Josef Reischig

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis.

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.

Catecholaminergic Neurons in the Rat Intrinsic Cardiac Nervous System

Immunoreactivities (IR) for catecholamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH), phenylethanolamine N-methyl transferase (PNMT), serotonin-synthesizing enzyme tryptophan hydroxylase, and neuropeptide Y were investigated in the intrinsic cardiac nervous system of 27–40–day-old rats using fluorescent immunohistochemistry. Individual neurons were identified by the general neuronal marker protein gene product 9.5. The presence of DβH and PNMT in the atrial specimens was verified using reverse transcriptase-polymerase chain reaction. Two types of catecholamine-handling intrinsic ganglion neurons were observed: small intensely fluorescent (SIF) cells and large-diameter neurons. SIF cells exhibited TH- and tryptophan hydroxylase-IR, but they were not positive for DβH. In contrast, large-diameter intrinsic TH-positive neurons, showing in majority also NPY-IR, displayed also DβH- and PNMT-IR, thus indicating the capacity for the synthesis of norepinephrine and epinephrine, respectively. In conclusion, the SIF cells are most probably dopaminergic and serotonergic neurons, whereas large-diameter intrinsic cells seem to represent a subpopulation of norepinephrine- and/or epinephrine-secreting neurons.

Expression of neuropeptide Y and its receptors Y1 and Y2 in the rat heart and its supplying autonomic and spinal sensory ganglia in experimentally induced diabetes

Diabetic cardiomyopathy, involving both cardiomyocytes and the sensory and autonomic cardiac innervation, is a major life-threatening complication in diabetes mellitus. Here, we induced long-term (26-53 weeks) diabetes in rats by streptozotocin injection and analyzed the major cardiac neuropeptide signaling system, neuropeptide Y (NPY) and its receptors Y1R and Y2R. Heart compartments and ganglia supplying sympathetic (stellate ganglion) and spinal sensory fibers (upper thoracic dorsal root ganglia=DRG) were analyzed separately by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Ventricular, but not atrial innervation density by NPY-immunoreactive fibers was diminished, and preproNPY expression was transiently (26 weeks) reduced in left atria, but remained unchanged in sympathetic neurons and was not induced in DRG neurons. In all ganglia and heart compartments, Y1R expression dominated over Y2R, and Y1R-immunoreactivity was observed on cardiomyocytes and neuronal perikarya. Atrial, but not ventricular Y1R expression was up-regulated after 1 year of diabetes. Collectively, these data show that a disturbance of the cardiac NPY-Y1R/Y2R signaling system develops slowly in the course of experimentally induced diabetes and differentially affects atria and ventricles. This is in parallel with the clinically observed imbalances of the cardiac autonomic innervation in diabetic cardiac autonomic neuropathy.

Distribution of vasoactive intestinal polypeptide in the rat heart: effect of guanethidine and capsaicin

Vasoactive intestinal polypeptide (VIP) is believed to coexist with acetylcholine in postganglionic parasympathetic neurones. However, the presence of VIP in extrinsic nerves and/or other types of intrinsic cardiac neurones has not been excluded. The aim of our study was to examine the distribution and origin of VIP-ergic innervation in the rat heart atria using immunocytochemistry and radioimmunoassay (RIA) combined with two types of denervation: sympathectomy, which was produced by guanethidine treatment and sensory denervation achieved by capsaicin administration. In whole-mount preparations of the intact atria, VIP-immunoreactive (IR) nerve fibres and ganglionic cells were found, the latter being much more numerous in the left atria (LA) than in the right ones. Some of VIP-IR nerve fibres forming bundles appeared to be extrinsic in origin. VIP-IR concentrations determined by RIA in the intact rats were significantly higher in the LA than in the right ones (p < 0.01). However, no changes in VIP-IR levels were found in either atrium after both guanethidine and capsaicin treatment protocols, thus indicating that VIP-immunoreactivity is not associated with either sympathetic or sensory innervation. In conclusion, the ganglionated plexus of the rat atria may comprise at least 3 different neuronal populations expressing VIP-positivity: 1. extrinsic preganglionic parasympathetic fibres, 2. intrinsic postganglionic parasympathetic neurones and 3. intrinsic local circuit neurones that do not express a cholinergic phenotype.

Structure of herpes simplex virus DNA: topography of the molecule. I. Absence of circularly permuted sequences.

Only linear structures were produced by re-annealing denatured herpes simplex type 1 virus (HSV)-DNA molecules, while T4-DNA molecules (known to be circularly permuted and therefore used as controls in the parallel tests) formed circles. These results suggested that HSV-DNA is a nonpermuted collection of sequences. Unfortunately, most of the linear structures observed after re-annealing were not full-length duplexes, thus making this test not quite satisfactory. Therefore another permutation test, the central region deletion experiment, was employed. Its results indicated that short fragments liberated from the ends of HSV-DNA by enzymatic treatment contained only a portion of the sequences present in the complete molecules. On the other hand, the presence of apparently all sequences was found in the short end-fragments of the control T4-DNA. These data also provide evidence that HSV-DNA molecules are not formed by circularly permuted collections of sequencies.

