Dynamin Isoforms Decode Action Potential Firing for Synaptic Vesicle Recycling

Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways, having distinct rate and time constants with physiological AP frequencies. Dynamin 3 resupplied the readily releasable pool with slow kinetics independently of the AP frequency but acted quickly, within 20 ms of the incoming AP. Under high-frequency firing, dynamin 1 regulated recycling to the readily releasable pool with fast kinetics in a slower time window of greater than 50 ms. Dynamin 2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.     

Proteomic signature of human cancer cells

We assessed proteomic profiles as biomarkers for monitoring cell phenotypes. Protein expression profiles were obtained by fluorescence two-dimensional difference gel electrophoresis (2-D-DIGE), in which quantitative ability is improved by labeling proteins with fluorescent dyes prior to electrophoresis. Integrated protein spot intensities were analyzed by a statistical approach. The proteomic data of two groups of cell lines: (1) adenocarcinoma (AC) cell lines derived from lung, pancreas and colon tissues and (2) lung cancer cell lines with different histological backgrounds, including AC, squamous cell carcinoma and small cell carcinoma, were assessed on the basis of prior biological information. Hierarchical clustering analysis and principal component analysis were used to divide the cell lines into subgroups on the basis of similarities between their protein expression profiles. The majority of cell lines were grouped according to their organ of origin or histological background. A machine-learning algorithm selected 32 protein spots that were responsible for the classification. The results indicate that proteomic data generated by 2-D-DIGE can provide a signature of essential cell phenotypes, suggesting that it might be possible to apply this technique to developing tumor markers that could identify the organ of origin of metastatic tumors and contribute to the differential diagnosis of lung cancer.     

Mitochondrial dysfunction and increased reactive oxygen species impair insulin secretion in sphingomyelin synthase 1-null mice

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic β-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic β-cells.     

Neuronal regulation by which microglia enhance the production of neurotrophic factors for GABAergic, catecholaminergic, and cholinergic neurons

A phenomenon-in which microglia are activated in axotomized rat facial nucleus suggests that a certain neuronal stimulus triggers the activation of microglia. However, how the microglial characteristics are regulated by this neuronal stimulus has not previously been determined. In this study, therefore, the regulation of microglial properties by neurons was characterized in vitro from a neurotrophic perspective. To evaluate the neurotrophic effects of microglia stimulated with neurons, the effects of conditioned medium (CM) of microglia stimulated with neuronal CM (NCM) were assessed in neuronal cultures. The amounts of tyrosine hydroxylase (TH) in neuronal culture exposed to CM of microglia stimulated with NCM was much more than those in neurons exposed to CM of control microglia, suggesting that neuronal stimulus enhances the production of neurotrophic factors for catecholaminergic neurons in microglia. Therefore, the neurotrophic effects of CM of microglia stimulated with NCM were analyzed in detail. The immunocytochemical and biochemical experiments revealed that the CM of microglia stimulated with NCM enhances the survival/maturation of GABAergic and catecholaminergic neurons. The levels of choline acetyltransferase specific to cholinergic neurons also significantly increased in response to stimulation with the same microglial CM. These results allowed us to investigate the production of neurotrophic factors in the CM of microglia stimulated with NCM. The results indicated that NCM induces nerve growth factor (NGF), and enhances neurotrophin-4/5 (NT-4/5), transforming growth factor beta1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), interleukin-3 (IL-3), and IL-10 in microglia. The promoted neurotrophic effects of CM of microglia stimulated with NCM were significantly abrogated by deprivation of neurotrophic factors by means of an immunoprecipitation method. Taken together, neuronal stimulus was found to activate microglia to produce more neurotrophic factors as above, thereby changing microglia into more neurotrophic cells.     

