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The Hippo and mTOR signaling cascades are major regulators of cell growth and division. Aberrant regulation of these pathways has been demonstrated to contribute to gliomagenesis and result in enhanced glioblastoma proliferation and invasive characteristics. Several crosstalk mechanisms have been described between these two pathways, although a complete picture of these signaling interactions is lacking and is required for effective therapeutic targeting. Here we report the ability of mTORC2 to directly phosphorylate YAP at serine 436 (Ser436) positively regulating YAP activity. We show that mTORC2 activity enhances YAP transcriptional activity and the induction of YAP-dependent target gene expression while its ablation via genetic or pharmacological means has the opposite affects on YAP function. mTORC2 interacts with YAP via Sin1 and mutational analysis of serine 436 demonstrates that this phosphorylation event affects several properties of YAP leading to enhanced transactivation potential. Moreover, YAP serine 436 mutants display altered glioblastoma growth, migratory capacity and invasiveness both in vitro and in xenograft experiments. We further demonstrate that mTORC2 is able to regulate a Hippo pathway resistant allele of YAP suggesting that mTORC2 can regulate YAP independent of Hippo signaling. Correlative associations between the expression of these components in GBM patient samples also supported the presence of this signaling relationship. These results advance a direct mTORC2/YAP signaling axis driving GBM growth, motility and invasiveness.

The purpose of this paper is to prove the reliability of artificial hip joints’ surface generated by the electrolytically treated grinding process; the evaluation was carried out in a hip simulator and animal tests. The evaluation indicated that the fabricated hyper-hemispherical shaped artificial hip joints presented a high quality smoother surface than 10 nm in Ra. The fabricated surface also showed its ability to prevent crystalline phase transition during the hip simulation test, suggesting that the stably modified layer was generated via grinding process. The results of animal test showed that the fabricated surface could have a highly-improved biocompatibility.

mTORC2 is a multiprotein kinase composed of mTOR, mLST8, PRR5, mSIN1 and Rictor. The complex is insensitive to rapamycin and has demonstrated functions controlling cell growth, motility, invasion and cytoskeletal assembly. mTORC2 is the major hydrophobic domain kinase which renders Akt fully active via phosphorylation on serine 473. We isolated Hsp70 as a putative Rictor interacting protein in a yeast two-hybrid assay and confirmed this interaction via co-immunoprecipitation and colocalization experiments. In cells expressing an antisense RNA targeting Hsp70, mTORC2 formation and activity were impaired. Moreover, in cells lacking Hsp70 expression, mTORC2 activity was inhibited following heat shock while controls demonstrated increased mTORC2 activity. These differential effects on mTORC2 activity were specific, in that mTORC1 did not demonstrate Hsp70-dependent alterations under these conditions. These data suggest that Hsp70 is a component of mTORC2 and is required for proper assembly and activity of the kinase both constitutively and following heat shock.

The applicability of numerical simulation to the sheet metal drawing by Maslennikov's technique is examined. A circular cup drawing process is numerically simulated using the dynamic explicit finite element code DYNA3D. The effect of the forming parameters, such as dimension and hardness of the rubber ring, frictional coefficient of the interface between the sheet and the rubber ring and mechanical properties of the sheet, on the sheet deformation is investigated. The increment in the cup depth obtained by each drawing operation tends to decrease with the number of times of the operations. The effect of frictional coefficient of the interface between the sheet and the rubber ring on the sheet deformation is not prominent. The sheet drawing with a harder rubber produces the deeper cup. On the other hand, the sallower cup is obtained with a thicker rubber ring. These demonstrated deformation behaviors are considered qualitatively adequate, therefore, it is concluded that the numerical simulation may be helpful in determining the forming conditions for the sheet metal drawing by Maslennikov's technique.

