Membrane microheterogeneity: Förster resonance energy transfer characterization of lateral membrane domains

Lateral membrane heterogeneity, in the form of lipid rafts and microdomains, is currently implicated in cell processes including signal transduction, endocytosis, and cholesterol trafficking. Various biophysical techniques have been used to detect and characterize lateral membrane domains. Among these, Förster resonance energy transfer (FRET) has the crucial advantage of being sensitive to domain sizes smaller than 50-100 nm, below the resolution of optical microscopy but, apparently, similar to those of rafts in cell membranes. In the last decade, several formalisms for the analysis of FRET in heterogeneous membrane systems have been derived and applied to the study of microdomains. They are critically described and illustrated here.     

Phylogeography of the cactophilic species Drosophila gouveai: demographic events and divergence timing in dry vegetation enclaves in eastern Brazil

Aim  The aim of this study was to assess the causal mechanisms underlying populational subdivision in Drosophila gouveai , a cactophilic species associated with xeric vegetation enclaves in eastern Brazil. A secondary aim was to investigate the genetic effects of Pleistocene climatic fluctuations on these environments.
Location  Dry vegetation enclaves within the limits of the Cerrado domain in eastern Brazil.
Methods  We determined the mitochondrial DNA haplotypes of 55 individuals (representing 12 populations) based on sequence data of a 483-bp fragment from the cytochrome c oxidase subunit II (COII) gene. Phylogenetic and coalescent analyses were used to test for the occurrence of demographic events and to infer the time of divergence amongst genetically independent groups.
Results  Our analyses revealed the existence of two divergent subclades (G1 and G2) plus an introgressed clade restricted to the southernmost range of D. gouveai . Subclades G1 and G2 displayed genetic footprints of range expansion and segregated geographical distributions in south-eastern and some central highland regions, east and west of the Paraná River valley. Molecular dating indicated that the main demographic and diversification events occurred in the late to middle Pleistocene.
Main conclusions  The phylogeographical and genetic patterns observed for D. gouveai in this study are consistent with changes in the distribution of dry vegetation in eastern Brazil. All of the estimates obtained by molecular dating indicate that range expansion and isolation pre-dated the Last Glacial Maximum, occurring during the late to middle Pleistocene, and were probably triggered by climatic changes during the Pleistocene. The current patchy geographical distribution and population subdivision in D. gouveai is apparently closely linked to these past events.     

Untersuchungen über den Gehörsinn bei Characiniden und Gymnotiden und den Bau Ihres Labyrinthes

Ohne Zusammenfassung     

LIFE HISTORY AND GROWTH OF LAURENCIA BRACHYCLADOS (RHODOPHYTA,CERAMIALES)1

Laurencia brachyclados Pilger from Hawai'i completed a “Polysiphonia-type” tri-phasic life history in 21 weeks in laboratory culture. Tetraspores developed into gametophytes in a nearly 1:1:1 ratio of females: males: non-reproductive. Carpospores were released as early as 21 days after mixing virgin female and male gametophytes. Cultured thalli showed a “guerilla type” growth form. Other Hawaiian Laurencia species in culture had longer maturation times or remained non-reproductive. Variation in life history schedules may influence Laurencia species coexistence and algal community structure.     

Effect of transforming growth factor beta (TGF-β) on ATP citrate lyase in isolated hepatocytes

Summary Transforming growth factor beta (TGF-) activates ATP citrate lyase in freshly isolated rat liver hepatocytes in a time dependent manner. Maximal stimulation of the enzyme occurred with less than thirty minutes of incubation of the cells with TGF-. The half maximal effect on the enzyme determined in hepatocytes incubated with TGF- for 10 min at 37°C was elicited by TGF- concentrations in the 10^–11 – 10^–12 M range. The potential role of TGF- stimulation of ATP citrate lyase activity in new membrane synthesis is discussed.     

Sensory organs of the thoracic legs of the moth Manduca sexta

Summary The thoracic legs of the moth Manduca sexta acquire a new form and develop a new complement of sensory organs and muscles during metamorphosis from larva to adult. Because of our interest in the reorganization of neural circuitry and the acquisition of new behaviors during metamorphosis, we are characterizing sensory elements of larval and adult legs so that we may determine the contribution of new sensory inputs to the changes in behaviors. Here we describe the sensory structures of adult legs using scanning electron microscopy to view the external sensilla and cobalt staining to examine innervation by underlying sensory neurons. We find that, in contrast to larval legs, the adult legs are covered with a diverse array of sensilla. All three pairs of thoracic legs contain scattered, singly innervated scalelike sensilla. Campaniform sensilla occur singly or in clusters near joints. Hair plates, consisting of numerous singly innervated hairs, are also present near joints. Other more specialized sensilla occur on distal leg segments. These include singly innervated spines, two additional classes of singly innervated hairs, and three classes of multiply innervated sensilla. Internal sensory organs include chordotonal organs, subgenual organs, and multipolar joint receptors.     

