Phorbol ester induced trafficking-independent regulation and enhanced phosphorylation of the dopamine transporter associated with membrane rafts and cholesterol

We examined the mechanisms involved in protein kinase C (PKC)-dependent down-regulation of dopamine transporter (DAT) activity and cell surface expression by treating heterologously expressing cells with the clathrin-mediated endocytosis inhibitor concanavalin A (Con A) or the cholesterol depleter/membrane raft disrupter methyl-β-cyclodextrin (MβC) prior to treatment with the PKC activator phorbol 12-myristate, 13-acetate (PMA). Con A blocked PMA-induced surface reductions of DAT but only partially inhibited down-regulation, while MβC partially blocked down-regulation but did not inhibit loss of cell surface DAT, demonstrating that PKC-induced DAT down-regulation occurs by a combination of trafficking and non-trafficking processes. Using density-gradient centrifugation, we found that DATs are distributed approximately equally between Triton-insoluble, cholesterol-rich membrane rafts and Triton-soluble non-raft membranes. DATs in both populations are present at the cell surface and are active for dopamine and cocaine binding. PMA-induced loss of cell surface DAT occurred only from non-raft populations, demonstrating that non-raft DATs are regulated by trafficking events and indicating the likelihood that the cholesterol-dependent non-trafficking regulatory mechanism occurs in rafts. PMA did not affect the DAT raft-non-raft distribution but stimulated the phosphorylation of DAT to a substantially greater level in rafts than non-rafts. These findings reveal a previously unknown role for cholesterol in DAT function and demonstrate the presence of distinct subcellular DAT populations that possess multiple regulatory differences that may impact dopaminergic neurotransmission.     

Plant growth and development influenced by transgenic insertion of bacterial chitinolytic enzymes

The role of the chitinolytic enzymes in plants is not necessarilyrestricted to plant defense. Tomato plants transformed with an endochitinaseand a chitobiosidase gene from Streptomyces albidoflavus andgrowth under greenhouse conditions showed a significant reduction in plantheight, and reduced time to flowering compared with the control(non-transformed) plants. The levels of chitobiosidase and endochitinaseactivity in the transgenic tomato plants were positively correlated with earlyflowering, and negatively correlated with plant height. We have not determinedwhether these effects are exclusively due to the expression of the transgenesof endochitinase and chitobiosidase from S. albidoflavus orthe additive effect of these 2 enzymes combined with the endogenouschitinolytic enzymes produced by the plants. However, when control plants were trimmed,early flowering was observed compared with the controls that were not trimmed, whichindicates that wound induced proteins such as chitinolytic enzymes affect thetime of flowering. In addition, the expression of the endochitinase andchitobiosidase genes significantly increased the number of flowers and fruit onthe plants, resulting in an increase in yield of fruit. One of the primarygoals of crop breeding programs is to increase the productivity of plants. These twogenes were directly associated with plant productivity, and should be studied further.     

Ecological and Biogeochemical Interactions Constrain Planktonic Nitrogen Fixation in Estuaries

Many types of ecosystems have little or no N₂ fixation even when nitrogen (N) is strongly limiting to primary production. Estuaries generally fit this pattern. In contrast to lakes, where blooms of N₂-fixing cyanobacteria are often sufficient to alleviate N deficits relative to phosphorus (P) availability, planktonic N₂ fixation is unimportant in most N-limited estuaries. Heterocystic cyanobacteria capable of N₂ fixation are seldom observed in estuaries where the salinity exceeds 8–10 ppt, and blooms have never been reported in such estuaries in North America. However, we provided conditions in estuarine mesocosms (salinity over 27 ppt) that allowed heterocystic cyanobacteria to grow and fix N₂ when zooplankton populations were kept low. Grazing by macrozooplankton at population densities encountered in estuaries strongly suppressed cyanobacterial populations and N₂ fixation. The cyanobacteria grew more slowly than observed in fresh waters, at least in part due to the inhibitory effect of sulfate (SO₄ ²⁻), and this slow rate of growth increased their vulnerability to grazing. We conclude that interactions between physiological (bottom–up) factors that slow the growth rate of cyanobacteria and ecological (top–down) factors such as grazing are likely to be important regulators excluding planktonic N₂ fixation from most Temperate Zone estuaries. Received 26 April 2002; Accepted 12 July 2002.     

Towards an ecological understanding of biological nitrogen fixation

Biogeochemistry - N limitation to primary production and other ecosystem processes is widespread. To understand the causes and distribution of N limitation, we must understand the controls of...     

H<Superscript>N</Superscript>, N,C<Superscript>α</Superscript> and C<Superscript>β</Superscript> assignments of the two periplasmic domains of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> DsbD

DsbD is a disulfide bond reductase present in the inner membrane of many Gamma-Proteobacteria. In the human pathogen Neisseria meningitidis, DsbD is required for viability and represents a potential target for the development of antibiotics. Here we report the chemical shift assignments (H^N, N, C^α and C^β) for the reduced and oxidized forms of the two periplasmic domains of N. meningitidis DsbD, n-NmDsbD and c-NmDsbD. The backbone amide resonances in all four forms were completely assigned, and the secondary structures for the core regions of the proteins were calculated using ¹³C^αβ shifts. The reduced and oxidized forms of each domain have similar secondary shifts suggesting they retain the same fold. We anticipate that these data will provide an important basis for studying the interaction between n-NmDsbD and c-NmDsbD, which is required for electron transfer across the bacterial cytoplasmic membrane.     

