Let-7, Mir-98 and Mir-181 as Biomarkers for Cancer and Schizophrenia

Recent evidence supports a role of microRNAs in cancer and psychiatric disorders such as schizophrenia and bipolar disorder, through their regulatory role on the expression of multiple genes. The rather rare co-morbidity of cancer and schizophrenia is an old hypothesis which needs further research on microRNAs as molecules that might exert their oncosuppressive or oncogenic activity in the context of their role in psychiatric disorders. The expression pattern of a variety of different microRNAs was investigated in patients (N = 6) suffering from schizophrenia termed control, patients with a solid tumor (N = 10) and patients with both schizophrenia and tumor (N = 8). miRNA profiling was performed on whole blood samples using the miRCURY LNA microRNA Array technology (6th & 7th generation). A subset of 3 microRNAs showed a statistically significant differential expression between the control and the study groups. Specifically, significant down-regulation of the let-7p-5p, miR-98-5p and of miR-183-5p in the study groups (tumor alone and tumorand schizophrenia) was observed (p<0.05). The results of the present study showed that let-7, miR-98 and miR-183 may play an important oncosuppressive role through their regulatory impact in gene expression irrespective of the presence of schizophrenia, although a larger sample size is required to validate these results. Nevertheless, further studies are warranted in order to highlight a possible role of these and other micro-RNAs in the molecular pathways of schizophrenia.     

Heart valve cryopreservation: protocol for addition of dimethyl sulphoxide and amelioration of putative amphotericin B toxicity

Aim

To investigate the need for stepwise addition of dimethyl sulphoxide to heart valves and amelioration of putative amphotericin B toxicity.

Methods

There were four groups: an untreated control (Group 1) and three experimental groups. For the latter, porcine heart valves were exposed to the antibiotic/antimycotic mixture used for disinfecting heart valves in the Bristol Heart Valve Bank, for 24 h at 22 °C. Dimethyl sulphoxide (Me₂SO, 10% v/v) was added either in two steps (5% then 10%) (Group 2) or in a single step. For single-step addition, valves were either first placed in Hanks’ balanced salt solution for 10 min before transfer to the cryoprotectant solution (Group 3) or immersed directly in the 10% cryoprotectant solution (Group 4). The valve leaflets were dissected from the valves and frozen in 10% Me₂SO in multi-well tissue culture plates at 1 °C/min to −80 °C. After storage overnight, the valve leaflets were warmed at approximately 11 °C/min and the cryoprotectant was removed by single-step dilution in excess Hartmann’s solution. Each leaflet was then divided into four pieces, which were placed in separate wells of a culture plate. Outgrowth of cells from the explants was monitored daily and graded according to the extent of cell growth.

Results

After freezing and thawing, only 77% of the explants from valves placed directly into 10% Me₂SO (Group 4) showed outgrowth of cells after freezing compared with 89% with two-step addition of Me₂SO (Group 2) and 95% with one-step addition after the extra rinse in Hanks’ solution (Group 3) (χ², p = 0.001). 92% of unfrozen control explants showed outgrowth of cells (Group 1). Only 37% of Group 4 explants reached confluence compared with 63 and 56%, respectively, of Groups 2 and 3 explants (χ², p = 0.007). The rates of cell growth in Group 2 (two-step addition of Me₂SO) and Group 3 (one-step addition of Me₂SO with additional Hanks’ solution rinse) were similar and faster than the Group 4 (one-step addition of Me₂SO without the additional Hanks’ rinse).

Conclusion

Single-step addition of Me₂SO before freezing gave similar results to two-step addition provided an additional rinse in Hanks’ solution was introduced after exposure to the antibiotic/antimycotic mixture. This suggests that antibiotic/antimycotic carryover may have been harmful during freezing and that the additional rinse in Hanks before one-step addition of Me₂SO, and the 5% Me₂SO step in the two-step protocol, merely served to reduce this carryover.     

Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story

Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.     

