Chemical characterization of a new Japanese variant of carbonic anhydrase I,CA INagasaki 1 (76 Arg→Gln)

A new inherited variant of carbonic anhydrase I (CA I), designated CA I_{Nagasaki 1} (CA I_{NGS 1}), was discovered during a survey of hemolysates from 5852 individuals from the cities of Hiroshima and Nagasaki in Japan. Analysis of the amino acid composition of a tryptic peptide from the CA I_{NGS 1} variant indicated that a glutaminyl residue was substituted for an arginyl residue at position 76. Heat degradation studies showed that the CA I_{NGS 1} variant was less stable than normal CA I. The CO₂ hydrase and esterase activities of the normal and variant carbonic anhydrases I, as well as the relative amounts of the two enzymes in heterozygotes, were similar.This work was supported in part by Contract E(11-1)-1552 with the Energy Research and Development Administration, Washington, D.C. (to J. V. Neel), and by U.S. Public Health Service Grant GM-24681.     

Reduced tendency to form a beta turn in peptides from the P22 tailspike protein correlates with a temperature-sensitive folding defect

A family of mutants of the P22 bacteriophage tailspike protein has been characterized as temperature sensitive for folding (tsf) by King and co-workers [King, J. (1986) Bio/Technology 4, 297-303]. There is substantial evidence that the tsf mutations alter the folding pathway but not the stability of the final folded protein. Several point mutations are known to cause the tsf phenotype; most of these occur in regions of the tailspike sequence likely to take up reverse turns. Hence, it has been hypothesized that the correct folding of the P22 tailspike protein requires formation of turns and that the mutations causing tsf phenotypes interfere at this critical stage. We have tested this hypothesis by study of isolated peptides corresponding to a region of the P22 tailspike harboring a tsf mutation. Comparison of the tendencies of wild-type and tsf sequences to adopt turn conformations was achieved by the synthesis of peptides with flanking cysteine residues and the use of a thiol-disulfide exchange assay. We find that the wild-type sequence, either as a decapeptide (Ac-CVKFPGIETC-CONH2) or as a dodecapeptide (Ac-CYVKFPGIETLC-CONH2), has a 3-5-fold greater tendency for its termini to approach closely enough to form the intramolecular disulfide than do the peptide sequences corresponding to the tsf mutant sequences, which have a Gly----Arg substitution (Ac-CVKFPRIETC-CONH2 or Ac-CYVKFPRIETLC-CONH2). A peptide with a D-Arg substituted for the Gly has a slightly higher turn propensity than does the wild type. Together with data from nuclear magnetic resonance analysis of the oxidized peptides, this suggests that a type II beta turn is favored by the wild-type sequence. Our results on isolated peptides from the P22 tailspike protein support the model for its folding that includes reverse turn formation as a critical step.     

Transmission of Megatrypanum Trypanosomes to Cervus dama by Tabanidae1

Four fallow deer, Cervus dama, became infected with Trypanosoma (Megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

Distinct mechanisms of action of selective estrogen receptor modulators in breast and osteoblastic cells

Raloxifene and idoxifene areselective estrogen receptor modulators (SERMs) that exhibittissue-specific agonist or antagonist properties via interactions withthe estrogen receptor (ER). Both compounds are similarlyosteoprotective in the ovariectomized rat in vivo as assessed bymeasurement of bone mineral density, urinary pyridinium cross-links,and serum osteocalcin, suggesting a similar mechanism of action.However, we have identified a fundamental difference in this mechanismvia the estrogen response element (ERE) in osteoblast-like cells. Withthe use of ERE-luciferase reporter constructs, raloxifene, like thecomplete ER-antagonist ICI-182780, acts as an antagonist viathe ERE in osteoblastic cells. In contrast, idoxifene, like17-estrogen itself and 4-OH-tamoxifen, acts as an agonist inosteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780and raloxifene inhibited the ERE-dependent agonist activity of17-estradiol and idoxifene in osteoblastic cells. In contrast, inbreast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 hadno agonist activity and, indeed, raloxifene and idoxifene were potentantagonists of ERE-mediated 17-estradiol action, indicating anERE-dependent mode of action in these cells. Although these SERMsexhibit a similar antagonist activity profile in breast cells, they canbe distinguished mechanistically in osteoblastic cells.

     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

Two-Phase Slug Flow Heat Exchanger for Microbial Thermal Inactivation Research

A continuous two-phase (air-liquid), slug flow, tubular heat exchanger was developed for microbial thermal inactivation research to give exposure times and temperatures in the range of high-temperature, short-time milk pasteurization as well as heat-treated sample volumes of at least 2 ml. The use of air to compartmentalize the liquid in the capillary tubing prevented the development of laminar flow, which enabled precise identification of the residence time of the fastest flowing particles in the heating, holding, and cooling sections of the instrument. Residence time distributions were quantitated by measuring the degree of spreading of radioactive tracers for water, whole milk, chocolate milk, cream, and ice-cream mix with holding temperatures from 50 to 72 C, holding times from 2 to 60 sec, and heating and cooling times of 1.7 sec each. For a holding time of 60 sec and a fastest particle velocity of 10.2 cm/sec, the velocity ratios of the fastest moving particle to the median particle were 1.05, 1.05, 1.10, and 1.13 for whole milk, chocolate milk, cream, and ice-cream mix, respectively. With shorter holding times, these velocity ratios were even closer to unity. These velocity ratios indicated that the instrument would be an effective tool for thermal inactivation research on microorganisms suspended in homogeneous fluids with a viscosity of 15 centipoises or less at the exposure temperature.     

Bilateral testicular infarction and orchiectomy as a complication of polyarteritis nodosa

We report an unusual case of a 28-year-old male with constitutional symptoms and bilateral testicular pain. After diagnosis of cytomegalovirus (CMV) hepatitis, his constitutional symptoms and testicular pain worsened despite treatment for epididymoorchitis. Ultrasound was concerning for infarction. Exploration in the operating room revealed bilateral testicular infarction requiring bilateral orchiectomy with subsequent androgen hormone replacement. Pathologic diagnosis was polyarteritis nodosa (PAN). PAN is a rare systemic vasculitis that affects multiple organs. There are no previous reports of PAN-induced vasculitis leading to bilateral testicular infarction and bilateral orchiectomy.