Lipase-mediated homotopic and heterotopic double resolutions of a planar chiral organometallic alcohol

Summary (±)-Tricarbonyl ⁶-3-methylbenzyl alcohol)chromium was resolved to of 100%e.e. and of 92%e.e. by lipase-catalyzed transesterifications arranged in homotopic and heterotopic double resolutions.     

Energy metabolism of sea urchin spermatozoa, with phosphatidylcholine as the preferred substrate

Phosphatidylcholine content in the spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, decreased rapidly during incubation with sea water. Sea urchin sperm contained approx. 85% phospholipid in total lipid. Phosphatidylcholine (PC) was the principal lipid. Other phospholipids, however, remained at constant levels during incubation. Although the free fatty acid content gradually increased following dilution of dry sperm in sea water, the amounts of triacylglycerol and cholesterol ester were extremely low. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in PC to be polyenoic. PC composed in part of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Furthermore, 1-palmitoyl-2-[1-14C]linoleoylphosphatidylcholine was transformed to 14C-labelled free fatty acid in a subcellular system. Thus, possibly, phospholipase A2 is present in sea urchin sperm. Also, [1-14C]oleic acid was immediately oxidized to 14CO2 by sperm. It is thus concluded that sea urchin sperm use phosphatidylcholine as a substrate for energy metabolism.     

Establishment of a Sensitized Immunoblotting Method for Measuring Plant Tubulin Content

A sensitized immunoblotting method was established for measuringsmall amounts of plant tubulin. The method involves electrophoretictransfer of protein including tubulin from SDS-polyacrylamidegels onto nitrocellulose paper, successive incubation of thenitrocellulose paper with a mouse monoclonal antibody to - orß-tubulin of chicken brain, an antibody to mouse IgGas the second antibody and the radioactive iodinated proteinA, and determination of the radioactivities of the bands onthe nitrocellulose paper thus probed. The radioactivities werelinearly proportional to the amounts of - or ß-tubulinfrom dark-grown Vigna mungo seedlings within a range of 4 to56 ng or of 4 to 32 ng, respectively. This method was used to estimate the tubulin contents of severalplant species using Vigna tubulin as a standard. -Tubulin contentsthus estimated were 25, 9, 19 and 11 µg-equivalents ofVigna tubulin per mg protein for Vigna seedlings, Daucus suspensioncells, Catharanthus suspension cells and Mougeolia cells, respectively.ß-Tubulin contents of Vigna, Daucus, Catharanthusand Mougeotia cells were 29, 10, 13 and 5 µg-equivalentsof Vigna tubulin per mg protein, respectively. (Received August 6, 1985; Accepted December 5, 1985)     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

Fibrinolysis: Its initiation and regulation

Fibrinolytic system is one of the major proteolytic pathways in vivo and primarily responsible for dissolution of thrombi. Two enzymes are primarily involved in this proteolytic system; plasminogen activator (PA) and plasmin. Plasmin is formed by a limited proteolysis of plasminogen by PA, which is mainly synthesized by and secreted from vascular endothelial cells. This proteolytic process proceeds physiologically only on the surface of fibrin. Thus, initiation and progression of the fibrinolytic process depend on the function of endothelial cells and fibrin formation. Endothelial cells may also synthesize and excrete PA inhibitor (PAI) which inhibits immediately, PA once released. The rates of synthesis and excretion of PA and PAI by endothelial cells are regulated by various factors. Among them, thrombin stimulates the release of PA whereas activated protein C may decrease the release of PAI. Thus, both enzymes enhance fibrinolytic potential. PA which has escaped from inhibition by PAI binds to fibrin. ₂-Plasmin inhibitor (₂PI) inhibits the binding of plasminogen to fibrin, thereby suppressing this fibrin-associated plasminogen activation. A part of ₂PI is cross-linked to fibrin by activated factor XIII when fibrin is formed, and the ₂PI thus cross-linked to fibrin inhibits in situ plasmin formed on fibrin. Thus, ₂PI as well as PAI plays a central role in inhibition of fibrinolysis.     

Response properties of FM-FM combination-sensitive neurons in the auditory cortex of the mustached bat

