Activity and mechanism of action of insect oostatic peptides in flesh fly

The relationship between structure and activity of insect oostatic decapeptide (Aed-TMOF) analogues in flesh fly was analyzed. The highest oostatic activity was exhibited by the pentapetide and tetrapeptide analogues, H-Tyr-Asp-Pro-Ala-Pro-OH and H-Tyr-Asp-Pro-Ala-OH, respectively. The tetrapeptide, either native or tritiated, was used to study its metabolism in the ovaries and hemolymph and to detect putative binding sites in the flesh fly ovaries and head. A high metabolism of the tetrapeptide with a half-life in the hemolymph and ovaries less than 1h was determined. The initial limiting step in the degradation is tyrosine(1) cleavage. Other degradation products were detected only transiently in low quantities. Using tritiated tetrapeptide, we found that only very low specific binding was detected in the homogenates of ovaries and in the rough membrane preparation in the presence and absence of protease inhibitors.     

Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm

A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct pDDR101 comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter. The E1alpha sequence was engineered to include an N-terminal His-tag. When protein samples were subjected to immobilized metal ion affinity chromatography, the alpha- and beta-subunits co-eluted, indicating association. When the recombinant protein sample was analyzed further by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha2beta2 heterotetramer. Recombinant E1 was able to decarboxylate [1-14C]pyruvate and was a substrate for in vitro phosphorylation by E1-kinase.     

Bacillus subtilis mutant with temperature-sensitive net synthesis of phosphatidylethanolamine.

Bacillus subtilis mutants with temperature-sensitive growth on complex media were screened for defects in phospholipid metabolism. One mutant was isolated that showed temperature-sensitive net synthesis of phosphatidylethanolamine. The mutant did not accumulate phosphatidylserine at the nonpermissive temperature. In the presence of hydroxylamine, wild-type B. subtilis accumulated phosphatidylserine at both 32 and 45 degrees C, whereas the mutant did only at 32 degrees C. In vitro phosphatidylethanolamine synthesis with bacterial membranes is no more temperature sensitive with mutant membranes than with wild-type membranes. The mutation probably affects the synthesis indirectly, possibly by altering a membrane protein. The mutant bacteria grew at the nonpermissive temperature, 45 degrees C, in a phosphate buffer-based minimal medium, although net synthesis of phosphatidylethanolamine was also temperature sensitive in this medium. One mutation caused both temperature-sensitive growth on complex media and temperature-sensitive net synthesis of phosphatidylethanolamine. The mutation is linked to aroD by transformation.     

N-acetylation of amino sugar methyl glycosides for gas-liquid chromatographic analysis.

A procedure is described for rapid and quantitative N-acetylation of amino sugars, particularly suitable for gas-liquid chromatographic analysis of constituent carbohydrates in glycoproteins.     

Effects of agrobacterial oncogenes in kidney vetch (Anthyllis vulneraria L.)

Kidney vetch seedlings were induced to form hairy roots by inoculating their mesocotyls with the wild-type strain 15834 of Agrobacterium rhizogenes or with the A. tumefaciens strain C58C1 containing a binary vector system (the pRiA4b as a helper and the vector pCB1346 bearing a pTiC58-derived isopentenyl transferase gene (ipt, cytokinin biosynthetic gene) under control of its native regulatory sequences). Transgenic lines of three distinct phenotypes were selected:

Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion

Bacillus subtilis succinate dehydrogenase (SDH) is composed of two unequal subunits designated Fp (Mr, 65,000) and Ip (Mr. 28,000). The enzyme is structurally and functionally complexed to cytochrome b 558 (Mr, 19,000) in the membrane. A total of 21 B. subtilis SDH-negative mutants were isolated. The mutants fall into five phenotypic classes with respect to the presence and localization of the subunits of the SDH-cytochrome b558 complex. One class contains mutants with an inactive membrane-bound complex. Membrane-bound enzymatically active SDH could be reconstituted in fused protoplasts of selected pairs of SDH-negative mutants. Most likely reconstitution is due to the assembly of preformed subunits in the fused cells. On the basis of the reconstitution data, the mutants tested could be divided into three complementation groups. The combined data of the present and previous work indicate that the complementation groups correspond to the structural genes for the three subunits of the membrane-bound SDH-cytochrome b558 complex. A total of 31 SDH-negative mutants of B. subtilis have now been characterized. The respective mutations all map in the citF locus at 255 degrees on the B. subtilis chromosomal map. In the present paper, we have revised the nomenclature for the genetics of SDH in B. subtilis. All mutations which give an SDH-negative phenotype will be called sdh followed by an isolation number. The designation citF will be omitted, and the citF locus will be divided into three genes: sdhA, sdhB, and sdhC. Mutations in sdhA affect cytochrome b558, mutations in sdhB affect Fp, and mutations in sdhC affect Ip.     

