Oleate and linoleate enhance the growth-promoting effects of insulin-like growth factor-I through a phospholipase D-dependent pathway in arterial smooth muscle cells

Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis.     

Specific sequences determine the stability and cooperativity of folding of the C-terminal half of tropomyosin

Tropomyosin is a flexible 410 A coiled-coil protein in which the relative stabilities of specific regions may be important for its proper function in the control of muscle contraction. In addition, tropomyosin can be used as a simple model of natural occurrence to understand the inter- and intramolecular interactions that govern the stability of coiled-coils. We have produced eight recombinant tropomyosin fragments (Tm(143-284(5OHW),) Tm(189-284(5OHW)), Tm(189-284), Tm(220-284(5OHW)), Tm(220-284), Tm(143-235), Tm(167-260), and Tm(143-260)) and one synthetic peptide (Ac-Tm(215-235)) to investigate the relative conformational stability of different regions derived from the C-terminal region of the protein, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that the fragments that include the last 24 residues of the molecule (Tm(143-284(5OHW)), Tm(189-284(5OHW)), Tm(220-284(5OHW)), Tm(220-284)) are completely dimerized at 10 microm dimer (50 mm phosphate, 100 mm NaCl, 1.0 mm dithiothreitol, and 0.5 mm EDTA, 10 degrees C), whereas fragments that lack the native C terminus (Tm(143-235),Tm(167-260), and Tm(143-260)) are in a monomer-dimer equilibrium under these conditions. The presence of trifluoroethanol resulted in a reduction in the [theta](222)/[theta](208) circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of urea-induced unfolding. Temperature and urea denaturation experiments show that Tm(143-235) is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e' positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of beta-branched residues located at d positions.     

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.     

Projection structure of full length connexin 43 by electron cryo-crystallography

We previously used electron cryo-crystallography to determine the three-dimensional structure of recombinant gap junction channels formed by a C-terminal truncation mutant of Cx43 (11). The dodecameric channel is formed by the end-to-end docking of two hexameric connexons, each comprised of 24 transmembrane alpha-helices. We have now generated two-dimensional crystals of the recombinant, full-length channel, as well as crystals in which the C-tail has been completely removed by trypsin digestion. Projection density maps at 7.5 A resolution closely resemble our previous analysis of the C-terminal truncation mutant (9). A difference map between the full length and trypsin-treated channels suggests that there are small but significant shifts in protein density upon removal of the C-tail.     

Mapping the domain of troponin T responsible for the activation of actomyosin ATPase activity. Identification of residues involved in binding to actin

The in vitro Ca(2+) regulation of the actomyosin Mg(2+)-ATPase at physiological ratios of actin, tropomyosin, and troponin occurs only in the presence of troponin T. We have previously demonstrated that a polypeptide corresponding to the first 191 amino acids of troponin T (TnT-(1-191)) activates the actomyosin Mg(2+)-ATPase in the presence of tropomyosin. In order to further characterize this activation domain, we constructed troponin T fragments corresponding to residues 1-157 (TnT-(1-157)), 1-76 (TnT-(1-76)), 77-157 (TnT-(77-157)), 77-191 (TnT-(77-191)), and 158-191 (TnT-(158-191)). Assays using these fragments demonstrated the following: (a) residues 1-76 do not bind to tropomyosin or actin; (b) residues 158-191 bind to actin cooperatively but not to tropomyosin; (c) the sequence 77-157 is necessary for troponin interaction with residue 263 of tropomyosin; (d) TnT-(77-191) on its own activates the actomyosin ATPase activity as described previously for TnT-(1-191). TnT-(1-157), TnT-(1-76), TnT-(77-157), TnT-(158-191), and combinations of TnT-(158-191) with TnT-(1-157) or TnT-(77-157) showed no effect on the ATPase activity. We conclude that the activation of actomyosin ATPase activity is mediated by a direct interaction between amino acids 77 and 191 of troponin T, tropomyosin, and actin.     

Is Fusarium culmorum isotrichodermin-15-hydroxylase different from other fungal species?

Fusarium spp. are ubiquitous fungi infecting cereals and grains, and therefore constitute a major problem for agriculture. Their trichothecene metabolites, and in particular deoxynivalenol and its 3-acetylated derivative, are the mycotoxins involved. The major metabolite produced by Fusarium culmorum is 3-acetyldeoxynivalenol. Studies in vivo with Fusarium culmorum have established that its tricyclic intermediate, isotrichodermin, is a major biosynthetic precursor, which is hydroxylated at position 15 to give 15-deacetylcalonectrin, prior to being converted to the product. In a preliminary in vitro investigation of the cell-free system involved in this transformation, we suggested that cytochrome P450 enzymes are not involved. In this paper, the isotrichodermin-15-hydroxylase from the microsomal fraction of Fusarium culmorum was solubilized and partially purified (60 fold). Our studies with cofactors indicate that this enzyme is a flavoprotein, and the inducers tested highly indicate that indeed the hydroxylase is not attached to cytochrome P450. This is particularly interesting, since the only other enzyme catalyzing the same reaction isolated from Fusarium sporotrichiodes is attached to cytochrome P450.     