Electron microscopy of binding of Epstein-Barr virus (ebv) nuclear antigen (EBNA-1) to ebv DNA

Specific binding sites for Epstein-Barr virus (EBV) nuclear antigen (EBNA-1), isolated and semipurified from EBV-transformed nonproductive Raji cells, were visualized on the molecule of EBV DNA by electron microscopy and mapped. Two measures had to be applied to counteract the limited purity of the EBNA-1 preparation: (i) EBV DNA/EBNA-1 complexes were specifically enlarged by binding with anti-EBNA-1 (IR-3) IgG antibody. (ii) DNA-binding proteins that did not react with the anti-EBNA-1 antibody were eluted from EBV DNA with 1.5 M NaCl, taking advantage of the resistance of DNA/EBNA-1/anti-EBNA-1 antibody complexes to the high-salt treatment. EBNA-1 bound at the highest relative frequency (greater than 30%) to the EBV DNA map positions of 8.8 +/- 0.3, 10.3 +/- 0.5, and 46.6 +/- 1.2 kb. It bound with a lower but still statistically significant frequency (16%) to the map positions of 64.5 +/- 1.0, 89.7 +/- 1.6, 129.6 +/- 1.1 kb.

Structure of herpes simplex virus DNA: topography of the molecule. II. Partial denaturation map.

Abstract A partial denaturation map of herpes simplex virus type 1 DNA was constructed after treatment of the DNA at pH 11.3. The map was asymmetrical, showing preferential strand separation at the following fractional lengths: 0.01, 0.04, 0.16, 0.23, 0.28, 0.54, 0.56, 0.57, 0.62, 0.66, 0.68, 0.75, 0.87, 0.91, 0.95, 0.96 and 0.99. After 90-min denaturation, single-stranded interruptions were detected within denatured parts of molecules.

Epstein-Barr virus nuclear antigen type 1 binding: electron microscopy

Epstein-Barr virus (EBV) nuclear antigen type-1 (EBNA-1) was extracted and purified from Raji cells by chromatography on DNA-Sepharose and Blue-dextran Sepharose. Its complexes with plasmid pM765-10 derived from EBV (strain M-ABA) DNA were visualized by electron microscopy. The criteria of specificity were as follows: (1) preferential binding of EBNA-1 to the ori-P region of pM765-10; (2) specific enlargement of EBV DNA/EBNA-1 complexes with anti-EBNA-1 (IR-3) IgG antibody; and (3) resistance of the resulting EBV DNA/EBNA-1/anti-EBNA-1 antibody complexes to treatment with 1.5 M NaCl. The optimal conditions for the formation of EBV DNA/EBNA-1 complexes were 50 to 150 mM NaCl and pH 6.0. A balanced equilibrium of EBNA-1 and pM765-10 was necessary to achieve both a high yield and specificity of EBV DNA/EBNA-1 complexes.

Establishment and characterization of clonal cell lines derived from a fibrosarcoma of the H2-K/v-jun transgenic mouse: A model of H2-K/v-jun mediated tumorigenesis

We used fibrosarcoma from an H2-K/v-jun transgenic mouse to derive a series of three immortal cell lines (JUN-1, -2, and -3). The cell lines exhibit strikingly different behavior regarding phenotype transformation. Features examined include contact inhibition and density limitation of growth, proliferation, invasiveness, motility, and organization of the microfilament system. Overall, JUN-2 and JUN-3 represent extreme phenotypes, with JUN-2 having a phenotype indicative of low-level cellular transformation and JUN-3 meeting all the criteria of the transformed phenotype. JUN-1 cells can also be regarded as transformed, but to a lesser extent than JUN-3. Their phenotype is in the majority of characteristics intermediary between JUN-2 and JUN-3. The transformation status is inversely related to the expression of the v-jun transgene, which is the highest in JUN-2, lower in JUN-1 and very low in JUN-3. This might be related to the MHC class I promoter driving its expression and to the general observation of repression of MHC class I genes coupled with cellular transformation. Based on this premise, we present a model of H2-K/v-jun-mediated tumorigenesis, in which v-jun-conditioned transformation represents merely an initial phase of tumorigenesis. Later during tumor progression, additional oncogenes are activated and/or tumor suppressor genes inactivated, leading on the one hand to further exacerbation of the transformed phenotype, and on the other hand to the repression of the H2-K/v-jun transgene (fixed in JUN-3). We believe that the system of JUN cell lines can be valuable for further molecular analysis of transformation-related traits.