Fluorescence characteristics of Yb 3+ :Ce 3+ :Er 3+ -codoped fluoride glasses is reported. Yb 3+ codoping improved the excitation efficiency at 980 nm pumping and Ce 3+ codoping improved the branching ratio of the Er 3+ 4 I 11/2 – 4 I 13/2 transition level, resulting in an enhancement in the fluorescence quantum yield at 1550 nm. The energy transfer dynamics of the Yb 3+ :Ce 3+ :Er 3+ -codoped fluoride glass was also analyzed based on a rate equation model. The separately measured energy transfer rates between Yb 3+ and Er 3+ , and between Er 3+ and Ce 3+ agreed to some extent with the theoretical simulation based on the rate equation model at a lower Yb 3+ concentration (

Dynamics of forward energy transfer and backward energy transfer processes in fluorozirconate glass doubly doped with Yb3+ and Er3+ have been explained by “rate equation” and “fluorescence transfer function” models. From the rate equation analysis, macroscopic energy transfer parameters, the cross-relaxation coefficients, were determined to be 1.36×10−17 and 3.38×10 −17 s −1 cm 3 for the forward energy transfer (Yb3+→Er3+) and the backward transfer (Er3+→Yb3+), respectively. Based on the fluorescence “transfer-function” model, the kinetics of the energy transfer processes between Yb3+ and Er3+ ions has been analyzed, and the microscopic energy transfer parameters of the Yb:Er-codoped system, the critical radii, were derived as 13.0 and 18.0 A for the donor–acceptor (Yb3+→Er3+) and the acceptor–donor (Er3+→Yb3+), respectively. The 1.55 μm band emission property of the rare-earth-codoped fluoride glass systems has been discussed.

We investigated the population structure and spatial pattern of major trees in a subalpine old-growth coniferous forest stand in the Ontake Forest Reserve of central Japan to examine the coexistence strategies of different species. We mapped all stems ≥5 cm in diameter at breast height (dbh) on a 2 ha plot. The stand contained nine woody plant species and 1508 living stems with a combined basal area of 55.6 m2 ha−1. The four major species were divided into two groups based on density and size/age structure. Group A (Abies mariesii and Abies veitchii) had a lower maximum age and higher density than group B, as well as reverse J- or L-shaped dbh distribution of live stems. Species in group B (Picea jezoensis var. hondoensis and Tsuga diversifolia) had very few stems in the understory, higher maximum ages than group A, and bell-shaped dbh distributions. Group B species may compensate for having fewer stems in the understory by having a longer lifespan, while species in group A may compensate for lower longevity by having numerous young stems in the understory. Canopy and understory stems of the four major coniferous species were patchily distributed throughout the plot. The distribution of canopy stems of species in group B was negatively associated with that of group A, but positively associated with understory stems in their own group. Similarly, the distribution of canopy stems of species in group A was negatively associated with that of understory stems of group B species. These results suggest species differences in favourable canopy and forest floor condition. Differences in life history strategies and site preferences may explain the coexistence of these species.

The regeneration mode of major tree species (Chamaecyparis obtusa (Sieb. et Zucc.) Endl., Thujopsis dolabrata Sieb. et Zucc., Chamaecyparis pisifera (Sieb. et Zucc.) Endl., Quercus mongolica Fischer ex Turcz., Magnolia obovata Thunb. and Betula grossa Sieb. et Zucc.) in relation to four canopy conditions (closed evergreen conifer canopy, closed evergreen conifer canopy adjacent to canopy gap (gap-adjacent), canopy gap, and closed deciduous broad-leaved tree (DBL) species canopy) was studied from 1988 to 1998 in a 4-ha permanent plot in the Akasawa Forest Reserve, central Japan. The vertical distribution of stems and the major species composition in four height (canopy; ≥25 m tall overtopping neighboring trees, subcanopy; ≥25 m tall, middle; ≥10 m tall, and understory