Adhesion-mediating molecules of human monocytes

Adhesion of monocytes to each other and to T cells and substrates is increased by phorbol esters. In the presence of these compounds monocyte aggregation was almost completely inhibited (greater than 90%) by monoclonal antibody 60.3. This antibody recognizes GP90 (CD18), a leukocyte surface glycoprotein which is separately and noncovalently associated to either GP160 (CD11a), GP155 (CD11b), or GP130 (CD11c). Anti-LFA-1 antibody (CD11a) was only partially inhibitory (35%) while antibodies 60.1 (CD11b) and anti-Leu-M5 (CD11c) had a minimal inhibitory effect (10%). Antibody LB-2 recognizing a single glycoprotein distinct from the GP90-GP160 complex and expressed on activated B and T cells, monocytes, and vascular endothelial cells was partially inhibitory (22%). Monoclonal antibodies anti-C3bR (CD35), T29/33 (CD45, leukocyte common antigen 200). TA-1 (CD11a), OKM1 (CD11b), F10-44-2 (brain-leukocyte antigen), OKM5 (monocyte-endothelial cell antigen) and to class I or class II molecules exerted no inhibition on the monocyte aggregation. Fab fragments of antibody 60.3 efficiently inhibited not only monocyte aggregation in the absence or presence of phorbol esters but also adhesion of these cells to autologous or allogeneic T lymphocytes and, to a lesser extent, to plastic surfaces. It is thus concluded that GP90, either alone or associated to the larger glycoproteins, and LB-2 antigen mediate monocyte adhesion.     

Molecular cloning of DNA complementary to bovine adrenal P450scc mRNA

P450_scc is the rate-limiting hormonally regulated enzyme that cleaves the cholesterol side chain. Translation of bovine adrenocortical mRNA and immunoprecipitation with rabbit anti-bovine P450_scc indicates P450_scc mRNA represents 1% of the total. DNA complementary to bovine adrenocortical mRNA was cloned in the $\text{PstI}$ site of pBR322 by dC·dG tailing and high-efficiency transformation. A clone containing sequences complementary to P450_scc mRNA was identified by hybrid-selected translation only when plasmid DNA was first purified by CsCl gradient centrifugation. As is often the case with hybrid-selected translation, the clone identified contains a small insert.     

Localization during development of alternatively spliced forms of cytotactin mRNA by in situ hybridization

Cytotactin, an extracellular glycoprotein found in neural and nonneural tissues, influences a variety of cellular phenomena, particularly cell adhesion and cell migration. Northern and Western blot analysis and in situ hybridization were used to determine localization of alternatively spliced forms of cytotactin in neural and nonneural tissues using a probe (CT) that detected all forms of cytotactin mRNA, and one (VbVc) that detected two of the differentially spliced repeats homologous to the type III repeats of fibronectin. In the brain, the levels of mRNA and protein increased from E8 through E15 and then gradually decreased until they were barely detectable by P3. Among the three cytotactin mRNAs (7.2, 6.6, and 6.4 kb) detected in the brain, the VbVc probe hybridized only to the 7.2-kb message. In isolated cerebella, the 220-kD polypeptide and 7.2-kb mRNA were the only cytotactin species present at hatching, indicating that the 220-kD polypeptide is encoded by the 7.2-kb message that contains the VbVc alternatively spliced insert. In situ hybridization showed cytotactin mRNA in glia and glial precursors in the ventricular zone throughout the central nervous system. In all regions of the nervous system, cytotactin mRNAs were more transient and more localized than the polypeptides. For example, in the radial glia, cytotactin mRNA was observed in the soma whereas the protein was present externally along the glial fibers. In the telencephalon, cytotactin mRNAs were found in a narrow band at the edge of a larger region in which the protein was wide-spread. Hybridization with the VbVc probe generally overlapped that of the CT probe in the spinal cord and cerebellum, consistent with the results of Northern blot analysis. In contrast, in the outermost tectal layers, differential hybridization was observed with the two probes. In nonneural tissues, hybridization with the CT probe, but not the VbVc probe, was detected in chondroblasts, tendinous tissues, and certain mesenchymal cells in the lung. In contrast, hybridization with both probes was observed in smooth muscle and lung epithelium. Both epithelium and mesenchyme expressed cytotactin mRNA in varying combinations: in the choroid plexus, only epithelial cells expressed cytotactin mRNA; in kidney, only mesenchymal cells; and in the lung, both of these cell types contained cytotactin mRNA. These spatiotemporal changes during development suggest that the synthesis of the various alternatively spliced cytotactin mRNAs is responsive to tissue-specific local signals and prompt a search for functional differences in the various molecular forms of the protein.