Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3′ end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.     

PR39, a peptide regulator of angiogenesis

Although tissue injury and inflammation are considered essential for the induction of angiogenesis, the molecular controls of this cascade are mostly unknown. Here we show that a macrophage-derived peptide, PR39, inhibited the ubiquitin-proteasome-dependent degradation of hypoxia-inducible factor-1alpha protein, resulting in accelerated formation of vascular structures in vitro and increased myocardial vasculature in mice. For the latter, coronary flow studies demonstrated that PR39-induced angiogenesis resulted in the production of functional blood vessels. These findings show that PR39 and related compounds can be used as potent inductors of angiogenesis, and that selective inhibition of hypoxia-inducible factor-1alpha degradation may underlie the mechanism of inflammation-induced angiogenesis.     

Induction of growth hormone (GH) mRNA by pulsatile GH-releasing hormone in rats is pattern specific

Growth hormone-releasing hormone (GHRH) is a main inducer of growth hormone (GH) pulses in most species studied to date. There is no information regarding the pattern of GHRH secretion as a regulator of GH gene expression. We investigated the roles of the parameters of exogenous GHRH administration (frequency, amplitude, and total amount) upon induction of pituitary GH mRNA, GH content, and somatic growth in the female rat. Continuous GHRH infusions were ineffective in altering GH mRNA levels, GH stores, or weight gain. Changing GHRH pulse amplitude between 4, 8, and 16 microg/kg at a constant frequency (Q3.0 h) was only moderately effective in augmenting GH mRNA levels, whereas the 8 microg/kg and 16 microg/kg dosages stimulated weight gain by as much as 60%. When given at a 1.5-h frequency, GHRH doubled the amount of GH mRNA, elevated pituitary GH stores, and stimulated body weight gain. In the rat model, pulsatile but not continuous GHRH administration is effective in inducing pituitary GH mRNA and GH content as well as somatic growth. These studies suggest that the greater growth rate, pituitary mRNA levels, and GH stores seen in male compared with female rats are likely mediated, in part, by the endogenous episodic GHRH secretory pattern present in males.     

Choline Availability Modulates the Expression of TGFβ1 and Cytoskeletal Proteins in the Hippocampus of Developing Rat Brain

Choline availability influences long-term memory in concert with changes in the spatial organization and morphology of septal neurons, however little is known concerning the effects of choline on the hippocampus, a region of the brain also important for memory performance. Pregnant rats on gestational day 12 were fed a choline control (CT), choline supplemented (CS), or choline deficient (CD) diet for 6 days and fetal brain slices were prepared on embryonic day 18 (El8). The hippocampus in these brain slices was studied for the immunohistochemical localization of the growth-related proteins transforming growth factor beta type 1 (TGF1) and GAP43, the cytoskeletal proteins vimentin and microtubule associated protein type 1 (MAP1), and the neuronal cell marker neuron specific enolase (NSE). In control hippocampus, there was weak expression of TGF1 and vimentin proteins, but moderately intense expression of MAP1 protein. These proteins were not homogeneously distributed, but were preferentially localized to cells with large cell bodies located in the central (CA1–CA3) region of the hippocampus, and to the filamentous processes of small cells in the fimbria region. Feeding a choline-supplemented diet decreased, whereas a choline-deficient diet increased the intensity of immunohistochemical labeling for these proteins in El8 hippocampus. GAP43 and NSE were localized to peripheral nervous tissue but not hippocampus, indicating that the maturation of axons and neurite outgrowth in embryonic hippocampus were unaffected by the availability of choline in the diet. These data suggest that the availability of choline affects the differentiation of specific regions of developing hippocampus.     

hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'NTR of the HCV RNA genome

Like other members of the Flaviviridae family, the 3' non-translated region (NTR) of the hepatitis C virus (HCV) is believed to function in the initiation and regulation of viral RNA replication by interacting with components of the viral replicase complex. To inves-tigate the possibility that host components may also participate in this process, we used UV cross-linking assays to determine if any cellular proteins could bind specifically to the 3'NTR RNA. We demonstrate the specific interaction of two host proteins with the extensive pyrimidine-rich region within the HCV 3'NTR. One host protein migrates as a doublet with a molecular weight of 57 kDa and is immunoreactive with antisera specific for polypyrimidine tract-binding protein (PTB), and the other protein (35 kDa) is recognized by a monoclonal antibody specific for heterogeneous nuclear ribonucleoprotein C (hnRNP C). These results suggest that recognition of the large pyrimidine-rich region by PTB and hnRNP C may play a role in the initiation and/or regulation of HCV RNA replication.     

Resistin induces monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression in endothelial cells via p38MAPK-dependent pathway

Resistin, firstly reported as an adipocyte-specific hormone, is suggested to be an important link between obesity and diabetes. Recent studies have suggested an association between resistin and atherogenic processes. The adhesion of circulating monocytes to endothelial cells is a critical step in the early stages of atherosclerosis. The purpose of the present study was to investigate the effect of resistin on the adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Our results showed that resistin caused a significant increase in monocyte adhesion. In exploring the underlying mechanisms of resistin action, we found that resistin-induced monocyte adhesion was blocked by inhibition of p38MAPK activation using SB203580 and SB202190. Furthermore, resistin increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by HUVECs and these effects were also p38MAPK-dependent. Resistin-induced monocyte adhesion was also blocked by monoclonal antibodies against ICAM-1 and VCAM-1. Taken together, these results show that resistin increases both the expression of ICAM-1 and VCAM-1 by endothelial cells and monocyte adhesion to HUVECs via p38MAPK-dependent pathways.     

Growth hormone-releasing factor releases ACTH from an AtT-20 mouse pituitary tumor cell line but not from normal pituitary cells

Corticotropin-releasing factor (CRF) and both human pancreatic growth hormone-releasing factor (hp-GRF) and rat hypothalamic GRF (rh-GRF) stimulated ACTH release from neoplastic AtT-20 mouse pituitary tumor cells in a dose-dependent fashion, with CRF inducing a 10-fold increase and GRF a maximal increment of approximately one-half that of CRF. Neither rh-GRF nor hp-GRF induced ACTH release in normal anterior pituitary cells. Pretreatment with either dexamethasone or somatostatin prior to the addition of rh-GRF inhibited the increase in ACTH release. Both ovine CRF and rh-GRF stimulated adenosine 3,5-monophosphate production in AtT-20 cells. The weak but clearly discernible effect of GRF on ACTH release from AtT-20 cells may be due to an abnormality in the AtT-20 cell receptor.     

A method for purification of bacterial R-type lipopolysaccharides (lipooligosaccharides)

A new gel filtration method was developed for purification of R-type lipopolysaccharides (lipooligosaccharides) from some nonenteric gram-negative bacteria, including Neisseria meningitidis, Haemophilus influenzae, and Bordetella pertussis. These wild-type lipooligosaccharides are poorly extractable by the phenol-chloroform-ether extraction method of C. Galanos, O. Luderitz, and O. Westphal [1969) Eur. J. Biochem. 9, 245-249) and therefore a new procedure was developed for their isolation. The lipooligosaccharides (LOS) were first extracted by hot phenol-water, treated with RNase, then disaggregated in deoxycholic acid, and purified by gel filtration on Sephadex G-75. By comparison the conventional hot phenol-water purification method using repeated ultracentrifugations yielded less LOS. The yield of LOS by gel filtration was 30 to 108% higher and the purity was better.