Apelin activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas

Apelin was recently found to be an inotropic polypeptide in isolated rat hearts, and intravenous injection of apelin can induce a transient decrease in blood pressure. To illustrate the mechanism of apelin-induced vasodilation, we observed the in vitro effects of apelin on the L-arginine (L-Arg)/nitric oxide (NO) pathway in the incubated, isolated rat aorta. Apelin stimulated vascular NO(2)(-) product and NOS activation in a concentration- and time-dependent manner. Compared with no apelin treatment, incubation with apelin (10(-9), 10(-8), and 10(-7)mol/L) increased NO(2)(-) product by 33%, 46%, and 69% (all p<0.01), respectively, and Ca(2+)-dependent constitutive NOS (cNOS) activity by 200%, 460%, and 550% (all p<0.01), respectively. However, Ca(2+)-independent NOS (iNOS) activity was not significantly altered (p>0.05). Apelin incubation (10(-9), 10(-8), and 10(-7)mol/L) increased L-Arg uptake by 130%, 180%, and 240% (all p<0.01), respectively. The mRNA level of cationic amino acid transporters, CAT-1 and CAT-2B, in rat aortic tissues treated with 10(-7)mol/L apelin was increased by 110% and 128%, respectively (both p<0.01). Incubation with 10(-7)mol/L apelin elevated eNOS mRNA and protein levels, by 53% (p<0.05) and 319% (p<0.01), respectively. Collectively, these results demonstrate that apelin directly activated the vascular L-Arg/NOS/NO pathway, which could be one of the important mechanisms of apelin-regulated vascular function.     

Normal cellular prion protein is a ligand of selectins: binding requires Le(X) but is inhibited by sLe(X)

The normal PrP(C) (cellular prion protein) contains sLe(X) [sialyl-Le(X) (Lewis X)] and Le(X). sLe(X) is a ligand of selectins. To examine whether PrP(C) is a ligand of selectins, we generated three human PrP(C)-Ig fusion proteins: one with Le(X), one with sLe(X), and the other with neither Le(X) nor sLe(X). Only Le(X)-PrP(C)-Ig binds E-, L- and P-selectins. Binding is Ca(2+)-dependent and occurs with nanomolar affinity. Removal of sialic acid on sLe(X)-PrP(C)-Ig enables the fusion protein to bind all selectins. These findings were confirmed with brain-derived PrP(C). The selectins precipitated PrP(C) in human brain in a Ca(2+)-dependent manner. Treatment of brain homogenates with neuraminidase increased the amounts of PrP(C) precipitated. Therefore the presence of sialic acid prevents the binding of PrP(C) in human brain to selectins. Hence, human brain PrP(C) interacts with selectins in a manner that is distinct from interactions in peripheral tissues. Alternations in these interactions may have pathological consequences.     

Comparison of the effects of acylation and amidation on the antimicrobial and antiviral properties of lactoferrin

AIMS: To compare amidation and acylation of lactoferrin (LF) from bovine milk, as a means of enhancing its antimicrobial and antiviral properties. METHODS AND RESULTS: LF was chemically modified by amidation with a 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) in the presence of ammonium ions or by acylation with either succinic or acetic anhydride. In the test systems used, amidation substantially enhanced the activity of LF against Pseudomonas fluorescens in comparison with native LF. However, increasing the net negative charge of LF by acylation had no effect on the activity of LF against P. fluorescens, and abrogated the antimicrobial activity of LF against Bacillus subtilis and Saccharomyces cerevisiae. Increasing the net negative charges of LF by acylation eliminated its antimicrobial and antiviral effects against poliovirus and feline calicivirus (nonenveloped viruses). CONCLUSIONS: The addition of positive charges to LF via amidation enhanced antimicrobial properties in contrast to increasing the negative charges by acylation, which abolished both the antimicrobial and antiviral properties of LF. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of charge alteration of LF determined in this study provides a basis for further development of LF formulations with enhanced antimicrobial effectiveness for use in food process hygiene, veterinary and health-care applications.     

Skp2B stimulates mammary gland development by inhibiting REA, the repressor of the estrogen receptor

Skp2B, an F-box protein of unknown function, is frequently overexpressed in breast cancer. In order to determine the function of Skp2B and whether it has a role in breast cancer, we performed a two-hybrid screen and established transgenic mice expressing Skp2B in the mammary glands. We found that Skp2B interacts with the repressor of estrogen receptor activity (REA) and that overexpression of Skp2B leads to a reduction in REA levels. In the mammary glands of MMTV-Skp2B mice, REA levels are also low. Our results show that in virgin transgenic females, Skp2B induces lobuloalveolar development and differentiation of the mammary glands normally observed during pregnancy. As this phenotype is identical to what was observed for REA heterozygote mice, our observations suggest that the Skp2B-REA interaction is physiologically relevant. However, in contrast to REA(+/-) mice, MMTV-Skp2B mice develop mammary tumors, suggesting that Skp2B affects additional proteins. These results indicate that the observed expression of Skp2B in breast cancer does contribute to tumorigenesis at least in part by modulating the activity of the estrogen receptor.     

