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.     

Single-locus control of the mast cell population in mouse skin

The number of mast cells in connective tissue from dorsal skin varied markedly among mouse strains. Inbred strains of mice were typed into three groups, high (NC and NZB mice), low (B6, B10, and BALB/c mice), and intermediate (C3H/He and DBA/2 mice), by their mast cell content in the skin. However, the strain differences in the number of mast cells was marginal at the age of weaning but became distinct with age. This could be explained mainly by the frequently observed clustering of mast cells in adult NC and NZB mice and the rarely observed clustering in younger mice as well as in adult B10 and BALB/c mice. The breeding experiment revealed that the difference in the number of mast cells between NC and B10 mice was controlled by a single autosomal dominant locus, for which we propose the designation Mcr (mast cell regulator). The role of the Mcr locus with regard to the frequency of the mast cell population in connective tissue is discussed.     

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.     

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.     

Assignment of the gene locus for the sixth component (C6) of complement in mice to chromosome 15

A structural locus (C-6) for the sixth component of complement in mice is assigned to chromosome 15. Three-point linkage analysis indicated that the order of loci is C-6, Gpt-1, Gdc-1, and that the map distances are 25.9±4.9 between C-6 and Gpt-1, and 36.4±5.5 between C-6 and Gdc-1. Since Gdc-1 is more distal than Gpt-1, and C-6 is 26 cM away from Gpt-1, it is estimated that the C-6 is proximal to the centromere. In addition, a new C6 form found in AKR mice is described. We propose the designation C6B for it and C-6 ^b for the allele encoding C6B.Abbreviations used in this paper IEF isoelectric focusing - GPT glutamic-pyruvic transaminase - GDC L-glycerol 3-phosphate dehydrogenase - cM centimorgan     

Distribution of Leu 7 (HNK-1) antigen in human digestive organs: an immunohistochemical study with monoclonal antibody

Summary Leu 7 immunoreactivity was demonstrated with the indirect peroxidase-labelled antibody method on frozen and paraffin-embedded tissue sections of human digestive organs. Anti-Leu 7 monoclonal antibody, which allegedly detects mononuclear cells with natural killer or killer activity, recognized lymphoid cells among intestinal epithelial cells and in the germinal centres of solitary lymphoid follicles of small and large intestine, and a few in gallbladder, liver and the lamina propria of the intestine. In addition, peripheral nerve fibres, endocrine cells in the gut and pancreas and carcinoid and islet cell tumours were also positively stained. At the ultrastructural level, Leu 7 antigen was localized on the plasma membrane of granulated lymphoid cells in the gut mucosa and on the secretory granules of intestinal endocrine cells. In normal pancreas, Leu 7 immunoreactivity was demonstrated in most cells containing pancreatic polypeptide and in many cells containing somatostatin or glicentin. Insulin-containing cells, however, lacked Leu 7 immunoreactant. These findings were obtained in both frozen sections and paraffin-embedded sections. The possible cross-reactivities of monoclonal antibodies are discussed as they raise an important caveat in immunohistochemical studies using these antibodies.