Modulation of fibroblast response to maitotoxin along the cell division cycle

Maitotoxin (MTX) induces an increase of [Ca²⁺]_i and of phosphoinositide breakdown in various cell types. The [Ca²⁺]_i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the signal induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca²⁺]_i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10–20 min (G1) or more (G2). No [Ca²⁺]_i change could be detected during mitosis. The [Ca²⁺]_i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.Abbreviations MTX maitotoxin - [Ca²⁺]_i intracellular calcium concentration - IP₃ inositol triphosphate     

Identification of two human sperm populations using flow and image cytometry

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley-Liss, Inc.     

Linkage of a new mutation in the proteolipid protein (PLP) gene to Pelizaeus-Merzbacher disease (PMD) in a large Finnish kindred.

The purpose of this study was to confirm linkage of the proteolipid protein gene (PLP) and Pelizaeus-Merzbacher disease (PMD). A T-->A transversion in nucleotide pair 35 of exon 4 of PLP was found in a large Finnish kindred with PMD. This mutation results in the substitution Val165-->Glu165. We used a combination of single-strand conformational polymorphism and PCR primer extension to determine the presence or absence of the point mutation in family members. A lod score of 2.6 (theta = 0) was found for linkage of the gene and the disease. We examined 101 unrelated X chromosomes and found none with the transversion. This is the second report of linkage of PMD to a missense mutation in PLP. These findings support the hypothesis that PMD in this family is a result of the missense mutation present in exon 4 of PLP.     

Carbohydrate distribution and cellular injuries in acid rain and cold-treated spruce needles

Summary The cellular structures of acid rain-irrigated needles of several provenances of Norway spruce (Picea abies L. Karst) seedlings were studied after winter experimental freezing. Frost injuries and recovery were characterized by visual damage scoring and classification of mesophyll cell alterations, also using histochemical methods for carbohydrate fluorescent staining. The treatment with-30° C during the late dormancy period was sufficient to cause significant injuries and intracellular degradation in the tissues of the green needles. The most affected seedlings in terms of visual injury scoring were found among those treated with clean water or at pH 3, while freezing injury, defined as an occlusion of phenolic substances in the central vacuole of the mesophyll cells, was most abundant in the needles from spruces irrigated either with clean water or at pH 4 or pH 3. Electron microscopy revealed the details of the injury, e. g. thinning out of the cytoplasm and chloroplast stroma, darkening of the chloroplasts and eventually swelling of the chloroplasts and protoplast. PAS and ConA reactions in the needle tissue revealed intense starch accumulation in the mesophyll and transfusion tissues as early as in March, with a tendency to increase, especially in the untreated needles during the recovery period. Plasma membrane disturbances were indicated by histochemical identification of callose deposits in the mesophyll cell walls, these being most abundant in the acid rain-treated needles. All these findings suggest that freezing at –30° C was more deleterious to the seedlings pretreated with acid or clean water than to those not given additional irrigation.     

Nucleolar association of fibroblast growth factor 3 via specific sequence motifs has inhibitory effects on cell growth.

The dual subcellular fate of fibroblast growth factor 3 (FGF3) is determined by the competing effects of amino-terminal signals for nuclear localization and secretion (P. Kiefer, P. Acland, D. Pappin, G. Peters, and C. Dickson, EMBO J. 13:4126-4136, 1994). Mutation analysis has implicated additional basic domains in the carboxy-terminal region of the protein as necessary for nuclear uptake and the association of FGF3 with the nucleoli. Immunogold electron microscopy shows that FGF3 is predominantly within the dense fibrillar component of the nucleolus. A form of FGF3 that localizes exclusively in the nucleus and nucleolus was generated by removing signals for secretion, and expression of this nonsecreted FGF3 in a mammary epithelial cell line resulted in slowly growing colonies of enlarged cells. Thus, nuclear import and nucleolar association of FGF3 are determined by the concerted interaction of several distinct motifs, and the exclusive production of the nuclear isoform can inhibit DNA synthesis and cell proliferation.     

Augmented sympathetic tone alters muscle metabolism with exercise: lack of evidence for functional sympatholysis

Shoemaker, J. Kevin, Prasant Pandey, Michael D. Herr, DavidH. Silber, Qing X. Yang, Michael B. Smith, Kristen Gray, and LawrenceI. Sinoway. Augmented sympathetic tone alters muscle metabolismwith exercise: lack of evidence for functional sympatholysis. J. Appl. Physiol. 82(6):1932-1938, 1997.It is unclear whether sympathetic tone opposesdilator influences in exercising skeletal muscle. We examined highlevels of sympathetic tone, evoked by lower body negative pressure(LBNP, 60 mmHg) on intramuscular pH and phosphocreatine (PCr)levels (³¹P-nuclear magnetic resonance spectroscopy) duringgraded rhythmic handgrip (30 contractions/min; ~17, 34, 52 and 69%maximal voluntary contraction). Exercise was performedwith LBNP and without LBNP (Control). At the end of exercise, LBNPcaused lower levels of muscle pH (6.59 ± 0.09) comparedwith Control (6.78 ± 0.05; P < 0.05). PCr recovery, an index of mitochondrial respiration, was lessduring the recovery phase of the LBNP trial. Exercise mean arterialpressure was not altered by LBNP. The protocols were repeated withmeasurements of forearm blood flow velocity and deep venous samples(active forearm) of hemoglobin (Hb) saturation, pH, and lactate. WithLBNP, mean blood velocity was reduced at rest, during exercise, andduring recovery compared with Control (P < 0.05). Also, venous Hbsaturation and pH levels during exercise and recovery were lower withLBNP and lactate was higher compared with Control(P < 0.05). We concludethat LBNP enhanced sympathetic tone and reduced oxygen transport. Athigh workloads, there was a greater reliance on nonoxidativemetabolism. In other words, sympatholysis did not occur.

