Regulation of S100A2 expression by TGF-β-induced MEK/ERK signalling and its role in cell migration/invasion

The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.     

GABA receptors and nitric oxide ameliorate constrictive collagen remodeling in hyperhomocysteinemia

Elevated plasma levels of homocysteine (Hcy) are associated with vascular dementias and Alzheimer's disease. The role of Hcy in brain microvascular endothelial cell (MVEC) remodeling is unclear. Hcy competes with muscimol, an gamma-amino butyric acid (GABA)-A receptor agonist. GABA is the primary inhibitory neurotransmitter in the brain. Our hypothesis is that Hcy induces constrictive microvascular remodeling by altering GABA-A/B receptors. MVEC from wild type, matrix metalloproteinase-9 (MMP-9) knockout (-/-), heterozygote cystathionine beta synthase (CBS-/+), and endothelial nitric oxide synthase knockout (eNOS-/-) mouse brains were isolated. The MVEC were incorporated into collagen (3.2 mg/ml) gels and the decrease in collagen gel diameter at 24 h was used as an index of constrictive MVEC remodeling. Gels in the absence or presence of Hcy were incubated with muscimol or baclofen, a GABA-B receptor agonist. The results suggested that Hcy-mediated MVEC collagen gel constriction was ameliorated by muscimol, baclofen, MMP-9, and eNOS gene ablations. There was no effect of anti-alpha 3 integrin. However, Hcy-mediated brain MVEC collagen constriction was abrogated with anti-beta-1 integrin. The co-incubation of Hcy with L-arginine ameliorated the Hcy-mediated collagen gel constriction. The results of this study indicated amelioration of Hcy-induced MVEC collagen gel constriction by induction of nitric oxide through GABA-A and -B receptors.     

Bipolar disorder: an update

Bipolar disorder (BPD) is one of the most severe forms of mental illness and is characterized by swinging moods. It affects both sexes equally in all age groups and its worldwide prevalence is approximately 3-5%. The clinical course of illness can vary from a mild depression to a severe form of mania. The condition has a high rate of recurrence and if untreated, it has an approximately 15% risk of death by suicide. It is the third leading cause of death among people aged 15-24 years and is a burden on society and families. The pathophysiology of the disorder is poorly understood. However, a variety of imaging studies suggests the involvement of structural abnormalities in the amygdala, basal ganglia and prefrontal cortex. There are two main biological models that have been proposed for depression. These are called the serotonin and norepinephrine hypotheses. Multiple lines of evidence support both of them. It is a life-long disease and runs in families but has a complex mode of inheritance. Family, twin and adoption studies suggest genetic factors but the candidate susceptibility genes, which when mutated can account for a substantial portion of BPD patients, have not yet been conclusively identified. There have been an increasing number of new generation antidepressant drugs developed to treat BPD. However, lithium salt is only the drug that is most efficient in long-term preventive treatment and it also has an anti-suicidal effect. The condition can be well managed by physicians and psychiatrists along with family and patient education. Identification of risk genes in the future may provide a better understanding of the nature of pathogenesis that may lead to a better therapeutic target.     

Recurrent missense (R197C) and nonsense (Y89X) mutations in the XLRS1 gene in families with X-linked retinoschisis

Congenital retinoschisis (RS) is a hereditary eye disorder characterized by intraretinal schisis and central and peripheral retinal lesion. The gene responsible for the X-linked retinoschisis (XLRS1) has recently been isolated and found to contain mutations in affected members of several families. In this communication, two families with X-linked RS were analyzed for possible disease-causing mutations by polymerase chain reaction amplification of exons followed by DNA sequencing. Our analyses reveal a missense mutation at codon 197 in exon 6 and a nonsense mutation in exon-4 of XLRS1 gene. These changes resulted in the replacement of a highly conserved arginine by a cysteine residue and introduced a premature termination signal at codon 89, respectively. These mutations, which are transmitted through three generations, cosegregated with the disease, and are not found in the unaffected family members and 150 normal X-chromosomes, are likely to be pathogenic in these families.     

More to learn from gene knockouts

Gene targeting by homologous recombination in mouse embryonic stem cells is a powerful technique to determine the physiological function of any gene product in embryonic and postnatal development and in molecular pathogenesis. Although the technique is very demanding and still in its developing stage several knockout mice carrying disrupted genes, which were once thought important for the development or molecular pathogenesis of certain tissues, have given unexpected results. A gene/function redundancy or superfluous and on-functional theory has been advanced by many investigatiors to explain the unexpected results. These surprising results may teach us a new lesson and lead to a revision of the strongly held view that highly conserved and abundantly expressed genes have a prominent role and function in cell physiology and development Additional, they may also support the notion that molecular cross-talk among the genes may play an important role in determining the minimal phenotype.     

Use of dithiothreitol for improved visibility of the F body in human Y-bearing spermatozoa

Dithiothreitol was used to decondense the nuclear chromatin before staining human spermatozoa with quinacrine for the F body present in Y-bearing spermatozoa. In unseparated fractions and those enriched in X- or Y-bearing spermatozoa, the number of spermatozoa with F bodies increased by 2-9% after dithiothreitol treatment.     

Engineering of a wheat germ expression system to provide compatibility with a high throughput pET-based cloning platform

Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT^® SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354–359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two ¹⁵N-, ¹³C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline.