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.     

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.     

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.     

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.     

Butyrate-induced cell differentiation of cell-cycle mutants and ''wild-type'' mastocytoma cells: Histamine, 5-hydroxytryptamine and metachromatic granules as independently regulated differentiation markers

In cultures of heat-sensitive (hs; arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (cs; arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants that had been isolated from the same subclone (K21) of the murine P-815-X2 mastocytoma line, the degree of cell differentiation was assessed by determining the cellular histamine and 5-hydroxytryptamine (5-HT) content as well as the number of metachromatic granules per cell. The findings were compared with those obtained for 'wild-type' K21 and P-815-X2 cells. The addition of butyrate to 'wild-type' cells or to mutant cells maintained at the respective permissive temperature resulted in a relative increase in the level of all three differentiation markers. In cs mutant cells, essentially the same pronounced increase in granule numbers was observed during butyrate treatment at 39.5 degrees C and during incubation at 33 degrees C without butyrate, thereby suggesting that butyrate induces morphological cell differentiation in cs mutants via the same mechanisms as exposure to the nonpermissive temperature. In contrast, the histamine and 5-HT levels reached in hs and cs mutant cells in the presence of butyrate were higher than those observed during incubation at the nonpermissive temperature. Large quantitative differences were detected with respect to the potential of individual cell lines to express the three differentiation parameters. High levels of histamine were characteristic of 'wild-type' P-815-X2 cells treated at 33 degrees C with butyrate, while low amine levels and small numbers of granules were observed in K21 cells (i.e., the parent line of hs and cs mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cardiac sodium channel shows a regular substate pattern indicating synchronized activity of several ion pathways instead of one

Cardiac sodium channel substates were induced by using different gating modifiers, namely S-DPI 201-106 (s), toxin II from Anemonia sulcata (a), veratridine (v) and mixtures of these agents (s + v, a + v). Current ratios (normalized substate currents), slope conductances, reversal potentials and saturation characteristics were evaluated for the individual channel substates. The results can be summarized as follows: (i) Current ratios fell into a pattern of six equidistant values (I to VI) irrespective of the modification applied (0.20, 0.34, 0.51, 0.69, 0.85, 1.00). Slope conductances, determinable for substates II, V and VI (4.8, 11.7 and 14.0, respectively), are also consistent with six conductance substates which are integer multiples of a smallest conductance (state I). (ii) The permeability ratio PNa+/PK+ (i.e., reversal potential of substate currents) of the sodium channel was conserved both for different modifications, i.e., by s, a, s + v and a + v, and for the different substates (at least for II, IV and VI) observed for each modification. (iii) Sodium binding to the channel is substate independent. Analysis of slope conductances of states II and VI for three sodium chloride concentrations (71.5, 140 and 303 mM) revealed different maximal conductances (geVImax = 2.9.geIImax) but similar apparent affinities for sodium (KNa + VI = 286 mM; KNa + II = 303 mM). These findings are shown to seriously challenge the commonly unquestioned conception that 'single-current events' reflect ion passage through only one single pathway. The alternative view, that not one pore, but either six or three pores with synchronized gating ('oligochannel') underlie 'single-channel events', is shown to readily account for the observed substate properties and appears not to contradict known properties of 'the sodium channel'. This fundamentally new view of the sodium channel aims to invoke further efforts to distinguish between conceptually distinct models of structure-function relationships for a variety of channels which show multiple substates and conserved ion selectivity.     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

In goodeid teleosts, prolonged embryonic development takes place within the ovarian cavity. Apposed maternal and embryonic epithelia interface via a nutritive liquid (embryotrophe) and facilitate aplacental matrotrophy. The role of the internal ovarian epithelium (IOE) in providing proteins for the embryotrophe has been studied using transmission electron-microscopic examinations of both the resting and the active ovarian lining, and isoelectric focusing of embryotrophe and maternal blood serum. The simple IOE is apparently composed of only one, filament-containing cell-type. In the non-gravid ovary these cells are cuboidal to columnar in shape, and are either compact and electron-dense or oedematous and light. During gestation, swelling of the ovarian connective tissue gives rise to dovetailing of the IOE with the subjacent capillary plexus. Part of the IOE overlying the capillaries becomes stretched, resulting in a thin endothelium-like demarcation. The nuclei and the bulk of the cytoplasm are usually recessed between the meshes of the protruding capillary network. The blood-embryotrophe pathway is thus reduced in places, to less than one m. The active form of the IOE contains a well-developed vacuolar apparatus composed of small vesicles, vacuoles, multivesicular bodies, and a few lysosomes. Elements of the RER are sparsely distributed throughout the cytoplasm. Endocytotic activity is observable at the apical and basolateral plasma membrane. Isoelectric focusing of both serum and embryotrophe produces numerous bands each between pI 4–8, which reveal many homologies. The intensity of corresponding bands varies considerably. It is concluded that the cells of the IOE provide a transport pathway for serum-derived macromolecular substances rather than produce proteinaceous secretions.