THE INTERACTION PHENOTYPE IN THE DROSOPHILA WILLISTONI–SPIROPLASMA SYMBIOSIS

Both the population and coevolutionary dynamics of hereditary male-lethal endosymbionts, found in a wide range of insect species, depend on host fitness and endosymbiont transmission rates. This paper reports on fitness effects and transmission rates in three lines of Drosophila willistoni infected with either male-lethal spiroplasmas or a spontaneous nonmale-lethal mutant. Overall fitness measures were reduced or unaffected by the infection; however, some infected females produced more offspring in early broods. Maternal transmission rates were high, but imperfect, and varied with a female's age, host line, and spiroplasma type. No evidence for paternal or horizontal transmission was found. If an altered temporal pattern of reproduction is not a factor in countering the loss of spiroplasma hosts through imperfect maternal transmission, persistence of this endoparasitism remains unexplained. Tolerance of the infection and ability to transmit bacteria varied with both host and spiroplasma line. Analysis of the interaction between the spontaneous nonmale-lethal mutant and its host suggests this symbiosis has undergone coevolution under laboratory culture.     

Gamma interferon is critical for resistance to Theiler's virus-induced demyelination.

Administration of neutralizing monoclonal antibody to gamma interferon increased Theiler's virus-induced demyelination and virus antigen persistence in the spinal cord in susceptible SJL/J mice and completely abrogated resistance such that all C57BL/10SNJ mice developed demyelination. These experiments support the hypothesis that gamma interferon is critically important for resistance to Theiler's virus-induced disease but is not required for myelin destruction.     

Effects of Auxin and Phenobarbital on Morphogenesis and Production of Digitoxin in Digitalis Callus

Pieces of callus obtained from seedlings of Digitalis purpureawere grown on solid Murashige-Skoog's medium supplemented with1 mg liter^–1 BA and 0.1 mg liter^–1 IAA or NAA, withor without phenobarbital (40 mg liter^–1). The replacementof the natural auxin IAA by the synthetic auxin NAA increasedcallus growth and inhibited organogenesis, whereas the additionof phenobarbital had the opposite effect. Morphometric measurementsrevealed a high ratio of vacuole to cytoplasm (v/v) in calluscells. This ratio was affected by the different treatments inthe same way as the fresh weight. The activity of mitochondrialcytochrome P450scc (the enzyme that provides the precursor,pregnenolone, for the biosynthesis of cardenolide in foxgloveplants) was detected in the relevant fraction of callus grownunder all experimental conditions, and its activity was increasedby the addition of phenobarbital. The different treatments testedincreased the cardenolide content and quantifiable amounts ofdigitoxin were detected in all callus tissues. It is of specialinterest that phenobarbital added to the culture medium increasedthe accumulation of digitoxin. The mechanism affecting the developmentand production of cardenolide in callus tissues of D. purpureaby phenobarbital and the replacement of IAA by NAA is discussed. (Received July 18, 1994; Accepted December 14, 1994)     

Mevalonate dependency of the early cell cycle mitogenic response to epidermal growth factor and prostaglandin F2α in Swiss mouse 3T3 cells

Lovastatin (LOV), a hydroxy-methylglutaryl-coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)— or prostaglandin F_2α (PGF_2α)—induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2–80 μM concentration range, with both mitogens attaining 50% inhibition at 7.5 μM. LOV exerted its effect within 0–8 h following mitogenic induction. Mevanolactone (10–80 μM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV-induced blockage of PGF_2α response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF- or PGF_2α-stimulated cells, LOV did not inhibit [³H]leucine or [³H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N′ glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N′ glycosylation process. These findings strongly suggest that either EGF or PGF_2α stimulations generate early cell cycle signals which induce mevalonate formation, N′ glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed. © 1995 Wiley-Liss, Inc.     

