Diel emigration and colonization responses of blackfly larvae (Diptera: Simuliidae) to ultraviolet radiation

1. Total counts of blackfly larvae densities over 30- and 57-h periods in experimental channels during May of 1996 & 1997 indicate that ultraviolet radiation (UV; 290–400 nm) may be important in stimulating emigration.
2. Under experimentally controlled solar UV exposure, larval densities at dawn in UV-shielded channels were 161% and 168% higher than in the UV-exposed channels. Larval densities in UV-exposed channels then decreased by 68.2% and 81.1% between dawn and early afternoon of the two days; density decreases in UV-shielded channels were slight, and not statistically significant, during the same periods.
3. Larvae within UV-exposed channels occupied shaded microhabitats during hours of intense solar radiation, suggesting that simuliid larvae can detect and respond to UV radiation over very short periods of time.
4. A cyclical pattern of UV-induced emigration during hours of increasing solar flux (06.30–13.30) and net immigration in the hours of decreasing solar flux and at night emerged. Thus stream invertebrates may be very sensitive to environmental changes, resulting in either increased UV flux or decreased shading of streams. Diel cycles in invertebrate densities should be taken into account in research designs and sampling protocols in order to identify and interpret correctly results of both periodic surveys and experiments.     

Alterations in nuclear anatomy by chemical modification of proteins in isolated rat liver nuclei

Whole rat liver nuclei were treated with citraconic anhydride, a reagent specific for primary amines. Dramatic changes were observed in nuclear morphology and light scattering properties. An analysis for DNA and RNA content suggested that DNA was released from the nuclei with a short half-time, approximately 2-4s demonstrating a biphasic release profile. RNA was similarly released but with a monophasic profile. Analysis of SDS-PAGE gels of modified nuclei demonstrated a progressive enrichment of nuclear matrix (lamins) polypeptides with extent of modification. H1 histone was quantitatively lost as a function of modification reagent concentration, while approx. 50% of the nucleosomal histones cosedimented with DNA- and RNA-free nuclei. Modification in the presence of 2 mM EGTA released all the DNA and RNA [less than or equal to 1% remaining) while retaining structures characteristic of nuclear matrix, nucleoli, and ribonucleoprotein (predominantly hnRNA group A and B). These nucleic acid-deficient structures have been termed nuclear fossils to differentiate them from high salt detergent-prepared empty nuclear sacks, nuclear remnants, or nuclear scaffolds. Modification in the presence of 2% Triton X-100 results in structures similar to the nuclear fossils (EGTA treatment), but missing the double bilayer and a 51K polypeptide that is a major component of the other structures. The use of chemical modification on the nucleus provides an experimental approach for examining the role of ionic interactions in controlling nuclear structure. Citraconylation may thus serve two functions: (a) as a protein-specific perturbant of nuclei capable of simply and rapidly preparing a range of structural variants for the analysis of nuclear interactions; (b) offer a paradigm for control of nucleic acid-polypeptide interactions based on post-translational alterations in protein charge.     

Protein transport in intact, purified pea etioplasts

We have developed a method to isolate intact, purified pea etioplasts. These etioplasts were capable of recognizing, transporting, and processing the precursor form of the small subunit of the ribulose-1,5-bisphosphate carboxylase, a protein which is not detectable at this developmental stage. Transport of proteins was completely dependent on ATP and could not be substituted for or stimulated by light. The transported precursor protein was processed to its proper molecular weight. The mature form of the small subunit was assembled with the large subunit of the ribulose-1,5-bisphosphate carboxylase already present at this stage to form an oligomer. Protein transport was completely abolished using the phosphatase inhibitor sodium fluoride. This is the first time protein transport has been demonstrated in isolated, purified etioplasts.     

Enhanced sensitivity to naltrexone is associated with an up-regulation in GABA receptor function.

Rats were made sensitive to the effects of the opioid antagonist naltrexone by treating them once weekly with cumulative doses of the drug (1, 3, 10, 30 and 100 mg/kg). Sensitization was monitored by measuring salivation following naltrexone administration. During the first week of treatment, no salivation was noted following any dose of naltrexone. Over a period of 8 weeks, however, increasing amounts of salivation were noted, with the most salivation occurring at the higher doses. Animals treated for 8 weeks with saline never salivated following injections. Following the development of sensitivity to naltrexone, the rats were sacrificed and their brains were assayed for GABA receptor function. GABA-stimulated chloride uptake, a measure of GABA receptor function, was unchanged in the cortex, but was increased in the cerebellum. These results suggest that the effects of naltrexone on cerebellar GABA receptors may be involved in the development of enhanced sensitivity to opioid antagonists.     