Neurexophilin 3 is highly localized in cortical and cerebellar regions and is functionally important for sensorimotor gating and motor coordination

Neurexophilin 3 (Nxph3) is a specific ligand of synaptic alpha-neurexins that are essential for efficient neurotransmitter release. Previous biochemical work demonstrated that Nxph3 interacts with an extracellular domain of alpha-neurexins in a tight complex; however, no information is available on the localization or functional role of Nxph3 in the brain. Here, we generated lacZ reporter gene knock-in mice to investigate the distribution of Nxph3 at the single-cell level and Nxph3 knockout mice to examine its functional importance. Nxph3 expression was restricted mostly to subplate-derived neurons in cortical layer 6b, granule cells in the vestibulocerebellum, and Cajal-Retzius cells during development. Colabeling experiments demonstrated that neurons expressing Nxph3 do not belong to a uniform cell type. Morphological analyses and systematic behavioral testing of knockout mice revealed no anatomical defects but uncovered remarkable functional abnormalities in sensory information processing and motor coordination, evident by increased startle response, reduced prepulse inhibition, and poor rotarod performance. Since Nxph3-deficient mice behaved normally while performing a number of other tasks, our data suggest an important role for Nxph3 as a locally and temporally regulated neuropeptide-like molecule, presumably acting in a complex with alpha-neurexins in select neuronal circuits.     

Equal volumes of high and low intensity of eccentric exercise in relation to muscle damage and performance

We examined differences in muscle damage and muscle performance perturbations in relation to the same volumes of high (HI) and low intensity (LI) of eccentric exercise. Untrained young healthy men (n = 12) underwent 2 isokinetic quadriceps eccentric exercise sessions, 1 on each randomly selected leg, separated by a 2-week interval. In the first session subjects performed HI exercise (i.e., 12 sets of 10 maximal voluntary efforts). In the second session, volunteers were subjected to continuous exercise of LI (50% of peak torque) until the total work done was approximately equal to that generated during HI. Muscle damage (serum creatine kinase concentration [CK], delayed onset of muscle soreness, and range of motion) and muscle performance (eccentric [EPT] and isometric peak torque [IPT]) indicators were assessed pre-exercise and 24, 48, 72, and 96 hours postexercise. Compared to baseline data, changes in muscle damage indicators were significantly different (p < 0.05) at almost all postexercise time points in both conditions. However, apart from the significant elevation of CK at 24 hours after HI (p < 0.05), no other significant differences were observed between the 2 exercise conditions (p > 0.05). The main finding in relation to muscle performance was that decrements following HI exercise were significantly greater (p < 0.05) compared to LI. Compared with baseline data, the EPT values following HI and LI exercise were as follows: 24 hours, 72.1% vs. 92%; 48 hours, 81.9% vs. 94.8%; 72 hours, 77.7% vs. 100.6%; 96 hours, 86.8% vs. 107.9%. The corresponding data for IPT were as follows: 24 hours, 86.4% vs. 102.8%; 48 hours, 84.2% vs. 107%; 72 hours, 84.8% vs. 109.2%; 96 hours, 86.8% vs. 114.4%. These results indicate that matching volumes of HI and LI eccentric exercise have similar effects on muscle damage, but HI has a more prominent effect on muscle performance.     

Chemical defense and antifouling activity of three Mediterranean sponges of the genus Ircinia

The defense roles and the antifouling activity of the organic extracts and the major metabolites of the sponges Ircinia oros, I. variabilis and I. spinosula were investigated. The antifeedant activity was tested in experimental aquaria on the generalist predator fish Thalassoma pavo as well as in coastal ecosystems rich in fishes. Some of the major metabolites exhibited high levels of antifeedant activity. The antifouling activity was tested in laboratory assays, against representatives of the major groups of fouling organisms (marine bacteria, marine fungi, diatoms, macroalgae and mussels). All extracts showed promising levels of activity. As was expected, no single extract was active in all tests and some fractions that were effective against one organism showed little or no activity against the others. The high but variable level of antifouling activity in combination with the absence of toxicity (tested on the development of oyster and sea urchin larvae) shows the potential of these metabolites to become ingredients in environmentally friendly antifouling preparations.     

