Characterization of eight new members of the calmodulin-like domain protein kinase gene family from Arabidopsis thaliana

A family of calcium-responsive protein kinases is abundant in plant cell extracts but has not been identified in animals and fungi. These enzymes have a unique structure consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. In this report, we present the amino acid sequences for eight new Arabidopsis cDNA clones encoding isoforms of this enzyme. Three isoforms were expressed as fusion proteins in Escherichia coli and exhibited calcium-stimulated protein kinase activity. We propose CPK as the gene designation for this family of enzymes and describe a phylogenetic analysis for all known isoforms.     

An evaluation of two methods used for microscopic analysis of airborne fungal spore concentrations from the Burkard Spore Trap

The Burkard Volumetric Spore Trap is a common and efficient instrument used to collect outdoor air samples. In North America, two slide counting methods have been widely used by aerobiologists: the single longitudinal traverse method and the twelve transverse traverse method. The purpose of this study was to compare the two counting methods by assessing fungal spore concentrations of ascospores, basidiospores, smut teliospores, Cladosporium, Alternaria, Epicoccum, Curvularia, Drechslera, Pithomyces, other spores, and total spores at two metropolitan Tulsa, Oklahoma sites (Tulsa and Hectorville) during September 1996. Results showed that both methods were sensing parallel fluctuations in average daily spore concentration, although the twelve transverse traverse method usually resulted in higher concentrations. At the Tulsa site, the twelve transverse traverse method gave statistically higher concentrations than the single longitudinal traverse method except for Epicoccum, Pithomyces, smut teliospores, and other spores. At the Hectorville site, however, only Cladosporium and basidiospores showed that the twelve transverse traverse method was statistically higher than the single longitudinal traverse method. Comparison with concentrations obtained by counting the total slide surface of two slides indicated that neither method was equivalent to the total slide spore count, although the twelve transverse traverse method gave a lower absolute percent difference from the total slide surface concentration. While the twelve transverse traverse method gave slightly better approximations of the spore concentration, the increase in accuracy may not justify the extra effort required to analyze with this method.     

Immunohistochemical assessment of the peripheral benzodiazepine receptor in human tissues.

Exhaustive analysis of the location of the peripheral benzodiazepine receptor (PBR) both at the subcellular and the tissue level is warranted to gain a better understanding of its biological roles. To date, many studies have been performed in animal models, such as rat, mouse, and pig, that yielded important information. However, only a few reports were dedicated to the analysis of PBR expression in humans. To enlarge on previous studies, we investigated PBR expression in different human organs using the monoclonal antibody 8D7 that specifically recognized the human PBR. First, we performed electron microscopic analysis that for the first time unambiguously demonstrated the localization of the PBR on the outer mitochondrial membrane. Second, focusing our analysis on human tissues for which information on PBR expression is sparse (lung, stomach, small intestine, colon, thyroid, adrenal gland, pancreas, breast, prostate, ovary), we found that PBR exhibits selective localization. This characterization of PBR localization in human tissues should provide important insights for the understanding of PBR functions.     

Genetic elements near the structural gene modulate the level of dopa decarboxylase during Drosophila development

Summary Measurement of dopa decarboxylase (DDC) levels in 109 strains of Drosophila melanogaster isogenic for second chromosomes isolated independently from natural populations was undertaken. One of the most extreme variants, designated Ddc ⁺⁴, was shown to have about 20% more DDC activity at adult eclosion than a standard laboratory strain used for comparison. The DDC overproduction was shown to segregate with the second chromosome and was mapped to a position within 0.15 map units of the DDC structural gene. The variant was shown to be an underproducer of DDC activity at pupariation and the genetic element responsible for this trait mapped in an identical fashion to that causing overproduction. The temporal phenotype described above was observed in the epidermis but DDC activity levels in neural tissue were normal. Examination of CRM levels at pupariation and eclosion revealed that altered DDC protein levels were responsible for the variant DDC activity levels. Electrophoretic analysis under both denaturing and non-denaturing conditions indicated that the DDC molecules in Ddc ⁺⁴ and the laboratory strain were indistinguishable. These results suggest that alterations in a genetic element (or elements) lying in close proximity to the structural gene are responsible for the complex temporal phenotype of DDC activity exhibited in the variant Ddc ⁺⁴.Abbreviations CRM cross-reacting material - DDC dopa decarboxylase - PTU phenylthiourea     

Redox thermodynamics of mutant forms of the rubredoxin from Clostridium pasteurianum: identification of a stable FeIII(S-Cys)3(OH) centre in the C6S mutant

