Insight into the mechanism by which metoclopramide improves immune functions after trauma-hemorrhage

Although studies have shownthat prolactin (Prl) and metoclopramide (Mcp) administration restoresthe depressed cell-mediated immune functions after hemorrhage, theunderlying mechanism responsible for the immunostimulatory effects ofMcp remains unknown. We hypothesized that Mcp improves immune responsesby upregulating the secretion of Prl. To test this hypothesis, maleC3H/HeN mice were subjected to sham operation or laparotomy (i.e., softtissue trauma) and hemorrhagic shock (Hem; 35 ± 5 mmHg for 90 min) and then resuscitated. Plasma Prl levels were determined 30 minafter Mcp (1 µg/g body wt sc at end of Hem) or vehicle (Veh)treatment in sham and Hem mice. The results indicate that plasma Prllevels increased significantly in Mcp-treated mice (sham-Veh 249.9 ± 5.3, Hem-Veh 229.9 ± 7.6, Hem-Mcp 596.9 ± 73.1 ng/ml,one-way ANOVA, P < 0.05 vs. Veh). To determine whetherMcp produces its salutary effects directly or indirectly via increasedPrl secretion, splenocyte proliferation and splenocyte interleukin(IL)-2 and IL-3 release from untreated sham or Hem mice were determinedin the presence of increasing concentrations of mouse Prl or Mcp. Theaddition of Mcp had no effect on splenocyte immune functions in vitro.However, the addition of Prl restored the hemorrhage-induced depressedsplenocyte proliferation as well as splenocyte IL-2 and IL-3 release invitro in a dose-dependent manner. Thus the beneficial effects of Mcp onimmune functions after Hem appear to be mediated by Prl. Because Mcpincreases plasma levels of the immunoenhancing hormone Prl, this agentshould be considered a useful adjunct for the treatment ofimmunodepression in trauma victims.

     

The female reproductive cycle is an important variable in the response to trauma-hemorrhage

Although immune functions in proestrus females are maintained after hemorrhage as opposed to decreased responses in males, it remains unknown whether such a sexual dimorphism also exists with regard to cardiovascular and hepatocellular functions under those conditions. To study this, male and female (estrus and proestrus) rats underwent a 5-cm midline laparotomy and were bled to and maintained at a mean blood pressure of 40 mmHg until 40% of the maximal bleed-out volume was returned in the form of Ringer lactate (RL). Rats were then resuscitated with four times the shed blood volume with RL. At 24 h thereafter, cardiac index; heart performance; hepatocellular function; and plasma estradiol, testosterone, and prolactin levels were measured. Cardiovascular and hepatocellular functions were depressed in males and estrus females (P < 0.05) but were not depressed in proestrus females after resuscitation. Plasma estradiol and prolactin levels were highest in proestrus females (P < 0.05), whereas males had high testosterone and the lowest estradiol levels (P < 0.05). Thus the female reproductive cycle is an important variable in the response to hemorrhage. Because low testosterone and high estradiol and prolactin levels appear to be beneficial for organ functions after trauma-hemorrhage, antagonism of testosterone receptors and/or increases in estradiol and prolactin levels in males and estrus females, respectively, may be novel approaches for improving organ functions under such conditions.     

Preinduction of heat shock proteins protects cardiac and hepatic functions following trauma and hemorrhage

Although studies have shown that induction of the heat shock proteins (HSPs), such as HSP-70, has various beneficial effects after ischemia-reperfusion, it remains unknown whether prior induction of HSP-70 has any salutary effects on cardiovascular and hepatocellular functions after trauma-hemorrhage and resuscitation. Male rats were exposed to heat stress (41 degrees C, 15 min) and then allowed to recover for 24 h at room temperature (21 degrees C). The rats then underwent laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the maximal shed blood volume was returned in the form of Ringer lactate. Animals were then resuscitated with four times the volume of shed blood with Ringer lactate over 60 min. The maximal rate of the left ventricular pressure increase or decrease was measured up to 4 h after resuscitation. Cardiac output, hepatocellular function, plasma levels of tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were determined at 4 h after resuscitation. Cardiac and hepatic tissue were examined for HSP-70 by Western blot analysis. Left ventricular performance, cardiac output, and hepatocellular function decreased significantly following trauma-hemorrhage. Plasma levels of TNF-alpha and IL-6 were also significantly increased. However, prior heat stress attenuated cardiovascular and hepatocellular dysfunction, decreased circulating levels of proinflammatory cytokines following trauma-hemorrhage, and was associated with an increased abundance of HSP-70 in the heart and liver. Our data, therefore, suggest that preinduction of HSP-70 protects cardiovascular and hepatocellular functions following trauma-hemorrhage and resuscitation.     