Expression levels of ACAT1 and ACAT2 genes in the liver and intestine of baboons with high and low lipemic responses to dietary lipids

Acyl–coenzyme A:cholesterol acyltransferase (ACAT) 1 and ACAT2 play an important role in cellular cholesterol esterification and thus modulate intestinal cholesterol absorption and hepatic lipoprotein secretion. The relative expression levels of ACAT1 and ACAT2 in human tissues differ from those in other animals, including nonhuman primates. The present study compared the relative expression levels of ACAT1 and ACAT2 in baboons with high and low lipemic responses to dietary lipids. We isolated RNA and prepared cDNA from frozen liver and small intestine from high- and low-responding pedigreed baboons necropsied after consuming a high-cholesterol and high-fat diet for 18 months. The expression of ACAT1 and ACAT2 was measured by TaqMan real-time quantitative PCR normalized to 18s ribosomal RNA. The expression of ACAT1 was higher than that of ACAT2 in the liver, whereas the expression of ACAT2 was higher than that of ACAT1 in the duodenum and jejunum. There was no difference in the expression of ACAT1 or ACAT2 in the liver and intestine between high- and low-responding baboons except that the expression of ACAT1 was higher in the duodenum of high responders than in that of low responders. Western blot analysis also showed a higher level of ACAT1 protein in the duodenum of high responders than in that of low responders. There was a significant correlation between duodenal ACAT expression levels and total plasma cholesterol concentration in baboons. These results suggest that differences in ACAT1 expression may affect plasma cholesterol concentration and partly affect diet-induced hyperlipidemia.     

Arachidonic acid release by cPLA2 may be causally related to NO-induced apoptosis in vascular smooth muscle cells

Apoptosis has been shown to occur in vascular smooth muscle cells during the development of atherosclerosis. In order to investigate the possible role of arachidonic acid during apoptosis in vascular smooth muscle, we induced apoptosis in cultured rat aortal smooth muscle cells (SMCs) by treatment with either UV (ultraviolet) radiation, tumor necrosis factor-alpha (TNF-alpha) or NO donor drugs (sodium nitroprusside, or S-nitroso-N-acetyl-D-penicillamine, SNAP). Apoptosis was detected by either DNA fragmentation analysis or by TUNEL analysis. UV radiation, TNF-alpha and NO were observed to stimulate apoptosis in the cells as well as to stimulate arachidonate release from the cells. NO also increased levels of cPLA2 in the cells, which is an enzyme that is frequently activated in cells that release arachidonate. These agents stimulated arachidonate release somewhat earlier than they stimulated apoptosis in the cells. The inhibition of cPLA2 by arachidonyl trifluoromethyl ketone (AACOCF3) also led to the inhibition of arachidonate release from the cells as well as the inhibition of nitroprusside stimulated apoptosis. Arachidonic acid itself could induce apoptosis in the cultured cells. These observations provide evidence that arachidonate may be involved in apoptosis in vascular smooth muscle.     

Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the -glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.     

Cognitive status and behavioral problems in older hospitalized patients

OBJECTIVES: (a) To determine the quantity and quality of behavioral problems in older hospitalized patients on acute care units; (b) to determine the burden of these behaviors on staff; and (c) to identify predictors of behavioral problems. METHODS: Upon admission, patients performed the Mini-Mental State Exam (MMSE), the Geriatric Depression Scale (GDS), and information was obtained on age, ethnicity, level of education, living arrangement, and psychiatric history. Two days post-admission, a clinical staff member caring for each patient, performed the Neuropsychiatric Inventory-Questionnaire (NPI-Q) to assess patients' behavioral problems and staff distress. PARTICIPANTS AND SETTING : Forty-two patients, over 60 years of age, admitted to medical and surgical units of the Veterans Affairs Hospitals in Palo Alto and San Francisco, participated. RESULTS: Twenty-three of 42 (55%) patients exhibited behavioral problems. Anxiety, depression, irritability, and agitation/aggression were the most frequently observed behaviors. The severity of the behavioral problems was significantly correlated with staff distress. Lower performance on the MMSE at admission was significantly associated with higher NPI-Q ratings. Specifically, of those cases with scores less than or equal to 27 on the MMSE, 66% had behavioral problems during hospitalization, compared to only 31% of those with scores greater than 27. CONCLUSION: Behavioral problems in older hospitalized patients appear to occur frequently, are a significant source of distress to staff, and can result in the need for psychiatric consultation. Assessment of the mental status of older adults at admission to hospital may be valuable in identifying individuals at increased risk for behavioral problems during hospitalization.