Dynamics of major conifer and deciduous broad-leaved tree (DBL) species in an old-growth (ca. 300 years old) Chamaecyparis obtusa (Sieb. et Zucc.) Endlicher forest was monitored using stem mapping method for 10 years (1988–1998) in a 4 ha permanent plot of the Akasawa Forest Reserve in central Japan. The plot was dominated by C. obtusa, followed by Thujopsis dolabrata Sieb. et Zucc. and Chamaecyparis pisifera (Sieb. et Zucc.) Endlicher, which are evergreen conifers, and DBL species such as Quercus mongolica Fischer ex Turcz., Magnolia obovata Thumb., and Betula grossa Sieb. et Zucc.. We focused the dynamics of stems ≥5 cm dbh (diameter at 1.3 m above ground) of these six major tree species. Out of 1443 stems ha−1 of these species which were alive in 1988, 154 were dead and 67 new stems recruited during 10 years. The recruitment rate was lower than the mortality rate for the conifers, but the reverse was almost true for the DBL species; the rates of recruitment and mortality of C. obtusa were 0.05 and 0.27% yr−1, respectively, and those of T. dolabrata were 0.53 and 1.48% yr−1, respectively. The recruitment rate of conifers was clearly lower than that of DBL species. The basal area of the total six major tree species was 64.2 m2 ha−1 at the beginning and increased to 65.9 m2 ha−1 in 1998. The gain rate of basal area was higher than the loss rate for each of the conifers and the DBL species. The gain rate of M. obovata (2.26% yr−1) and the loss rate of T. dolabrata (0.77% yr−1) were highest among major tree species. The mortality of stems was size dependent for all major tree species, which implies that smaller stems had higher mortality. Standing dead was first, especially for DBL species, and stem broken was next dominant state of the stem mortality. The recruitment and mortality of some species related to forest floor conditions or canopy gaps. The DBL species significantly recruited and died under canopy gaps. The mortality of some species depended on the local density of understory stems. These results clearly indicate that this old-growth forest has not still reached a equilibrium state.

A biological method was developed for quantifying and cloning of infectious virions of human immunodeficiency virus type 1 (HIV-1). Virus preparations were mixed with permissive cells for binding, and the cells were cast in an agarose gel. After incubation for 9 days viral particles released from infected cells propagating from each initially infected cell were transferred on nylon membrane and subjected to hybridization using a radioactive HIV-1 DNA probe. Infectious centers of HIV-1 were detected as hybridization spots on autoradiographs regardless of cytopathic effects or syncytium formation. Three different CD4+ cell lines (MT-4, MOLT-4 and U937) and peripheral blood mononuclear cells from healthy donors were used as recipient cells. Infectious virions were recovered from a portion of agarose gel corresponding to each hybridization spot. This assay is suitable for quantifying infectious HIV-1 virions with different cell tropisms and for investigating the relationship between the phenotype and genotype of HIV-1 at a clonal level.

Distributions of line-intensity ratios that correspond to the electron temperature of a laser-created fusion plasma were measured by means of toroidally bent Bragg crystals. Those were determined by seeding the fuel with a tiny amount of argon and by obtaining monochromatic image of Ar+17 (1s−3p) Lyβ and Ar+16 (1 s2−1s3p) Heβ lines. Furthermore, the experimental distribution of the line ratio of Lyβ to Heβ is remarkably different from the post-processed outputs from the one-dimensional implosion code ILESTA-1D with the help of the RATION code.

Summary The authors performed Hallux valgus operations on ten patients in 20 feet using Ilizarov external fixator, which is a useful apparatus for treating severe foot deformities. This fixator consists of a half-ring which is made of carbon, six male or female posts, six wire-fixing bolts, and three olive wires 1.5 mm in diameter. The total weight of the instrument is only about 200 g. Instrument fixation lasted around 5–6 weeks. The method of osteotomy comprised 20 distal soft tissue procedures, and the basal metatarsal osteotomy was fixed by two Kirschner (K)-wires 1.8 mm in diameter. The deformity of the Hallux valgus was successfully corrected in all 20 feet. The authors found that all patients were able to walk without crutches immediately after the operation, and the forefoot transverse arch was reconstructed.

