Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.     

Mechanisms of HIV-1 evasion to the antiviral activity of chemokine CXCL12 indicate potential links with pathogenesis

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses’ receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.     

Detection of myocardial ischemia by epicardial detection of potassium ion activity]

Early detection of myocardial ischaemia is a central problem in cardiological and cardiosurgical intensive care. A new approach is the use of ion-selective electrodes implanted directly on the myocardium, enabling detection of increased potassium activity as an indication of general hypoxia. After a comprehensive study of the electrode parameters, an animal experiment was carried out, in which it was found that respiration-induced hypoxia resulted in an increase in epicardial potassium activity (p < 0.01). Blood gas analysis performed simultaneously revealed reduced arterial pO2, but no acidosis. Haemodynamic data evidenced hypoxic depression of circulatory parameters. Histological examinations of the myocardium beneath the electrodes revealed typical lymphocytic infiltration. Electron microscopy demonstrated crystolysis in the mitochondria as an early sign of hypoxia, thus confirming the sensitivity of these electrodes. This underscores the potential of ion-selective electrodes for the detection of myocardial ischaemia, and they should now be investigated in the clinical setting.     

Aluminum,Fe, Ca,Mg, K,Mn, Cu,Zn and P in above- and belowground biomass. I.Abies amabilis andTsuga mertensiana

In a mature mixed subalpine stand ofTsuga mertensiana andAbies amabilis, significantly higher Al levels were found in foliage, branch and root tissues ofT. mertensiana.Tsuga mertensiana had significant increases in Al, Ca and Mn levels with increasing foliage age. In current foliage,T. mertensiana had lower levels of Ca, similar levels of Mg and P, and higher levels of Mn thanA. amabilis. Both tree species had Cu and Fe present at higher levels in branch than foliage tissues. Fine roots had the highest concentrations of Al, Fe and Cu but the lowest Ca and Mn concentrations of all tissues analyzed. In the roots of both species, phloem tissues always had significantly higher Al levels than xylem. Fine roots (< 1 and 1–2 mm) ofT. mertensiana had higher Al levels than were found inA. amabilis. Roots greater than 2 mm in diameter exhibited no significant differences in Al levels in phloem or xylem tissue betweenA. amabilis andT. mertensiana. The two species show a clear difference in their ability to accumulate specific elements from the soil.     

Biosynthesis of Astacus protease, a digestive enzyme from crayfish

For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalloenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.     

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.     

Fish oil affects phosphoinositide turnover and thromboxane A metabolism in cultured vascular muscle cells

Fish oil has been reported as having beneficial effects on cardiovascular diseases. Elevated serum lipoproteins, prostaglandins and intracellular free calcium concentrations [( Ca2+]i) of the vasculature and thus the phosphoinositide (PI) turnover may be involved in the pathogenesis of these disorders. Therefore, the effect of fish oil on the potency of both low-density lipoprotein (LDL) and angiotensin II (AII) to stimulate the PI turnover in cultured rat vascular smooth muscle cells (VSMC) has been studied. Furthermore, a possible link between PI turnover activity and thromboxane A2 (TXA2) metabolism in these cells has been investigated. In VSMC cultured for up to 7 weeks with either fish oil or n-3 eicosapentaenoic acid (EPA) a decrease to 5-48% of the LDL-induced inositol trisphosphate (IP3) formation (= 100%) was found. A similar range of decreased IP3 synthesis was observed, when AII was used instead of LDL. Both LDL- and AII-stimulated TXA2 synthesis was suppressed concomitantly within the range 34-60%. Blockade of VSMC TXA2 biosynthesis with either indomethacin or TXA2 synthetase blocker (SQ-80338) inhibited LDL-induced formation of IP3 in a dose-dependent manner. Similar results were obtained, when TXA2 receptor coupling antagonists (SQ-27427 or BM-13177) were used. However, blockers of TXA2 synthesis and of TXA2 receptor binding failed to affect AII-induced formation of IP3.     

A “new” genetic polymorphism of a human serum protein: inter-alpha-trypsin-inhibitor

Summary A new genetic polymorphism of a human serum glycoprotein, the inter--trypsin-inhibitor (ITI), has been demonstrated by population and family studies. Sera were examined after neuraminidase treatment by isoelectric focusing on agarose gels followed by immunoblotting or by immunfixation with specific ITI-antiserum. Using this method, three common ITI phenotypes 1, 1–2 and 2, as well as two further rare ITI types 1–3 and 2–3 were disclosed. Genetically, these phenotypes are controlled by three allelic genes that determine a total of six phenotypes. These alleles are designated ITI^*1, ITI^*2 and ITI^*3. The homozygous form of the third allele ITI^*3 has not been found, as yet. The frequencies of ITI were examined in two population samples from Southern Germany (n=248) and from Tyrol, Austria (n=124). The gene frequencies of the common alleles ITI^*1 and ITI^*2 were 0.575 and 0.417, respectively, in Southern Germany, and 0.577 and 0.423, respectively, in Tyrol, Austria. The third allele ITI^*3 was found only in the sample from Southern Germany, thus far, and was calculated to be 0.008.