Differential scanning calorimetric study of the thermal unfolding of myosin rod,light meromyosin,and subfragment 2

The thermal unfolding of myosin rod, light meromyosin (LMM), and myosin subfragment 2 (S-2) was studied by differential scanning calorimetry (DSC) over the pH range of 6.5–9.0 in 0.5M KCl and either 0.20M sodium phosphate or 0.15M sodium pyrophosphate. Two rod samples were examined: one was purified by Sephadex G-200 without prior denaturation (native rod), and the other was purified by a cycle of denaturation-renaturation followed by Sephacryl S-200 chromatography (renatured rod). There were clearly distinguishable differences in the calorimetric behavior of these two samples. At pH 7.0 in phosphate the DSC curves of native rod were deconvoluted into six endothermic two-state transitions with melting temperatures in the range of 46–67°C and a total enthalpy of 4346 kJ/mol. Under identical conditions the melting profile of LMM was resolved into five endothermic peaks with transition temperatures in the range of 45–66°C, and the thermal profile of long S-2 was resolved into two endotherms, 46 and 57°C. Transition 4 observed with native rod was present in the deconvoluted DSC curve for long S-2, but absent in the DSC curve for LMM. This transition was identified with the high-temperature transition detected with long S-2 and attributed to the melting of the coiled-coil α-helical segment of subfragment 2 (short S-2). The low-temperature transition of long S-2 was attributed to the unfolding of the hinge region. The smallest transition temperatures observed for all three fragments were 45–46°C. It is suggested that the most unstable domain in rod (domain 1) responsible for the 46°C transition includes both the hinge region, which is the C-terminal segment of long S-2, and a short N-terminal segment of LMM. This domain, accounting for 21% of the rod structure, contains the S-2/LMM junction, and upon proteolytic cleavage yields the C-terminal and N-terminal ends of long S-2 and LMM, respectively. Over the pH range of 6.5–7.5, the observed specific heat of denaturation of rod was approximately equal to the sum of the specific heats of LMM and S-2. This finding provides an additional argument for the existence of independent domains in myosin rod.     

Insertional inactivation of a chromosomal locus that modulates expression of potential virulence determinants in Staphylococcus aureus.

A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this locus is distinct from agr, xpr, and sar, three previously described global regulatory loci. Transduction experiments demonstrated that the genotype associated with mutant 1E3 could be transferred back into the parental strain ISP479C. The transductant 1E3-2 displayed a phenotypic profile similar to that of the original mutant. Northern (RNA) blot studies showed that this locus may be involved in modulating target genes at the mRNA level. In the rabbit endocarditis model, there was a significant decrease in both the infectivity rate and intravegetation bacterial density with mutant 1E3 compared with the parent at an inoculum of 10(3) CFU. Since protein A and the fibrinogen-binding protein(s) are major surface proteins that may mediate bacterial adhesion to host tissues, this locus may be an important genetic element involved in the expression of virulence determinants in S. aureus.     

Apoptosis Induced via AMPA-Selective Glutamate Receptors in Cultured Murine Cortical Neurons

Abstract: We have investigated the mechanisms of cell death induced by long-term exposure to the glutamate receptor agonist ( S )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate [( S )-AMPA]. Using primary cultures of pure neurons (95%) grown in serum-free conditions, we found that 24-h exposure to ( S )-AMPA (0.01–1,000 µ M ) induced concentration-dependent neuronal cell death (EC₅₀ = 3 ± 0.5 µ M ) with cellular changes including neurite blebbing, chromatin condensation, and DNA fragmentation, indicative of apoptosis. ( S )-AMPA induced a delayed cell death with DNA fragmentation occurring in ∼50% of cells at concentrations between 100 and 300 µ M detected using terminal transferase-mediated dUTP nick end-labeling (TUNEL) and agarose gel electrophoresis. Apoptotic chromatin condensation was detected using 4,6-diamidino-2-phenylindole, a fluorescent DNA binding dye. Cell death induced by ( S )-AMPA was attenuated by the AMPA receptor-selective antagonist LY293558 (10 µ M ) and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 µ M ), yielding EC₅₀ values of 73 ± 5 and 265 ± 8 µ M , respectively, and was unaffected by the NMDA receptor antagonist MK-801 (10 µ M ). The number of apoptotic nuclei induced by 300 µ M ( S )-AMPA (57%) was also reduced substantially by the antagonists LY293558 and CNQX, with only 20% and 18% of neurons, respectively, staining TUNEL-positive at 24 h. In addition, cycloheximide (0.5 µg/ml) also inhibited ( S )-AMPA-induced DNA fragmentation and cell death. Our results show that long-term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons, suggesting a wide involvement of AMPA-sensitive glutamate receptors in excitotoxic injury and neurodegenerative pathologies.     

