The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

Isolation of renal brush-border membrane vesicles by a low-speed centrifugation; effect of sex hormones on Na+-H+ exchange in rat and mouse kidney

Na+-H+ exchange in rat and mouse renal brush-border membrane vesicles was studied by fluorescence quenching of the delta pH indicator, acridine orange. Brush-border membrane vesicles were isolated by a modified Mg/EGTA-precipitation method at low speed centrifugation (8000 X g). The enzymatic characteristics of these membrane vesicles were similar to those obtained by the original high-speed centrifugation method (Biber et al. (1981) Biochim. Biophys. Acta, 647, 169-176). The rates of Na+-H+ exchange in renal brush-border membrane vesicles from male and female rats were similar. Neither ovariectomy nor treatment of ovariectomized rats with estradiol or testosterone changed the activity of Na+-H+ exchanger. The rates of Na+-H+ exchange in the mouse were smaller than in the rat indicating the existence of species differences. Na+-H+ exchange in mouse renal brush-border membranes exhibit strong sex differences, the rates in the male being higher than in the female. Castration of male mice led to a decrease in Na+-H+ exchange to values found in females. Treatment of castrated mice with estradiol had no effect. In contrast, treatment with testosterone increased the rate of the exchanger by more than 100%. The effect of testosterone was restricted to the Vmax of the Na+-H+ exchanger, whereas the apparent Km for Na+ remained unchanged. Na+-dependent D-glucose transport in mouse renal luminal membranes exhibited also sex differences due to the potent stimulatory effect of testosterone. Therefore, Na+-H+ exchange and Na+-dependent D-glucose transport in the mouse kidney are under control of androgen hormones. This effect could be in close connection with the wellknown renotropic action of androgens in the mouse.     

On the localization of genes on certain autosomes of man through chromosome aberrations

Summary Chromosome aberrations permit the assignment to and the exclusion of genes on certain chromosomes or definite segments. Only exclusion methods are discussed. All relevant data are compiled in a table from which it can be determined, which genetic systems are excluded from certain autosomal segments.

---

Zusammenfassung Angeborene Chromosomenaberrationen ermöglichen die Zuordnung und den Ausschluß von Genen auf dem betroffenen Segment. Nur die für Lokalisierungsausschlüsse verwendbaren Methoden werden diskutiert. Aus einer Zusammenstellung der bisherigen Befunde wird ermittelt, welche genetischen Systeme von einer Lokalisierung auf bestimmten Autosomen(segmenten) des Menschen ausgeschlossen werden können.

---

---

Direktor: Prof. Dr. Dr. H. Baitsch

---

Supported by the Deutsche Forschungsgemeinschaft.     

Single chloride channels in endosomal vesicle preparations from rat kidney cortex

Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H⁺ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl^– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl^–=Br^–=I^–>SO ₄ ^2– F^–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl^–-flux measurements and studies on the H⁺ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H⁺ uptake into the endosomes.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

DNA translocation by the restriction enzyme from E. coli K

The restriction endonuclease Eco K binds to a host specificity site and then proceeds to cleave the DNA at sites that may to several thousand bases away. It does this by translocating the DNA past the enzyme in an ATP-dependent reaction that results in the formation of highly twisted loop intermediates. DNA cleavage can occur on either side of the host specificity site.     

A compact from of methylated DNA is solutions containing poly (ethylene glycol).