Palmar and plantar pads and flexion creases of genetic polydactyly mice (Pdn)

Attempts to gain a better understanding of the relationship between the epidermal ridge patterns (dermatoglyphics) and flexion creases on the volar aspects of human hands and feet and specific medical disorders led to a search for a suitable animal model, allowing studies of the fetal development of the pertinent structures. A common experimental animal, the rat (Rattus norvegicus), was found to be an excellent candidate, owing to the strong resemblance of the volar pads and flexion creases on its palmar and plantar surfaces to those of human subjects. A hereditary preaxial polydactyly mouse (Pdn) provides an opportunity to study the effects of this malformation on the surrounding morphological structures and, specifically, on the volar pads, i.e., the sites over which the dermatoglyphic patterns develop. The hands and feet of the wild‐type (+/+) mice show no anomalies, and their major pad and flexion crease configurations correspond to those of normal rats. The heterozygous (Pdn/+) mice, in spite of having a thumb/big toe with a duplicated distal phalanx on their hands/feet, did not display any alterations in palmar/plantar pads. The homozygous (Pdn/Pdn) mice have a protrusion in the thenar area and one to three supernumerary digits on the preaxial portion of both the hands and feet. The effect of these anomalies was found to be limited to the pad and flexion crease configurations in the preaxial areas; the postaxial sites were not affected. The original number of pads on the thenar/first interdigital areas of Pdn/Pdn mice was apparently identical to that of the +/+ and Pdn/+ mice. The preaxial protrusion, however, affected the number, size, and location of the pads observed in the newborn mice, resulting in varying pad configurations, such as fused and scattered pads or a pad cluster formed by gathering the neighboring pads. These pad modifications were induced by the preaxial plantar/palmar protrusion only and were not affected by the presence of supernumerary preaxial digits. In view of the similarities in the morphology and fetal development of human and mouse distal limbs, the present study is relevant to human subjects, particularly to the understanding of the significance of dermatoglyphic variations in individuals with specific medical disorders. Future studies of naturally occurring or experimentally induced limb malformations in mice or rats should provide valuable insights into the development of human hands and feet and into factors contributing to their congenital anomalies. J. Morphol. 239:87–96, 1999. © 1999 Wiley‐Liss, Inc.     

Fetal and postnatal development of palmar,plantar, and digital pads,and flexion creases of the rat (Rattus norvegicus)

Fetal development of the hands and feet of rats was investigated to determine the feasibility of using rats as an experimental model for studying the factors influencing early development of the hands and feet, and especially the dermatoglyphics in humans. Eighty rat fetuses of 14–21 days gestational age and 80 newborn rats of 0–7 days of age were used to study the morphological features of the palmar, plantar, and digital areas and to determine the timing of appearance and the location of the volar pads and flexion creases. Comparisons between analogous developmental stages of rat and human fetuses demonstrate striking similarities in overall fetal development. Marked differences, however, were found between rat and human fetuses in the timing of developmental milestones and in some morphological features. The results indicate that rats can serve as a useful experimental model in studies of the utility of the epidermal ridge configurations and flexion creases in medical disorders, provided that the differences in the timing of development are taken into consideration. © 1996 Wiley-Liss, Inc.     

A screen for nigericin-resistant yeast mutants revealed genes controlling mitochondrial volume and mitochondrial cation homeostasis

Little is known about the regulation of ion transport across the inner mitochondrial membrane in Saccharomyces cerevisiae. To approach this problem, we devised a screening procedure for facilitating the identification of proteins involved in mitochondrial ion homeostasis. Taking advantage of the growth inhibition of yeast cells by electroneutral K(+)/H(+) ionophore nigericin, we screened for genetic mutations that would render cells tolerant to this drug when grown on a nonfermentable carbon source and identified several candidate genes including MDM31, MDM32, NDI1, YMR088C (VBA1), CSR2, RSA1, YLR024C, and YNL136W (EAF7). Direct examination of intact cells by electron microscopy indicated that mutants lacking MDM31 and/or MDM32 genes contain dramatically enlarged, spherical mitochondria and that these morphological abnormalities can be alleviated by nigericin. Mitochondria isolated from the Deltamdm31 and Deltamdm32 mutants exhibited limited swelling in an isotonic solution of potassium acetate even in the presence of an exogenous K(+)/H(+) antiport. In addition, growth of the mutants was inhibited on ethanol-containing media in the presence of high concentrations of salts (KCl, NaCl, or MgSO(4)) and their mitochondria exhibited two- (Deltamdm31 and Deltamdm32) to threefold (Deltamdm31Deltamdm32) elevation in magnesium content. Taken together, these data indicate that Mdm31p and Mdm32p control mitochondrial morphology through regulation of mitochondrial cation homeostasis and the maintenance of proper matrix osmolarity.