The thermal induced helix--beta transition of poly(N epsilon-methyl-L-lysine) and poly(N delta-ethyl-L-ornithine) in aqueous solution

Uncharged poly(N^ε-methyl-L -lysine) (PMLL) and its isomer, poly(N^δ-ethyl-L -ornithine) (PELO), in alkaline solution (pH ca. 12) undergo a helix-to-β transition upon mild heating at 50°C or higher in a manner similar to that of poly(L -lysine) (PLL). The rate of conversion follows the order: PMLL < PELO < PLL. The helix can be regenerated upon cooling near zero degrees, for instance, after more than 12 hr at 2°C. At concentrations less than 0.02% the β form is intramolecular, but at higher concentrations both intra- and intermolecular β forms are generated. Poly(N^δ-methyl-L -ornithine) (PMLO), an isomer of PLL, behaves like poly(L -ornithine); uncharged PMLO in alkaline solution is partially helical and becomes disordered at elevated temperatures.     

A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

Interactions of chloride and amiloride with the renal Na+/H/ antiporter

Amiloride is a reversible inhibitor of the Na+/H+ antiporter which acts at the external aspect of the transport system. The kinetics of inhibition of the Na+/H+ antiporter with amiloride have been controversial, with the usual finding of simple competitive inhibition, but with other reports of mixed and noncompetitive inhibition of the transporter by amiloride. The present experiments demonstrate that the chloride content of the external transport buffer affects the kinetics of amiloride inhibition. Either simple competitive or mixed inhibition by amiloride was observed in the same vesicle preparations depending on the presence of chloride or gluconate in the buffer. The effect of chloride on the inhibitory effect of amiloride was dependent on the concentration of chloride and amiloride. Similar effects were observed with more potent analogues of amiloride. These findings suggest that the external aspect of the antiporter has a site or sites at which the inhibitory effects of amiloride on the Na+/H+ antiporter can be modified by chloride, even though chloride has only slight effects on the kinetics of the Na+/H+ antiporter in the absence of amiloride.     

Clearance of rat C-reactive protein in vivo and by perfused liver.

The clearance in vivo of rat C-reactive protein (CRP) was studied: (i) in the whole animal and (ii) by using a rat liver perfusion system. Rat CRP is a glycosylated serum protein containing a complex-type biantennary carbohydrate structure on each of its five subunits. The half-life of rat asialo CRP was approximately 5 min. More than 75% of the radioactivity associated with rat asialo CRP and asialo alpha 1-acid glycoprotein (AGP) was recovered in the liver. A small amount of radioactivity (0.8%) associated with rat CRP and rat asialo CRP was found in the lungs. Competitive inhibition of the clearance of 125I-labelled rat asialo CRP from the circulation by asialo AGP was dose dependent, and resulted in a corresponding decrease in the recovery of radioactivity associated with rat asialo CRP in the liver. This indicated that asialo AGP and rat asialo CRP were cleared by the hepatic asialoglycoprotein receptor. This observation was confirmed when the clearance of rat asialo CRP was studied using a rat liver perfusion system. Using this system, the clearance of rat asialo CRP and asialo AGP from the perfusate was inhibited by N-acetylgalactosamine, but not by phosphorylcholine, a ligand through which most of the CRP reactions are mediated. This study provides an example of a circulating serum glycoprotein containing a biantennary carbohydrate structure that is cleared by the asialoglycoprotein receptor.     

BJ-HCC-20, a potential novel cancer-testis antigen.

In an effort to identify novel Cancer-Testis genes, we analyzed the sequence in the q26-28 region of human X chromosome by several on-line tools. The candidate sequences were then confirmed by experiments. We have obtained a novel Cancer-Testis gene, BJ-HCC-20. In vivo, it was found to have two isoforms. In samples of liver, colon, gastric and lung cancer tested, the expression frequency of BJ-HCC-20 is 25%, 17%, 21% and 15%, respectively. Full-length cDNAs of both BJ-HCC-20 isoforms were isolated and their gene structures and promoter regions were characterized. BJ-HCC-20 might have implications in theoretical and practical tumor biology.     

Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin 2

The systemic administration of high-dose recombinant IL 2 mediated significant reductions of established 3-day pulmonary micrometastases from both weakly immunogenic and nonimmunogenic sarcomas. However, when treatment with IL 2 was delayed for 10 days after the injection of tumor cells in an attempt to treat grossly visible pulmonary macrometastases, only those established from weakly immunogenic sarcomas remained susceptible. Established 10-day pulmonary nodules from the nonimmunogenic sarcomas became refractory to IL 2 therapy. We utilized selective depletion of lymphocyte subsets in vivo by the systemic administration of specific monoclonal antibodies to cells bearing either the L3T4 or Lyt-2 marker or a heteroantiserum to cells bearing the ASGM-1 glycosphingolipid to identify lymphocytes involved in IL 2-induced tumor regression. Cells with potent lymphokine-activated killer (LAK) activity against fresh tumor targets in vitro were identified in the lungs of IL 2-treated mice. By flow cytometry analysis, the majority of these effector cells were Thy-1+, L3T4-, Lyt-2-, ASGM-1+. Depletion in vivo of ASGM-1+ cells before the onset of IL 2 administration eliminated the successful therapy of 3-day pulmonary metastases from nonimmunogenic sarcomas, with concurrent elimination of LAK cell activity in the lungs. In mice with 3-day pulmonary metastases from weakly immunogenic sarcomas, both Lyt-2+ cells and ASGM-1+ cells were involved in IL 2-mediated tumor regression, but Lyt-2+ cells appeared to be the more potent mediator in the response. Lyt-2+ cells were also involved in the elimination of grossly visible 10-day macrometastases from these weakly immunogenic tumors. Depletion of L3T4+ cells had no effect on tumor regression. Thus, although LAK effectors derived from ASGM-1+ precursors can eliminate pulmonary micrometastases regardless of tumor immunogenicity, Lyt-2+ cells are predominant effectors in the elimination of both pulmonary micro- and macrometastases from weakly immunogenic sarcomas.     

Enantioselective hydrolysis of oxazepam 3-acetate by esterases in human and rat liver microsomes and rat brain S9 fraction

Rates of hydrolysis of racemic and enantiomeric oxazepam 3-acetates (OXA) by esterases in human and rat liver microsomes and rat brain S9 fraction were compared. When rac-OXA was the substrate, esterases in human and rat liver microsomes were highly enantioselective toward (R)-OXA. In contrast, esterases in rat brain S9 fraction were highly enantioselective toward (S)-OXA. Hydrolysis rates of rac-OXA were highly dependent on the amount of esterases used. At 0.05 mg protein equivalent of esterases and 150 nmol of rac-OXA per ml of incubation mixture, the (R)-OXA was hydrolyzed 3.6-fold and 18.5-fold faster than (S)-OXA by rat and human liver microsomes, respectively. The specific activities (nmol of OXA hydrolyzed/mg microsomal protein/min) of liver microsomes in the hydrolysis of enantiomerically pure (R)-OXA were approximately 120 (rat) and 1,980 (human), and in the hydrolysis of enantiomerically pure (S)-OXA were 4 (rat) and 7 (human), respectively. In the incubation of rac-OXA with rat brain S9 fraction, (S)-OXA was hydrolyzed approximately 6-fold faster than (R)-OXA. Results also indicated an enantiomeric interaction in the hydrolysis of rac-OXA by esterases in rat and human liver microsomes; the presence of (R)-OXA stimulated the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA inhibited the hydrolysis of (R)-OXA. In rat brain S9 fraction, the presence of (R)-OXA inhibited the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA appeared to have stimulated the hydrolysis of (R)-OXA.