Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Therapeutic efficacy of Stephania tetrandra S. Moore for treatment of neovascularization of retinal capillary (retinopathy) in diabetes--in vitro study.

The present study was designed to examine therapeutic efficacy of the root extract of Stephania Tetrandra S. Moore (STMS) (traditional Chinese medicine; Han Fang Ji) for treatment of neovascularization of the retinal capillary (retinopathy) in streptozotocin (STZ)-induced diabetic rats (STZ diabetic rats) in culture. Recently we have established the culture system in which fetal bovine serum (FBS) in Dulbecco modified Eagle medium (DMEM) induced neovascularization of the retinal capillary and choroidal capillary in normal rats in culture. STZ diabetic rats showed more neovascularization of the retinal capillary and choroidal capillary than did normal rats in culture. In this study, the retinal tissue was removed for the posterior ocular region and cultured in DMEM containing FBS. The choroidal tissue of the posterior ocular region was also removed and cultured as an internal reference. Administration of STSM (0.91, 9.1 and 91 microg/ml) significantly suppressed neovascularization of the retinal capillary in both STZ diabetic rats and normal rats in a dose-dependent manner. Similar results were obtained with the choroidal capillary; administration of STSM suppressed neovascularization of the choroidal capillary in both STZ diabetic rats and normal rats. In order to determine the component of STSM inhibiting neovascularization of the retinal capillary, tetrandrine (a major chemical constituent of STSM) was administered and neovascularization of the retinal capillary was examined in culture. The effect of tetrandrine on the choroidal capillary was also examined as an internal reference. Administration of tetrandrine (0.1, 1.0 and 10 microM) suppressed neovascularization of the retinal capillary in both STZ diabetic rats and normal rats in a dose-dependent manner. Similar results were obtained with the choroidal capillary of both STZ diabetic rats and normal rats. We infer, therefore, that STSM has a direct effect on the retinal capillary of posterior ocular region and suppresses neovascularization of retinal capillary in STZ diabetic rats through the activation of tetrandrine. These results suggest that STSM may prevent for delay the progression of retinopathy in diabetic patients.     

Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase

Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.     

Flow cytometric quantification of rat spermatogenic cells after hypophysectomy and gonadotropin treatment

DNA flow cytometry was evaluated as a tool to analyze stage-specific changes that occur in absolute cell numbers in the testes. Hypophysectomy was selected as a model system for perturbing testicular cell types, since the cytological sequelae of this treatment post-hypophysectomy in the rat are well documented in the literature. Rat spermatogenic cells in stages II-V, VII, and IX-XIII of the seminiferous epithelial cycle (as defined by Leblond and Clermont, 1952) were quantified in numbers per standard length of seminiferous tubule by DNA flow cytometry after hypophysectomy and subsequent gonadotropin treatment. In agreement with previous histological studies, we found that acrosome- and maturation-phase spermatids disappeared from the seminiferous epithelium after 17 days post-hypophysectomy, whereas meiosis and early spermiogenesis continued at least 164 days. The number of meiotic cells and round spermatids gradually decreased after hypophysectomy. Changes were observed as early as Day 6 post-hypophysectomy. Treatment with human chorionic gonadotropin (hCG) alone maintained most cell numbers within normal limits, and follicle-stimulating hormone (FSH) was needed in addition to hCG to maintain the normal number of cells with the amount of DNA contained in primary spermatocytes and spermatogonia in G2/M-phase (4C) in stages IX-XIII and elongated spermatids (1C') in stages II-V of the epithelial cycle. The absolute numbers of spermatogenic cells at different phases of maturation provide a useful reference for quantitative studies of spermatogenesis. Pathological changes in the seminiferous epithelium can be detected and quantified by DNA flow cytometry.     

