Dual Effects of Hydrogen Sulfide Donor on Meiosis and Cumulus Expansion of Porcine Cumulus-Oocyte Complexes

Hydrogen sulfide (H₂S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H₂S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H₂S donor, Na₂S, accelerated oocyte in vitro maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H₂S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H₂S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H₂S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.     

Comparison of simian immunodeficiency virus SIVagmVer replication and CD4+ T-cell dynamics in vervet and sabaeus African green monkeys

The simian immunodeficiency viruses (SIV) naturally infect a wide range of African primates, including African green monkeys (AGM). Despite moderate to high levels of plasma viremia in naturally infected AGM, infection is not associated with immunodeficiency. We recently reported that SIVagmVer90 isolated from a naturally infected vervet AGM induced AIDS following experimental inoculation of pigtailed macaques. The goal of the present study was to evaluate the replication of this isolate in two species of AGM, sabaeus monkeys (Chlorocebus sabaeus) and vervets (C. pygerythrus). Inoculation of sabaeus AGM with SIVagmVer90 resulted in low and variable primary and set-point viremia (<10(2) to 10(4) copies/ml). In contrast, inoculation of vervet AGM with either SIVagmVer90 or blood from a naturally infected vervet (Ver1) resulted in high primary viremia and moderate plateau levels, similar to the range seen in naturally infected vervets from this cohort. CD4(+) T cells remained stable throughout infection, even in AGM with persistent high viremia. Despite the lack of measurable lymphadenopathy, infection was associated with an increased number of Ki-67(+) T cells in lymph node biopsies, consistent with an early antiviral immune response. The preferential replication of SIVagmVer in vervet versus sabaeus AGM shows that it is critical to match AGM species and SIV strains for experimental models of natural SIV infection.     

Physiological and immunological changes in the brain associated with lethal eastern equine encephalitis virus in macaques

Aerosol exposure to eastern equine encephalitis virus (EEEV) can trigger a lethal viral encephalitis in cynomolgus macaques which resembles severe human disease. Biomarkers indicative of central nervous system (CNS) infection by the virus and lethal outcome of disease would be useful in evaluating potential medical countermeasures, especially for therapeutic compounds. To meet requirements of the Animal Rule, a better understanding of the pathophysiology of EEEV-mediated disease in cynomolgus macaques is needed. In this study, macaques given a lethal dose of clone-derived EEEV strain V105 developed a fever between 2–3 days post infection (dpi) and succumbed to the disease by 6 dpi. At the peak of the febrile phase, there was a significant increase in the delta electroencephalography (EEG) power band associated with deep sleep as well as a sharp rise in intracranial pressure (ICP). Viremia peaked early after infection and was largely absent by the onset of fever. Granulocytosis and elevated plasma levels of IP-10 were found early after infection. At necropsy, there was a one hundred- to one thousand-fold increase in expression of traumatic brain injury genes (LIF, MMP-9) as well as inflammatory cytokines and chemokines (IFN-γ, IP-10, MCP-1, IL-8, IL-6) in the brain tissues. Phenotypic analysis of leukocytes entering the brain identified cells as primarily lymphoid (T, B, NK cells) with lower levels of infiltrating macrophages and activated microglia. Massive amounts of infectious virus were found in the brains of lethally-infected macaques. While no infectious virus was found in surviving macaques, quantitative PCR did find evidence of viral genomes in the brains of several survivors. These data are consistent with an overwhelming viral infection in the CNS coupled with a tremendous inflammatory response to the infection that may contribute to the disease outcome. Physiological monitoring of EEG and ICP represent novel methods for assessing efficacy of vaccines or therapeutics in the cynomolgus macaque model of EEEV encephalitis.     

Non-equivalent role of inter- and intramolecular hydrogen bonds in the insulin dimer interface

Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 μM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 μM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain β-strand to the core of the molecule. The release of the B-chain β-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability.     

These squatters are not innocent: the evidence of parasitism in sponge-inhabiting shrimps

Marine sponges are frequently inhabited by a wide range of associated invertebrates, including caridean shrimps. Symbiotic shrimps are often considered to be commensals; however, in most cases, the relationship with sponge hosts remains unclear. Here we demonstrate that sponge-inhabiting shrimps are often parasites adapted to consumption of sponge tissues. First, we provide detailed examination of morphology and stomach contents of Typton carneus (Decapoda: Palaemonidae: Pontoniinae), a West Atlantic tropical shrimp living in fire sponges of the genus Tedania. Remarkable shear-like claws of T. carneus show evidence of intensive shearing, likely the result of crushing siliceous sponge spicules. Examination of stomach contents revealed that the host sponge tissue is a major source of food for T. carneus. A parasitic mode of life is also reflected in adaptations of mouth appendages, in the reproduction strategy, and in apparent sequestration of host pigments by shrimp. Consistent results were obtained also for congeneric species T. distinctus (Western Atlantic) and T. spongicola (Mediterranean). The distribution of shrimps among sponge hosts (mostly solitary individuals or heterosexual pairs) suggests that Typton shrimps actively prevent colonisation of their sponge by additional conspecifics, thus protecting their resource and reducing the damage to the hosts. We also demonstrate feeding on host tissues by sponge-associated shrimps of the genera Onycocaris, Periclimenaeus, and Thaumastocaris (Pontoniinae) and Synalpheus (Alpheidae). The parasitic mode of life appears to be widely distributed among sponge-inhabiting shrimps. However, it is possible that under some circumstances, the shrimps provide a service to the host sponge by preventing a penetration by potentially more damaging associated animals. The overall nature of interspecific shrimp-sponge relationships thus warrants further investigation.     

Internal standard-based analysis of microarray data2--analysis of functional associations between HVE-genes

In this work we apply the Internal Standard-based analytical approach that we described in an earlier communication and here we demonstrate experimental results on functional associations among the hypervariably-expressed genes (HVE-genes). Our working assumption was that those genetic components, which initiate the disease, involve HVE-genes for which the level of expression is undistinguishable among healthy individuals and individuals with pathology. We show that analysis of the functional associations of the HVE-genes is indeed suitable to revealing disease-specific differences. We show also that another possible exploit of HVE-genes for characterization of pathological alterations is by using multivariate classification methods. This in turn offers important clues on naturally occurring dynamic processes in the organism and is further used for dynamic discrimination of groups of compared samples. We conclude that our approach can uncover principally new collective differences that cannot be discerned by individual gene analysis.     

Improved Detection of Deletions and Duplications in the DMD Gene Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method

The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.