Summary For echolocation, the mustached bat,Pteronotus parnellii rubiginosus, emits orientation sounds (pulses) and listens to echoes. Each pulse is made up of 8 components, of which 4 are constant frequencies (CF_1–4) and 4 are frequency-modulated (FM_1–4). Target-range information, conveyed by the time delay of the echo FM from the pulse FM, is processed in this species by specialized neurons in a part of the auditory cortex known as the FM-FM area. These cortical neurons are responsive to pulse-echo pairs at specific echo delays (Fig. 1). The essential components in the sound pair include the pulse FM₁ followed by an echo FM_n (n=2, 3 or 4). Downward sweeping FM₁-FM_n sounds that are similar to those the animal naturally hears during echolocation are the most effective in evoking facilitative responses. Most FM-FM neurons, however, still exhibit facilitative responses to stimulus pairs consisting of upward sweeping FM sounds and/or pure tones at frequencies found in FM sweeps (Figs. 2 and 3). The magnitude of facilitation is altered by changes in echo rather than pulse amplitude (Figs. 5 and 6). Neurons characterized by shorter best delays (or echoes from closer targets) do not require larger best echo amplitudes for facilitation.Abbreviations CF constant frequency - FM frequency modulation - H _n CF — FM harmonics of the mustached bat biosonar signal - CF _n CF components of the harmonics - FM _n FM components of the harmonics - PCF _n pulse CF_n - ECF _n echo CF_n - PFM _n pulse FM_n - EFM _n echo FM_n - PH _n pulse H_n - EH _n echo H_n - BA best amplitude for facilitation - BD best delay for facilitation - PST peri-stimulus-time - PSTC peri-stimulus-time-cumulative - dB SPL dB re 20 Pa     

Dormancy inDioscorea: Different temperature adaptation of seeds,bulbils and subterranean organs in relation to north-south distribution

Temperature dependencies of sprouting and germination were compared for subterranean perennating organs and seeds of ten closely related species of the genusDioscorea (Dioscoreaceae), a group of monocotyledonous summer perennials which are distributed from the tropics to the subarctic. The species used wereD. nipponica Makino,D. tokoro Makino,D. japonica Thunb.,D. tenuipes Franch. et Savat.,D. septemloba Thunb.,D. quinqueloba Thunb.,D. izuensis Akahori,D. bulbifera L. f.spontanea (Makino) Makino et Nemoto,D. pentaphylla. L. andD. alata L.; they are distributed from cold northern areas to warmer southern areas approximately in this order in and around Japan. Bulbil sprouting was also studied in those forming bulbils. Subterranean organs of the tropical species sprouted faster without any prior temperature treatment, whereas those of species from the more northern areas sprouted after prechilling. Northern species required longer, periods of prechilling for sprouting. On the other hand, with seeds or bulbils, the southern species required longer periods of prior temperature treatment for dormancy breaking. This difference in the length of dormant periods between seeds or bulbils and subterranean organs among the ten species may be related to their size and position of shedding; seeds or bulbils are small and are shed on the ground surface, whereas subterranean organs are large and are located below the surface. It is important to determine in other perennials whether the above relation between dormant features of seeds or bulbils and subterranean organs are common properties or not.     

Prophage Origin of a Virulent Phage Appearing on Fermentations of Lactobacillus casei S-1

For protection from the abnormal fermentation of Lactobacillus casei S-1 caused by contamination of a virulent phage, FSV, the origin of this phage was studied. Morphologies, viral structural proteins, and DNA structures of three independent isolates of FSV were compared with those of FSW, which is lysogenized in strain S-1. The results showed (i) that the morphology of FSV phages is indistinguishable from that of FSW and (ii) that all viral structural components found in FSW are present in the particles of FSV's. In addition, restriction endonuclease analyses of viral DNA showed that the HindIII-digested fragments of FSW DNA, the sum of which covered at least 94.7% of this phage genome, were conserved in the FSV DNA digests. Results of Southern filter hybridization of the S-1 and prophage-cured cell (C239) DNAs with FSV DNA as a probe revealed that C239 had lost most of the FSV DNA sequence, whereas S-1 had about one copy of the FSV DNA sequence. These results indicate that virulent phage FSV is derived from the lysogenized phage FSW. Therefore, the appearance of FSV can be eliminated by using the prophage-cured derivative of S-1.     

Electron microscopic histochemistry of acetylcholinesterase of rat brain by Karnovsky's method

Summary Karnovsky's electron microscopic acetylcholinesterase method was successfully applied to rat brain fixed by vascular perfusion with either 2% glutataldehyde or 4% formaldehyde. 2% glutaraldehyde showed better fine structure but worse preservation of the enzyme than 4% formaldehyde.In the neuropil of the caudate nucleus, locus coeruleus and dorsal nucleus of the vagus, AChE activity was most intensely demonstrated on the plasma membranes of preterminal axons and somewhat less strongly on those of axon terminals and contacting dendritic branches. The axoplasm and synaptic vesicles were usually negative, while the cytoplasm and neurotubules of the dendritic branches showed some activity. In the nodule and uvula of the cerebellum moderate activity was exhibited on the synaptic contacts between the mossy fiber endings and granule cell dendrites. In the hypothalamus and other autonomic regions the characteristic coexistence of AChE and granulated vesicles of axon terminals could be demonstrated.In the perikaryon of positive nerve cells, AChE was observed strongly in the cytoplasm, disseminated irregularly or attached to the endoplasmic reticulum, while it was absent in the mitochondria and lysosomal dense bodies.     

Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.