Studies on the interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C solid state NMR

The effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on model phospholipid membranes were studied using 13C and 2H solid-state nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) with specific 13C and 2H labels as endogenous probes at the carbonyl and the C-7 methylene groups, respectively, of the sn-2 chain were used to study the conformational and dynamical properties of the bilayer as a function of temperature. There were no significant changes between the 13C and 2H spectra of the DPPC preparation containing the inactive steroid and that of DPPC with no drug. However, the physiologically active steroid produces significant spectral 2H and 13C changes. These changes include a reduction of the main phase transition temperature and a broadening of that transition. Alphaxalone also increases the relative number of gauche conformers in the liquid-crystalline phase of DPPC and increases the rate of axial diffusion in both the gel and liquid-crystalline phase. The thermotropic properties of the above preparations, as monitored by differential scanning calorimetry, were congruent with the spectroscopic data.     

Opioid peptides are substrates for the bifunctional enzyme LTA4 hydrolase/aminopeptidase

We determined if any naturally occurring peptides could act as substrates or inhibitors of the bifunctional, Zn²⁺ metalloenzyme LTA₄ hydrolase/aminopeptidase (E.C. 3.3.2.6). Several opioid peptides including met⁵-enkephalin, leu⁵-enkephalin, dynorphin_1–6, dynorphin_1–7, and dynorphin_1–8 competitively inhibited the hydrolysis of L-proline-p-nitroanilide by leukotriene A₄ hydrolase/ aminopeptidase, consistent with an interaction at its active site. The enzyme catalyzed the N-terminal hydrolysis of tyrosine from met⁵-enkephalin with K_m =450 ± 58 μM and V_max =4.9 ± 0.6 nmol-hr⁻¹-ug⁻¹ and from leu⁵-enkephalin with K_m =387 ± 90 μM and V_max =6.2 ± 2.5 nmol-hr⁻¹-ug⁻¹. Bestatin, captopril and carnosine inhibited the hydrolysis of the enkephalins. It is noteworthy that the bifunctional catalytic traits of this enzyme include generation of an hyperalgesic substance, LTB₄, and inactivation of analgesic opioid peptides.     

Sample entropy analysis of neonatal heart rate variability

Abnormal heart rate characteristics of reduced variability and transient decelerations are present early in the course of neonatal sepsis. To investigate the dynamics, we calculated sample entropy, a similar but less biased measure than the popular approximate entropy. Both calculate the probability that epochs of window length m that are similar within a tolerance r remain similar at the next point. We studied 89 consecutive admissions to a tertiary care neonatal intensive care unit, among whom there were 21 episodes of sepsis, and we performed numerical simulations. We addressed the fundamental issues of optimal selection of m and r and the impact of missing data. The major findings are that entropy falls before clinical signs of neonatal sepsis and that missing points are well tolerated. The major mechanism, surprisingly, is unrelated to the regularity of the data: entropy estimates inevitably fall in any record with spikes. We propose more informed selection of parameters and reexamination of studies where approximate entropy was interpreted solely as a regularity measure.     

Effects of DCPA,EPTC, and Chlorpropham on Pathogenicity of Meloidogyne hapla to Alfalfa

Treatments wilh the herbicides chlorpropham (isopropyl m-chlorocarbinilate), DCPA (dimethyl tetrachloroterephthalate), and El''TC (S-ethyl dipropylthiocarbamate), alone or in combination with Meloidogyne hapla Chilwood, significantly reduced the growth of both nemalode-resistant ''Nev Syn XX'' atttt susceptible ''Ranger" alfalfa (Medicago saliva L.) seedlings. M. hapla infection of both alfalfas was reduced by all herbicides because of fewer available infective courts in the treated plants. EPTC, however, reduced resistance to M. hapla, as indicated by increased galling of ''Nev Syn XX'' plants.     

Colonization of rye green manure and peanut fruit debris by Aspergillus falvus and Aspergillus niger group in field soils.

Aspergillus flavus and Aspergillus niger group colonization of deep-plowed, decomposing rye green manure cover crops in peanut field soils was studied in four fields during 1972 and 1973; colonization of decomposing peanut fruits was studied in 1972 in two fields. A. flavus colonization of rye and peanut fruits was greater in soils of heavy texture, and an A. flavus population as high as 165 propagules per g of soil was observed in soil adjacent to rye, whereas A. flavus populations in soils not associated with rye were 18 propagules per g of soil or lower. Highest A. flavus populations in soil adjacent to decomposing peanut fruits were usually comparable to populations associated with rye. Little decomposing rye or peanut fruit colonization was generally observed by the A. flavus competitor, A. niger group. A. flavus may maintain or increase its inoculum potential by colonization of these and other moribund plant tissues.     

CYP3A4-V and prostate cancer in African Americans: causal or confounding association because of population stratification?

