Slit-2 induces a tumor-suppressive effect by regulating beta-catenin in breast cancer cells

SLIT-2 is considered as a candidate tumor suppressor gene, because it is frequently inactivated in various cancers due to hypermethylation of its promoter region and allelic loss. However, the exact mechanism of its tumor-suppressive effect has not been elucidated. Here, we observed that Slit-2-overexpressing breast cancer cells exhibited decreased proliferation and migration capabilities compared with control cells under in vitro conditions. These results were confirmed in vivo in mouse model systems. Mice injected with MCF-7/Slit-2 cells showed a 60-70% reduction in tumor size compared with mice injected with MCF-7/VC cells both in the absence and presence of estrogen. Upon further elucidation, we observed that Slit-2 mediates the tumor-suppressive effect via a coordinated regulation of the beta-catenin and PI3K signaling pathways and by enhancing beta-catenin/E-cadherin-mediated cell-cell adhesion. Our study for the first time reveals that Slit-2-overexpressing breast cancer cells exhibit tumor suppressor capabilities through the novel mechanism of beta-catenin modulation.     

Genes encoding members of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene family from <Emphasis Type="Italic">Azadirachta indica</Emphasis> and correlation with azadirachtin biosynthesis

Azadirachta indica (A. Juss) commonly known as Neem is an important source of valuable natural products and occupies an important place in traditional healthcare system. Naturally, this plant synthesizes a number of tetranortriterpenoids utilizing isoprenoid as substrate flux. Although various phytochemical and pharmacological studies in A. indica have been carried out, but very limited information is available about the biosynthetic pathway as well as structural and regulatory genes involved in synthesis of bioactive molecules. In this study, we have cloned and characterized two genes, AiHMGR1 and AiHMGR2, encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34) catalyzing the rate limiting step of the isoprenoid biosynthesis. Two isoforms, AiHMGR1 and AiHMGR2, contain an open reading frame of 1707 and 1695 bp encoding polypeptides of 568 and 545 amino acid residues, respectively. The nucleotide and encoded amino acid sequence analyses suggest that both genes encode polypeptides with necessary structural domains present in other plant HMGRs, however, have different genomic organization. The relative expression analysis suggests that two genes express differentially in various tissues. Out of the two genes, expression of AiHMGR2 showed a direct correlation with azadirachtin accumulation in fruit tissue. The common as well as unique cis-regulatory elements present in both genes might be responsible for differential expression of both the genes in various tissues. The color complementation assay in Escherichia coli suggests that though both AiHMGR1 and AiHMGR2 encode functional proteins, AiHMGR2 is more active as compared to AiHMGR1.     

Contrasting effects of ischemia on the kinetics of membrane voltage and intracellular calcium transient underlie electrical alternans

Repolarization alternans has been considered a strong marker of electrical instability. The objective of this study was to investigate the hypothesis that ischemia-induced contrasting effects on the kinetics of membrane voltage and intracellular calcium transient (Ca(i)T) can explain the vulnerability of the ischemic heart to repolarization alternans. Ischemia-induced changes in action potential (AP) and Ca(i)T resulting in alternans were investigated in perfused Langendorff guinea pig hearts subjected to 10-15 min of global no-flow ischemia followed by 10-15 min of reperfusion. The heart was stained with 100 microl of rhod-2 AM and 25 microl of RH-237, and AP and Ca(i)T were simultaneously recorded with an optical mapping system of two 16 x 16 photodiode arrays. Ischemia was associated with shortening of AP duration (D) but delayed upstroke, broadening of peak, and slowed decay of Ca(i)T resulting in a significant increase of Ca(i)T-D. The changes in APD were spatially heterogeneous in contrast to a more spatially homogeneous lengthening of Ca(i)T-D. Ca(i)T alternans could be consistently induced with the introduction of a shorter cycle when the upstroke of the AP occurred before complete relaxation of the previous Ca(i)T and generated a reduced Ca(i)T. However, alternans of Ca(i)T was not necessarily associated with alternans of APD, and this was correlated with the degree of spatially heterogeneous shortening of APD. Sites with less shortening of APD developed alternans of both Ca(i)T and APD, whereas sites with greater shortening of APD could develop a similar degree of Ca(i)T alternans but slight or no APD alternans. This resulted in significant spatial dispersion of APD. The study shows that the contrasting effects of ischemia on the duration of AP and Ca(i)T and, in particular, on their spatial distribution explain the vulnerability of ischemic heart to alternans and the increased dispersion of repolarization during alternans.     

Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli

Actin-based protrusions can form prominent structures on the apical surface of epithelial cells, such as microvilli. Several cytoplasmic factors have been identified that control the dynamics of actin filaments in microvilli. However, it remains unclear whether the plasma membrane participates actively in microvillus formation. In this paper, we analyze the function of Drosophila melanogaster cadherin Cad99C in the microvilli of ovarian follicle cells. Cad99C contributes to eggshell formation and female fertility and is expressed in follicle cells, which produce the eggshells. Cad99C specifically localizes to apical microvilli. Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length. Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length. This study reveals Cad99C as a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process.     

