Evolution of the Larval Cuticle Proteins Coded by the Secondary Sex Chromosome Pair: X2 and Neo-Y of Drosophila miranda: I. Comparison at the DNA Sequence Level

The larval cuticle protein genes (Lcps) represent a multigene family located at the right arm of the metacentric autosome 2 (2R) in Drosophila melanogaster. Due to a chromosome fusion the Lcp locus of Drosophila miranda is situated on a pair of secondary sex chromosomes, the X2 and neo-Y chromosome. Comparing the DNA sequences from D. miranda and D. melanogaster organization and the gene arrangement of Lcp1–Lcp4 are similar, although the intergene distances vary considerably. The greatest difference between Lcp1 and Lcp2 is due to the occurrence of a pseudogene in D. melanogaster which is not present in D. miranda. Thus the cluster of the four Lcp genes existed already before the separation of the melanogaster and obscura group. Intraspecific homogenizations of different cluster units must have occurred repeatedly between the Lcp1/Lcp2 and Lcp3/Lcp4 sequence types. The most obvious example is exon 2 of the Lcp3 gene in D. miranda, which has been substituted by the corresponding section of the Lcp4 gene rather recently. The homogenization must have occurred before the translocation which generated the neo-Y chromosome. Lcp3 of D. melanogaster has therefore no orthologous partner in D. miranda. Rearrangements in the promoter regions of the D. miranda Lcp genes have generated new, potentially functional CAAT-box motifs. Since three of the Lcp alleles on the neo-Y are not expressed and Lcp3 is expressed only at a reduced level, it is suggestive to speculate that the rearrangements might be involved as cis-regulatory elements in the up-regulation of the X2-chromosomal Lcp alleles, in Drosophila an essential process for dosage compensation. The Lcp genes on the neo-Y chromosome have accumulated more base substitutions than the corresponding alleles on the X2. Received: 27 December 1995 / Accepted: 30 April 1996     

Population genetics ofEulemur macaco macaco (Primates: Lemuridae) on the islands of Nosy-Be and Nosy-Komba and the Peninsula of Ambato (Madagascar)

Analysis for genetic variation of insular and mainland populations ofEulemur macaco has revealed: (1) a different degree of genetic variation between populations; and (2) the phylogenetic relationships between groups, on the islands of Nosy-Be and Nosy-Komba, and in the Peninsula of Ambato (Madagascar). Eleven systems of blood proteins from 157 animals were used as genetic markers. The genetic variation was lower on the island of Nosy-Komba than in the mainland of Ambato. This is consistent with the expectation that genetic variation is lower on islands than on mainlands. In contrast, the genetic variation on the island of Nosy-Be was the highest of the three populations. This finding can best be explained by assuming that the sample of Nosy-Be consists of individuals of several small isolated groups, where genetic drift computation showed the population of Nosy-Be to be distinct, and the populations of Nosy-Komba and Ambato to be close within the same branch of the dendrogram. These findings give an insight into the population history of the island of Nosy-Komba, which might have been populated by mainland groups from Ambato.     

Localized delivery of epidermal growth factor improves the survival of transplanted hepatocytes

Hepatocyte transplantation may provide a new approach for treating a variety of liver diseases if a sufficient number of the transplanted cells survive over an extended time period. In this report, we describe a technique to deliver growth factors to transplanted hepatocytes to improve their engraftment. Epidermal growth factor (EGF) was incorporated (0.11%) into microspheres (19 +/- 12 mum) fabricated from a copolymer of lactic and glycolic acid using a double emulsion technique. The incorporated EGF was steadily released over 1 month in vitro, and it remained biologically active, as determined by its ability to stimulate DNA synthesis, cell division, and long-term survival of cultured hepatocytes. EGF-containing microspheres were mixed with a suspension of hepatocytes, seeded onto porous sponges, and implanted into the mesentery of two groups of Lewis rats. The first group of animals had their portal vein shunted to the inferior vena cava prior to cell transplantation (portal-caval shunt = PCS), and the second group of animals did not (non-PCS). This surgical procedure improves the survival of transplanted hepatocytes. The engraftment of transplanted hepatocytes in PCS animals was increased two-fold by adding EGF microspheres, as compared to adding control microspheres that contained no growth factors. Devices implanted into non-PCS animals had fewer engrafted hepatocytes than devices implanted into PCS animals, regardless of whether blank or EGF-containing microspheres were added. These results first indicate that it is possible to design systems which can alter the microenvironment of transplanted hepatocytes to improve their engraftment. They also suggest that hepatocyte engraftment is not improved by providing single growth factors unless the correct environment (PCS) is provided for the transplanted cells. (c) 1996 John Wiley & Sons, Inc.     

