Calmodulin oxidation and methionine to glutamine substitutions reveal methionine residues critical for functional interaction with ryanodine receptor-1

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.     

Characterization and pathogenic potential of Listeria monocytogenes isolates from the smoked fish industry

This study was designed to evaluate the hypothesis that some of the Listeria monocytogenes subtypes associated with foods, specifically smoked fish, may have an attenuated ability to cause human disease. We tested this hypothesis by using two different approaches: (i) comparison of molecular subtypes found among 117 isolates from smoked fish, raw materials, fish in process, and processing environments with subtypes found among a collection of 275 human clinical isolates and (ii) the evaluation of the cytopathogenicity of industrial isolates. Ribotyping and PCR-restriction fragment length polymorphism typing of the hlyA and actA genes differentiated 23 subtypes among the industrial isolates and allowed classification of the isolates into three genetic lineages. A significantly higher proportion of human isolates (69.1%) than industrial isolates (36.8%) were classified as lineage I, which contains human sporadic isolates and all epidemic isolates. All other industrial isolates (63.2%) were classified as lineage II, which contains only human sporadic isolates. Lineage I ribotypes DUP-1038B and DUP-1042B represented a significantly higher proportion of the human isolates than industrial isolates (5.1%). Lineage II ribotypes DUP-1039C, DUP-1042C, and DUP-1045, shown previously to persist in the smoked fish processing environment, represented nearly 50% of the industrial isolates, compared to 7.6% of the human isolates. Representatives of each subtype were evaluated with a tissue culture plaque assay. Lineage I isolates formed plaques that were significantly larger than those formed by lineage II isolates. Isolates from the smoked fish industry representing three ribotypes formed no plaques or small plaques, indicating that they had an impaired ability to infect mammalian cells. While L. monocytogenes clonal groups linked to human listeriosis cases and outbreaks were isolated, our data also suggest that at least some L. monocytogenes subtypes present in ready-to-eat foods may have limited human-pathogenic potential.     

Comparing quantitative measures of erythema,pigmentation and skin response using reflectometry

We measured a number of pigmentation and skin response phenotypes in a sample of volunteers (n=397) living in State College, PA. The majority of this sample was composed of four groups based on stated ancestry: African-American, European-American, Hispanic and East Asian. Several measures of melanin concentration (L*, melanin index and adjusted melanin index) were estimated by diffuse reflectance spectroscopy and compared. The efficacy of these measures for assessing constitutive pigmentation and melanogenic dose-response was evaluated. Similarly, several measures of erythema (a*, erythema index and adjusted erythema index) were compared and evaluated in their efficacy in measuring erythema and erythemal dose-response. We show a high correspondence among all of the measures for the assessment of constitutive pigmentation and baseline erythema. However, our results demonstrate that evaluating melanogenic dose-response is highly dependent on the summary statistic used: while L* is a valid measure of constitutive pigmentation it is not an effective measure of melanogenic dose-response. Our results also confirm the use of a*, as it is shown to be highly correlated with the adjusted erythema index, a more advanced measure of erythema based on the apparent absorbance. Diffuse reflectance spectroscopy can be used to quantify the constitutive pigmentation, melanogenic dose-response at 7 d and erythemal dose-response at both 24 h and 7 d postexposure.     

Skin responses to ultraviolet radiation: effects of constitutive pigmentation,sex, and ancestry