Molecular determinants of infectious pancreatic necrosis virus virulence and cell culture adaptation

Infectious pancreatic necrosis viruses (IPNVs) exhibit a wide range of virulence in salmonid species. In previous studies, we have shown that the amino acid residues at positions 217 and 221 in VP2 are implicated in virulence. To pinpoint the molecular determinants of virulence in IPNV, we generated recombinant IPNV strains using the cRNA-based reverse-genetics system. In two virulent strains, residues at positions 217 and 247 were replaced by the corresponding amino acids of a low-virulence strain. The growth characteristics of the recovered chimeric strains in cell culture were similar to the low-virulence strains, and these viruses induced significantly lower mortality in Atlantic salmon fry than the parent strains did in in vivo challenge studies. Furthermore, the virulent strain was serially passaged in CHSE-214 cells 10 times and was completely characterized by nucleotide sequencing. Deduced amino acid sequence analyses revealed a single amino acid substitution of Ala to Thr at position 221 in VP2 of this virus, which became highly attenuated and induced 15% cumulative mortality in Atlantic salmon fry, compared to 68% mortality induced by the virulent parent strain. The attenuated strain grows to higher titers in CHSE cells and can be distinguished antigenically from the wild-type virus by use of a monoclonal antibody. However, the virulent strain passaged 10 times in RTG-2 cells was stable, and it retained its antigenicity and virulence. Our results indicate that residues Thr at position 217 (Thr217) and Ala221 of VP2 are the major determinants of virulence in IPNV of the Sp serotype. Highly virulent isolates possess residues Thr217 and Ala221; moderate- to low-virulence strains have Pro217 and Ala221; and strains containing Thr221 are almost avirulent, irrespective of the residue at position 217.     

Different cellular localization,translocation, and insulin-induced phosphorylation of PKBalpha in HepG2 cells and hepatocytes

Protein kinase B (PKB), a serine/threonine protein kinase, prevents apoptosis and promotes cellular transformation. PKB activity is stimulated by insulin. In this report, we examined the relative amounts of expression, location, and translocation upon insulin stimulation of PKBalpha in normal primary hepatocytes and carcinoma cells, HepG2 cells. Non-phosphorylated PKBalpha was present in both types of unstimulated cells. The phosphorylated form of the enzyme was present in the nucleus of unstimulated HepG2 cells but not in normal hepatocytes. In the cytoplasm, PKBalpha was found in greater abundance in the hepatocytes as compared in HepG2 cells. Insulin induced the translocation of phosphorylated PKBalpha from the nucleus to the nuclear membrane in HepG2 cells. In contrast, insulin caused translocation and phosphorylation of PKBalpha from the cytosol to the plasma membrane in normal hepatocytes. In addition, there is a higher expression of PKBalpha in the HepG2 cells as compared to normal primary hepatocytes. These findings provide an important distinction between hepatocellular HepG2 cells and normal liver cells and suggest that the presence of constitutively active nuclear PKB in the transformed cells might be an important contributor in cell transformation and immortality of hepatoma cells.     

Protamine is a low molecular weight polycationic amine that produces actions on cardiac muscle

Protamine is a polycationic amine used clinically to reverse heparin overdose. Here we characterized the actions of protamine on the cardiovascular system of anesthetized rats and in isolated Langendorff rat hearts in order to define a possible mechanism of action on cardiovascular tissue. In anesthetized rats, protamine reduced blood pressure in a dose-dependent fashion and reduced heart rate. Only at a dose of 32 mg/kg did protamine increase the Q-aT interval of the electrocardiogram (EKG) to 62 +/- 6 msec from a control of 54 +/- 5 msec (p < 0.05). Protamine dose-dependently reduced cardiac output by 74 +/- 5% and stroke volume by 62 +/- 15 %, suggesting that it directly affects cardiac contractility. An analysis of blood chemistry suggests that protamine does not alter plasma electrolyte or serum enzyme levels at the doses administered. Protamine produced aberrant rhythms in normal rat hearts when administered between 1-32 mg/kg. The P-Q segment of the EKG for each of the arrhythmic complexes was reduced to 24 +/- 1 msec compared to 32 +/- 3 msec in normal EKG complexes suggestive of anomalous atrio-ventricular or pre-excitation conduction. Isolated rat heart studies confirmed that protamine produced a reduction in cardiac contractility. Our studies suggest that the cardiovascular depressant actions of protamine result from a direct effect on the heart and that protamine may produce aberrant conduction within the heart which may result in deleterious effects in heart function, especially conditions associated with myocardial disease.