"The nuclear envelope,a meiotic jack-of-all-trades"

The nucleus is one of the membrane-bound organelles that are a distinguishing feature between eukaryotes and prokaryotes. During meiosis, the nuclear envelope takes on functions beyond separating the nucleoplasm from the cytoplasm. These include associations with meiotic chromosomes to mediate pairing, being a sensor for recombination progression, and supportive of enormous nuclear growth during oocyte formation. In this review, we highlight recent results that have contributed to our understanding of meiotic nuclear envelope function and dynamics.     

Genomic potential of Marinobacter aquaeolei, a biogeochemical "opportunitroph"

The genus of Marinobacter is one of the most ubiquitous in the global oceans and assumed to significantly impact various biogeochemical cycles. The genome structure and content of Marinobacter aquaeolei VT8 was analyzed and compared with those from other organisms with diverse adaptive strategies. Here, we report the many "opportunitrophic" genetic characteristics and strategies that M. aquaeolei has adopted to promote survival under various environmental conditions. Genome analysis revealed its metabolic potential to utilize oxygen and nitrate as terminal electron acceptors, iron as an electron donor, and urea, phosphonate, and various hydrocarbons as alternative N, P, and C sources, respectively. Miscellaneous sensory and defense mechanisms, apparently acquired via horizontal gene transfer, are involved in the perception of environmental fluctuations and antibiotic, phage, toxin, and heavy metal resistance, enabling survival under adverse conditions, such as oil-polluted water. Multiple putative integrases, transposases, and plasmids appear to have introduced additional metabolic potential, such as phosphonate degradation. The genomic potential of M. aquaeolei and its similarity to other opportunitrophs are consistent with its cosmopolitan occurrence in diverse environments and highly variable lifestyles.     

Chronomes, time structures, for chronobioengineering for "a full life"

Week-long or longer monitoring of blood pressure and heart rate, coupled to time-structure analyses, can help detect disease-risk elevations, as a warning of the need for a preventive prehabilitation. Within the normal range of physiologic variation, computer methods quantify time structures, or chronomes, that can serve as reference values. The major applied purpose for mapping chronomes is the detection of disease-risk syndromes such as blood pressure "overswinging" and heart rate "underswinging." Too much blood pressure variability (circadian hyperamplitude tension; CHAT), is a risk factor for vascular disease. Other risk syndromes are chronome alterations of heart rate variability (CAHRVs), consisting of a loss of "jitter", i.e., a reduced standard deviation of heart rate or of alterations in the spectral element of the heart-rate-variability chronome, such as in the correlation dimension, an endpoint of deterministic chaos. These alterations can again serve for prehabilitation. On the basic side, the spectral element of the heart-rate-variability chronomes extends from focus on the heartbeat's period of about 1 second to periods in heart rate and its standard deviation that are numerical equivalents of about 10.5- and about 21-year cycles of solar activity. A seemingly unnatural physiologic rhythm or pattern (such as one of 81.6 hours) may correspond numerically to a purely physical environmental rhythm. For example, interplanetary magnetic storms, with their cycles as external chronome components, trigger myocardial infarctions, strokes, and traffic accidents. The systematic monitoring of external rhythms along with physiologic ones for the concurrent analysis of rhythms with longer and longer periods could detect alterations anywhere in and between the 1 cycle/sec and the 1 cycle/10.5- or 21-years regions of the spectrum. Chronobiomimetic engineering for discovering both instantaneous and long-term chronorisk alterations can provide warnings of increased risk. If risk-lowering therapy is then instituted automatically, instrumented health care will be extended beyond the pacemaker-cardioverter-defibrillator, which focuses on the frequency of 1 cycle/sec. Instrumentation that automatically detects blood pressure that varies too much and heart rate that varies too little is needed for prompting prophylactic CHAT and CAHRV treatment. A database of reference values that can be used for chronodiagnosis is now accumulating.     

Evaluation of a semi-closed underwater breathing apparatus, the "eOBA"

Semi-closed underwater breathing apparatus has commonly been used among military and commercial divers, but never available for recreational divers because of complicated operations, difficulties of maintenance, and expensiveness. Nippon Sanso K.K. has newly deployed a semi-closed circuit underwater breathing apparatus called the "eOBA". It is especially designed for divers to enjoy shallow (max. 5 m) and short (10 min.) dives. This eOBA was evaluated from manned testing based on physiological requirements for the apparatus. Four male and four female subjects participated in the three test trials. Subjects maintained their position at the depths of 2 m and 4 m for 10 min. and e exercised on the underwater ergometer at the depth of 2.4 m. The pressure at the mouthpiece, tidal volume, breathing rate, the inspired O2 level, CO2 level were monitored on breath-by-breath basis. The inspired O2 level ranged from 45% (at exercise) to 60% (at rest) and the inspired CO2 level were kept less than 1.0% in the most cases. The P-V loop indicated relatively little external work of breathing (less than 0.1 kg.m/l) and allowable peak pressures (less than 25cmH2O). Results show that the eOBA meets the standards sufficiently under the test conditions and is a safe apparatus for recreational divers if it is properly used.     