Role of eIF3a (eIF3 p170) in intestinal cell differentiation and its association with early development

Eukaryotic initiation factor 3a (eIF3a) has been suggested to play a regulatory role in mRNA translation. Decreased eIF3a expression has been observed in differentiated cells while higher levels have been observed in cancer cells. However, whether eIF3a plays any role in differentiation and development is currently unknown. Here, we investigated eIF3a expression during mouse development and its role in differentiation of colon epithelial cells. We found that eIF3a expression was higher in fetal tissues compared with postnatal ones. Its expression in intestine, stomach, and lung abruptly stopped on the 18th day in gestation but persisted in liver, kidney, and heart throughout the postnatal stage at decreased levels. Similarly, eIF3a expression in colon cancer cell lines, HT-29 and Caco-2, drastically decreased prior to differentiation. Enforced eIF3a expression inhibited while knocking it down using small interference RNA promoted Caco-2 differentiation. Thus, eIF3a may play some roles in development and differentiation and that the decreased eIF3a expression may be a pre-requisite of intestinal epithelial cell differentiation.     

Aging effects on the habitual expression of HSP70 mRNA in the hippocampus of rats

Heat shock proteins are induced by stressful stimuli and have been shown to protect cells and organs from such stresses both in vitro and in vivo. This study examined the regulation of HSP70 mRNA expression and detected the effect of aging on RNA expression in hippocampus of rats. The stress models were built by using forced-swimming in 25 degrees C and 4 degrees C water, respectively. Two groups of male rats, 2-month-old and 16-month-old, respectively, were randomly divided into three subgroups: acute stress (AS) model, chronic habituation stress (CHS) model and chronic dishabituation stress (CDS) model. Observation of exploratory behavior in an open-field (OF) test indicated stress levels. The expression of HSP70 mRNA in hippocampus was measured by RT-PCR after 0, 30, 60, 180, and 360 min of stress, respectively. Results showed that the number of quadrant crossing in both aged CHS and young CHS groups decreased gradually with the process of stress, reflecting an adaptation to the stress condition. Repeated swimming in warm water resulted in habitual expression of HSP70 mRNA in both young and aged CHS group, indicating an adaptation to the stress. The RNA expression of young CHS group was significantly stronger than that of the aged CHS group at 30, 60, 180, and 360 min after stress (P < 0.05). Meanwhile, in an intensive stress level in which the rats swam in 4 degrees C water, a high expression level of HSP70 mRNA was achieved in CDS groups, producing a dishabituation that proved the habitual expression from the other side. These results showed that senescence dramatically affected both exploratory behavior and HSP70 mRNA expression in rats' hippocampus. The results also suggested that chronic stress could lead to the habituational expression of HSP70 mRNA, but high intensive stress could reverse the habituational state and lead to the dishabituational expression. Moreover, the duration of stimuli is one of the important factors that affect the level of HSP70 mRNA expression.     

Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein

Imidazole is a reagent widely used in protein purifying processes. Here, we reveal a novel chaperone-like activity for imidazole using enhanced green fluorescent protein (EGFP) as a model protein. Experimental results showed that imidazole acted as an effective catalyst for refolding of the chemically denatured EGFP and suppressor for the heat-induced aggregation of EGFP. The refolding kinetics was determined in real time. Both the recovering yield and refolding rate of denatured EGFP in the presence of imidazole were increased. The studies on elucidating the mechanism show that imidazole may catalyze the prolyl cis/trans isomerization and the possible mechanism was discussed. To our knowledge, there are no data on the effect of imidazole on protein folding. Considering the prolyl isomerization is the rate-limited step for refolding of most proteins and aggregation is a universal serious problem for biotechnology, imidazole thus represents a previous unknown type of protein-folding catalyst.     

Homochiral metal-organic coordination networks from l-typtophan

Three homochiral metal-organic coordination networks [Co₂(l-Trp)₂(Py)₆] · Py · (ClO₄)₂ (1), [Ni(l-Trp)(Py)₃] · H₂O · ClO₄ (2) and [Co₂(l-Trp)(INT)₂(H₂O)₂(ClO₄)] (3), all containing natural amino acid l-HTrp (l-typtophan), were hydrothermally synthesized and structurally characterized. The compounds 1 and 2 crystallize in the orthorhombic space group C222₁, with a = 10.731(2) Å, b = 19.709(4) Å, c = 27.365(6) Å and Z = 4 for 1 and a = 10.710(10) Å, b = 20.088(18) Å, c = 27.63(3) Å and Z = 8 for 2, respectively. The compound 3 has the monoclinic space group P2₁, with a = 8.1934(14) Å, b = 13.209(2) Å, c = 12.464(2) Å, β = 104.107(3)° and Z = 2. Both 1 and 2 consist of 1D helical chains. Compound 3 is composed of 2D networks, which further assemble into a 3D supramolecular structure via weak interlayer interactions. The optically pure amino acid l-HTrp plays an important role leading to homochiral structures reported here.