Heme oxygenase (HO) exists as two isoenzymes designated heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). HO-1 has been identified as a heat shock or stress protein and is inducible whereas HO-2 is largely refractory to induction. HO-2 is the predominant isoenzyme in normal brain and appears to have a predominantly neuronal distribution in cerebral cortex. Cortical stab wound injury resulted in HO-1 induction as determined by Western blot analysis. Immunohistochemical analysis suggested that induced HO-1 was largely restricted to reactive astrocytes and macrophage-like cells. Enhanced HO-1 immunoreactivity was observed in hypertrophied, GFAP+ reactive astrocytes near the wound margin as early as 12 h after injury. Very rarely were HO-1+ neurons observed and then only up to 6 h after stabbing. Maximal numbers of HO-1+ astrocytes were found 3 days after stabbing. Their numbers declined thereafter. By 5 days after stab injury few HO-1+ reactive astrocytes were observed although GFAP+ reactive astrocytes were still prominent near the wound margin. HO-1+ macrophage-like cells were initially observed between 1 and 3 days after injury and they persisted in the margin of the wound for at least 14 days. The proximity of HO-1+ cells to the wound margin suggests that factors associated with injury contribute to the regulation of HO-1 in injured cortex.

This study is a follow-up of previous work which demonstrated that cultured cortical neurons did not synthesize HSP70i immediately after heat stress when compared with cultured cortical astrocytes. We have extended the period of observation for HSP70i induction of cultured cortical neurons and astrocytes up to 24 h after heat stress. Cultured rat cortical neurons derived from 16-day-old fetal rats respond differently to heat stress than cultured rat astrocytes derived from newborn rats. They showed a delayed HSP70i induction in the majority of cultured neurons and the response was heterogeneous and was absent in most smaller neurons. The delayed neuronal induction was accompanied by a prolonged activation of heat-shock transcription factor 1 (HSF-1) and prolonged transcription of HSP70i mRNA. In comparison astrocytes showed a marked early induction of HSP70i mRNA and protein. In addition the induction of HSP70i in astrocytes was followed by translocation of the protein into the nucleus, a finding which we failed to demonstrate in neurons. Immunostaining for HSP70i was more uniform in astrocytes than neurons. Many neurons did not stain for up to 24 h after heat shock in this study. Immunocytochemical staining of HSF-1 and 2 showed major differences between neurons and astrocytes. Astrocytes showed localization of HSF-1 to the nucleus before and after heat stress, while neurons showed HSF-1 localization to the cytoplasm and nucleus before and after heat stress. Finally HSF-2 was undetectable in neurons when compared with astrocytes by Western immunoblot analysis. However, astrocytes and neurons revealed weak immunostaining of HSF-2 in the cytoplasm and nucleus. The staining in the neurons was likely secondary to cross-reactivity to an unidentified protein. We conclude that HSP70i expression after heat shock is delayed in rat cortical neurons when compared with rat cortical astrocytes. In addition most small neurons did not synthesize HSP70i after heat shock. This difference in induction of HSP70i may be secondary to localization and activation of HSF-1 but not HSF-2. Neuronal susceptibility to injury may be related to the delayed induction of HSP70i and also the possible failure of newly synthesized HSP70i to translocate into the nucleus.

A high speed half-tracked truck using a new type of metal-coreless rubber track for traveling and load-carrying both on- and off-road was developed. In this application a conventional embedded-metal rubber track was examined in a first step, and was found to be unsuitable because of its chain-type structure. Then a new type of positive drive metal-coreless rubber track with endless spiral cable reinforcement was developed to meet all of the desired performance, i.e. low driving resistance, low heat build-up due to flexing, low noise, low vibration, light weight, good flexibility, low wear, etc. The development of this track, along with the use of properly matched undercarriage and suspension, has renewed interest in the use of half-tracked trucks. This truck is based on a conventional four-wheel-drive dump truck of two tonnes payload with its rear wheels converted into rubber tracks. Both high mobility off-road and traveling speed faster than 60 km/h on-road are obtained without detracking of the rubber tracks from the undercarriage. Therefore, the truck is capable of transporting its load between on- and off-road without transshipment. An identical driver's license for the same category as the original truck has been granted by the Japanese Ministry of Transportation since 1992. In this paper, the structure of the truck, the metal-coreless rubber track developed especially for high-speed traveling, the test results, and some market applications are reported.