The proteome of Mycoplasma pneumoniae, a supposedly "simple" cell

This review covers progress in proteome research on Mycoplasma pneumoniae made over the last 5 years. This bacterium is one of the smallest known self-replicating bacteria. With fewer than 700 proposed proteins, it is well suited to a comprehensive proteome analysis. While all of the proposed genes are transcribed, thus far 620 proteins, about 90% of the predicted proteome, have been identified experimentally. To study the proteome organization of M. pneumoniae, 178 soluble protein complexes were isolated under non-denaturing conditions by tandem affinity chromatography and their composition determined by SDS-PAGE and mass spectrometry. The 62 homomultimeric and 116 heteromultimeric protein complexes could be classified according to 12 different COG functional categories. The complexes interacted with each other to some extent, forming larger assemblies. Protein complexes that were large enough and had specific structures (e.g. ribosomes or DNA-dependent RNA polymerase) were visible and countable in their natural environment by cryo-electron tomography. In addition to characterization of the soluble complexes, the analysis of the Triton X-100 insoluble fraction has a major role in the elucidation of the cytoskeleton-like structure, because by analogy with eukaryotic cells, almost all of the structural proteins involved in its formation, and enriched sub-cellular structures, can be found in this fraction.     

Development of a semi-closed underwater breathing apparatus, the "eOBA"

Nippon Sanso K.K developed a compact semi-closed underwater breathing apparatus, the eOBA. It consists of a mouthpiece, manifold with a purge valve, two spring-loaded flexible tubes, a small CO2 absorbent canister (net wt. = 190g), and two compact high pressure bottles (50ccx2: 190kg/cm2: 80%O2, 20%N2) with a regulator which supplies the gas at the constant flow rate of 1.5 l/min and lasts for 10 min. Thus, a counterlung is not incorporated. However, spring-loaded tubes act as a counterlung since its volume increases to 3.5 l when fully inflated. Dives to a depth of 5m are also recommended because of no bypass valve. This new eOBA was tested using the mechanical breathing machine and CO2 supply system to the circuit. For the various combinations of tidal volumes (0.5-2.5 l) and respiratory rates (10-20 breaths/min), the pressure at the mouthpiece, respiratory volume and the CO2 level were continuously monitored. The CO2 absorption rates were then calculated. The thin sloping P-V loops demonstrate that the eOBA is a flow dependent type of apparatus. It was found that the external work of breathing (0.1 kg.m/l at 30 l/min) were allowable. The CO2 absorption rates were sufficient when minute ventilation increased to 30 l/min. Thus, results show that the eOBA must be suitable for shallow and short dives.     

Three-dimensional model for stellacyanin, a "blue" copper-protein.

A three-dimensional model of the "blue" copper-glycoprotein stellacyanin from Rhus vernicifera has been derived by computer graphics, energy minimization and molecular dynamics techniques. The initial atomic co-ordinates were obtained by making substitutions and insertions in the known structure of another blue copper-protein, cucumber basic protein (CBP), which is 46% homologous with stellacyanin and has similar spectroscopic properties. An important difference between CBP and stellacyanin is that the latter lacks methionine, a residue that forms an exceptionally long bond to the copper atom in all blue copper-proteins of known structure. In the aligned amino acid sequences, stellacyanin has glutamine 97 at the position that corresponds to the copper-binding methionine 89 in CBP. The hypothesis that the copper atom in stellacyanin is co-ordinated by the side-chain functional groups of histidine 46, cysteine 87, histidine 92 and glutamine 97 leads to a model that enables the spectroscopic properties, redox potential and electron-transfer kinetics of the protein to be rationalized. The present model for stellacyanin is more plausible than an antecedent model derived from the structure of plastocyanin. This demonstrates that the output from molecular modeling calculations is strongly dependent on the input, and that sequence homology with the target molecule is an important criterion for the selection of a starting model.     

Sarcolemmal and mitochondrial effects of a KATP opener, P-1075, in "polarized" and "depolarized" Langendorff-perfused rat hearts

We investigated consequences of cardiac arrest on sarcolemmal and mitochondrial effects of ATP-sensitive potassium channel (KATP) opener, P-1075, in Langendorff-perfused rat hearts. Depolarised cardiac arrest (24.7 mM KCl) did not affect glibenclamide-sensitive twofold activation of rubidium efflux by P-1075 (5 microM) from rubidium-loaded hearts, but eliminated uncoupling produced by P-1075 in beating hearts: 40% depletion of phosphocreatine and ATP, 50% increase in oxygen consumption, and reduction of cytochrome c oxidase. Depolarized cardiac arrest by calcium channel blocker, verapamil (5 microM), also prevented uncoupling. Lack of P-1075 mitochondrial effects in depolarized hearts was not due to changes in phosphorylation potential, because 2,4-dintrophenol (10 microM) reversed the [PCr]/[Cr] increase and Pi decrease, characteristic of KCl-arrest, but did not restore uncoupling. In agreement with this conclusion, pyruvate (5 mM) increased [PCr]/[Cr] and decreased Pi, but did not prevent uncoupling in beating hearts. A decrease in mean [Ca2+] in KCl-arrested hearts could not account for lack of P-1075 mitochondrial effects, because calcium channel opener, S-(-)-Bay K8644 (50 nM), and beta-agonist, isoproterenol (0.5 microM), did not facilitate uncoupling. In contrast, in adenosine (1 mM)-arrested hearts (polarized arrest), P-1075 caused 40% phosphocreatine and ATP depletion. In isolated rat liver mitochondria, P-1075 (20 microM) decreased mitochondrial membrane potential (DeltaPsi) by approximately 14 mV (demonstrated by redistribution of DeltaPsi-sensitive dye, rhodamine 800) in a glibenclamide-sensitive manner. We concluded that cell membrane depolarization does not prevent activation of sarcolemmal KATP by P-1075, but it plays a role in mitochondrial uncoupling effects of P-1075.     