Some peculiarities of compactization of double-stranded DNA molecules containing methylated nitrogen bases have been studied in water-salt solutions of PEG. It is shown that the methylation of N7-atoms of guanyl residues in original DNA molecules does not prevent the formation of DNA compact particles, but results in a decrease of the amplitude of the negative band in the CD spectrum of compact particles. The influence of N7-guanine methylation on the shape of the CD spectrum being the greater, the lower is the concentration of PEG. The dependence of the negative band amplitude in the CD spectrum on the content of methylated guanyl residues is practically the same for low-molecular weight DNA's from different sources. The observed decrease in the negative band amplitude is interpreted as a result of alterration of guanyl residue orientation relative to the helix axis which leads to diminished optical activity of the "microcrystalline" domains of compact particles. The evidence obtained suggests that changes in the secondary structure of DNA lead to considerable difference between CD spectra of compact particles of methlated DNA and psi-form of DNA. (The changes in the CD spectrum of the DNA compact particles occur also as a result of methylation of C5-atoms of cytosine residues). It is suggested that the negative band in the CD spectrum can be used a criterion for detection of negligible alterations in the DNA secondary structure.     

Differentiation in cultures derived from embryonic chicken muscle: I. Muscle-specific enzyme changes before fusion in EGTA-synchronized cultures

It is known that myoblast fusion fails to occur in cultures containing EGTA (a calcium-specific chelator) but occurs very rapidly after EGTA medium is replaced with standard high-calcium medium. On the basis of a careful analysis of the time course of fusion in cultures switched from EGTA to standard medium, it is proposed that this method of synchronization be used routinely in studies of the timing of different processes during in vitro myogenesis. The kinetics of accumulation of total enzyme activity for creatine kinase and fructose diphosphate aldolase indicate that the increases characteristic of terminal muscle differentiation begin prior to the experimentally imposed onset of fusion in EGTA-synchronized cultures. Additionally, the accumulation of M-creatine kinase subunits, also typical for muscle differentiation, is shown by microcomplement fixation to begin before the switch from EGTA to standard medium. Creatine kinase isoenzyme patterns also show that the transition from B- to M-subunit-containing creatine kinases occurs in EGTA cultures not switched to standard medium. Like EGTA, 5-bromodeoxyuridine (BrdUrd) reversibly prevents myoblast fusion. By adding EGTA and BrdUrd in different sequences to muscle cell cultures, it is shown that they act at different stages in the course of in vitro myogenesis. Cells cultured in EGTA from 23 to 69 hr after plating fused very rapidly when switched to medium containing BrdUrd. In the reverse experiment, in which BrdUrd preceded EGTA, no fusion occurred. Parallel experiments with 5-fluorodeoxyuridine suggest that cell division is necessary to reverse the inhibitory effect of BrdUrd, but not that of EGTA; this is consistent with the observed kinetics of fusion after switching to standard medium. These data strongly support a model of myogenesis in vitro in which two processes (one BrdUrd-sensitive, the other EGTA-sensitive) occur sequentially. In the first process, myogenic cells give rise to cells capable of producing molecules necessary for (terminal) skeletal muscle differentiation, including both those required for cell fusion and specific isoenzymes. The second process, fusion itself, can occur in the presence of BrdUrd or in the absence of cell division.     

Possibilities of Triplex-Formation by 21-Mer Oligodeoxyribonucleotides as Analogs of Tetrahymena Pre-rRNA Fragments

Abstract

A potential DNA triple helix of 21-mer oligodeoxyribonucleotides was synthesized and characterized. The strands were chosen to study the interaction of internal guide and intervening sequences analogs as well as adjacent 3′and 5′exon parts around the splicing site of Tetrahymena pre-rRNA. Further in parallel works a series of different RNA and DNA strands was synthesized and combined yielding a suitable order of stability. Here we want to show an isolated examination of a DNA-strand triple helix with defined sequences containing a central mismatched base arrangement and T-A-T bases at the ends.     

Binding of Bis-linked Netropsin Derivatives in the Parallel-Stranded Hairpin Form to DNA

Abstract

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5′-CCTATATCC-3′ in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis -diammine Pt(II)- bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5′-CCTATATCC-3′ (I), 5′-CCTTAATCC-3′ (II), 5′-CCTTATTCC-3′ (III), 5′-CCTTTTTCC-3′ (IV) and 5′-CCAATTTCC-3′ (V) decreases in the order I = II > III > IV> V. The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.