Firing properties of accessory olfactory bulb mitral/tufted cells in response to urine delivered to the vomeronasal organ of gray short-tailed opossums

In comparison with many mammals, there is limited knowledge of the role of pheromones in conspecific communication in the gray short-tailed opossum. Here we report that mitral/tufted (M/T) cells of the accessory olfactory bulb (AOB) of male opossums responded to female urine but not to male urine with two distinct patterns: excitation followed by inhibition or inhibition. Either pattern could be mimicked by application of guanosine 5'-O-3-thiotriphosphate and blocked by guanosine 5'-O-2-thiodiphosphate, indicating that the response of neurons in this pathway is through a G-protein-coupled receptor mechanism. In addition, the inhibitor of phospholipase C (PLC), U73122, significantly blocked urine-induced responses. Male and female urine were ineffective as stimuli for M/T cells in the AOB of female opossums. These results indicate that urine of diestrous females contains a pheromone that directly stimulates vomeronasal neurons through activation of PLC by G-protein-coupled receptor mechanisms and that the response to urine is sexually dimorphic.     

Uterine epithelial cells specifically induce interferon-stimulated genes in response to polyinosinic-polycytidylic acid independently of estradiol

Interferon β (IFNβ) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNβ and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I:C) induced the mRNA expression of IFNβ, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNβ or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNβ upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I:C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I:C), had no effect on IFNβ or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNβ partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I:C) had no effect on IFNβ and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens.     

Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein

Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.     

Survival of Escherichia coli under lethal heat stress by L-form conversion

Transition of bacteria to cell wall deficient L-forms in response to stress factors has been assumed as a potential mechanism for survival of microbes under unfavorable conditions. In this article, we provide evidence of paradoxal survival through L-form conversion of E. coli high cell density population after lethal treatments (boiling or autoclaving). Light and transmission electron microscopy demonstrated conversion from classical rod to polymorphic L-form shape morphology and atypical growths of E. coli. Microcrystal formations observed at this stage were interpreted as being closely linked to the processes of L-form conversion and probably involved in the general phenomenon of protection against lethal environment. Identity of the morphologically modified L-forms as E. coli was verified by species specific DNA-based test. Our study might contribute to a better understanding of the L-form phenomenon and its importance for bacterial survival, as well as provoke reexamination of the traditional view of killing strategies against bacteria.     

Enhancement of alkaline phytase production in Pichia pastoris: influence of gene dosage, sequence optimization and expression temperature

Supplementation of animal feed with phytases has proven to be an effective strategy to alleviate phosphorous contamination of soil and water bodies. The inability of non-ruminant animals to digest phytates in corn and soybeans contributes to environmental contamination. Alkaline phytase from lily pollen (LlALP) exhibits unique catalytic and thermal stability properties that could be useful as a feed supplement. rLlALP2 was successfully expressed in Pichia pastoris; however, enzyme yields were modest (8-10 mg/L). In this paper, we describe our efforts to enhance rLlALP2 yield by investigating the influence of the following potential limiting factors: transgene copy number, codon bias, sequence optimization, and temperature during expression. Data presented indicate that increasing rLlAlp2 copy number was detrimental to heterologous expression, clones with one copy of wt-rLlAlp2 produced the highest activity, clones with two, four and seven or more copies produced 70%, 25% and 10% respectively, of enzyme activity implying that gene dosage is not limiting rLlALP2 yield. Use of a sequence-optimized rLlAlp2 increased the yield of the active enzyme by 25-50% in one/two copy clones, suggesting that translational efficiency is not a major bottleneck for rLlALP2 expression. Reducing the temperature during heterologous expression led to increases of 1.2-20-fold suggesting that protein folding and post-translational processes may be the dominant factors limiting rLlALP2 expression. Early knowledge of the transgene copy number allowed us to develop a more rational strategy for yield enhancement. Cumulatively, sequence optimization and temperature reduction led to the doubling of rLlALP2 enzyme activity in P. pastoris.     

Regurgitation and reingestion in captive gorillas: Description and intervention

Direct observation of regurgitation and reingestion (r/r) of captive gorillas was used to describe and catalog this behavior and thus lay the groundwork for possible intervention in the r/r cycle. Analysis of questionnaires regarding 117 gorillas at 17 zoos suggests that social deficits during early development contribute to the occurrence of r/r later in life. Wild-caught and captive-born, hand-reared gorillas show higher incidence of r/r than that of mother-reared infants. R/r increases the daily ingestion time. Feeding browse decreases r/r and increases time spent feeding from about 11% of the day to 27% of the day.     

Embryonic skin development and repair

Fetal cutaneous wounds have the unique ability to completely regenerate wounded skin and heal without scarring. However, adult cutaneous wounds heal via a fibroproliferative response which results in the formation of a scar. Understanding the mechanism(s) of scarless wound healing leads to enormous clinical potential in facilitating an environment conducive to scarless healing in adult cutaneous wounds. This article reviews the embryonic development of the skin and outlines the structural and functional differences in adult and fetal wound healing phenotypes. A review of current developments made towards applying this clinical knowledge to promote scarless healing in adult wounds is addressed.