Adrenal cortex and stomach lesions associated with stress in wild male African green monkeys (Cercopithecus aethiops) in the post-capture period

The objective of this study was to look for early pathological changes in stress target organs, adrenal glands, and stomachs in captured wild African green monkeys (AGMs). Three wild-caught male AGMs and seven singly housed wild AGMs were euthanized on day 1 and day 45 post-capture, respectively, and compared with four wild males euthanized with a rifle as controls. Morphometric analyses of the adrenal cortices and the cortical zones were done using an image analyzer. By day 45, the confined animals were clinically healthy, but had lost 47% mean body weight despite ad libitum feeding. The width of zona fasciculata in the controls was significantly smaller compared with that of 45-day monkeys (P < 0.05). Numerous acidophilic, hyperplastic and hypertrophic cells were present in the zona fasciculata of the 1-day confined AGMs. In the 45-day monkeys, there was glandular hyperplasia in the zona glomerulosa and the acini were distended and vacuous; yellow, granular pigmentation was distributed in the zona fasciculata. Acute stomach lesions represented by petechiation were seen in one monkey on day 1. Deep, circular, mucosal erosions, one to five in number and measuring from 0.5 to 1 mm in diameter, were present in three monkeys on day 45 post-capture. There were no adrenal cortex or stomach lesions in the rifle-shot monkeys. In conclusion, pathological lesions in the adrenal glands, and stomachs of the wild AGMs and weight loss occurred within the initial 45-day period following capture and confinement.     

Reversal of Ketamine/Xylazine combination anesthesia by Atipamezole in olive baboons (Papio anubis)

Background  The potential of Atipamezole (ATI) to reverse Ketamine/Xylazine (KET/XYL) anesthesia in the Olive baboon ( Papio anubis ) was studied.
Methods  Anesthesia was induced with 10 mg/kg KET and 0.5 mg/kg XYL intramuscularly. Mean arousal time (MAT), heart rate (HR), systolic arterial blood pressure (SAP), rectal temperature, respiratory rate (RR), and hemoglobin oxygen saturation (SpO₂) were monitored. Baboons were treated with: KET/XYL only, KET/XYL followed by 100 μg/kg ATI or by 200 μg/kg ATI administered 25 minutes after KET/XYL.
Results  Atipamezole rapidly reversed depressed HR and SAP (10 ± 5.2 minutes), RR (5 ± 2 minutes) and SpO₂ (3 ± 6 minutes) and significantly decreased MAT (13 ± 2.2 minutes) vs. KET/XYL alone (35 ± 5 minutes). Emesis was absent and salivation was observed after administration of 200 μg/kg ATI only.
Conclusions  Atipamezole at 100 μg/kg is sufficient for rapid and smooth reversal of KET/XYL anesthesia in the Olive baboon with minimal side effects.     

Studies on the protective effect of dietary fish oil on uranyl-nitrate-induced nephrotoxicity and oxidative damage in rat kidney

Human and animal exposure demonstrates that uranium is nephrotoxic. However, attempts to reduce it were not found suitable for clinical use. Dietary fish oil (FO) enriched in ω-3 fatty acids reduces the severity of cardiovascular and renal diseases. Present study investigates the protective effect of FO on uranyl nitrate (UN)-induced renal damage. Rats prefed with experimental diets for 15 days, given single nephrotoxic dose of UN (0.5 mg/kg body weight) intraperitoneally. After 5 d of UN treatment, serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport were analyzed in rat kidney. UN nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. UN increased the activity of lactate dehydrogenase and NADP-malic enzyme whereas decreased malate, isocitrate and glucose-6-phophate dehydrogenases; glucose-6-phophatase, fructose-1, 6-bisphosphatase and BBM enzyme activities. UN caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation, activities of superoxide dismutase, glutathione peroxidase and decreased catalase activity. Feeding FO alone increased activities of enzymes of glucose metabolism, BBM, oxidative stress and Pi transport. UN-elicited alterations were prevented by FO feeding. However, corn oil had no such effects and was not similarly effective. In conclusion, FO appears to protect against UN-induced nephrotoxicity by improving energy metabolism and antioxidant defense mechanism.     

Association between Alcohol Consumption and Pancreatic Cancer Risk: A Case-Control Study

Purpose

Evidence is inconsistent regarding alcohol and pancreatic cancer risk, although heavy drinking may increase risk.

Methods

A population-based case-control study was conducted using 345 pancreas cancer cases diagnosed 2011–2012 and 1,285 frequency-matched controls from Ontario, Canada. Logistic regression was used to evaluate alcohol consumption and pancreatic cancer risk; data was also stratified by sex and smoking status to assess interaction.

Results

Alcohol consumption was not associated with pancreatic cancer risk (age-adjusted odds ratio=0.78, 95% CI: 0.58, 1.05 for 1 - 3 drinks/week; age-adjusted odds ratio=0.86, 95% CI: 0.63, 1.17 for 4 - 20 drinks/week), however there was a non-significant increased risk for heavy drinkers consuming ≥21 drinks/week (age-adjusted odds ratio=1.35, 95% CI: 0.81, 2.27). Cigarette smoking modified the alcohol-cancer relationship; among current smokers, heavy alcohol consumption was associated with a significantly increased pancreatic cancer risk (age-adjusted odds ratio=4.04, 95% CI: 1.58, 10.37), whereas this significant association with heavy drinking was not observed among non-smokers (age-adjusted odds ratio=2.01, 95% CI: 0.50, 8.18). Furthermore, light – moderate alcohol intake was associated with increased pancreas cancer risk among current smokers.

Conclusions

While alcohol was not significantly associated with pancreatic cancer risk, smoking status modified this relationship such that among current smokers, alcohol intake was associated with a greater than two-fold increased risk of pancreatic cancer. The results should be interpreted with caution due to small sample sizes within subgroups and correction for multiple comparisons should be considered. These findings should be replicated in larger studies where more precise estimates of risk can be obtained.