     

Genetic Modulation of RNA Metabolism in Drosophila. I. Increased Rate of Ribosomal RNA Synthesis

It has been suggested that a particular Y chromosome which is rDNA-deficient (YbbSuVar-5) may be associated with an increased utilization of rDNA template in adult testes (Shermoen and Kiefer 1975). To extend the observations on this chromosome, experiments were designed to determine if the chromosome has an effect on rRNA synthesis in bobbed adults and on classic bobbed phenotypes (shortened and thinner scutellar bristles and delayed development). Specific activity measurements were made on rRNA extracted from adult males of the genotypes car bb/YbbSuVar-5, which are rDNA-deficient to the same extent, and from Samarkand+ isogenic (Sam+ iso), which is a wild-type stock. The resulting data demonstrated that the presence of the YbbSuVar-5 chromosome increases the rate of ribosomal RNA synthesis in adult flies. In addition, it was found that the presence of this particular Y chromosome restores wild-type bristle phenotype and development time. Appropriate genetic crosses indicate that the observed effects (increased rRNA synthesis, restoration of wild-type phenotype) are a function of this particular Y chromosome, and are not due to autosomal factors. The results of these experiments suggest that the rate of rRNA accumulation is under genetic control.     

Cell cycle-specific glucocorticoid growth regulation of a human cell line (NHIK 3025)

It has been reported that the human cell line NHIK 3025 has a specific cytoplasmic glucocorticoid receptor. When these cells were exposed to glucocorticoids, the cell cycle time was prolonged. Cells, synchronized by mitotic selection, were subjected to the synthetic glucocorticoid dexamethasone throughout the cell cycle. Only cells exposed in the first half of G1 phase had a lengthened cell cycle time. Most of the prolongation was also located within the G1 phase. The dexamethasone growth inhibition was reversible and could be detected only in the cell cycle where the cells were exposed to the steroid. DNA-histograms of asynchronous cells were recorded by flowcytometry at various times after steroid exposure. These histograms also showed G1 phase sensitivity and G1 phase prolongation after exposure to dexamethasone. Our results thus indicate that these cells have a dexamethasone-sensitive restriction point in mid-G1 phase of the cell cycle.     

Die Morphologie der Schleimsekretion im Fruchtknoten vonAptenia cordifolia

Zusammenfassung Der Fruchtknoten vonAptenia cordifolia enthÄlt wÄhrend der Samenentwicklung einen proteinreichen Polysaccharidschleim. Verschieden alte schleimproduzierende Placentarpapillen werden einer elektronenmikroskopischen Analyse unterzogen. Kurz vor dem Einsetzen der Schleimproduktion ist das rauhe ER noch spÄrlich entwickelt. Der Golgi-Apparat ist unauffÄllig und wenig aktiv. Zu Beginn der Schleimbildung sind als hauptsÄchliche Strukturkomponenten hypersekretorische Dictyosomen und ER-umschlossene Vakuolen (storage vesicles) zu beobachten. Es wird angenommen, da\ diese Komplexe aus rauhem ER und vermutlich mitèinander verschmolzenen Golgi-Vesikeln die charakteristischen Synthese-Einheiten für den Polysaccharid-Protein-Schleim darstellen, da sie nachweislich neben Polysacchariden auch Proteine enthalten. Membranfusionen zwischen Vesikeln und dem Plasmalemma deuten auf Exocytose-Prozesse unter Beteiligung des Golgi-Apparates hin. Daneben wird eine holocrine Ausscheidung des in den storage vesicles zunÄchst gespeicherten Polysaccharid-Protein-Schleimes bei Degeneration des Protoplasten vermutet.

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Morphology of slime secretion in the seed vessels ofAptenia cordifolia

Summary During seed development the gynaeceum ofAptenia cordifolia produces a mucilage rich in carbohydrates and protein. The mucilage-producing placentary papillae are analyzed in different developmental stages by electron microscopy. Just before mucilage production is started, the rough ER occurs but sparsely. At that time the dictyosomes are inconspicuous and of low activity. When mucilage production commences, one can observe hypersecretory dictyosomes and ER-ensheathed vacuoles (storage vesicles) as the main structural components. It is suggested that the complexes of rough ER and probably fused Golgi vesicles are the synthetizing units of the carbohydrate protein mucilage, since in these complexes both components can be identified cytochemically. Fusion sites of plasmalemma and vesicles indicate processes of exocytosis-probably involving the Golgi apparatus. In addition, a holocrine excretion of the mucilage initially enclosed in the storage vesicles via degeneration of the protoplast is assumed.

     

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10⁻⁸ moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10⁻⁷ moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with ³H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.