Purification and Characterization of a Membrane-Bound Enkephalin-Forming Carboxypeptidase, "Enkephalin Convertase"

Enkephalin convertase, the enkephalin-synthesizing carboxypeptidase B-like enzyme, has been purified to apparent homogeneity from bovine pituitary and adrenal chromaffin granule membranes. The membrane-bound enkephalin convertase can be solubilized in high yield with 0.5% Triton X-100 in the presence of 1 M NaCl. Extensive purification is achieved by affinity chromatography with p-aminobenzoyl-L-arginine linked to Sepharose 6B. Enzyme purified from both pituitary and adrenal chromaffin granule membranes shows a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with an apparent molecular weight of 52,500, whereas enkephalin convertase purified from soluble extracts of these tissues has an apparent molecular weight of 50,000. The regional distribution of the membrane-bound enzyme in the rat brain differs from that of the soluble enzyme. While the soluble enzyme shows 10-fold variations, resembling somewhat the enkephalin peptides, membrane-bound enkephalin convertase is more homogeneously distributed throughout the brain. In rat pituitary glands, membrane-bound enzyme activity is similar in the anterior and posterior lobes, whereas the soluble enzyme is enriched in the anterior lobe. Membrane-bound and soluble forms of enkephalin convertase isolated from either bovine pituitary glands or adrenal chromaffin granules show identical substrate and inhibitor specificities. As with the soluble enzyme, membrane-bound enkephalin convertase hydrolyzes [Met]- and [Leu]enkephalin-Arg6 and -Lys6 to enkephalin, with no further degradation of the pentapeptide.     

实施“三高”教学,创建“微生物学实验”课程新体系

深化微生物实验教学改革,实施“高起点、高效率、高质量”教学,从调整教学内容,改革教学方法,建立和完善考评机制入手,创建“以基础知识和基本技能为基础性内容,以创新能力培养为核心内容”的微生物学实验课程新体系,激发了学生对微生物学实验的兴趣,培养了学生独立思考、创新思维和综合动手的能力。     

Isolation of Mitochondria from Orobanche, a "Total" Root Angiospermic Parasite

Mitochondria were isolated from the scapes of Orobanche cernua. On the basis of comparative studies with four media, a mannitol grinding medium was selected at a slightly alkaline pH and supplemented with EDTA, metabisulphite, BSA and soluble PVP. Various intermediates of the Krebs cycle, pyruvate, glutamate and NADH were oxidized by the isolated particles, though with different efficiencies. The rate of oxidation was steady for every intermediate tested over a 60 min period, except for NADH. In the majority of cases, the oxidation was accompanied by efficient coupling with phosphorylation. Antimycin A led to significant reduction in the oxygen uptake in the presence of succinate and complete suppression of phosphate-esterification. The amount of protein recovered in the mitochondrial fraction was comparable with the reported recoveries from a number of plant tissues. The biochemical integrity of mitochondria in the particulate fraction appeared possible from: (a) the absence of respiratory response to added cytochrome c during the oxidation of succinate, (b) insignificant oxidation of exogenously added pyruvate in the absence of sparker and (c) the ability to satisfactorily couple phosphorylation to the oxidation of a number of substrates.     

Minimally disturbed,multicycle, and reproducible synchrony using a eukaryotic "baby machine"

A eukaryotic "baby machine" has been developed that produces synchronized cultures that display up to four synchronous cell cycles. That such cells can be produced implies that methods unable to produce successive synchronized cell cycles may not actually synchronize cells. But most important, the baby machine method now opens the way for the study of the cell cycle of minimally disturbed, artifact-free, well-synchronized, mammalian cells.     

"Action potentials" in Neurospora crassa, a mycelial fungus.

Occasional spontaneous "action potentials" are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5-20 mV depolarized from the normal resting potential (near-180 mV). The "action potentials" are long-lasting, 1-2 min and at the peak reach a membrane potential near-40 mV. A 2-to 8-fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H+ or Cl- and (b) periodic decreases in activity of the major electrogenic pump (H+) or the Neurospora membrane, coupled with a nonlinear (inverse signoid) current-boltage relationship. Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.