Effects of growth hormone on diaphragmatic recovery from malnutrition.

A 25% weight loss was induced in adult Fisher 344 rats by nutritional deprivation. Subsequently, normal feeding was resumed. Refed animals were divided into three groups and received recombinant human growth hormone (rhGH) injections during 5 wk of refeeding, saline injections during 5 wk of refeeding, or 9 wk of refeeding without injections. The effects of nutritional deprivation and the various refeeding protocols on the cross-sectional areas (CSA) of each of the four types of myofibers [typed immunohistochemically with antibodies against four myosin heavy chain (MHC) isoforms known to be present in the rat diaphragm] were determined. Malnutrition decreased the CSA of myofibers containing MHC2X, MHC2B, and MHC2A (i.e., fast myofibers), with the greatest effect on muscle mass being due to the atrophy of fibers containing MHC2X. Fibers containing MHC beta/slow failed to undergo malnutrition-induced atrophy. Whereas refeeding for 5 wk in the absence of rhGH allowed the recovery of CSA of fibers containing MHC2A, fibers containing MHC2B and MHC2X remained smaller than fibers of similar type in control animals. In contrast, 5 wk of refeeding supplemented with rhGH returned all fiber CSAs to control values. Even when refeeding alone was extended to 9 wk to allow for weight stabilization, the CSA of the fibers containing MHC2B and MHC2X remained smaller than similar fibers in control muscle. Serum insulin-like growth factor, a marker of malnutrition (R. Reeves and J. Elders, J. Nutr. 109: 613-620, 1979), was significantly decreased after nutritional deprivation and returned to normal after 5 wk of refeeding and GH supplementation.     

Violet Red Bile 2 Agar for Stressed Coliforms

Counts on a new, autoclave-sterilizable violet red bile (VRB-2) agar were compared with counts on freshly boiled VRB agar. Yields on VRB-2 agar averaged 217, 180, 130, and 112% of counts obtained on the control medium for samples of water, cottage cheese, frozen vegetables, and raw milk, respectively. The general principle used for the development of VRB-2 agar could be applied to many other kinds of selective plating media.     

Neue Beiträge zur Kenntnis der Systrophe des Protoplasmas

Ohne ZusammenfassungMeinem hochverehrten Lehrer, Herrn Prof. Dr. E. Küster, bin ich für die vielseitigen Anregungen und Beratungen, die er mir bei meinen Untersuchungen zuteil werden ließ, zu großem Dank verpflichtet.     

Mitotic catastrophe triggered in human cancer cells by the viral protein apoptin

Mitotic catastrophe is an oncosuppressive mechanism that senses mitotic failure leading to cell death or senescence. As such, it protects against aneuploidy and genetic instability, and its induction in cancer cells by exogenous agents is currently seen as a promising therapeutic end point. Apoptin, a small protein from Chicken Anemia Virus (CAV), is known for its ability to selectively induce cell death in human tumor cells. Here, we show that apoptin triggers p53-independent abnormal spindle formation in osteosarcoma cells. Approximately 50% of apoptin-positive cells displayed non-bipolar spindles, a 10-fold increase as compared to control cells. Besides, tumor cells expressing apoptin are greatly limited in their progress through anaphase and telophase, and a significant drop in mitotic cells past the meta-to-anaphase transition is observed. Time-lapse microscopy showed that mitotic osteosarcoma cells expressing apoptin displayed aberrant mitotic figures and/or had a prolonged cycling time during mitosis. Importantly, all dividing cells expressing apoptin eventually underwent cell death either during mitosis or during the following interphase. We infer that apoptin can efficiently trigger cell death in dividing human tumor cells through induction of mitotic catastrophe. However, the killing activity of apoptin is not only confined to dividing cells, as the CAV-derived protein is also able to trigger caspase-3 activation and apoptosis in non-mitotic cancer cells.     

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut⁺ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio^® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L^?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016     

Pituitary adenylate cyclase-activating peptide: a pivotal modulator of steroid-induced reproductive behavior in female rodents

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates the secretion of GnRH into the hypothalamic hypophysial portal system and sensitizes the pituitary for release of hormones that trigger ovulation. Because reproductive behavior is synchronized with GnRH release, the present study was undertaken to determine whether PACAP in the ventromedial nucleus (VMN) plays a role in receptivity. To this end, we used rat and mouse reproductive behavioral models to determine the biological relationship between PACAP and steroid receptor function in females. We provide evidence for the requirement of PACAP in the VMN for progesterone (P)-dependent sexual behavior in estrogen (E)-primed females. We clarify the biological and molecular mechanisms of PACAP activity by showing 1) that inhibition of endogenous PACAP suppresses P receptor (PR)-dependent sexual behavior facilitated by the steroid P or D1-like agonist SKF38393 and 2) that PR, steroid receptor coactivators-1 and -2, and new protein synthesis are essential for ligand independent PACAP-facilitated behavior. These findings are consistent with convergence of PACAP-mediated cellular signals on PR for genomic activation and subsequent behavioral changes. Further, we show that steroids regulate both endogenous PACAP mRNA in the VMN and immunoreactive PACAP in the medial basal hypothalamus and cerebral spinal fluid for ligand-dependent, steroid receptor-dependent receptivity. The present findings delineate a novel, steroid-dependent mechanism within the female hypothalamus by which the neuropeptide PACAP acts as a feed-forward, paracrine, and/or autocrine factor for synchronization of behavior coordinate with hypothalamic control of ovulation.     

