hospital outpatient settings using nationally representative data from the 1997 National Hospital Ambulatory Medical Care Survey (NHAMCS). After controlling for diagnosis and other factors, we found that African Americans were less likely than whites to receive mental health counseling and psychotherapy, but more likely than whites to receive pharmacotherapy. We also found that substance abuse clinics were more likely than primary care and specialty mental health clinics to provide mental health counseling and psychotherapy. However, specialty mental health clinics were the only clinics to provide pharmacotherapy. Future research should examine racial disparities in a variety of settings, controlling for diagnosis as well as other factors.     

Mitogen-activated protein kinases in the acute diabetic myocardium

Diabetes mellitus (DM) causes myocardial remodeling on the subcellular level and alterations in the function of the cell membranes ion transport systems resulting in contractile dysfunction. The present study was aimed to investigate the expression and activation of mitogen-activated protein kinases (MAPKs) and their possible role in the acute diabetic rat hearts. Rats were injected with single dose of streptozotocin (45 mg/kg, i.v.), and after 1 week the disease was manifested by hyperglycemia and cardiac dysfunction. The Langendorff-perfused hearts were subjected to ischemia (5 or 30 min occlusion of LAD coronary artery). The protein pattern in cytosolic fraction of the heart tissue was determined after electrophoretic separation. The levels and activation of MAPKs were determined by Western blot analysis using specific antibodies. No differences between the diabetics and controls in the level of ERKs were found at baseline. However, in DM samples ERKs phosphorylation was markedly increased, and further changes occurred during ischemia. Also content of phoshorylated c-Raf kinase (an upstream activator of ERKs) was slightly increased at baseline conditions in the diabetic samples. In contrast, no significant changes in the contents and phosphorylation of p38-MAPK were observed at baseline. But some differences in the p38-MAPK phosphorylation were found during ischemia.The results show that differential pattern of protein kinase cascades activation in the diabetic hearts might be account for the modulation of their response to ischemia.     

HASTY,the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis

Loss-of-function mutations of HASTY (HST) affect many different processes in Arabidopsis development. In addition to reducing the size of both roots and lateral organs of the shoot, hst mutations affect the size of the shoot apical meristem, accelerate vegetative phase change, delay floral induction under short days, adaxialize leaves and carpels, disrupt the phyllotaxis of the inflorescence, and reduce fertility. Double mutant analysis suggests that HST acts in parallel to SQUINT in the regulation of phase change and in parallel to KANADI in the regulation of leaf polarity. Positional cloning demonstrated that HST is the Arabidopsis ortholog of the importin beta-like nucleocytoplasmic transport receptors exportin 5 in mammals and MSN5 in yeast. Consistent with a potential role in nucleocytoplasmic transport, we found that HST interacts with RAN1 in a yeast two-hybrid assay and that a HST-GUS fusion protein is located at the periphery of the nucleus. HST is one of at least 17 members of the importin-beta family in Arabidopsis and is the first member of this family shown to have an essential function in plants. The hst loss-of-function phenotype suggests that this protein regulates the nucleocytoplasmic transport of molecules involved in several different morphogenetic pathways, as well as molecules generally required for root and shoot growth.     

Dental Ontogeny in Pliocene and Early Pleistocene Hominins

Until recently, our understanding of the evolution of human growth and development derived from studies of fossil juveniles that employed extant populations for both age determination and comparison. This circular approach has led to considerable debate about the human-like and ape-like affinities of fossil hominins. Teeth are invaluable for understanding maturation as age at death can be directly assessed from dental microstructure, and dental development has been shown to correlate with life history across primates broadly. We employ non-destructive synchrotron imaging to characterize incremental development, molar emergence, and age at death in more than 20 Australopithecus anamensis, Australopithecus africanus, Paranthropus robustus and South African early Homo juveniles. Long-period line periodicities range from at least 6–12 days (possibly 5–13 days), and do not support the hypothesis that australopiths have lower mean values than extant or fossil Homo. Crown formation times of australopith and early Homo postcanine teeth fall below or at the low end of extant human values; Paranthropus robustus dentitions have the shortest formation times. Pliocene and early Pleistocene hominins show remarkable variation, and previous reports of age at death that employ a narrow range of estimated long-period line periodicities, cuspal enamel thicknesses, or initiation ages are likely to be in error. New chronological ages for SK 62 and StW 151 are several months younger than previous histological estimates, while Sts 24 is more than one year older. Extant human standards overestimate age at death in hominins predating Homo sapiens, and should not be applied to other fossil taxa. We urge caution when inferring life history as aspects of dental development in Pliocene and early Pleistocene fossils are distinct from modern humans and African apes, and recent work has challenged the predictive power of primate-wide associations between hominoid first molar emergence and certain life history variables.