Comparison of marker types and map assumptions using Markov chain Monte Carlo-based linkage analysis of COGA data

We performed multipoint linkage analysis of the electrophysiological trait ECB21 on chromosome 4 in the full pedigrees provided by the Collaborative Study on the Genetics of Alcoholism (COGA). Three Markov chain Monte Carlo (MCMC)-based approaches were applied to the provided and re-estimated genetic maps and to five different marker panels consisting of microsatellite (STRP) and/or SNP markers at various densities. We found evidence of linkage near the GABRB1 STRP using all methods, maps, and marker panels. Difficulties encountered with SNP panels included convergence problems and demanding computations.     

Metformin Targets the Metabolic Achilles Heel of Human Pancreatic Cancer Stem Cells

Pancreatic ductal adenocarcinomas contain a subset of exclusively tumorigenic cancer stem cells (CSCs), which are capable of repopulating the entire heterogeneous cancer cell populations and are highly resistant to standard chemotherapy. Here we demonstrate that metformin selectively ablated pancreatic CSCs as evidenced by diminished expression of pluripotency-associated genes and CSC-associated surface markers. Subsequently, the ability of metformin-treated CSCs to clonally expand in vitro was irreversibly abrogated by inducing apoptosis. In contrast, non-CSCs preferentially responded by cell cycle arrest, but were not eliminated by metformin treatment. Mechanistically, metformin increased reactive oxygen species production in CSC and reduced their mitochondrial transmembrane potential. The subsequent induction of lethal energy crisis in CSCs was independent of AMPK/mTOR. Finally, in primary cancer tissue xenograft models metformin effectively reduced tumor burden and prevented disease progression; if combined with a stroma-targeting smoothened inhibitor for enhanced tissue penetration, while gemcitabine actually appeared dispensable.     

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.     

Repetitive DNAs and differentiation of sex chromosomes in neotropical fishes

The processes working on sex chromosome differentiation are still not completely understood. However, the accumulation of repetitive DNA sequences has been shown to be one of the first steps in the early stages of such differentiation. In addition, regions with suppressed or no recombination have a potential to accumulate these DNA sequences and, for this reason, the absence of recombination between the sex chromosomes favors, by itself, the accumulation of repetitive sequences on these chromosomes during evolution. The diversity of sex-determining mechanisms in fish, alongside with the absence of heteromorphic sex chromosomes in many species, makes this group a useful model to better understand evolutionary processes of sex chromosomes in vertebrates, considering that fish occupy the basal position in the phylogeny of this group. In this review we draw attention to a preferential accumulation and enrichment in repetitive DNAs in sex chromosomes of many neotropical fish species in comparison with autosomes. This phenomenon has been observed between both morphologically differentiated and nascent sex chromosome systems, which highlight the potential role of these sequences in the differentiation of fish sex chromosomes generating differences in morphology and size between them.     

Influence de l'âge des grand-parents sur l'apparition des mâles chez le Rotifère Notommata copeus Ehr.

When parental females (♀♀P = mothers) of the rotifer Notommata copeus are placed in light conditions inducing the appearance of mictic females in their offspring (F₁), the age of the parents of these parental females significantly affects the ratio of mictic females in the F₁ generation.

The preparental females (♀♀PP = grandmothers), the parental females and the F₁ females are isolated in a medium changed at each generation and, in the second experimental series, changed daily: the preparental age effect implicates the transmission of substances on two generations.

This influence of the preparental age is rhythmic: the ratio of mictic females in F₁ related to this age varies in a sinusoidal manner. This influence is endogenous: it persists, always in a sinusoidal form, when the medium in which each grandmother is placed is changed daily.

Furthermore, the net reproduction ratio, Ro, does not vary significantly with the preparental age during these experiments.     

Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway.

Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, I(SOC), in A549 and endothelial cells. Twenty different 'chimeric' 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a I(SOC), which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 microM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca(2+) response to thapsigargin by 25% and similarly reduced I(SOC) by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion.