Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role of Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation.     

Frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates

The recognition that AIDS originated as a zoonosis heightens public health concerns associated with human infection by simian retroviruses endemic in nonhuman primates (NHPs). These retroviruses include simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus (STLV), simian type D retrovirus (SRV), and simian foamy virus (SFV). Although occasional infection with SIV, SRV, or SFV in persons occupationally exposed to NHPs has been reported, the characteristics and significance of these zoonotic infections are not fully defined. Surveillance for simian retroviruses at three research centers and two zoos identified no SIV, SRV, or STLV infection in 187 participants. However, 10 of 187 persons (5.3%) tested positive for SFV antibodies by Western blot (WB) analysis. Eight of the 10 were males, and 3 of the 10 worked at zoos. SFV integrase gene (int) and gag sequences were PCR amplified from the peripheral blood lymphocytes available from 9 of the 10 persons. Phylogenetic analysis showed SFV infection originating from chimpanzees (n = 8) and baboons (n = 1). SFV seropositivity for periods of 8 to 26 years (median, 22 years) was documented for six workers for whom archived serum samples were available, demonstrating long-standing SFV infection. All 10 persons reported general good health, and secondary transmission of SFV was not observed in three wives available for WB and PCR testing. Additional phylogenetic analysis of int and gag sequences provided the first direct evidence identifying the source chimpanzees of the SFV infection in two workers. This study documents more frequent infection with SFV than with other simian retroviruses in persons working with NHPs and provides important information on the natural history and species origin of these infections. Our data highlight the importance of studies to better define the public health implications of zoonotic SFV infections.     

Neuraminidase fromPasteurella haemolytica

A soluble extracellular antigen produced byPasteurella haemolytica serotype 1 possesses neuraminidase activity. The metal ions Mn²⁺, Mg²⁺, and Ca²⁺ stimulate enzyme activity, while the heavy metal ions Hg²⁺ and Fe³⁺ inhibit neuraminidase activity. Extensive dialysis of the enzyme with 50 mM ethylenediaminetetraacetic acid does not result in loss of enzyme activity. Antiserum prepared againstPasteurella haemolytica serotype 1 inhibits neuraminidase activity 2.0- to 2.7-fold, suggesting that neuraminidase is part of the antigenic complex. Initial rate kinetics of neuraminidase reveals that the Michaelis constant for fetuin is 3.84 mg/ml (96 M) and the maximal velocity (V _max) is 0.53 mmol/min/mg of protein.     

Large scale separation of protease inhibitors from malignant human breast tissue

Summary ₁-Antitrypsin was isolated as an electrophoretically homogeneous protein in preparative quantities from malignant human breast tissue by a two-step procedure of affinity chromatography on Sepharose-chymotrypsin, followed by polyacrylamide gel electrophoresis. Additionally, ₂-macroglobulin and ₁-antichymotrypsin were bound to, and subsequently eluted from the column. Antithrombin III and ₁-acid glycoprotein, which were also components of the tissue extracts, were not bound, and appeared in the breakthrough peak. ₁-Antitrypsin appeared to be partially modified immunologically by Sepharose-chymotryppin chromatography. However, ₂-macroglobulin remained immunologically unaltered. Upon subsequent polyacrylamide gel electrophoresis, the ₁-antitrypsin regained its capacity to give an immunological reaction of complete identity. Inhibitory activity toward proteases is also observed in filtrates of 1000 molecular weight cut-off membrane filters. Endogenous caseinolytic activity was observed in several eluate fractions. However, only N-benzoyl-L-tyrosine-ethyl ester, of the model compounds tested, was cleaved. The esterase activity is attributed to a breast tissue component since the column was free from dissociable chymotrypsin. Affinity chromatography on Sepharose-chymotrypsin represents a means of rapidly separating 37.5% of the total antichymotryptic activity from breast tissue extracts in high purity, in a single step.     

Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock-controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.     

Circadian regulation of hippocampal long-term potentiation

The goal of this study is to investigate the possible circadian regulation of hippocampal excitability and long-term potentiation (LTP) measured by stimulating the Schaffer collaterals (SC) and recording the field excitatory postsynaptic potential (fEPSP) from the CA1 dendritic layer or the population spike (PS) from the soma in brain slices of C3H and C57 mice. These 2 strains of mice were of interest because the C3H mice secrete melatonin rhythmically while the C57 mice do not. The authors found that the magnitude of the enhancement of the PS was significantly greater in LTP recorded from night slices compared to day slices of both C3H and C57 mice. They also found significant diurnal variation in the decay of LTP measured with fEPSPs, with the decay slower during the night in both strains of mice. There was evidence for a diurnal rhythm in the input/output function of pyramidal neurons measured at the soma in C57 but not C3H mice. Furthermore, LTP in the PS, measured in slices prepared during the day but recorded during the night, had a profile remarkably similar to the night group. Finally, PS recordings were carried out in slices from C3H mice maintained in constant darkness prior to experimentation. Again, the authors found that the magnitude of the enhancement of the PS was significantly greater in LTP recorded from subjective night slices compared to subjective day slices. These results provide the 1st evidence that an endogenous circadian oscillator modulates synaptic plasticity in the hippocampus.     

Beneficial effect of short-term exposure of human NK cells to IL15/IL12 and IL15/IL18 on cell apoptosis and function

Monokines IL12, IL15, and IL18 have been shown to activate NK cell function, however with high apoptosis induced by their combination within 48 h. Here, we demonstrate for the first time that CD56+ cells incubated for only 18 h with the combination of IL15/IL12 or IL15/IL18, then washed, and further cultured in plain medium, exhibit low levels of apoptosis. These shortly activated CD56+ cells show high killer activity against NK- and LAK-sensitive tumor targets that persists over a culture period of 18 days after two additional 6 h cycles of exposure to the monokines applied every 8 days and also retain their ability for high cytokine production during each exposure. Moreover, these repetitive short-term exposures of CD56+ cells to the monokine combinations result in long-lived CD56+ cells with slower rates of FcgammaRIII receptor (CD16) decline, therefore exhibiting higher antibody depended cytotoxicity, as opposed to the continuous incubation with the monokine combinations. In conclusion, short-term exposure of CD56+ cells to IL15/IL12 or IL15/IL18 at 8-day intervals may hold a promise for improved clinical results in cellular adoptive cancer immunotherapy and for the in vivo injections of the monokines.     

Grass pollen immunotherapy induces mucosal and peripheral IL-10 responses and blocking IgG activity

T regulatory cells and IL-10 have been implicated in the mechanism of immunotherapy in patients with systemic anaphylaxis following bee stings. We studied the role of IL-10 in the induction of clinical, cellular, and humoral tolerance during immunotherapy for local mucosal allergy in subjects with seasonal pollinosis. Local and systemic IL-10 responses and serum Ab concentrations were measured before/after a double-blind trial of grass pollen (Phleum pratense, Phl P) immunotherapy. We observed local increases in IL-10 mRNA-positive cells in the nasal mucosa after 2 years of immunotherapy, but only during the pollen season. IL-10 protein-positive cells were also increased and correlated with IL-10 mRNA(+) cells. These changes were not observed in placebo-treated subjects or in healthy controls. Fifteen and 35% of IL-10 mRNA signals were colocalized to CD3(+) T cells and CD68(+) macrophages, respectively, whereas only 1-2% of total CD3(+) cells and 4% of macrophages expressed IL-10. Following immunotherapy, peripheral T cells cultured in the presence of grass pollen extract also produced IL-10. Immunotherapy resulted in blunting of seasonal increases in serum allergen Phl p 5-specific IgE, 60- to 80-fold increases in Phl p 5-specific IgG, and 100-fold increases in Phl p 5-specific IgG4. Post-immunotherapy serum exhibited inhibitory activity, which coeluted with IgG4, and blocked IgE-facilitated binding of allergen-IgE complexes to B cells. Both the increases in IgG and the IgG "blocking" activity correlated with the patients' overall assessment of improvement. Thus, grass pollen immunotherapy may induce allergen-specific, IL-10-dependent "protective" IgG4 responses.     

Functional and structural similarities between the internal ribosome entry sites of hepatitis C virus and porcine teschovirus, a picornavirus

Initiation of protein synthesis on picornavirus RNA requires an internal ribosome entry site (IRES). Typically, picornavirus IRES elements contain about 450 nucleotides (nt) and use most of the cellular translation initiation factors. However, it is now shown that just 280 nt of the porcine teschovirus type 1 Talfan (PTV-1) 5' untranslated region direct the efficient internal initiation of translation in vitro and within cells. In toeprinting assays, assembly of 48S preinitiation complexes from purified components on the PTV-1 IRES was achieved with just 40S ribosomal subunits plus eIF2 and Met-tRNA(i)(Met). Indeed, a binary complex between 40S subunits and the PTV-1 IRES is formed. Thus, the PTV-1 IRES has properties that are entirely different from other picornavirus IRES elements but highly reminiscent of the hepatitis C virus (HCV) IRES. Comparison between the PTV-1 IRES and HCV IRES elements revealed islands of high sequence identity that occur in regions critical for the interactions of the HCV IRES with the 40S ribosomal subunit and eIF3. Thus, there is significant functional and structural similarity between the IRES elements from the picornavirus PTV-1 and HCV, a flavivirus.     

Roles of molecular regions in determining differences between voltage dependence of activation of CaV3.1 and CaV1.2 calcium channels

Voltage-dependent calcium channels are classified into low voltage-activated and high voltage-activated channels. We have investigated the molecular basis for this difference in voltage dependence of activation by constructing chimeras between a low voltage-activated channel (Ca(V)3.1) and a high voltage-activated channel (Ca(V)1.2), focusing on steady-state activation properties. Wild type and chimeras were expressed in oocytes, and two-electrode voltage clamp recordings were made of calcium channel currents. Replacement of domains I, III, or IV of the Ca 3.1 channel with the corresponding domain of Ca(V)1.2 led (V)to high voltage-activated channels; for these constructs the current/voltage (I/V) curves were similar to those for Ca(V)1.2 wild type. However, replacement of domain II gave only a small shift to the right of the I/V curve and modulation of the activation kinetics but did not lead to a high voltage-activating channel with an I/V curve like Ca 1.2. We also investigated the role of the voltage sensor (V)S4 by replacing the S4 segment of Ca(V)3.1 with that of Ca 1.2. For domain I, there was no shift in the I/V curve (V)as compared with Ca(V)3.1, and there were relatively small shifts to the right for domains III and IV. Taken together, these results suggest that domains I, III, and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca 3.1 and Ca(V)1.2. However, the S4 segments in domains I, (V)III, and IV did not account for this difference in voltage dependence.