Age structure,growth and reproduction ofRutilus lemmingii in an intermittent stream of the Guadalquivir river basin,southern Spain

The age, growth and reproduction ofRutilus lemmingii (Steindachner, 1866), an endemic cyprinid from the Iberian Peninsula, was studied for over a period of two years in a small seasonal tributary of the Guadalquivir river basin. Approximately 65 % of the total growth in length occured in the first year of life. Males reached a maximum age of 3+ yr (Fork Length, F.L. = 114 mm) and females 4+ yr (F.L. = 144 mm). Both sexes matured during their second year of life (1 +). The overall sex ratio (334 males to 389 females) differed significantly from unity. Somatic condition decreased markedly during the reproductive period of March to May.R. lemmingii is a multiple spawner and releases two batches of eggs per female each year. Mean egg diameter of the first batch was larger than the second one. The regression between fecundity and Fork Length (mm) was: Fec = 0.014 F.L.Z.^2.858 Compared with available information, thisR. lemmingii population, located at a lower latitude, is characterized by fast growth, early maturity, high level of reproductive effort, and a short life-span. These life-history characteristices are typical of species in unstable environments, where adult mortality is high, variable or unpredictable.     

Toxicology of vanadium compounds in diabetic rats: The action of chelating agents on vanadium accumulation

The possible use of vanadium compounds in the treatment of diabetic patients is now being evaluated. However, previously to establish the optimal maximum dose for diabetes therapy, it should be taken into account that vanadium is a highly toxic element to man and animals. The toxic effects of vanadium are here reviewed. The tissue vanadium accumulation, which would mean an additional risk of toxicity following prolonged vanadium administration is also discussed. Recently, it has been shown that coadministration of vanadate and TIRON, an effective chelator in the treatment of vanadium intoxication, reduced the tissue accumulation of this element, decreasing the possibility of toxic side effects derived from chronic vanadium administration without diminishing the hypoglycemic effect of vanadium. However, previously to assess the effectiveness of this treatment in diabetic patients, a critical reevaluation of the antidiabetic action of vanadium and its potential toxicity is clearly needed.     

Inequalities in income and long-term disability in Spain: analysis of recent hypotheses using cross sectional study based on individual data.

OBJECTIVE: To compare the relation between inequalities in long-term disability and income in the 17 regions of Spain. DESIGN: Data were taken from the survey on impairments, disabilities, and handicaps that was carried out in Spain in 1986. For each region the inequality in long-term disability associated with income was calculated as the odds ratio associated with reducing monthly household income by 10,000 pesetas (about Ponds 50) (estimate of effect of inequality of income) and the odds ratio for the inequality in long-term disability between those at the bottom and those at the top of the income hierarchy (relative index of inequality). MAIN OUTCOME MEASURE: Prevalence of long-term disability. RESULTS: Five of the eight regions where lowering income had a greater effect on long-term disability were among those with the lowest income per head, while six of the remaining nine regions where the effect was smaller were among those with the highest income per head. Three regions with the highest estimate of relative index of inequality had the highest estimate of effect, and another three regions with the lowest estimate of relative index of inequality had the lowest estimate of effect. In contrast, the relative position of the remaining 11 regions varied from one measure to another. CONCLUSIONS: These results support the theory that additional increments in material wellbeing have a negligible effect on health in countries with high socioeconomic development. However, inequality in income distribution did not determine inequality in health between those at the bottom and those at the top of the income hierarchy in many Spanish regions.     

Dismantling Cell–Cell Contacts during Apoptosis Is Coupled to a Caspase-dependent Proteolytic Cleavage of β-Catenin

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis.

In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.

     