A nuclear specific glycoprotein representative of a unique pattern of glycosylation

Whole rat liver nuclei were reacted with UDP-[14C]galactose in the presence of bovine beta(1----4) galactosyltransferase. The reaction mixture was electrophoresed on a reducing sodium dodecyl sulfate-polyacrylamide gel. Autoradiograms of the gel demonstrated a major labeled broad band migrating with an apparent molecular weight of 65,000-66,000. A number of other less prominently labeled bands were also present. The labeled 65,000-66,000 band when cut from the gel and subjected to alkaline reduction while in the gel matrix exclusively yielded a 14C-labeled disaccharide that co-migrated with a [14C]Gal-GlcNAcol standard in descending paper chromatography. Treatment of this disaccharide with beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) from Aspergillus niger removed all the [14C]galactose label. Treatment of the labeled 65,000-66,000 polypeptide with Endoglycosidase F, however, did not remove the [14C]galactose label. Western transfer blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels performed with horseradish peroxidase-labeled succinyl wheat germ agglutinin, a lectin specific for GlcNAc, on unlabeled nuclei revealed a dominant band at 63,000-64,000. Subjecting 14C-labeled nuclei to this procedure resulted in a shift of the major horseradish peroxidase-labeled succinyl wheat germ agglutinin band to 65,000-66,000. The shifted band was coincident with the [14C]galactose band as visualized on an autoradiogram. A survey of other rat tissue nuclei revealed the same spectrum of [14C]galactose acceptor proteins with a dominant 65,000-66,000 galactose-labeled band.     

Specific silver staining of experimentally undercondensed chromosome regions

Treatment of human and mouse cell cultures with DNA binding AT-specific compounds and with some base analogues induced distinct undercondensations in several heterochromatic chromosome regions. All those heterochromatic regions undercondensed by AT-specific DNA ligands (distamycin A, DAPI, Hoechst 33258) could be heavily labeled with the silver(Ag)-staining technique; but the heterochromatic regions undercondensed with the cytidine analogue 5-azacytidine were Ag-negative. In metaphase chromosomes from BrdU-treated human cell cultures, the bifilarly substituted chromatids, which show a slight undercondensation, were also Ag-negative. Cytochemical analyses of the Ag-stained undercondensed heterochromatic regions showed that the Ag-stainable material consisted of nonhistone proteins. The mechanism of Ag staining in the undercondensed heterochromatic regions was compared with Ag staining of the nucleolus organizer regions.     

Microspectrophotometric and scanning microphotometric studies of carp (Cyprinus carpio L.) erythrocytes

Carp (Cyprinus carpio) hemoglobin readily autoxidizes in blood smears. Quantification of Soret-band absorbance in individual erythrocytes by means of scanning cytophotometry therefore requires more elaborate methods of preparation of blood samples. Of the fixatives that have been tested, suspension of whole blood in isotonic salt solutions containing glutaraldehyde was most suitable. Glutaraldehyde-fixed red blood cells are totally resistant to hemolysis. In the course of fixation, hemoglobin is transformed to methemoglobin. Spectrophotometry indicated extensive similarities between glutaraldehyde-fixed carp methemoglobin and human methemoglobin. In aqueous solutions, the intensity of the Soret-peak was pH-dependent. The allosteric modifier organic polyphosphate caused an R----T transition, resulting in increased molar extinctions. Dried preparations showed Soret-spectra that were not influenced from either pH or organic polyphosphate concentration of the aqueous suspensions in which the erythrocytes had been stored. The same was true for slide preparations of cyanomethemoglobin, easily derived from methemoglobin on addition of potassium cyanide. In the absence of oxygen fresh blood cells from carp slowly transform their hemoglobin into deoxyhemoglobin. Spectra of the intermediate stages of deoxygenation, Hb4(O2)3, Hb4(O2)2 and Hb4(O2), as well as mixtures of these intermediates, could be monitored.     

In vitro and in vivo activation of human mononuclear phagocytes by interferon-gamma. Studies with normal and AIDS monocytes

To determine the potential immunotherapeutic role of interferon-gamma (IFN-gamma) as a mononuclear phagocyte-activating agent, we examined the effector cell function of peripheral blood monocytes from healthy donors and acquired immunodeficiency syndrome (AIDS) patients after either in vitro and/or in vivo treatment with recombinant (r) IFN-gamma. When assayed immediately after a 24-hr in vitro pulse with 300 U/ml, normal and AIDS monocytes behaved similarly with little augmentation of their intrinsically high levels of H2O2 release and activity against Toxoplasma gondii; in contrast, activity toward the more resistant intracellular pathogen, Leishmania donovani, was appreciably enhanced by rIFN-gamma. In addition, upon testing 4 to 6 days after in vitro pulsing, both normal and AIDS monocytes showed clear evidence of persistent activation in all three assays. The capacity of IFN-gamma to similarly activate monocytes in vivo was confirmed in all ten treated AIDS patients by examining cells before and after 24-hr infusions of 0.03 and 0.5 mg of rIFN-gamma/square meter (M2) of body surface area. For postinfusion monocytes tested after 1 day in culture, H2O2 release and antitoxoplasma activity were essentially unchanged, but antileishmanial effects were augmented. After 5 to 7 days in culture, monocytes from treated patients showed 3.2- to 5.9-fold increases in H2O2-releasing capacity and increases of 49 to 68% and 35 to 61% in intracellular activity against T. gondii and L. donovani, respectively. These results indicate that the human monocyte can be induced by rIFN-gamma to express signs of both immediate and persistent activation and suggest that, as a direct activator of mononuclear phagocytes, rIFN-gamma may also have potential as an immunotherapeutic agent for patients with intracellular infections.