Homopolymer mutational hot spots mediate herpes simplex virus resistance to acyclovir.

In the majority of cases, the mechanism underlying the resistance to acyclovir (ACV) of herpes simplex viruses (HSVs) is thymidine kinase (TK) deficiency. Plaque isolates from eight ACV-resistant (ACVr) clinical isolates from AIDS patients, of which five reactivated, were sequenced to determine the genetic lesion within the tk gene conferring resistance and whether this may have correlated with reactivation potential. Mutations were clustered within two homopolymer nucleotide stretches. Three plaque isolates (1737-14, 90-150-3, and 89-650-5) had insertion mutations within a stretch of 7 guanosines, while two isolates (89-063-1 and 89-353-1) had frameshift mutations within a stretch of 6 cytosines (a deletion and an insertion, respectively). Mutations resulted in premature termination codons, and the predicted 28- and 32-kDa truncated TK products were detected by Western blot analysis of virus-infected cell extracts. The repair of one homopolymer frameshift mutation (in isolate 1737-14) restored TK activity, demonstrating that this mutation is the basis of TK deficiency. Of the five reactivated isolates, four were TK deficient and contained frameshift mutations while the fifth retained TK activity because of its altered-TK or Pol- phenotype. These data demonstrate that the majority of ACVr clinical isolates contain frameshift mutations within two long homopolymer nucleotide stretches which function as hot spots within the HSV tk gene and produce nonfunctional, truncated TK proteins.     

Nucleotide sequence of the VP1 gene of the foot-and-mouth disease virus strain A Venceslau

The VP1 coat protein of FMDV strain A Venceslau (Aven) consists of 213 amino acid residues. Serum neutralization tests demonstrated that strain Aven is closely related to strain A Argentina/79 (A₇₉) but significantly different from strain A₂₄ Cruzeiro (A₂₄). There is a strong correlation between the amino acid sequences and the serological data. Nucleotide and amino acid sequence analyses of VP1 showed that serologically related viruses (Aven and A₇₉) differ less in this region of the genome than those of serologically distinct viruses (Aven vs. A₂₄). The most significant variation between Aven and A₂₄ occurs at amino acid positions 43 to 46, in which all four residues are different.     

Development of a hemolytic plaque assay for glutamic acid, lysine-containing polypeptides: demonstration that nonresponder mice produce antibodies to these peptides when conjugated to an immunogenic carrier.

Fowl gamma-globulin, when chemically conjugated to GLO or GL, functions as a T-dependent immunogenic carrier and stimulates anti-GLO and anti-GL antibody production in nonresponder mice. The conjugation procedure utilizes the Schiff base reaction. The anti-GL and anti-GLO responses were detected by hemagglutination and hemolytic plaque assays by using GL-coated erythrocytes. The coupling of GL to erythrocytes utilizes a novel procedure in which a palmitoyl derivative of GL is adsorbed onto red blood cells. The optimal conditions for preparing the palmitoyl derivative and for coupling to SRBC are presented. With the hemolytic plaque assay, we have verified that GLO responder animals make both IgM and IgG responses, whereas nonresponder mice fail to make either IgM or IgG plaque-forming cells.     

A rapid quantitative method for measuring UTP, UDP, and UMP in the picomole range.

A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [¹⁴C]glucose 1-phosphate catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase yielding UDP-[¹⁴C]glucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 m Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.     