Entomopathogenic fungi in predatory beetles (Col.: Carabidae andStaphylinidae) from agricultural fields

Prevalence of entomopathogenic fungi was studied in overwintering ground beetles (Col.: Carabidae) and rove beetles (Col.: Staphylinidae) collected from fields of lucerne, white cabbage and white cabbage undersown with white clover. In general infection levels in adult ground beetles and rove beetles were low (Carabidae: max. 7.6%,Staphylinidae: max. 7.0%). In comparison, prevalence of entomopathogenic fungi in carabid larvae was high (19–50%). At one study site an epizootic ofBeauveria bassiana was observed, infecting 67% of the staphylinidAnotylus rugosus and 37% of the staphylinidGyrohypnus angustatus. Beauveria bassiana was the predominant fungus isolated from ground beetles and rove beetles from all studied sites. Other fungal species included the hyphomycetesMetarhizium anisopliae, Paecilomyces farinosus andVerticillium lecanii as well asZoophthora radicans andZoophthora philonthi (Zygomycetes: Entomophthorales). Two individuals ofAnotylus rugosus were found to have a dual infection ofZoophthora philonthi andBeauveria bassiana.

---

Résumé La présence de champignons entomopathogènes a été recherchée chez des carabes (Col.: Carabidae) et des staphylins (Col.: Staphylinidae) récoltés dans des champs de luzerne, de chou et d'une culture mélangée chou-trèfle blanc. Chez les adultes, le taux d'infection dans les deux groupes d'insectes est en général très faible (Carabidae: max. 7,6%, Staphylinidae: max. 7%). Chez les larves de carabes par contre, le taux d'infection par les champignons est élevé (19–50%). Sur l'un des sites de l'étude, une épizootie àBeauveria bassiana a été observée, l'infection portant sur 67% des individus récoltés appartenant à l'espèceAnotylus rugosus et 37% desGyrohypnus angustatus. B. bassiana est le champignon prédominant isolé à partir des coléoptères de tous les sites étudiés. D'autres espèces fongiques ont été relevées: ce sont les hyphomycètesMetarhizium anisopliae, Paecilomyces farinosus etVerticillium lecanii ainsi queZoophthora radicans etZoophthora philonthi. Deux individus d'A. rugosus ont été retrouvés infectés à la fois parErynia etB. bassiana.

     

Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria.     

Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover.

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.     

The unexpected long period of elevated CH4 emissions from an inundated fen meadow ended only with the occurrence of cattail (Typha latifolia)

Drainage and agricultural use transform natural peatlands from a net carbon (C) sink to a net C source. Rewetting of peatlands, despite of high methane (CH₄) emissions, holds the potential to mitigate climate change by greatly reducing CO₂ emissions. However, the time span for this transition is unknown because most studies are limited to a few years. Especially, nonpermanent open water areas often created after rewetting, are highly productive. Here, we present 14 consecutive years of CH₄ flux measurements following rewetting of a formerly long-term drained peatland in the Peene valley. Measurements were made at two rewetted sites (non-inundated vs. inundated) using manual chambers. During the study period, significant differences in measured CH₄ emissions occurred. In general, these differences overlapped with stages of ecosystem transition from a cultivated grassland to a polytrophic lake dominated by emergent helophytes, but could also be additionally explained by other variables. This transition started with a rapid vegetation shift from dying cultivated grasses to open water floating and submerged hydrophytes and significantly increased CH₄ emissions. Since 2008, helophytes have gradually spread from the shoreline into the open water area, especially in drier years. This process was periodically delayed by exceptional inundation and eventually resulted in the inundated site being covered by emergent helophytes. While the period between 2009 and 2015 showed exceptionally high CH₄ emissions, these decreased significantly after cattail and other emergent helophytes became dominant at the inundated site. Therefore, CH₄ emissions declined only after 10 years of transition following rewetting, potentially reaching a new steady state. Overall, this study highlights the importance of an integrative approach to understand the shallow lakes CH₄ biogeochemistry, encompassing the entire area with its mosaic of different vegetation forms. This should be ideally done through a study design including proper measurement site allocation as well as long-term measurements.     

Individual day-to-day variations in plasma amino acid levels in healthy persons

Summary Fasting plasma levels of the large neutral amino acids (LNAA;l-forms) andl-tryptophan (TRP) ratios were determined in thirteen healthy volunteers (7 males, 6 females) on five consecutive mornings, and the same procedure was repeated for each individual three months later. We found characteristic overall ranges for the parameters studied, and, in addition to certain differences between the sexes, considerable inter- and intraindividual daily variations. Although the individuals showed statistically identifiable mean levels that, in the majority of subjects, were maintained over a period of three months, it is concluded that the degree of intraindividual variability does not allow us to regard a single value as characteristic for a given individual. This should be borne in mind in particular in follow-up studies of plasma LNAA in patients with, for example, depressive disorders.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.