Constitutive skin pigmentation and skin responses to ultraviolet radiation were measured on a sample of volunteers (n=250) living in State College, PA, USA. The sample was composed of individuals of European American (n=190), Hispanic (n=45), and East Asian ancestry (n=15). Constitutive pigmentation was measured using the Adjusted Melanin Index (AMI), Erythemal Dose Response (EDR) was measured using the slope of a* at 24 h (Deltaa*), and Melanogenic Dose-Response (MDR) was measured using DeltaAM, the slope of AMI at 7 d. The relationships between constitutive skin pigmentation, EDR, MDR, sex, age, and ancestry were investigated. European Americans showed a lower constitutive pigmentation, had a significantly higher burn response (EDR), and had a significantly lower tanning response (MDR) than Hispanics and East Asians. No significant difference is seen between Hispanics and East Asians for either constitutive pigmentation or EDR. Constitutive pigmentation in females was slightly lower than in males in all three samples, but the difference was not significant. While no differences were observed in MDR between sexes, males had a stronger EDR than females regardless of population or constitutive pigmentation level, and this difference was significant in European Americans and Hispanics. We observed no age-related differences in any of the populations or measures investigated. We evaluated the relationship between constitutive pigmentation, EDR and MDR. There was a strong inverse correlation between constitutive pigmentation and EDR in the three samples (European Americans, R2=0.176, P < 0.001; Hispanics, R2=0.204, P=0.009; East Asians, R2=0.223, P=0.098) and a strong direct correlation between constitutive pigmentation and MDR in European Americans and Hispanics (European Americans, R2=0.094, P < 0.001; Hispanics, R2=0.164, P=0.012). In other words, persons with lower constitutive pigmentation both burn more and tan less than persons with higher pigmentation. However, after controlling for constitutive pigmentation, EDR and MDR were significantly correlated in European Americans (R2=0.041 P=0.006). Thus, the general observation that persons who burn more tan less is probable because of the common link that these two phenotypes have with constitutive skin pigmentation and, in fact, once pigmentation has been adjusted for, there is a positive correlation between tanning response and burning response in European Americans.     

Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms

Complex glycoinositolphosphoryl ceramides (GIPCs) have been purified from a pathogenic encapsulated wild-type (WT) strain of Cryptococcus neoformans var. neoformans and from an acapsular mutant (Cap67). The structures of the GIPCs were determined by a combination of tandem mass spectrometry, nuclear magnetic resonance spectroscopy, methylation analysis, gas chromatography-mass spectrometry, and chemical degradation. The main GIPC from the WT strain had the structure Manp(alpha1-3)[Xylp(beta1-2)] Manp(alpha1-4)Galp(beta1-6)Manp(alpha1-2)Ins-1-phosphoryl ceramide (GIPC A), whereas the compounds from the acapsular mutant were more heterogeneous in their glycan chains, and variants with Manp(alpha1-6) (GIPC B), Manp(alpha1-6) Manp(alpha1-6) (GIPC C), and Manp(alpha1-2)Manp(alpha1-6)Manp(alpha1-6) (GIPC D) substituents linked to the nonreducing terminal mannose residue found in the WT GIPC A were abundant. The ceramide moieties of C. neoformans GIPCs were composed of a C(18) phytosphingosine long-chain base mainly N-acylated with 2-hydroxy-tetracosanoic acid in the WT GIPC while in the acapsular Cap67 mutant GIPCs, as well as 2-hydroxy-tetracosanoic acid, the unusual 2,3-dihydroxy-tetracosanoic acid was characterized. In addition, structural analysis revealed that the amount of GIPC in the WT cells was fourfold less of that in the acapsular mutant.     

Dehydroepiandrosterone protects the microcirculation of muscle flaps from ischemia-reperfusion injury by reducing the expression of adhesion molecules