Membrane-initiated steroid signaling (MISS): genomic steroid action starts at the plasma membrane

Plasma membrane (PM) steroid recognition sites are thought to be responsible only for rapid, non-genomic responses without any link to the nuclear receptor-mediated genomic effects of steroids. We focused on a PM "glucocorticoid-importer" (GC-importer) that imports GC into rat liver cells. This site interacts also with particular gestagens (progesterone, P; medroxyprogesterone, MP; ethynodiol, Ethy) and estrogens (ethinylestradiol, EE(2); mestranol), which do not bind to the nuclear GC receptor (GR). To elucidate the role of the GC-importer, we transfected a rat wild-type hepatocyte (CC-1) and a hepatoma cell line, unable to import GC (MH 3924), with a GC<-->GR-responsive luciferase (luc)-reporter gene. Selected steroids were tested for their ability to induce or inhibit luc expression. Corticosterone (B) and dexamethasone (Dex), but also the GC-antagonists cortexolone (Cortex), P and MP, induced luc. Even the PM-impermeable BSA-derivatives of B, Dex and Cortex did so to almost the same extent as the free steroids. MH 3924 cells respond stronger than CC-1 to luc inducing steroids. Luc expression was inhibited by RU 38 486, but also by EE(2) and Ethy. The thiol reactive mesylate-derivatives of B, Dex and Cortex induced to a considerably lesser extent than the free or BSA-steroids. The thiol reagent mersalyl blocks cellular entry of GC and inhibits luc induction in CC-1 cells. Incubation with EE(2) and B of PM-vesicles, isolated from liver cells, resulted in a decrease of the density of two 75 and 52kDa G-proteins reflecting a diminished exchange of GDP by GTP. CONCLUSION: the PM-residing GC-importer, now renamed "Steroid Hormone Recognition and Effector Complex" (SHREC) is an interdependent part of the complete GC signal propagation in which G-proteins are involved. Free SH-groups of SHREC are a prerequisite for genomic GC activity. Specific interactions between SHREC and GC-agonist/-antagonist trigger steroid-dependent signaling. However, import of the ligand into the cell terminates it. Thus, the PM-related non-genomic steroid responses are clearly linked to the GR-related genomic effects.     

Antimicrobial activity of organic extracts isolated from Aplysina fistularis (Demospongiae: Aplysinidae)

Organic extracts of the sponge Aplysina fistularis (Pallas 1766) were tested for antimicrobial activity against Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). The minimal inhibitory concentration (MIC) and toxic activity of extract were determined. Susceptibility trials of organic fractions obtained by VLC: Hexane, EtOAc and CHCl3 showed that EtOAc fraction has antibacterial activity against E. coli, while CHCl3 fraction inhibited E. coli and S. aureus growth. The later refractioning of EtOAc fraction and the biodirected assays showed that fractions F12 and F13 of EtOAc/Hex and EtOAc F14 were bioactive against Gram positive and Gram negative bacteria. Only EtOAc/MeOH Sf2 from subfractionig of EtOAc F14 produced inhibition for E. coli and S. aureus. In Sf2 EtOAc/MeOH, MIC was moderate for S. aureus (MIC > 256 g/ml). F4 CHCl3/MeOH produced a high inhibition in S. aureus (MIC = 0.125 g/ml) and for E. coli (MIC > 16 g/ml). F10 CHCl3/MeOH showed a moderate activity against S. aureus (MIC > 128 g/ml) and low activity against E. coli (MIC = 512 g/ml). F10 CHCL3/MeOH did no present toxic activity against Artemia salina. The fractiorts F4 CHCL3/MeOH and Sf2 EtOAc/MeOH were toxic for this organism when the concentration was higher than 100 microg/ml. LC50 in both cases was 548.4 and 243.4 microg/ml respectively. Secondary metabolites of medium polarity obtained from A. fistularis have a wide spectrum of anti bacterial activity. Toxicity analysis suggests that only F10 CHCL3/MeOH has potential as an antimicrobial agent for clinical use.     

Uncoupling Lipid Metabolism from Inflammation through Fatty Acid Binding Protein-Dependent Expression of UCP2

Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2.