Direct solid phase radioimmunoassay for chicken lipoprotein lipase.

A direct, noncompetitive immunoassay for chicken lipoprotein lipase (LPL) was developed. Antibodies to LPL were purified by immunoadsorption chromatography of goat antisera on an LPL-Sepharose column. Purified anti-LPL immunoglobulins were coupled covalently to hydrophilic polyacrylamide beads by a carbodiimide reagent. An excess amount of these beads was incubated with the sample or the standard to be assayed. The amount of LPL immobilized by the beads was then detected by an excess amount of ¹²⁵I-labeled anti-LPL immunoglobulin. A linear relationship was obtained between the radioactivity bound and the amount of highly purified LPL used as a standard. The range of the assay was from 0.1-1.1 ng LPL. The assay was specific for chicken LPL and showed no cross-reactivity with liver lipase. It does not distinguish heat-inactivated from catalytically active enzyme species. This assay should be useful in studies of lipoprotein lipase where both catalytic activity and enzyme mass need to be quantitated.     

Immune suppression in vivo with antigen-modified syngeneic cells. II. T cell-mediated nonresponsiveness to fowl gamma-globulin.

Intravenous administration of syngeneic spleen cells coupled with the palmitoyl derivative of fowl gammma-globulin (p-F gamma G) results in a profound state of F gamma G-specific tolerance in C57BL/6 mice. Administration of p-F gamma G coupled syngeneic cells specifically reduces both the primary and secondary hapten and carrier-specific PFC responses to TNP-F gamma G. Since the haptenic response is affected, the tolerance functions at the level of the F gamma G-specific helper T cell. As few as 10(3) p-F gamma G spleen cells carrying only 1 ng of p-F gamma G can induce tolerance. At least a 2-day-induction period is required. This nonresponsiveness is long lived, lasting over 120 days. Spleen cells from tolerized mice can transfer suppression to normal syngeneic recipients. Treatment of tolerant spleens with anti-Thy 1.2 antiserum + C eliminates the suppressor cell activity. In addition, thymocytes and purified splenic T cells from tolerized mice can transfer suppression to normal recipients. Thus, at least a component of this nonresponsiveness is mediated by suppressor T cells. The requirement of antigen association with cell membrane components and the general applicability of this method of inducing T cell nonresponsiveness are discussed.     

Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.

The purpose of this experiment was to characterize the high density lipoproteins (HDL) as a function of hydrated density. HDL was subfractionated on the basis of hydrated density by CsCl density gradient centrifugation of whole serum or the d 1.063-1.25 g/ml HDL fraction isolated from three men and three women. Apolipoprotein A-I and A-II quantitation by radial immunodiffusion showed that the A-I/A-II ratio varied with the lipoprotein hydrated density. The A-I/A-II molar ratio of HDL lipoproteins banding between d 1.106 and 1.150 g/ml was nearly constant at 2.2 +/- 0.2. In the density range 1.151-1.25 g/ml the A-I/A-II ratio increased as the density increased. On the other hand, in the density range between 1.077 and 1.105 the A-I/A-II ratio increased as the density decreased, ranging from 2.8 +/- 0.5 for the d 1.093-1.105 g/ml fraction to 5.6 +/- 1.3 for the d 1.077-1.082 g/ml fraction. The d 1.063-1.076 g/ml fraction and the d 1.077-1.082 g/ml fractions had comparable A-I/A-II ratios. Serum and the d 1.063-1.25 g/ml HDL fraction exhibited similar trends. The cholesterol/(A-I + A-II) ratio decreased as the density increased in all 12 samples (six serum and six HDL) examined. Gradient gel electrophoresis of the density gradient fractions showed that as the density increased from 1.063 to 1.200 g/ml the apparent molecular weight decreased from 3.9 x 10(5) to 1.1 x 10(5). HDL subfractions with the same hydrated densities had comparable molecular weights and A-I/A-II and cholesterol/(A-I + A-II) ratios when isolated from men or women. HDL contains subpopulations that differ in the A-I/A-II molar ratio.-Cheung, M. C., and J. J. Albers. Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.