Heme oxygenase-2 isozyme is the predominant form of heme oxygenase in rat brain by western blot analysis. Heme oxygenase-1 isozyme is not induced by light adaptation in rat retina by western blot analysis. Immunocytochemistry localizes heme oxygenase-2 in three areas of the retina: the retinal pigment epithelium, inner segment and external nuclear layers of the rat retina. Ganglion cells and cell bodies of the internal nuclear layer of the retina and Müller cells were largely unstained for heme oxygenase-2. The localization of heme oxygenase-2 in the retina implies that its function is not associated with phototransduction. Also, light adaptation does not appear to induce heme oxygenase-1, a measure of oxidative injury.

The induction of the highly inducible 70-kDa heat shock protein (HSP 70) is associated with thermotolerance and survival from many other types of stress. This investigation studied the pharmacological induction of HSP 68 (HSP 68 is the rat homolog of human HSP 70) by 1,10-phenanthroline in cultured rat astrocytes under conditions that activated heat shock transcription factor-1 without inducing HSP 68 synthesis. Two conditions that activate heat shock transcription factor-1 and promote its binding to the heat shock element without subsequent transcription of HSP 68 mRNA, intracellular acidosis and exposure to salicylate, showed synthesis of HSP 68 when 1,10-phenanthroline was added to culture medium after the activation of heat shock transcription factor-1. 1,10-phenanthroline mimicked heat shock by inducing HSP 68 mRNA and protein under both conditions. 1,10-phenanthroline added alone to culture medium did not induce the synthesis of HSP 68 or activate heat shock transcription factor-1. These findings strongly suggest a multistep activation for HSP 68 synthesis and also demonstrate that the synthesis of HSP 68 can be pharmacologically regulated.

Proximal tubular dysfunction is a main toxic sign of chronic cadmium (Cd) poisoning, but it is not clear why the proximal tubular epithelium is more susceptible than distal tubular cells to Cd toxicity. Kidney cell lines of proximal (LLC-PK 1 ) and distal (MDCK) tubular origins have been used to study comparative Cd toxicity. From cell morphology, cell numbers and cellular protein amounts, LLC-PK, cells were found to be more susceptible to Cd than MDCK cells. In LLC-PK 1 cells, cell number was decreased with time at concentrations of 25 μ m Cd or more, whereas MDCK cells proliferated even under 50 μ m Cd until hr 24. Cd accumulated in both cell types in a time- and dose-dependent manner; the accumulated Cd level was greater in LLC-PK 1 cells than in MDCK cells. The basal metallothionein (MT) amount was low and similar between the two cell lines; MT was induced in a dose- and time-dependent manner by Cd in MDCK cells whereas MT induction in LLC-PK 1 cells was observed only until Cd cytotoxicity overwhelmed cell viability. The vulnerability of LLC-PK 1 cells can be explained by the fact that they accumulate more Cd than MDCK cells, with insufficiently induced MT to sequester Cd ions.

We study tunnel magnetoresistance (TMR) through grain boundaries where tunneling electrons interact with localized spins via ferromagnetic exchange interaction. It is shown that spin–flip tunneling due to the exchange interaction gives rise to appreciable effects on TMR, and that TMR increases almost linearly with increasing magnetic field.

The heat shock response in a transformed astrocyte line was compared with nontransformed astrocytes. The synthesis of HSP 68, the major inducible heat shock protein (HSP 68) was induced by a non-lethal 45 degrees C, 10 min heat shock. Although the incorporation of [35S]methionine into HSP 68 suggested that similar amounts of protein were being synthesized after heat shock, Western immunoblotting demonstrated striking differences in the HSP immunostaining between the two cell types. By one- and 'two-dimensional gel electrophoresis the major 68 kDa heat shock protein (HSP 68) was similar in both cell types. However, HSP 68 from heat shocked, transformed astrocytes did not immunostain with the monoclonal antibody, C-92, which is specific for the major inducible heat shock protein of HeLa cells. In contrast HSP 68 from heat shocked, nontransformed astrocytes immunostained quite well. A polyclonal antibody raised against the inducible 72 kDa heat shock protein of HeLa cells immunostained the HSP 68 from both astrocytes and transformed astrocytes. Analysis of the mRNA from the two cell types after heat shock revealed two bands of approximately 2.5 and 2.8 kb in astrocytes but only a single 2.5 kb band in the heat shocked transformed astroglia. These results suggest that structural differences in the HSP 68 may be present in the transformed astrocytes compared to the normal astrocytes.

