Modelluntersuchungen zur Struktur des purpurnen Farbstoffkomplexes der Giemsa-Färbung

Summary Nuclei of Giemsa stained cells show a purple coloration, which is generated by a complex of DNA, azure B (AB) and eosin Y (EY). The structure of this complex is unknown. Its absorption spectrum shows a sharp and strong band at 18 100 cm^–1 (552 nm), the so called Romanowsky band (RB). It is possible to produce the complex outside of the cell, but it is cubersome to handle. Easier to handle is a purple complex composed of chondroitin sulfate (CHS), AB and EY, which also shows a sharp and strong RB at 18100 cm^–1 in the absorption spectrum. This CHS-AB-EY complex is a model for the DNA-AB-EY complex of Giemsa stained cell nuclei. We tried to investigate its structure.In the first step of the staining procedure CHS binds AB cations forming a stable CHS-AB complex. In the case of saturation each anionic SO ₄ ^– - and COO^–-binding site of CHS is occupied by one dye cation and the complex has 1:1 composition. It has a strong and broad absorption band with its maximum at ca. 18000 cm^–1 (556 nm). In the second step the CHS-AB complex additionally binds EY dianions forming the purple CHS-AB-EY complex with its RB at 18100 cm^–1. This band can be clearly distinguished from the broad absorption of the bound AB cations. RB is generated by the EY chromophore, whose absorption is shifted to longer wavelength by the interaction with the CHS-AB framework.The CHS chains of the CHS-AB and CHS-AB-EY complexes can be mechanically aligned in a preferred direction k. Fine films of highly orientated complexes were prepared with a special technique and studied with a microspectrophotometer equipped with a polarizer and an analyzer. They are birefringent and dichroic-the more birefringent, the better the mechanical orientation. The sites of best orientation within the film were selected according to the quality of the birefringence. We measured the absorption of these regions with linearly polarized light. By setting the polarizer (e _p parallel () or perpendicular () to k, we found that the transition moment m _AB of the long wave-length absorption of AB in the CHS-AB and the CHS-AB-EY complexes is polarized almost perpendicular to the preferred direction k, m _AB k. But the transition moment m _EY of EY in CHS-AB-EY is polarized parallel to k, m _EY k. The transition moments m _AB and m _EY lay in the molecular plane in the direction of the long axes of the AB and EY chromophores, respectively. Therefore, in both CHS-AB and CHS-AB-EY the long axes of the AB molecules are approximately perpendicular to the CHS chain; but in CHS-AB-EY the long axes of the EY chromophore are parallel to the chain of the biopolymer. This structure is somewhat surprising. In the CHS-AB-EY dye complex the chromophores of AB and EY are not parallel but approximately perpendicular to each other.     

In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

A demyelinating disease induced in C57B1/6N mice by intracranial injection of a coronavirus (murine hepatitis virus strain A59) is followed by functional recovery and efficient CNS myelin repair. To study the biological properties of the cells involved in this repair process, glial cells were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. Using three-color immunofluorescence combined with [3H]thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. We identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells with the O4 antibody. Cultures from demyelinated tissue differed in several ways from those of age-matched controls: first, the total number of O-2A lineage cells was strikingly increased; second, the O-2A population consisted of a higher proportion of O4-positive astrocytes and cells of mixed oligodendrocyte-astrocyte phenotype; and third, all the cell types within the O-2A lineage showed enhanced proliferation. This proliferation was not further enhanced by adding PDGF, basic fibroblast growth factor (bFGF), or insulin-like growth factor I (IGF-I) to the defined medium. However, bFGF and IGF-I seemed to influence the fate of O-2A lineage cells in cultures of demyelinated tissue. Basic FGF decreased the percentage of cells expressing galactocerebroside. In contrast, IGF-I increased the relative proportion of oligodendrocytes. Thus, O-2A lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. These properties may be linked to the efficient remyelination achieved in this demyelinating disease.     

Purinergic Modulation of Hippocampal Acetylcholine Release Involves α-Dendrotoxin-Sensitive Potassium Channels

Modulation of acetylcholine (ACh) release from superfused hippocampal slices was examined when the release of ACh was stimulated by exposure of slices to elevated K+ concentration. Evoked release was not sensitive to inhibition by 0.1 microM tetrodotoxin, but it could be inhibited in a dose-dependent manner by a muscarinic agonist (10-100 nM oxotremorine) and a purinergic agonist (10-100 nM 2-chloroadenosine). The alpha-dendrotoxin (100 nM), which selectively blocks voltage-gated inactivating K+ channels in nerve endings, did not affect the release of ACh under resting or depolarized conditions. However, alpha-dendrotoxin reduced the 2-chloroadenosine-induced inhibition of release, but did not alter the oxotremorine-induced inhibition. These results suggest that an alpha-dendrotoxin-sensitive K+ channel may be activated as an obligatory step in the modulation of ACh release by presynaptic purinergic receptor activation, but not in the modulation by presynaptic muscarinic receptors.     

