Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.     

Cytokine production in a model of wound healing: the appearance of MIP-1, MIP-2, cachectin/TNF and IL-1

Macrophages are essential for normal wound repair and many of their effects on healing wounds are likely to be mediated by the secretion of cytokines. This study examines the appearance of messenger RNA (mRNA) for cachectin/tumor necrosis factor (TNF), IL 1, and macrophage inflammatory proteins 1 and 2 (MIP-1 and MIP-2), as well as the mature peptides, in a model of wound healing using wound chambers. RNA for all four cytokines can be detected in wound inflammatory cells by polymerase chain reaction amplification throughout the first 7 days. Cachectin/TNF and IL 1 protein levels peaked on the first day after wound chamber implantation, and MIP-1 and MIP-2 were detected only on day 3. The data suggest that these cytokines participate in the early inflammatory response to wounding.     

13C NMR of methylated lysines of fd gene 5 protein: evidence for a conformational change involving lysine 24 upon binding of a negatively charged lanthanide chelate

Helical complexes formed between fd DNA and reductively methylated fd gene 5 protein were indistinguishable by electron microscopy from complexes formed with the nonmethylated protein. 13C NMR spectroscopy of 13C-enriched N epsilon, N epsilon-dimethyllsyl residues of the protein showed that three of these residues (Lys-24, Lys-46, and Lys-69) were selectively perturbed by binding of the oligomer d(pA)7. These were the same lysyl residues that we previously found to be most protected from methylation by binding of the protein to poly[r(U)] [Dick, L. R., Sherry, A. D., Newkirk, M. M., & Gray D. M. (1988) J. Biol. Chem. 263, 18864-18872]. Thus, these lysines are probably directly involved in the nucleic acid binding function of the protein. Negatively charged chelates of lanthanide ions were used to perturb the 13C NMR resonances of labeled lysyl and amino-terminal residues of the gene 5 protein. The terbium chelate was found to bind tightly (Ka approximately 10(5) M-1) to the protein with a stoichiometry of 1 chelate molecule per protein dimer. 13C resonances of Lys-24, Lys-46, and Lys-69 were maximally shifted by the terbium chelate and were maximally relaxed by the gadolinium chelate. Also, the terbium chelate was excluded by the oligomer d(pA)7. Computer fits of the induced chemical shifts of 13C resonances with those expected for various positions of the terbium chelate failed to yield a possible chelate binding site unless the chemical shift for Lys-24 was excluded from the fitting process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recent Advances in Geriatric Dentistry: Implications for Clinical Practice1

The clinical applications of recent research advances in oral health are discussed using a modified case presentation. Conversely, clinical care often highlights the research questions that must be answered to adequately address the oral health problems of the elderly. It is stressed that science transfer is and will continue to be necessary to take the findings from the basic and clinical research arena to the practice arena.     

Evidence for at least two dominant neutralization antigens on human rhinovirus 14.

A collection of 28 mutants of human rhinovirus 14, selected for resistance to 10 individual neutralizing monoclonal antibodies, was used to identify two major neutralization antigens, N-Ag I and N-Ag II. Isoelectric analysis showed that all 16 of the N-Ag I mutants analyzed were charge altered in VP1;8 of 12 N-Ag II mutants were altered in VP3. These results suggest that N-Ag I resides on VP1, whereas N-Ag II lies on VP3. The frequency of charge alterations was much higher than predicted by the genetic code, suggesting that charged amino acids on the antigenic sites play an important role in interaction with neutralizing antibody. Antibodies against N-Ag I and N-Ag II neutralize with widely different efficiencies.     

PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca

ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - NOS nopaline synthase - CCC covalently closed circular DNA - L linear DNA - PEG polyethylene glycol - HS herring sperm DNA - P protoplasts - PCM protoplast culture medium - MES morpholinoethane-sulfonic acid - Cm chloramphenicol - Ac acetylated - MUG 4-methyl umbelliferyl ß-D-glucuronide - TLC thin layer chromatography     

N-Methyl-D-Aspartate Receptor Activation and Ca2+ Account for Poor Pyramidal Cell Structure in Hippocampal Slices

The CA1 pyramidal cells appear damaged in micrographs of guinea pig hippocampal slices incubated in normal physiological buffer at 36–37°C. This is remedied if slices are incubated in modified buffers for the first 45 min. Cell morphology is improved if this buffer is devoid of added Ca²⁺ and much improved if it contains N-methyl-D-aspartate (NMDA) receptor antagonists or 0 mM Ca²⁺ and 10 mM Mg²⁺. The cells then appear similar to CA1 pyramidal cells in situ. These findings support the notion that NMDA receptor activation and Ca²⁺, acting in the period immediately after slice preparation, permanently damage CA1 pyramidal cells in vitro.     

Derivation and characterization of an efficiently myocarditic reovirus variant.

A reovirus variant, 8B, was isolated from a neonatal mouse which had been inoculated with a mixture of two reovirus strains: type 1 Lang (T1L) and type 3 Dearing (T3D) (E. A. Wenske, S.J. Chanock, L. Krata, and B. N. Fields, J. Virol. 56:613-616, 1985). 8B is a reassortant containing eight gene segments derived from the T1L parent and two gene segments derived from the T3D parent. Upon infection of neonatal mice, 8B produced a generalized infection characteristic of many reoviruses, but it also efficiently induced numerous macroscopic external cardiac lesions, unlike either of its parents. Microscopic examination of hearts from infected mice revealed myocarditis with necrotic myocytes and both polymorphonuclear and mononuclear cellular infiltration. Electron microscopy revealed viral arrays in necrotic myocytes and dystrophic calcification accompanying late lesions. Determination of viral titers in hearts from T1L-, T3D-, or 8B-infected mice indicated that growth was not the primary determinant of myocardial necrosis. Results from inoculations of athymic mice demonstrated that T cells were not a requirement for the 8B-induced myocarditis. Finally, 8B was more cytopathic than either of the parent viruses in cultured mouse L cells. Together, the data suggest that 8B-induced myocardial necrosis is due to a direct effect of reovirus on myocytes. Reovirus thus provides a useful model for the study of viral myocarditis.     

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.