Isolated iron-molybdenum cofactor of nitrogenase exists in multiple forms in its oxidized and semi-reduced states

Electrochemical and EPR spectroscopic experiments demonstrate that the isolated iron-molybdenum cofactor from the molybdenum-iron protein of nitrogenase from Azotobacter vinelandii exists in multiple forms in both its oxidized and semi-reduced states. The particular forms found in either oxidation state appear to be a function of the acid/base status of the solvent, N-methylformamide. In "alkaline" N-methylformamide, a single, detectable form of iron-molybdenum cofactor is observed for both oxidized and semi-reduced states. The semi-reduced form, termed R(s-r), is the one previously recognized with an S = 3/2 EPR spectrum with apparent g values of 4.6, 3.4, 2.0. Its oxidized counterpart, termed B(ox), is characterized electrochemically by a differential pulse voltammetric reduction peak at -0.37 V versus the normal hydrogen electrode. In "acidic" solvent, two distinct, previously unrecognized redox pairs of iron-molybdenum cofactor forms exist. The two semi-reduced forms, N(s-r) and W(s-r), are characterized by EPR spectra with g = 4.5, 3.6, 2.0 and g = 4.9, 3.1, 1.9, respectively. Their oxidized counterparts, A(ox) and C(ox), have differential pulse voltammetric reduction peaks at -0.32 and -0.43 V versus the normal hydrogen electrode, respectively. Manipulations of either the isolation protocol or the sample conditions affects both the type and distribution of forms present. Each form likely corresponds to a biologically significant state of the cofactor cluster within the protein.     

Influence of oestradiol on protein expression and methionine utilization during morphogenesis of Paracoccidioides brasiliensis

The temporal sequence of cytosolic protein expression during phase transition of Paracoccidioides brasiliensis was examined. Electrophoretic analysis of cytosol proteins by one-dimensional SDS-PAGE revealed numerous differences between the mycelial and yeast forms as well as alterations induced by 17 beta-oestradiol. Using either protein staining or fluorography of [35S]methionine-labelled proteins 30 phase-specific bands were detected, 12 mycelial-associated bands (range 30 to 140 kDa) and 18 yeast-associated bands (range 22 to 127 kDa). In cells undergoing mycelial to yeast transition after a shift from 25 degrees C to 37 degrees C, the protein patterns showed a temporal progression toward the yeast profile with the accumulation of yeast bands prior to observable morphogenesis. Five novel protein bands (range 23 to 50 kDa) were detected by silver staining during transition. Treatment of temperature-shifted mycelial cultures with 2.6 x 10(-7) M-oestradiol altered observed profiles; 4 of 12 mycelial-associated bands were maintained whereas the appearance of the 5 novel transition bands and 9 of 18 yeast-associated bands was blocked or delayed. Analysis of [35S]methionine-labelled proteins revealed that oestradiol induced label uptake by mycelial cells, blocked the synthesis of a 92 kDa yeast-specific band 72 h into transition, and diminished label incorporation 120 h into transition. In conjunction with these steroid-induced alterations of protein expression, little or no morphological transformation occurred. These results support our hypothesis that, analogous to mammalian steroid receptor action, the functional responses of P. brasiliensis to oestradiol are related to regulation of protein expression, presumably mediated via a specific binding protein-ligand complex.     

Contraception in mares heteroimmunized with pig zonae pellucidae

Ten fertile feral mares and 6 domestic horses (4 fertile mares, 1 infertile mare, 1 gelding) were immunized with heat-solubilized pig zonae pellucidae by 4 injections equivalent to 2000 or 5000 zonae each at 2-4-week intervals and a booster injection of 20,000 zonae 6-10 months after the last of the initial inoculations. The immune response was reflected by high antibody levels as measured by an enzyme-linked immunosorbent assay (ELISA) using immobilized pig zona antigen. In-vivo inhibition of fertility occurred in 12 (86%) of the 14 fertile mares studied and persisted for a minimum of 7 months. Repeated mating of the fertile domestic mares resulted in conception when anti-pig zona antibody concentrations had decreased from initial peak absorbance ratios (greater than 1.0) to relatively lower levels (0.64 or less with one exception). An indirect immunofluorescence assay, revealed a considerably lower cross-reactive antibody titre with horse oocytes as compared to pig oocytes. Clinical, endocrinological and histological analyses of the ovaries and their function following regained fertility after immunization revealed no abnormalities. One mare remained infertile.     