Chloroplast DNA variation inDesmodium subgenusPodocarpium (Leguminosae): Infrageneric phylogeny and infraspecific variations

Chloroplast DNA (cpDNA) restriction site variation was examined in five species ofDesmodium subgenusPodocarpium (Leguminosae; Papilionoideae; Desmodieae). Twenty four phylogenetically informative cpDNA mutations were scored. The cladistic analysis of characters based on the 24 mutations resulted in the most parsimonious tree which supports the monophyly of the subgenus.Desmodium elegans of subgenusDollinera was the sister group of subgenusPodocarpium in this tree. The groupings obtained from the cpDNA characters were consistent with the present infrageneric classification system for the subgenus except for the infraspecific taxa ofD. podocarpum. Three groups withinD. podocarpum, which were incongruent with the infraspecific classification of the species, were distinguished by a total of four site mutations. The first group consisted of subsp.podocarpum, subsp.fallax, and subsp.oxyphyllum var.oxyphyllum; the second subsp.oxyphyllum var.oxyphyllum; and the last subsp.oxyphyllum var.oxyphyllum and var.mandshuricum.     

An overview on chlorophylls and quinones in the photosystem I-type reaction centers

Minor but key chlorophylls (Chls) and quinones in photosystem (PS) I-type reaction centers (RCs) are overviewed in regard to their molecular structures. In the PS I-type RCs, the prime-type chlorophylls, namely, bacteriochlorophyll (BChl) a′ in green sulfur bacteria, BChl g′ in heliobacteria, Chl a′ in Chl a-type PS I, and Chl d′ in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a′)₂ and (BChl g′)₂ in anoxygenic organisms, or heterodimers, Chl a/a′ and Chl d/d′ in oxygenic photosynthesis. Conversions of BChl g to Chl a and Chl a to Chl d take place spontaneously under mild condition in vitro. The primary electron acceptors, A ₀, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, whereas the side chains may have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic PS I.     

A high-throughput screen for inhibitors of the prolyl isomerase,Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation

The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.     

Geographic variation in critical photoperiod for diapause induction and its temperature dependence in Hyphantria cunea Drury (Lepidoptera: Arctiidae)

The fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae), was introduced from North America to Japan half a century ago. The critical photoperiod for diapause induction and its temperature dependence, as defined by the difference in the critical photoperiod between 20 and 25°C, were investigated in order to understand the mechanisms behind a shift from bi- to trivoltine life cycles. The critical photoperiod for diapause induction was shorter in the southern trivoltine populations than in the northern bivoltine populations, and this was more marked at 25°C than at 20°C. Although the critical photoperiod showed a positive correlation with the original latitude, the correlation was relatively low at both temperatures. Conversely, temperature dependence of the critical photoperiod for diapause induction correlated negatively with the original latitude. The trivoltine populations showed greater temperature sensitivity than the bivoltine populations. These results suggest that an increase in temperature sensitivity of the diapause response to photoperiods was involved in the shift to a trivoltine life cycle. The crossing experiments suggested that the photoperiodic control of diapause induction and its temperature dependence are under polygenic control without sex-linkage. Received: 29 October 1996 / Accepted: 26 February 1997     

Phenotypic and genetic characterization of the Atp7a(Mo-Tohm) mottled mouse: a new murine model of Menkes disease

Mottled Tohoku (Atp7a(Mo-Tohm) or Mo(Tohm)) is an X-linked mutation with mottled pigmentation in heterozygous (Mo(Tohm)/+) females and is embryonic lethal at E11 in hemizygous (Mo(Tohm)/Y) males. Copper levels were low in the brain and high in the intestine of Mo(Tohm) mice. Two congenic strains with ICR or C57BL/6 (B6) background were produced for genetic and phenotypic analyses and revealed that Mo(Tohm)/+ females with ICR background survived until adulthood, while most with B6 background died within 2 days after birth. The Mo(Tohm)/Y males with both backgrounds died at around E11. Massive hemorrhage was shown in the yolk sac cavity with irregular attachment between the mesoderm and the endothelial cells of blood vessels in the embryos at E10.5, suggesting that this irregular attachment causes embryonic lethality. The Mo(Tohm) mutant had a 1440-bp deletion between intron 22 and exon 23 of the Atp7a gene. Mo(Tohm)/Y males with the wild-type Atp7a cDNA transgene were rescued from embryonic lethality, confirming that the Mo(Tohm) mutant is caused by the defect in the Atp7a gene. This mutant mouse is the most severe model of human Menkes disease in mottled mice established to date and one of the useful models for understanding the gene function of Menkes disease.