A 28-fold increase in secretory protein synthesis is associated with DNA puff activity in the salivary gland of Bradysia hygida (Diptera, Sciaridae)

When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that these polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7, C5, C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time (Spradling AC and Mahowald AP (1980) Proceedings of the National Academy of Sciences, USA, 77: 1096-1100).

The left ventricular contractility of the rat heart is modulated by changes in flow and a1-adrenoceptor stimulation

Myocardial contractility depends on several mechanisms such as coronary perfusion pressure (CPP) and flow as well as on a1-adrenoceptor stimulation. Both effects occur during the sympathetic stimulation mediated by norepinephrine. Norepinephrine increases force development in the heart and produces vasoconstriction increasing arterial pressure and, in turn, CPP. The contribution of each of these factors to the increase in myocardial performance needs to be clarified. Thus, in the present study we used two protocols: in the first we measured mean arterial pressure, left ventricular pressure and rate of rise of left ventricular pressure development in anesthetized rats (N = 10) submitted to phenylephrine (PE) stimulation before and after propranolol plus atropine treatment. These observations showed that in vivo a1-adrenergic stimulation increases left ventricular-developed pressure (P<0.05) together with arterial blood pressure (P<0.05). In the second protocol, we measured left ventricular isovolumic systolic pressure (ISP) and CPP in Langendorff constant flow-perfused hearts. The hearts (N = 7) were perfused with increasing flow rates under control conditions and PE or PE + nitroprusside (NP). Both CPP and ISP increased (P<0.01) as a function of flow. CPP changes were not affected by drug treatment but ISP increased (P<0.01). The largest ISP increase was obtained with PE + NP treatment (P<0.01). The results suggest that both mechanisms, i.e., direct stimulation of myocardial a1-adrenoceptors and increased flow, increased cardiac performance acting simultaneously and synergistically.

Detection of point mutations by non-isotopic single strand conformation polymorphism

We have developed a procedure for nonradioactive single strand conformation polymorphism analysis and applied it to the detection of point mutations in the human tumor suppressor gene p53. The protocol does not require any particular facilities or equipment, such as radioactive handling, large gel units for sequencing, or a semiautomated electrophoresis system. This technique consists of amplification of DNA fragments by PCR with specific oligonucleotide primers, denaturation, and electrophoresis on small neutral polyacrylamide gels, followed by silver staining. The sensitivity of this procedure is comparable to other described techniques and the method is easy to perform and applicable to a variety of tissue specimens.

Pancreatic nitric oxide and oxygen free radicals in the early stages of streptozotocin-induced diabetes mellitus in the rat

The objective of the present study was to explore the regulatory mechanisms of free radicals during streptozotocin (STZ)-induced pancreatic damage, which may involve nitric oxide (NO) production as a modulator of cellular oxidative stress. Removal of oxygen species by incubating pancreatic tissues in the presence of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) (1 U/ml) produced a decrease in nitrite levels (42%) and NO synthase (NOS) activity (50%) in diabetic but not in control samples. When NO production was blocked by N G-monomethyl-L-arginine (L-NMMA) (600 µM), SOD activity increased (15.21 ± 1.23 vs 24.40 ± 2.01 U/mg dry weight). The increase was abolished when the NO donor, spermine nonoate, was added to the incubating medium (13.2 ± 1.32). Lipid peroxidation was lower in diabetic tissues when PEG-SOD was added (0.40 ± 0.02 vs 0.20 ± 0.03 nmol/mg protein), and when L-NMMA blocked NOS activity in the incubating medium (0.28 ± 0.05); spermine nonoate (100 µM) abolished the decrease in lipoperoxide level (0.70 ± 0.02). We conclude that removal of oxygen species produces a decrease in pancreatic NO and NOS levels in STZ-treated rats. Moreover, inhibition of NOS activity produces an increase in SOD activity and a decrease in lipoperoxidation in diabetic pancreatic tissues. Oxidative stress and NO pathway are related and seem to modulate each other in acute STZ-induced diabetic pancreas in the rat.

