A new mode for heme-heme interactions in hemoglobin associated with distal perturbations.

The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.     

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.     

Monte Carlo simulation studies on the prediction of protein folding types from amino acid composition.

In the methodology development for statistical prediction of protein structures, the founders of different methods usually selected different sets of proteins to test their predicted results. Therefore, it is hard to make a fair comparison according to the results they reported. Even if the predictions by different methods are performed for the same set of proteins, there is still such a problem: a method better that the other for one set of proteins would not necessarily remain so when applied to another set of proteins. To tackle this problem, a Monte Carlo simulation method is proposed to establish an objective criterion to measure the accuracy of prediction for the protein folding type. Such an objective accuracy is actually corresponding to the asymptotical limit genereated during the Monte Carlo simulation process. Based on that, it has been found that the average objective accuracy for predicting the all-alpha, all-beta, alpha + beta, and alpha/beta proteins by the least Euclid's distance method (Nakashima, H., K. Nishikawa, and T. Ooi. 1986. J. Biochem. 99:152-162) is 73.0% and that by the least Minkowski's distance method (Chou, P.Y. 1989. Prediction in Protein Structure and the Principles of Protein Conformation. Plenum Press. New York. 549-586) is 70.9%, indicating that the former is better than the latter. However, according to the original reports, the latter claimed a rate of correct prediction with 79.7% but the former with only 70.2%, leading to a completely opposite conclusion. This indicates the necessity of establishing an objective criterion, and a comparison is meaningful only when it is based on the objective criterion. The simulation method and the idea developed here also can be applied to examine any other statistical prediction methods.     

Conformational perturbation of the anticancer nucleotide arabinosylcytosine on Z-DNA: molecular structure of (araC-dG)3 at 1.3 A resolution.

The left-handed Z-DNA structure of an araC-containing (where araC stands for arabinosylcytosine) hexamer, (araC-dG)3, has been solved by x-ray diffraction analysis at 1.3 A resolution. This hexamer was crystallized in the hexagonal P6(5)22 (a = b = 17.96 A, c = 43.22 A) space group in which the hexamers have statistically disordered packing arrangement along the 6(5) screw axis, yet the crystals diffract x-rays to high resolution. Its structure has been refined by the constrained least square refinement to a final R factor of 0.287 using 737 [> 3.0 sigma(F)] observed reflections. The asymmetric unit of the unit cell contains only a dinucleotide, 5'-p (araC)p(dG). The overall conformation resembles that of the canonical Z-DNA, but with some differences in details. The O2' hydroxyl groups of the araC residues form intramolecular hydrogen bonds with N2 of the 5'-guanine residues. In the deep groove of Z-DNA, these hydroxy groups replace the bridging water molecules that stabilize the guanine in the syn conformation. The results reinforce the earlier observation made by the structural analysis of another hexamer, d(CG[araC]GCG), with a mono-substitution of araC [M.-K. Teng, Y.-C. Liaw, G. A. van der Marel, J. H. van Boom, and A. H.-J. Wang (1989) Biochemistry, vol. 28, pp. 4923-4928]. These two structures show that araC residue can be incorporated readily into the Z structure and probably facilitates the B to Z transition, as supported by uv absorption spectroscopic studies in a number of araC-containing oligonucleotides. The potential biological roles of the araC-modified Z-DNA are discussed.     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

Binding of mutagenic pyrolyzates to fractions of intestinal bacterial cells.

The binding of mutagenic pyrolyzates to cell fractions from some gram-negative intestinal bacteria and to thermally treated bacterial cells was investigated. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were effectively bound by several of the bacterial cells. The cell wall skeletons of all bacteria effectively bound Trp-P-1 and Trp-P-2. Their cytoplasmic fractions retained Trp-P-1 and Trp-P-2, but to a lesser extent than the cell wall skeletons. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) was not found in their cytoplasmic fractions. These cell wall skeletons also bound 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-amino-5-phenylpyridine (Phe-P-1), IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQX). The amount of each mutagen bound differed with the type of mutagen and the bacterial strain used. The outer membrane of Escherichia coli IFO 14249 showed binding of about 123.7 micrograms/mg of Trp-P-2, and its cytoplasmic membrane bound 57.14 micrograms/mg. Trp-P-2 bound to the bacterial cells was extracted with ammonia (5%), methanol, and ethanol but not with water.     

Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.     

Characterization of the epidermal growth factor receptor in preimplantation pig conceptuses.

Embryos recovered from sows on Days 9-13 of pregnancy (Day 0 = first day of estrus) exhibited saturable and time-dependent specific binding of 125I-epidermal growth factor (EGF). The specific binding (pg/mg protein) was greater (P less than 0.001) for Day 13 elongated conceptuses than for conceptuses of earlier stages. Scatchard analyses showed two classes of binding sites (Kd = 7.0 +/- 2.6 x 10(-11) M, Bmax = 6.2 +/- 1.4 fmol/mg protein and Kd = 3.4 +/- 0.2 x 10(-8) M, Bmax = 420 +/- 80 fmol/mg protein). The EGF receptor in Day 13 conceptus membranes is a 170-kDa protein and was phosphorylated in the presence of EGF and adenosine triphosphate. EGF stimulated protein tyrosine kinase activity about 1.6-fold over basal levels. The results show that the preimplantation pig conceptus possesses EGF-binding sites with the properties of functional EGF-receptors.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.