Methanococcus jannaschii prolyl-cysteinyl-tRNA synthetase possesses overlapping amino acid binding sites

The protein translation apparatus of Methanococcus jannaschii possesses the unusual enzyme prolyl-cysteinyl-tRNA synthetase (ProCysRS), a single enzyme that attaches two different amino acids, proline and cysteine, to their cognate tRNA species. Measurement of the ATP-PP(i) exchange reaction revealed that amino acid activation, the first reaction step, differs for the two amino acids. While Pro-AMP can be formed in the absence of tRNA, Cys-AMP synthesis is tRNA-dependent. Studies with purified tRNAs indicate that tRNA(Cys) promotes cysteine activation. The k(cat) values of wild-type ProCysRS for tRNA prolylation (0.09 s(-1)) and cysteinylation (0.02 s(-1)) demonstrate that both aminoacyl-tRNAs are synthesized with comparable rates, the cysteinyl-tRNA synthetase activity being only 4.5-fold lower than prolyl-tRNA synthetase activity. Kinetic analysis of ProCysRS mutant enzymes, generated by site-directed mutagenesis, shows glutamate at position 103 to be critical for proline binding, and proline at position 100 to be involved in cysteine binding. The proximity in ProCysRS of amino acid residues affecting binding of either cysteine or proline strongly suggests that structural elements of the two amino acid binding sites overlap.     

Synergistic Combinations of Nucleoside Analog Drugs with Other Drugs Induce Greater Apoptosis in Human Leukemic T-Cells

Abstract

Combinations of nucleoside analog drugs such as 6-MP. ara-C. or F-araA are synergistic against human leukemic T-cells and induce apoptotic cell death. Addition of Taxotere or PEG-ASNase to the synergistic combination of nucleoside analog drugs augments the synergism several fold by enhancing cellular apoptosis.     

Population-based trends of pregnancy outcome in obese mothers: what has changed over 15 years

Maternal obesity is a growing concern worldwide. We aimed to determine trends of obesity in women of reproductive age over a 15-year period, the relationship between prepregnancy weight status and pregnancy-related outcomes, and possible changes over a 15-year period. Data was derived from two identical, cross-sectional National Perinatal Surveys (NPSs). Analysis was restricted to the mothers of healthy, full-term, singleton infants. Overall, the study population consisted of 18,752 mother-infant pairs (7,208 from the 1st NPS and 11,544 from the 2nd NPS). The prevalence of overweight and obese women prior to pregnancy increased significantly between the two surveys, from 12 to 15.3% and 2.1 to 4.7%, respectively. In both study periods, prepregnancy weight status was positively associated with pre-eclampsia (P < 0.001) and method of delivery (P < 0.001). After adjusting for potential confounders, overweight and obese women had higher odds for caesarian section (adjusted (odd ratio) OR = 1.87) and pre-eclampsia (adjusted OR = 2.59) than normal-weight women. These associations did not change between the study periods. In conclusion, prepregnancy maternal obesity is steadily increasing over time. The magnitude of the problem underlies the need for public health programs to focus on obesity prevention, particularly among women of reproductive age, and re-evaluate the guidelines for various perinatal practices.     

Solution conformation of the antibody-bound tyrosine phosphorylation site of the nicotinic acetylcholine receptor beta-subunit in its phosphorylated and nonphosphorylated states

Phosphorylation of the acetylcholine receptor (AChR) seems to be responsible for triggering several effects including its desensitization and aggregation at the postsynaptic membrane and probably initiates a signal transduction pathway at the postsynaptic membrane. To study the structural and functional role of the tyrosine phosphorylation site of the AChR beta-subunit and contribute to the in-depth understanding of the structural basis of the ion channel function, we synthesized four peptides containing the phosphorylated and nonphosphorylated sequences (380-391) of the human and Torpedo AChR beta-subunits and studied their interaction with a monoclonal antibody (mAb 148) that is known to bind to this region and that is capable of blocking ion channel function. All four peptides were efficient inhibitors of mAb 148 binding to AChR, although the nonphosphorylated human peptide was considerably less effective than the three others. We then investigated the conformation acquired by all four peptides in their antibody-bound state, which possibly illustrates the local conformation of the corresponding sites on the intact AChR molecule. The phosphorylated human and Torpedo peptides adopted a distorted 3(10) helix conformation. The nonphosphorylated Torpedo peptide, which is also an efficient inhibitor, was also folded. In contrast, the nonphosphorylated human peptide (a less efficient inhibitor) presented an extended structure. It is concluded that the phosphorylation of the AChR at its beta-subunit Tyr site leads to a significant change in its conformation, which may affect several functions of the AChR.