Influence of exercise on the activity and the distribution between free and bound forms of glycolytic and associated enzymes in tissues of horse mackerel

The effects of short-term burst (5 min at 1.8 m/s) swimming and long-term cruiser (60 min at 1.2 m/s) swimming on maximal enzyme activities and enzyme distribution between free and bound states were assessed for nine glycolytic and associated enzymes in tissues of horse mackerel, Trachurus mediterraneus ponticus. The effects of exercise were greatest in white muscle. The activities of phosphofructokinase (PFK), pyruvate kinase (PK), fructose-1,6-bisphosphatase (FBPase), and phosphoglucomutase (PGM) all decreased to 47, 37, 37 and 67%, respectively, during 60-min exercise and all enzymes except phosphoglucoisomerase (PGI) and PGM showed a change in the extent of binding to subcellular particulate fractions during exercise. In red muscle, exercise affected the activities of PGI, FBPase, PFK, and lactate dehydrogenase (LDH) and altered percent binding of only PK and LDH. In liver, exercise increased the PK activity 2.3-fold and reduced PGI 1.7-fold only after 5 min of exercise but altered the percent binding of seven enzymes. Fewer effects were seen in brain, with changes in the activities of aldolase and PGM and in percent binding of hexokinase, PFK and PK. Changes in enzyme activities and in binding interactions with subcellular particulate matter appear to support the altered demands of tissue energy metabolism during exercise.

The 5alpha-reductase type 1, but not type 2, gene is expressed in anagen hairs plucked from the vertex area of the scalp of hirsute women and normal individuals

The aim of the present study was to determine the expression of the genes for type 1 (SDR5A1) and type 2 (SDR5A2) 5alpha-reductase isoenzymes in scalp hairs plucked from 33 hirsute patients (20 with polycystic ovary syndrome and 13 with idiopathic hirsutism) and compare it with that of 10 men and 15 normal women. SDR5A1 and SDR5A2 expression was estimated by RT-PCR using the gene of the ubiquitously expressed protein ß2-microglobulin as an internal control. The results are expressed as arbitrary units in relation to ß2-microglobulin absorbance (mean ± SEM). SDR5A2 expression was not detected in any hair samples analyzed in this study. No differences were found in SDR5A1 mRNA levels between men and normal women (0.78 ± 0.05 vs 0.74 ± 0.06, respectively). SDR5A1 gene expression in the cells of hair plucked from the scalp of normal women (0.85 ± 0.04) and of women with polycystic ovary syndrome (0.78 ± 0.05) and idiopathic hirsutism (0.80 ± 0.06) was also similar. These results indicate that SDR5A1 gene expression in the follicular keratinocytes from the vertex area of the scalp seems not to be related to the differences in hair growth observed between normal men and women and hirsute patients. Further studies are needed to investigate the expression of the 5alpha-reductase genes in other scalp follicular compartments such as dermal papillae, and also in hair follicles from other body sites, in order to elucidate the mechanism of androgen action on the hair growth process and related diseases.

Naisjohtajat – miksi heitä ei ole ja mitä siitä? - miesjohtajien ja ei-johtavassa asemassa olevien naisten näkemyksiä case-yritys x:ssä