Cultured cortical neurons and astrocytes were compared for synthesis of the major inducible 68 kDa heat shock protein. By one- and two-dimensional electrophoresis the inducible 68 kDa protein appeared similar, but astrocytes produced greater amounts of the protein by 3 h than did neurons. Antibodies raised against HeLa cell inducible 72 and constitutive 73 kDa heat shock proteins were used to characterizes the inducible heat shock proteins in neurons and astrocytes. Unlike the gels, major differences were noted of the major inducible heat shock protein in astrocytes compared with neurons when analyzed by Western immunoblots. Heat shock protein 68 kDa mRNA induction in neurons was less than astrocytes suggesting an attenuated inducible 68 kDa heat shock protein response in neurons. The neuronal protein may be a different isoform of the 70 kDa family of heat shock proteins.

Introduction Adult T-cell leukaemia/lymphoma (ATL) caused by human retrovirus, HTLV-1, is a neoplasm of mature T-cell. In Japan, more than a thousand patients with ATL die every year because this disease still remains incurable. Acute crisis is a common phenomenon of chronic leukaemia and indolent haematological malignancy, in which proliferation of leukaemic cells is accelerated following a certain term of chronic phase. Although some genetic changes are thought to be responsible for this sudden change of characteristics in leukaemic cells, it has been unclear if they are the cause or the result of crisis. Methods We assessed the therapy and outcome of 79 newly diagnosed or referred ATL patients from 2005 to 2010. Serum sample of patient was preserved in freezer to measure the level of soluble proteins, sCD30 and sIL-2R, using ELISA. The sIL-2R concentration in serum was measureed by Cell freeN IL-2R (Kyowa Medex, Japan) and Determiner CL IL-2R (Kyowa Medex, Japan). The sCD30 concentration in serum was measured by Human sCD30 Platinum ELISA (eBioscience). In the present study, patients with ATL chronic type were investigated by monitoring changes in the serum levels of sIL-2R and sCD30. Results Six patients showed acute crisis from chronic phase (the observed periods of chronic phase are 31–866 days) and increasing sCD30 level as well as sIL-2R elevation was found as an earlier event of the acute crisis (average levels of sCD30 and sIL-2R were 2029 U/ml and 14035 U/ml in acute crisis; 341 U/ml and 5008 U/ml in chronic phase). In contrast, average of sCD30 and sIL-2R in 25 HTLV-1 carriers are 37 U/ml and 428 U/ml, respectively. Although the patterns and levels of soluble proteins elevation were unique to each case, serum sCD30 level predominantly elevated at the crisis. Interestingly, CD30 is not always detected on the surface of ATL cells. Chronic inflammation caused by constituvive activation of STATs may be associated with Matrix Metalloproteinase activation which sheds CD30 from the surface of ATL cells. Conclusion Our findings suggest that soluble protein levels in sera of patients are useful biological markers for diagnosis and therapy of ATL. Furthermore, since spontaneous remission was observed during the insufficinet level of serum sCD30 by comparison to the sIL-2R level, it is conceivable that the sCD30 protects CD30+ leukaemic stem cells from CD30 ligand-mediated cell death.