CYP3A4-V, an A to G promoter variant associated with prostate cancer in African Americans, exhibits large differences in allele frequency between populations. Given that the African American population is genetically heterogeneous because of its African ancestry and subsequent admixture with European Americans, case-control studies with African Americans are highly susceptible to spurious associations. To test for association with prostate cancer, we genotyped CYP3A4-V in 1376 (2 N) chromosomes from prostate cancer patients and age- and ethnicity-matched controls representing African Americans, Nigerians, and European Americans. To detect population stratification among the African American samples, 10 unlinked genetic markers were genotyped. To correct for the stratification, the uncorrected association statistic was divided by the average of association statistics across the 10 unlinked markers. Sharp differences in CYP3A4-V frequencies were observed between Nigerian and European American controls (0.87 and 0.10, respectively; P<0.0001). African Americans were intermediate (0.66). An association uncorrected for stratification was observed between CYP3A4-V and prostate cancer in African Americans (P=0.007). A nominal association was also observed among European Americans (P=0.02) but not Nigerians. In addition, the unlinked genetic marker test provided strong evidence of population stratification among African Americans. Because of the high level of stratification, the corrected P-value was not significant (P=0.25). Follow-up studies on a larger dataset will be needed to confirm whether the association is indeed spurious; however, these results reveal the potential for confounding of association studies by using African Americans and the need for study designs that take into account substructure caused by differences in ancestral proportions between cases and controls.     

Impact of the functional status of saeRS on in vivo phenotypes of Staphylococcus aureus sarA mutants

We investigated the in vivo relevance of the impact of sarA and saeRS on protease production using derivatives of the USA300 strain LAC. The results confirmed that mutation of saeRS or sarA reduces virulence in a bacteremia model to a comparable degree. However, while eliminating protease production restored virulence in the sarA mutant, it had little impact in the saeRS mutant. Additionally, constitutive activation of saeRS (saeRS^C) enhanced the virulence of LAC and largely restored virulence in the isogenic sarA mutant. Based on these results, together with our analysis of the representative virulence factors alpha toxin, protein A (Spa), and extracellular nucleases, we propose a model in which the attenuation of saeRS mutants is defined primarily by decreased production of such factors, while constitutive activation of saeRS increases virulence, and reverses the attenuation of sarA mutants, because it results in both increased production and decreased protease‐mediated degradation of these same factors. This regulatory balance was also apparent in a murine model of catheter‐associated infection, with the results suggesting that the impact of saeRS on nuclease production plays an important role during the early stages of these infections that is partially offset by increased protease production in sarA mutants.     

High-frequency plant regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.)

Shoot regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.) was tested on MS basal medium supplemented with four different growth regulators. Regeneration frequencies for medium supplemented with 10 M 2,4-dichlorophenoxyacetic acid (2,4-D), 60 M 4amino-3, 5,6-picolinic acid (picloram), or 30 M 3,6dichloro-o-anisic acid (dicamba) ranged from 0.4 to 4%. Medium supplemented with 30 M dicamba plus 10 M 6-benzylaminopurine (BA) resulted in regeneration of shoots from 20% of the calli tested. Higher rates of growth regulators (60 or 90 M dicamba, 20 M BA) resulted in regeneration of shoots from 45% of calli of the cultivar Baron. In a subsequent study, the response of 12 North American cultivars grown on these media was cultivar-specific, with mean frequencies of regeneration ranging from 4% to 40%.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,5,6-picolinic acid - BA 6-benzylaminopurine     

Ribonucleic Acid Synthesis During the Differentiation of Sporangia in the Water Mold Achlya

The role of ribonucleic acid (RNA) synthesis in the development of sporangia in the saprolegniaceous mold Achlya was studied. Methods were developed for growing and treating large populations of mycelia so that the hyphal tips would differentiate into sporangia with considerable synchrony. Under the starvation conditions imposed for the differentiation of sporangia, net RNA, deoxyribonucleic acid (DNA), and protein synthesis ceased. However, incorporation of radioactive precursors into RNA continued at a high rate throughout the period of differentiation, showing that the enzymatic mechanism for RNA synthesis was still in an active state. Actinomycin D inhibited the differentiation of sporangia and the incorporation of labeled precursors into RNA. The level of actinomycin used did not inhibit the normal outgrowth and branching of the mycelia that occurred during differentiation. Thus, DNA-dependent RNA synthesis was required for the differentiation of sporangia. Sucrose gradient analysis of newly synthesized RNA showed that only the ribosomal and soluble fractions of RNA were labeled during vegetative growth. During the differentiation of sporangia, ribosomal and soluble RNA fractions were also labeled, and, in addition, a heterodisperse fraction of labeled RNA which was heavier than ribosomal RNA appeared; this fraction was not evident in the newly synthesized RNA from vegetative mycelia.