Adhesion molecules contribute to ischemia-reperfusion injury by increasing the endothelial adhesion and extravasation of leukocytes. Scientific evidence suggests that presurgical treatment with dehydroepiandrosterone may protect the microvasculature against this damage, but the exact mechanism is not known. The purpose of this study was to investigate the effects of presurgical dehydroepiandrosterone treatment on microcirculatory hemodynamic parameters and the expression of adhesion molecules in a rat cremaster muscle flap model. Twenty male rats were randomly assigned to three experimental groups. In group I (n = 5), the muscle flaps did not receive presurgical treatment. In group II (n = 6), propylene glycol (30 mg/kg), the vehicle for dehydroepiandrosterone, was injected intravenously before ischemia was induced. In group III (n = 9), dehydroepiandrosterone (30 mg/kg) was injected intravenously before ischemia was induced. All flaps were subjected to 6 hours of ischemia and 90 minutes of reperfusion. Microcirculatory variables (functional capillary density, red blood cell velocity in the main flap arteriole, and numbers of rolling, sticking, and transmigrating leukocytes), blood levels of three adhesion molecules (L-selectin, Mac-1 integrin, and CD44), and the numbers of leukocytes expressing those molecules were analyzed. Analysis of the microcirculatory parameters revealed that dehydroepiandrosterone treatment before ischemia had significant preservative effects on the red blood cell velocity and functional capillary density 30 and 90 minutes after reperfusion, compared with the control and vehicle-treated groups. Leukocyte-endothelial cell interactions were also affected by dehydroepiandrosterone treatment, as reflected by significant decreases in the numbers of sticking and transmigrating leukocytes 30 and 90 minutes after reperfusion. In dehydroepiandrosterone-treated animals, leukocytes exhibited lower levels of expression of adhesion molecules after the onset of ischemia, compared with the control groups. In this study, intravenous dehydroepiandrosterone administration reduced the activation of leukocytes and improved red blood cell velocity and capillary perfusion in the muscle flap microcirculation during ischemia-reperfusion injury. This protective effect was most likely the result of delayed expression of Mac-1 integrin, L-selectin, and CD44 molecules on leukocytes.     

Backbone dynamics measurements on leukemia inhibitory factor, a rigid four-helical bundle cytokine

The backbone dynamics of the four-helical bundle cytokine leukemia inhibitory factor (LIF) have been investigated using 15N NMR relaxation and amide proton exchange measurements on a murine-human chimera, MH35-LIF. For rapid backbone motions (on a time scale of 10 ps to 100 ns), as probed by 15N relaxation measurements, the dynamics parameters were calculated using the model-free formalism incorporating the model selection approach. The principal components of the inertia tensor of MH35-LIF, as calculated from its NMR structure, were 1:0.98:0.38. The global rotational motion of the molecule was, therefore, assumed to be axially symmetric in the analysis of its relaxation data. This yielded a diffusion anisotropy D(parallel)/D(perpendicular) of 1.31 and an effective correlation time (4D(perpendicular) + 2D(parallel))(-1) of 8.9 ns. The average values of the order parameters (S2) for the four helices, the long interhelical loops, and the N-terminus were 0.91, 0.84, and 0.65, respectively, indicating that LIF is fairly rigid in solution, except at the N-terminus. The S2 values for the long interhelical loops of MH35-LIF were higher than those of their counterparts in short-chain members of the four-helical bundle cytokine family. Residues involved in LIF receptor binding showed no consistent pattern of backbone mobilities, with S2 values ranging from 0.71 to 0.95, but residues contributing to receptor binding site III had relatively lower S2 values, implying higher amplitude motions than for the backbone of sites I and II. In the relatively slow motion regime, backbone amide exchange measurements showed that a number of amides from the helical bundle exchanged extremely slowly, persisting for several months in 2H2O at 37 degrees C. Evidence for local unfolding was considered, and correlations among various structure-related parameters and the backbone amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four-helical bundle cytokines.     

β-Carotene inhibition of aflatoxin biosynthesis amongAspergillus flavus genotypes from Illinois corn

Thirty-nineAspergillus flavus genotypes (DNA fingerprinting) isolated from corn grown in a field near Kilbourne, Illinois were evaluated for their sensitivity to β-carotene (50 μg/ml) inhibition of aflatoxin B₁ biosynthesis. Inhibition of aflatoxin was greater than 90% for 28 of the genotypes and >70% for 38 of the 39 genotypes. FiveA. flavus strains (4 fingerprint groups) isolated from molded raw peanuts, NRRL 3239, NRRL 3357, NRRL 6514, NRRL 6515 and NRRL 13135, produced greater quantities of aflatoxin than all 39 genotypes isolated from corn, and were less sensitive to β-carotene inhibition.Aspergillus flavus NRRL 3357 is commonly used as inoculum in variety trials for aflatoxin resistance. Isolate identity and sensitivity to potential inhibitors in corn can be critical in assessing corn resistance to aflatoxin.