Redox thermodynamic data provide a detailed insight into control of the reduction potential E degrees' of the [Fe(S-Cys)4] site in rubredoxin. Mutant forms were studied in which specific structural changes were made in both the primary and secondary coordination spheres. Those changes have been probed by resonance Raman spectroscopy. The decrease of approximately 200 mV in E degrees' observed for the [Fe(S-Cys)3(O-Ser)]-/2- couples in the surface ligand mutants C9S and C42S is essentially enthalpic in origin and associated with the substitution of ligand thiolate by ligand olate. However, the pH dependence of the potentials below characteristic pKa(red) approximately equals 7 is an entropic contribution, plausibly associated with increased conformational flexibility induced by a longer Fe(II)-O(H)-Ser bond in the reduced form. The presence of a second surface Ser ligand in the new double mutant protein C9S/C42S affects the enthalpic term primarily for pH>pKa(red) > or = 9.3, but for pHpKa approximately 9: [Fe(III)(S-Cys)3(OH)]- + e- --> [Fe(II)(S-Cys)3(OH)]2-. pH [Fe(II)(S-Cys)3(OH2)]-.     

Comparison of sialyl- and alpha-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased beta-galactoside alpha-2,6-sialyltransferase.

Previous studies have indicated that transfection of NIH3T3 cells with the ras oncogene induced modifications of the terminal glycosylation of N-linked glycans which appeared in the early stage after transfection. These changes affected especially the terminal part of N-linked glycans which is substituted with alpha-1,3-Gal residues in NIH3T3 and with Neu5Ac residues in the ras-transformed counterpart. We have transformed NIH3T3 cells with the human c-Ha-ras oncogene, evaluated tumorigenicity and metastatic capacity in vivo and compared alpha-1,3-galactosyltransferase, alpha-2,3- and alpha-2,6-sialyltransferases activities. By using different specific acceptors, we detected the enhancement of sialic acid transfer in transformed cells while the activity of alpha-1,3-galactosyltransferase remained unchanged. We showed that the higher sialyltransferase activity was due to the increase of beta-galactoside alpha-2,6-sialyltransferase in ras-transfectant although alpha-2,3-sialyltransferase was weakly expressed in these cells. On the basis of binding of different lectins, we correlated these observations with changes of protein glycosylation. We concluded that altered glycosylation of ras-transformed NIH3T3 is the result of a competitive effect of the enzymes acting for terminal glycosylation of N-linked glycans and the reflection of the higher expression of alpha-2,6-sialyltransferase.     

Responses ofArabidopsis roots to auxin studied with high temporal resolution: Comparison of wild type and auxin-response mutants

We modified a video digitizer system to allow short-term high-resolution measurements of root elongation in intact seedlings ofArabidopsis thaliana (L.) Heynh. We used the system to measure the kinetics of promotion and inhibition of root elongation by applied auxin and to determine the dose-response relationship for auxin action on elongation in roots of wild-type seedlings and seedlings of mutants (axr1,aux1, andaxr2) with altered auxin responsiveness. Roots of the mutants showed less inhibition in the presence of inhibitory concentrations of auxin than did roots of the wild type. The latent period preceding the change in elongation rate after auxin application was the same foraxr1 andaxr2 as for the wild type whereas the latent period foraux1 was about twice as long as for the wild type. Low concentrations (ca. 10^–11 M) of auxin induced substantial promotion of root elongation in the wild type and inaxr2.We thank Linda Young and Roger Hangarter for helping to develop the system for mountingArabidopsis seedlings and Wendy Hankie, Julia Hufford, and Ruperto Villella for doing some of the experiments. We thank Roger Hangarter for valuable discussions of the data. This work was supported by National Science Foundation Grant No. DCB-9105807 and by National Aeronautics and Space Administration Grant No. NAG10-0084     

Daily patterns of courtship and mating behavior in the male Japanese quail

Crowing behavior was monitored constantly in male Japanese quail housed singly over 30 successive days. The photoperiod was 16h of light and 8 h of dark. A daily pattern in crowing was observed in which the frequencies were elevated in the afternoon and at the beginning of darkness. However, peak crowing occured 2 h prior to the onset of light. These rhythms were highly correlated among individuals and extremely repeatable over the sequential days of observation.In a second experiment, males which were paired with females were observed for frequencies of crowing, courtship, and mating behavior during the lighted portion of the day. In this experiment, the same photoperiod (16L:8D) was maintained. Paired males exhibited a daily pattern in crowing similar to that observed in the singly housed males. The frequency of mating was the highest between 1200 and 1300 h and lowest at 1400 h. Mating success was highest at midday, as were the number of males exhibiting mating behavior. These diurnal patterns in sexual behavior may depend on environmental cues such as photoperiod, which, in turn, may stimulate endocrine triggers.