The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na⁺– K⁺ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.Cell polarity (asymmetry) is a broadly distributed and highly conserved feature of many different cell types, from prokaryotes to higher eukaryotes (Nelson, 1992). In multicellular organisms it is more conspicuous in, but not restricted to, neurons and epithelial cells. In the latter, the plasma membrane is organized in two different domains, apical and basolateral. This characteristic enables epithelia to accomplish their most specialized roles including absorption and secretion and, in general, to perform the functions of organs with an epithelial parenchyma such as the kidney, liver, intestine, stomach, exocrine glands, etc. (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson, 1989).The acquisition and maintenance of epithelial polarity is based on multiple interrelated mechanisms that may work in parallel. Although the origin of polarization depends on the sorting of apical and basolateral membrane proteins at the trans-Golgi network (Simons and Wandinger-Ness, 1990), the mechanisms involved in the transport of apical or basolateral carrier vesicles, the specific fusion of such vesicles to the appropriate domain, and the retention of membrane proteins in their correct positions are also important (Wollner and Nelson, 1992). Various components of the cytoskeleton seem to be especially involved in these mechanisms (Mays et al., 1994). Among them, the microtubules, characteristically oriented in the apical–basal axis with their minus ends facing toward the apical domain, appear in a strategic position to transport carrier vesicles (Bacallao et al., 1989). This orientation is largely expected because of the apical distribution of centrioles and microtubule organizing centers in epithelial cells (Buendia et al., 1990). The molecular interactions responsible for that localization, however, are unknown.Actin is a widespread component of the membrane skeleton found under apical, lateral, and basal membranes in a nonpolarized fashion (Drenckhahn and Dermietzel, 1988; Vega-Salas et al., 1988). Actin bundling into microvillus cores in the presence of villin/fimbrin, on the other hand, is highly polarized to the apical domain (Ezzell et al., 1989; Louvard et al., 1992). In fact, different isoforms of plastins determine microvillus shape in a tissue-specific manner (Arpin et al., 1994b ). Why this arrangement is not found in other actin-rich regions of the cell is unclear (Louvard et al., 1992; Fath and Burgess, 1995).Fodrin, the nonerythroid form of spectrin, underlies the basolateral domain (Nelson and Veshnock, 1987a ,b) and is known to participate in the anchoring/retention of basolateral proteins (Drenckhahn et al., 1985; Nelson and Hammerton, 1989). Although different groups have found specific cytoskeletal anchoring of apical membrane proteins at the “correct” domain (Ojakian and Schwimmer, 1988; Salas et al., 1988; Parry et al., 1990), no specific apical counterpart of the basolateral fodrin cytoskeleton is known. This is especially puzzling since we showed that MDCK cells can maintain apical polarity in the absence of tight junctions, an indication that intradomain retention mechanisms are operational for apical membrane proteins (Vega-Salas et al., 1987a ).It is known that a network of intermediate filament (IF)¹, the major component of the terminal web, bridges the desmosomes under the apical membrane in brush border cells (Franke et al., 1979; Hull and Staehelin, 1979; Mooseker, 1985), although no specific protein has been identified with this structure. The observation of a remarkable resistance to extractions of apical proteins anchored to cytoskeletal preparations (Salas et al., 1988) comparable to that of intermediate filaments, led us to the study of cytokeratins in polarized cells. We developed an antibody against a 53-kD intermediate filament protein in MDCK cells. This protein was found to be distributed exclusively to the apical domain and to form large (2,900 S) multi-protein complexes with apical plasma membrane proteins. Internal microsequencing of the 53-kD protein showed very high (95– 100%) homology with two polypeptides in the rod domain of cytokeratin 19 (CK19; Moll et al., 1982) a highly conserved and peculiar intermediate filament protein (Bader et al., 1986). A complete identification however, could not be achieved (Rodriguez et al., 1994). The present study was undertaken to establish that identity and to determine the possible functions of this apical membrane skeleton. Because cytokeratins have been poorly characterized in canine cells, and no cytokeratin sequences are available in this species, we decided to switch from MDCK cells to two human epithelial cell lines, CACO-2, an extensively studied model of epithelial polarization that differentiates in culture to form brush border containing cells (Pinto et al., 1983), and MCF-10A (Tait et al., 1990), a nontumorigenic cell line derived from normal mammary epithelia, as a model of nonbrush border cells.To assess possible functions of cytokeratin 19, we chose to selectively reduce its synthesis using anti-sense phosphorothioate oligodeoxy nucleotides, an extensively used approach in recent years (e.g., Ferreira et al., 1992 ; Hubber et al., 1993; Takeuchi et al., 1994). Although we could not achieve a complete knock out, the steady-state levels of cytokeratin 19 were decreased to an extent that enabled us to detect significant changes in the phenotype of CACO-2 and MCF-10A cells.