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.     

LapB (YciM) orchestrates protein–protein interactions at the interface of lipopolysaccharide and phospholipid biosynthesis

The outer membrane (OM) of Gram-negative bacteria functions as an essential barrier and is characterized by an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet. The enzyme LpxC catalyzes the first committed step in LPS biosynthesis. It plays a critical role in maintaining the balance between LPS and phospholipids (PL), which are both derived from the same biosynthetic precursor. The essential inner membrane proteins YejM (PbgA, LapC), LapB (YciM), and the protease FtsH are known to account for optimal LpxC levels, but the mechanistic details are poorly understood. LapB is thought to be a bi-functional protein serving as an adaptor for FtsH-mediated turnover of LpxC and acting as a scaffold in the coordination of LPS biosynthesis. Here, we provide experimental evidence for the physical interaction of LapB with proteins at the biosynthetic node from where the LPS and PL biosynthesis pathways diverge. By a total of four in vivo and in vitro assays, we demonstrate protein–protein interactions between LapB and the LPS biosynthesis enzymes LpxA, LpxC, and LpxD, between LapB and YejM, the anti-adaptor protein regulating LapB activity, and between LapB and FabZ, the first PL biosynthesis enzyme. Moreover, we uncovered a new adaptor function of LapB in destabilizing not only LpxC but also LpxD. Overall, our study shows that LapB is a multi-functional protein that serves as a protein–protein interaction hub for key enzymes in LPS and PL biogenesis presumably by virtue of multiple tetratricopeptide repeat (TPR) motifs in its cytoplasmic C-terminal region.     

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:

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.     

Butyrate inhibits cytokine-induced VCAM-1 and ICAM-1 expression in cultured endothelial cells: the role of NF-kappaB and PPARalpha

Adhesion and migration of leukocytes into the surrounding tissues is a crucial step in inflammation, immunity, and atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. Butyrate, a natural short-chain fatty acid produced by bacterial fermentation of dietary fiber, has been attributed with anti-inflammatory activity in inflammatory bowel disease. Butyrate in vitro is active in colonocytes and several other cell types. We have studied the effect of butyrate on expression of endothelial leukocyte adhesion molecules by cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with butyrate-inhibited tumor necrosis factor-alpha (TNFalpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) in a time and concentration-dependent manner. Butyrate at 10 mM/L inhibited interleukin-1 (IL-1)-stimulated VCAM-1 and ICAM-1 expression. The effect of butyrate on cytokine-stimulated VCAM-1 expression was more pronounced than in the case of ICAM-1. Butyrate decreased TNFalpha-induced expression of mRNA for VCAM-1 and ICAM-1. Suppressed expression of VCAM-1 and ICAM-1 was associated with reduced adherence of monocytes and lymphocytes to cytokine-stimulated HUVEC. Butyrate inhibited TNFalpha-induced activation of nuclear factor-kappaB (NF-kappaB) in HUVEC. Finally, butyrate enhanced peroxisome proliferator-activated receptor-alpha (PPARalpha) expression in HUVEC. These results demonstrate that butyrate may have anti-inflammatory properties not only in colonocytes but also in endothelial cells. The anti-inflammatory and (perhaps) antiatherogenic properties of butyrate may partly be attributed to an effect on activation of NF-kappaB and PPARalpha and to the associated expression of VCAM-1 and ICAM-1. The present findings support further investigations on the therapeutic benefits of butyrate in several pathological events involving leukocyte recruitment.     

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.     

Results of selfmonitoring on glucometer systems Advance and Optium in daily routine

The aim of this prospective clinical study was to compare the results of B-glucose estimations performed simultaneously on glucometer Advance (with Micro-draw strips) and Optium (G3 strips) by lay healthy volunteers under non-standardized conditions of everyday life, to assess the difficulties dealing with lay-handling of these systems and to demonstrate the possibilities of the software Glucobalance (Hypoguard) and PC-Link (Medisense/Abbott) for the analysis of selfmonitoring. In the course of 5 days, a total of 721 pairs of measurements were carried out on 10 pairs of glucometer Advance and Optium by 10 healthy volunteers aged 16-40 years. The data transfer of all values into computer from glucometer Advance using the Glucobalance software and from glucometer Optium using the PC-Link was carried out to determine the results. The correlation of B-glucose measured on the glucometer Advance and Optium was strong (r = 0.73). Glucometer Advance brings values about 0.21 +/- 0.06 mmol/l lower than glucometer Optium. The average difference found within each pairs of glucometers Advance - Optium varied. Nevertheless, these differences are acceptable for routine selfmonitoring. The handling of glucometer Advance is not difficult for lay persons. The Glucobalance software simplifies the result evaluation by each tested person. Even though there are some advantages in comparison with the PC-Link, it should be further developed.