The precise radioimmunoassay of adenosine: minimization of sample collection artifacts and immunocrossreactivity.

Anti-adenosine antibodies were produced in rabbits immunized with N6-carboxymethyladenosine conjugated to methyl albumin. 125I-N6-Aminobenzyladenosine was synthesized and used as a high-specific-activity, high-affinity ligand. A radioimmunoassay (RIA) was developed that can detect 6.25 nM (312.5 fmol) of underivatized adenosine and cross-reacts less than 0.02% with adenine nucleotides and guanosine and not at all with 1 mM inosine. The sensitivity of the RIA can be increased to a detection limit of 0.125 nM (6.25 fmol) by derivitizing samples with benzyl bromide to form N6-benzyladenosine. The assay was adapted to an automated RIA procedure. Assay precision was increased by: (i) inhibiting slight adenosine deaminase activity present in anti-sera; (ii) treating buffers and albumin used in the RIA with charcoal to remove contaminating adenosine; and (iii) correcting for a small but variable component of immunoreactivity not attributable to adenosine. A second antibody prepared with a 2',3'-disuccinyladenosine-albumin conjugate was also found to detect some non-adenosine-mediated immunoreactivity in plasma samples. Immunointerference in human plasma was eliminated in samples treated with ZnSO4/Ba(OH)2 or partially purified over C18 Sep Paks to remove nucleotides and assayed after sample benzylation or succinylation. Human blood was mixed with a novel "stop" solution that was optimized to inhibit adenosine formation from AMP by greater than 99% and to inhibit adenosine uptake into red cells and degradation by greater than 94%. Human plasma/stop solution was assayed by RIA and HPLC with equivalent results.     

Gene-culture coevolutionary theory

Gene-culture coevolutionary theory is a branch of theoretical population genetics that models the transmission of genes and cultural traits from one generation to the next, exploring how they interact. These models have been employed to examine the adaptive advantages of learning and culture, to investigate the forces of cultural change, to partition the variance in complex human behavioral and personality traits, and to address specific cases in human evolution in which there is an interaction between genes and culture.     

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [³⁵S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 M KN-93, but binding is not affected by 5 M KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 M KN-93, but not by 5 M KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.Abbreviations CaM calmodulin - CaMK (II) Ca²⁺/calmodulin-dependent protein kinase (II) - CBP CaM-binding protein - CDPK Ca²⁺-dependent protein kinase - MCK1 maize homolog of mamalian CaMK This work is supported by the National Aeronautics and Space Administration grant No: NAGW 238.     

Polymorphic Admixture Typing in Human Ethnic Populations

A panel of 257 RFLP loci was selected on the basis of high heterozygosity in Caucasian DNA surveys and equivalent spacing throughout the human genome. Probes from each locus were used in a Southern blot survey of allele frequency distribution for four human ethnic groups: Caucasian, African American, Asian (Chinese), and American Indian (Cheyenne). Nearly all RFLP loci were polymorphic in each group, albeit with a broad range of differing allele frequencies (δ). The distribution of frequency differences (δ values) was used for three purposes: (1) to provide estimates for genetic distance (differentiation) among these ethnic groups, (2) to revisit with a large data set the proportion of human genetic variation attributable to differentiation within ethnic groups, and (3) to identify loci with high δ values between recently admixed populations of use in mapping by admixture linkage disequilibrium (MALD). Although most markers display significant allele frequency differences between ethnic groups, the overall genetic distances between ethnic groups were small (.066–.098), and <10% of the measured overall molecular genetic diversity in these human samples can be attributed to “racial” differentiation. The median δ values for pairwise comparisons between groups fell between .15 and .20, permitting identification of highly informative RFLP loci for MALD disease association studies.