Determination of guanine by high-performance liquid chromatography with electrochemical detection: application to DNA and RNA assays

A highly sensitive assay for guanine was developed using high-performance liquid chromatography with electrochemical detection (ECD). Guanine was susceptible to the electrochemical oxidation, and ECD response was proportional to the amount of guanine in the range 0.25-4 pmol of guanine. The ECD of guanine was applicable to the analysis of nucleic acids. DNA and RNA were hydrolyzed in 0.03 and 3 M HCl, respectively, and guanine liberated from the nucleic acids was separated on a reverse-phase column and determined by ECD. The method allowed detection of 0.2 ng of calf thymus DNA or tRNA. An application of the method is shown for DNA and RNA assays in trichloroacetic acid extracts of rat adrenal and liver.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Changes in Crassulacean Acid Metabolism of Kalanchoe blossfeldiana by Different Nitrogen Sources

Kalanchoe blossfeldiana Poelln. cv. Hikan (a Crassulacean acidmetabolism (CAM) plant) was grown in pots containing soil for6 months and then cultured in nutrient solution containing 10mM nitrate or ammonium as a sole nitrogen source for 2 or 3months, under a long-day (16 h) condition. Plant growth was better in the nitrate medium. Leaves of thenitrate-grown plants showed greater diurnal fluctuations intitratable acidity and malate content than those of the ammonium-grownplants. The diurnal patterns in CO₂ exchange of nitrate-grownplants were basically similar for both groups, but the amountof net CO₂ uptake at night was twice as large in the nitrate-grownplants. The leaves of the nitrate-grown plants had 1.3 to 2.5times higher activities of phosphoenolpyruvate carboxylase (PEPC),phosphofructokinase (PFK) and NAD glycelaldehyde-3-phosphatedehydrogenase (G3PDH). These results indicate that K. blossfeldianagrown in nitrate medium showed more CAM activity than thosein ammonium medium. (Received August 13, 1987; Accepted February 22, 1988)     

An examination of the method of sizing the spermatozoa of the domestic fowl and duck with an electronic counter

Although many estimations have been made electronically of mammalian sperm volume, detailed investigations have not been reported for avian spermatozoa with an electronic counter. In the present study, sizing of spermatozoa of fowls and Muscovy and Pekin drakes was examined using a Coulter counter (model ZB). In our preliminary work on fowl sperm volumes, we found mono- or di-morphic distribution displays that were modified depending on the combination of amplification (AMP) and aperture current (APC). Therefore, methodology to estimate the fowl and drake sperm volume was examined. Dilution of semen had no effect on the dimorphic distribution pattern of the sperm volume. Density-gradient centrifugation did not separate two kind of particles in the semen in either continuous or discontinuous Percoll gradients; therefore, we varied settings of AMP and APC, and found that the most suitable settings for measuring sperm volumes of these birds are 1 for AMP and 8 for APC. With these settings, mean volumes of spermatozoa were 5.1 μm³ for fowls, 5.7 μm³ for Muscovy drakes, and 5.6 μm³ for Pekin drakes.     

4-Hydroxyaminoquinoline 1-oxide metabolism and DNA adducts in the early stage of tumorigenesis in rats: comparison of target organ pancreas with non-target organ liver

In order to probe key early molecular events which might be responsible for the initiation of rat pancreatic tumorigenesis by 4-hydroxyaminoquinoline 1-oxide (4-HAQO), the uptake and metabolism of carcinogen and the formation and subsequent repair of DNA adducts were monitored under conditions of high and low tumorigenicity, respectively in partially pancreatectomized and non-operated animals, and in the liver, a non-target organ for this carcinogen. Although uptake of radioactively labelled 4-HAQO was higher in the liver than in the pancreas, generation of DNA adducts was 20 times greater in the latter organ. This discrepancy was probably due to a difference in the metabolic profile of 4-HAQO. The spectrum of the adducts was qualitatively similar in both organs. No qualitative or quantitative differences could be established under the high and low tumorigenicity conditions with regard to DNA adduct formation or persistence. The major difference was the presence of a relatively large extent of pancreatic DNA replication under the high tumorigenic condition. The results indicated that metabolic profile of 4-HAQO, quantity of DNA adducts and levels of DNA replication are key factors involved in initiation of tumorigenesis.