Neuronal Intermediate Filament Expression During Neurite Outgrowth from Explanted Goldfish Retina: Effect of Retinoic Acid

Regulation of the goldfish neuronal intermediate filament proteins ON1 and ON2 was investigated in a retinal explant system. The synthesis of these proteins in explanted retina decreased with increasing time in culture, despite continuing neurite outgrowth. Thus, ON1/ON2 neurofilament expression is regulated independently from neurite outgrowth. During regeneration of the goldfish optic nerve in vivo, the expression of these proteins increased during the later phase of the process, when growing axons make contact with the optic tectum. The declining synthesis of ON1 and ON2 during neurite outgrowth in culture suggests that factors extrinsic to the retina are necessary to support synthesis of these proteins. Treating retinal explants with retinoic acid stimulated the synthesis of the ON1/ON2 proteins in a dose-dependent manner. This stimulation was effective during a period of declining synthesis of the ON1/ON2 proteins, restoring their synthesis towards initial levels of expression. These results show that retinoic acid serves as a modulator of neurofilament expression in this in vitro model of nerve regeneration.     

Structural gene isolation and prepeptide sequence of gallidermin, a new lanthionine containing antibiotic

Peptide antibiotics containing lanthionine and 3-methyllanthionine bridges, named lantibiotics are of increasing interest. A new lantibiotic, gallidermin, has been isolated from Staphyloccus gallinarum. Here we report the isolation of its structural gene which we name gdmA. In all lantibiotics so far studied genetically, three peptides can be formally distinguished: (i) the primary translation product, which we call the prepeptide; (ii) the propeptide lacking the leader sequence and (iii) the mature lantibiotic. Unlike the plasmid-coded epidermin, gdmA is located on the chromosome. The gdmA locus codes for a 52 amino acid residue prepeptide, consisting of an alpha-helical leader sequence of hydrophilic character, which is separated from the C-terminus (propeptide) by a characteristic proteolytic processing site (Pro-2 Arg-1 Ile1). Although pro-gallidermin differs from pro-epidermin (a recently isolated lantibiotic) only by a single amino acid residue exchange. Leu instead of Ile, the N-terminus of the prepeptide differs by an additional two exchanges.     

The perennial taxa ofCrucianella (Rubiaceae) in SW. Asia and their eco-geographical differentiation

The perennial taxa ofCrucianella in Asia form a coherent group, apparently diploid (x = 11) and outbreeding throughout, and should be placed into sect.Roseae. This Irano-Turanian group has its centre of diversity in the mountain systems south of the Caspian Sea and reaches with outposts NE. and E. Anatolia, NE. Iraq, S. Iran and C. Asia. Four species and 13 subspecies (within the polymorphicC. gilanica) are recognized, described (partly as new), and illustrated (Figs. 1–6). Conspectus, keys and distribution maps (Figs. 7 and 8) as well as plesio- and apomorphic character states and data on size of areas are provided (Table 1). There is an obvious correlation between more plesiomorphic taxa with smaller areas in the distribution centre of the group, and more apomorphic taxa with larger areas towards its periphery (Fig. 9). These findings are linked to a working hypothesis on the evolution of the group.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.     

In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Physical Basis for Altered Stem Elongation Rates in Internode Length Mutants of Pisum

Biophysical parameters related to gibberellin (GA)-dependent stem elongation were examined in dark-grown stem-length genotypes of Pisum sativum L. The rate of internode expansion in these genotypes is altered due to recessive mutations which affect either the endogenous levels of, or response to, GA. The GA deficient dwarf L181 (ls), two GA insensitive semierectoides dwarfs NGB5865 and NGB5862 (Ika and Ikb, respectively) and the `slender' line L197 (la cry^[ill]), which is tall regardless of GA content, were compared to the wild-type tall cultivar, Torsdag. Osmotic pressure, estimated by vapor pressure osmometry, and turgor pressure, measured directly with a pressure probe, did not correlate with the differences in growth rate among the genotypes. Mechanical wall properties of frozen-thawed tissue were measured using a constant force assay. GA deficiency resulted in increased wall stiffness judged both on the basis of plastic compliance and plastic extensibility normalized for equal stem circumference. Plastic compliance was not reduced in the GA insensitive dwarfs, though Ika reduced circumference-normalized plasticity. In contrast, in vivo wall relaxation, determined by the pressure-block technique, differed among genotypes in a manner which did correlate with extension rates. The wall yield threshold was 1 bar or less in the tall lines, but ranged from 3 to 6 bars in the dwarf genotypes. The results with the ls mutant indicate that GA enhances stem elongation by both decreasing the wall yield threshold and increasing the wall yield coefficient. In the GA-insensitive mutants, Ika and Ikb, the wall yield threshold is substantially elevated. Plants possessing Ika may also possess a reduced wall yield coefficient.