Nest building by lowland gorillas in the Lopé Reserve,Gabon: Environmental influences and implications for censusing

We analyzed data from 373 fresh nest-sites (containing 2435 nests) of lowland gorillas (Gorilla g. gorilla)during a 4-year period in the Lopé Reserve, Gabon, to determine whether the observed variability in nest building was due to environmental influences. We recognized and defined seven types of nest in terms of the degree of construction and the raw materials used. Overall, nests built on the ground from herbaceous plants are the most common type (40%), followed by tree nests (35%). Frequencies of the different nest-types vary significantly between eight habitat-types. In habitat-types with high densities of understory herbs, ground nests predominated, but when herbs were rare, the majority of nests were in trees. A general preference for sleeping in herbaceous ground nests is indicated since trees are abundant in all habitat-types, except savanna. The frequency of nesting in trees shows a significant positive correlation with rainfall, but effects of climate are confounded by seasonal variation in use of different habitat-types. When elephants were attracted to the same localized food sources as gorillas, many tree nests were built even when herbs were available. We conclude that different nest-types reflect a variety of solutions to maximize comfort, depending on available raw materials and the probability of rainfall or disturbance by elephants or both factors. Nests are a powerful tool for population censuses and demographic studies of great apes, but problems exist in interpreting data on lowland gorilla nests. Results from this analysis show that only a third of nest-sites accurately reflects group size (of weaned individuals) and that 26% of all gorilla nest-sites could be mistaken for those of chimpanzees, as all nests, or all those visible from a transect, were in trees. Gorilla nests at Lopé were nonrandomly distributed with respect to habitat-types, and nest construction varied seasonally, thereby introducing sources of bias to transect nest counts. We discuss these problems and ones related to assessing the decay rate of nest-sites and make recommendations relevant to census work.     

The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signaling potential.

We report the cloning and primary structure of the Drosophila insulin receptor gene (inr), functional expression of the predicted polypeptide, and the isolation of mutations in the inr locus. Our data indicate that the structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The INR proreceptor (M(r) 280 kDa) is processed proteolytically to generate an insulin-binding alpha subunit (M(r) 120 kDa) and a beta subunit (M(r) 170 kDa) with protein tyrosine kinase domain. The INR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension which contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage which leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90 and a free 60 kDa C-terminus) from the inr gene. These subunits assemble into mature INR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation of both types of beta subunits, which in turn allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of INR have different signaling potentials. Finally, we show that loss of function mutations in the inr gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, render pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of inr appears to be required in the embryonic epidermis and nervous system among others, since development of the cuticle, as well as the peripheral and central nervous systems are affected by inr mutations.     

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.     

Incidence of heavy metals in the mangrove flora and sediments in Kerala, India

The mangroves of Kerala on the south-west coast of India are fast disappearing due to land reclamation and other anthropogenic disturbances. There are very few ecosystem level studies made in these much threatened biotopes in Kerala. The present study involves the measurement of heavy metals in the mangrove flora and sediments of three mangrove habitats along the Kerala coast. Sampling was carried out for a period of one year at bi-monthly intervals, with concentrations of Fe, Mn, Cu,Zn,Pb and Co analysed using atomic absorption spectrophotometry. An appreciable variation was observed in metal concentrations indifferent mangrove species. Cu, Zn and Pb were found to be in higher concentrations in Avicennia officinalis whereas higher levels of Fe, Mn and Co were observed in the species Barringtonia racemosa. The analysis of heavy metals indicated a high level of metal pollutants such as Fe, Cu, Zn and Pb in the mangrove habitats of Quilon and Veli compared to the relatively uncontaminated areas of Kumarakom. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Isolation of cDNA and genomic DNA clones encoding type II collagen.

A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs.     

Assembly of Bacillus subtilis phage phi29. 1. Mutants in the cistrons coding for the structural proteins.

The effect of mutations in the cistrons coding for the phage structural proteins has been studied by analyzing the phage-related structures accumulated after restrictive infection. Infection with susmutants in cistron 8, lacking both the major head and the fiber protein, does not produce any phage-related structure, suggesting a single route for the assembly of phage phi29; infection with ts mutants in this cistron produces isometric particles. Mutants is cistron 9, coding for the tail protein, TP1, produce DNA-free prolate heads with an internal core; these particles are abortive and contain the head proteins HPO, HP1 and HP3, the upper collar protein NP2 and the nonstructural proteins p7, p15 and p16. Mutants in cistron 10, coding for the upper collar protein, NP2, produce DNA-free isometric heads also with an internal core; they contain the head proteins and the nonstructural protein p7, suggesting that this protein forms the internal core. Mutants in cistrons 11 and 12, coding for the lower collar protein, NP3, and the neck appendages, NP1, respectively, give rise to the formation of DNA-containing normal capsids and DNA-free prolate particles, more rounded at the corners than the normal capsids and with an internal core; the DNA-containing 11-particles are formed by the head proteins and the upper collar protein; the DNA-free 11-particles contain, besides these proteins, the nonstructural protein p7 and a small amount of proteins p15 and 16. The DNA-containing 12-particles have all the normal phage structural proteins except the neck appendages, formed by protein NP1; the DNA-free particles are similar to the DNA-free 11-particles. After restricitive infection mutant sus14(1241) has a delayed lysis phenotype and produces a phage burst higher than normal, after artificial lysis. It produces DNA-containing particles, identical to wild-type phage, which have all the normal phage structural proteins, and DNA-free prolate particles, more rounded at the corners than the final phage particles and with an internal core; the last particles contain the same proteins as the DNA-free 11 or 12-particles. These particles could represent a prohead state, ready for DNA encapsulation. None of the DNA-containing particles have the nonstructural proteins p7, p15 or p16, suggesting that these proteins are released from the proheads upon DNA encapsulation.