Synthesis and antiviral property of allophenylnorstatine-based HIV protease inhibitors incorporating D-cysteine derivatives as P2/P3 moieties

We designed several HIV protease inhibitors with various d-cysteine derivatives as P(2)/P(3) moieties based on the structure of clinical drug candidate, KNI-764. Herein, we report their synthesis, HIV protease inhibitory activity, HIV IIIB cell inhibitory activity, cellular toxicity, and inhibitory activity against drug-resistant HIV strains. KNI-1931 showed distinct selectivity against HIV proteases and high potency against drug-resistant strains, surpassing those of Ritonavir and Nelfinavir.     

Alteration of the timing of implantation by in vivo gene transfer: delay of implantation by suppression of nuclear factor kappaB activity and partial rescue by leukemia inhibitory factor

Nuclear factor kappaB (NF-kappaB) is activated in the murine endometrium during implantation period [Am. J. Reprod. Immunol. 51 (2004) 16]. Transient transfection of IkappaBalpha mutant (IkappaBalphaM) cDNA into the mouse uterine cavity using hemagglutinating virus of Japan envelope vector suppressed uterine NF-kappaB activity less than half of that observed in control on days 3.5 and 4.5 p.c. IkappaBalphaM cDNA transfection led to significant delay of implantation. After IkappaBalphaM cDNA transfection, LIF mRNA expression in the uterus was significantly suppressed on days 3.5 and 4.5 p.c. Co-transfection of LIF cDNA with IkappaBalphaM cDNA in the uterus partially rescued the delay of implantation induced by suppression of NF-kappaB activity. Taken together, these findings indicate that NF-kappaB activation determines the timing of the implantation, at least in part, via control of LIF expression.     

Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of Clostridium cellulovorans EngE

engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been cloned and sequenced (Y. Tamaru and R. H. Doi, J. Bacteriol. 181:3270-3276, 1999). The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins. Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA. Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface. This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in Escherichia coli and cell wall preparations from C. cellulovorans. When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall. However, rEngEs without SLH domains lost their ability to bind to cell walls. When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE. However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions. These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin.     

Smad3 mediates TGF-beta1-induced collagen gel contraction by human lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key mediator in tissue repair and fibrosis. Using small interference RNA (siRNA), the role of Smad2 and Smad3 in TGF-beta stimulation of human lung fibroblast contraction of collagenous matrix and induction of alpha-SMA and the role of alpha-SMA in contraction were assessed. HFL-1 cells were transfected with Smad2, Smad3 or control-siRNA, and cultured in floating Type I collagen gels +/- -TGF-beta1. TGF-beta1 augmented gel contraction in Smad2-siRNA- and control-siRNA-treated cells, but had no effect in Smad3-siRNA-treated cells. Similarly, TGF-beta1 upregulated alpha-SMA in Smad2-siRNA- and control-siRNA-treated cells, but had no effect on Smad3-siRNA-treated cells. Alpha-SMA-siRNA-treated cells did not contact the collagen gels with or without TGF-beta1, suggesting alpha-SMA is required for gel contraction. Thus, Smad3 mediates TGF-beta1-induced contraction and alpha-SMA induction in human lung fibroblasts. Smad3, therefore, could be a target for blocking contraction of human fibrotic tissue induced by TGF-beta1.     

Identification of two novel GTP-binding protein alpha-subunits that lack apparent ADP-ribosylation sites for pertussis toxin

Molecular cloning of cDNAs encoding alpha-subunits of guanine nucleotide-binding regulatory proteins (G-proteins) has revealed the existence of nine species of alpha-subunits. We have identified two additional G-protein alpha-subunits, which we refer to as GL1 alpha and GL2 alpha, by isolating bovine liver cDNA clones that cross-hybridized at reduced stringency with bovine Gi1 alpha-subunit cDNA. The deduced amino acid sequences of GL1 alpha and GL2 alpha share 83% identity with each other and show 45-55% identity with those of other known G-protein alpha-subunits. Both GL1 alpha and GL2 alpha lack a consensus site for ADP-ribosylation by pertussis toxin. Messenger RNA corresponding to GL2 alpha was detected in all tissues examined, but GL1 alpha mRNA was detected only in liver, lung, and kidney. Antiserum prepared against a synthetic pentadecapeptide corresponding to the deduced carboxyl terminus of GL2 alpha specifically reacted with a 40-kDa protein in mouse liver, brain, lung, heart, kidney, and spleen. The amount of the 40-kDa protein was highest in brain and lung. We suggest that GL1 alpha and GL2 alpha are new members of a subfamily of pertussis toxin-insensitive G-proteins.     

Purification and Properties of a New l-Fucose-specific Lectin Produced by Streptomyces No. 100–2

The inhibitory effects of 3-nitro-2,4,6-trihydroxybenzamide derivatives on human 5-lipoxygenase (5-LO), a key enzyme in arachidonic acid cascades, were examined using 5-LO produced by Escherichia coli. Some of the tested compounds inhibited the conversion of arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), and in particular the N-phenylbutyl derivative was about 30 times more active (IC₅₀ = 35 μm) than caffeic acid (IC₅₀ = 1000 μm), a known selective 5-LO inhibitor.