It has been believed that autonomic nerve plays little role in HPV. However, these studies were done in the conditions with normal bronchial tone. Little has been known about the role of autonomic nerve on HPV during broncho-spasm as in the case with bronchial asthma, in which autonomic nerves are involved in pathogenesis and the altered tone of autonomic nerve may affect pulmonary circulation. To clarify the role of autonomic nerves on HPV when there is bronchospasm, we induced hypoxia while provoking bronchospasm by intratracheal administration of histamine or acetylcholine in isolated cat lungs (n=36) perfused with diluted autologous blood (Ht=l 0%). We used 30% 02 and 5% CO2 for noormoxia and 2% 02 and 5% CO2 for hypoxia. Hexamethonium (C6) as a ganglion blockaer, tetrodotoxin (TTX) as a blockade of nerve conduction, atropine a muscarinic receptor antagonist, and also selective or non-selective adrenergic inhibitors (8: propranolol, 131 ;acebutolol, metoprolol, 13-2;butoxamine, alpha; phentolamine, alpha-1 ; prazosin, alpha-2; yohimbin) were administered before induction of bronchospasm following control HPV. C6 and TTX did not affect parameters of pulmonary circulation when HPV was not accompanied by bronchospasm or when bronchospasm was induced during normoxia. Nevertheless, these agents attenuated HPV when there was bronchospasm. Atropine almost completely abolished HPV during bronchospasm. Beta and selective 13-2 inhibitors also completely abolished HPV during bronchospasm. Alpha blockades did not affect HPV even when there was bronchospasm. We conclude that autonomic nerves, especially vagal nerves and 13 adrenergic nerve play a crucial role in HPV when it occured during bronchospasm. These finding may indicate that many sympathetic agonist and anti-cholinergic drugs, used in the treatment of bronchial asthma, may affect ventilation-perfusion matching.

A sensitive and precise method for measuring endogenous phylloquinone (K1) and menaquinone (MK-n) in human liver was developed, based on gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection. Subnanogram levels of vitamin K compounds in 1 g of liver specimen were detectable. We measured vitamin K concentrations in 38 human resected livers. K1 and MK-4 to MK-13 were detected. The concentrations of MK-10 to MK-12 in livers with chronic hepatitis (n = 10) and cirrhosis (n = 22) were significantly lower than in normal livers (n = 6). It is suggested that the decreased concentrations indicate functional damage of the hepatocytes.

Phenytoin applied to developing neonatal mouse cerebellar cultures at concentrations of 9-46 micrograms/ml of nutrient medium from the day of explantation to 16 days in vitro induced cerebellar cortical degeneration. The degree of neurotoxicity correlated with drug concentration. Purkinje cells were the most susceptible of the cerebellar elements, and intracerebellar nucleus neurons were the most resistant. In contrast, mature mouse cerebellar explants were resistant to chronic exposure to high concentrations of phenytoin.

Homogenates of bovine thyroid gland contained the glycosyltransferases required for the in vitro formation of the disialoganglioside N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (G& and the trisialoganglioside N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]galactosylglucosylceramide (&lb) from lactosylceramide (GL-2). These enzyme activities were constituted into two separate biosynthetic pathways which culminated in the formation of Gnls and GJTlb, respectively. Based on substrate competition experiments, the last three reactions in each pathway appeared to be catalyzed by the same three glycosyltransferases. These three enzymes as well as CMP-AcNeu:GL-2 sialyltransferase were highly enriched in the Golgi apparatus fraction from bovine thyroid. The reaction product of the latter enzyme is Nacetylneuraminylgalactosylglucosylceramide (Gm), the first ganglioside intermediate in both pathways. In contrast, CMP-AcNeu:Gm sialyltransferase, which catalyzes the formation of N-acetylneuraminyl-Nacetylneuraminylgalactosylglucosylceramide (G&, the branch point between the two pathways, was not localized in the Golgi apparatus. Bovine thyroid, which contains a complex pattern of gangliosides, is capable of synthesizing these important membrane constituents. In addition, these results suggest that the separate subcellular localization of Gn3 synthesis may be involved in the regulation of ganglioside biosynthesis by the two pathways.

Cells of budding yeast, Saccharomyces cerevisiae, with n bud scars are said to belong to the stage class n. Previous linear theory on the stage structure of yeast populations is extended so as to include the nonlinear density effect on the population growth in a batch culture. Analysis of experimental data by means of this theory shows that the density effect is strongly stage dependent. The dependence is such that the virgin (n = 0) population is more vulnerable than their matured (n ≥ 1) counterparts.