Joidenkin tutkimusten mukaan naisten vähäinen määrä johdossa voi olla uhka organisaatiolle. Lasikattoilmiöllä tarkoitetaan naisten urakehityksen katkeamista tietylle tasolle ylimmän johdon alapuolelle ikään kuin naisten ja tuon ylimmän tason välissä olisi lasinen, näkymätön katto, sukupuolistereotypioiden muodostama este. Yksi yleinen lasikaton selitysten kolmijako on henkilökohtaiset, organisatoriset ja yhteiskunnalliset tekijät. (Lämsä & Hautala 2004, 252). Hoyt (2007, 270-278) tekee kolmijaon seuraavasti: inhimillinen pääoma, sukupuolierot ja ennakkoluulot. Yritys X:n keskijohdossa työskentelee yksi nainen, ylimmässä johdossa ei yhtäkään. Tutkimuksessa halu-taan selvittää miesjohtajien ja ei-johtavassa asemassa olevien naisten käsitystä siitä, onko yritys x:ssä lasi-kattoa, miksi naisjohtajia on niin vähän ja "mitä siitä" ts. onko mitään ongelmaa olemassakaan. Tässä tutkimuksessa pohditaan diskurssianalyysin keinoin, miten yritys X:ssä puhutaan naisjohtajuusaiheesta, millai-seksi sukupuolen merkitys työelämässä määritellään ja mitä ajatellaan naisten kykenevyydestä johtajiksi. Naturalisoiva diskurssi oli vahva niin miesjohtajien ja ei-johtavassa asemassa olevien naisten puheessa. Sen lisäksi hahmotellaan familistista, empiiristä, humanistista ja historiallista diskurssia naisjohtajuuspuheesta. Diskurssien yhteenkietominen hegemonisoimisstrategiana kuvaa tapaa, jolla palasia muista diskursseista käytetään tukemaan tiettyä toista diskurssia (Jokinen et al. 1993c, 95) Miesjohtajien puheessa naisten keskeiset, ominaisuudet - liiallinen tarkkuus ja huolellisuus yhdistettynä epävarmuuteen - ovat ongelmallisia johtajanuran kannalta. Jos näistä johtajuuden kannalta negatiivisista ominaisuuksista ei jostain syystä kuitenkaan muodostuisi uralla etenemisen estettä, äitiys ja perheellisyys "luonnollisesti" tekee tämän. Aiheet myös kietoutuvat yhteen: äitiys ja vastuu perheestä lisäävät naisten huolellisuutta, tarkkuutta ja epävarmuutta entisestään. Lisäksi äitiyslomat ja työhön käytettävissä oleva aika ja puut-tuva halu käyttää elämästä iso osa uranluomiseen ovat johtajaksi etenemisen esteitä. Miesjohtajien mukaan tämä on jossain määrin ongelma, kun heterogeenisyyttä johtamiseen kuitenkin tarvittaisiin, mutta loppujen lopuksi kuitenkin melko epäkiinnostava ja pieni ongelma; ongelma ei miesten mielestä johdu miesten tai yhteiskunnallisista asenteista, vaan naisista itsestään ja he tarvitsevat uralla edetäkseen tukea, rohkaisua ja henkilöstöpankkeja, joita miesjohtajat voivat tuottaa. Johtaminen ylipäänsä ei ole miesjohtajien mielestä hirveän kiinnostavaa. Jos naiset (kaikesta edellä sanotusta huolimatta) etenevät yritysten johtoon, eivät he tule siellä toimeen keskenään. Kaiken kaikkiaan koko naisjohtajuusaihe ei ole kovin kiinnostava ja naisjohtajuuden vähäisyyden (mahdollisen) ongelman ratkaisee aika uuden, tasa-arvoisemman sukupolven myötä. Naishaastateltujen näkökulmasta sen sijaan naisilla on pyrkyä johtotehtäviin - joskaan ei samassa määrin kuin miehillä. Naishaastateltujen mukaan miehet suosivat toisiaan työelämässä ja naiset kohtaavat asenteita, joita vastaan joutuvat taistelemaan ja tästä syystä johtajien joukossa on niin vähän naisia. Historialliset tekijät pitävät asenteita yllä. Perheellisyys on naisille suurempi uraeste kuin miehille, "luonnollisesti". Naishaastateltujen mielestä naisten vähäisyys johdossa on merkittävä ongelma, koska naisilla on erityislaatuisia ominaisuuksia, joista olisi hyötyä tehtävässä. Naishaastateltujen puheessa miesten ominaisuuksia vastaavasti vähäteltiin. Naisjohtajien vähäisyyden ongelmalle ei naishaastateltujen mielestä kuitenkaan ole tehtävissä paljonkaan: miesten ja yhteiskunnan asenteiden pitäisi muuttua, mutta keinoja tähän ei esitetä, sen sijaan naisten itsensä pitäisi vain "yrittää vielä kovemmin".

Kiinteistön välillinen omistaminen konsernirakenteessa kiinteistöstä saatavan tulon tuloverotuksen näkökulmasta

Pro gradu –tutkielman ensisijaisena tavoitteena on selvittää miksi kiinteistön omista-minen on perusteltua järjestää konsernissa kiinteistöyhtiön välityksellä. Tutkielman aiheen käsittely on rajattu koskemaan vain kotimaista lainsäädäntöä. Tutkimuson-gelman perusteella tutkielmassa keskitytään tarkastelemaan emoyhtiön näkökulmas-ta siihen kohdistuvia tuloverovaikutuksia, kun se omistaa kiinteistöyhtiön. Tutkielmas-sa on tutkimusmetodina sovellettu lainoppia ja tutkimus kuuluu vero-oikeuden alaan. Koska vero-oikeutta tulkitaan lain sanamuodon mukaisesti, tutkimuksessa annetaan runsaasti painoarvoa voimassa olevalle lainsäädännölle. Kuitenkin tulkinnanvaraisis-sa kysymyksissä tutkielmassa on myös hyödynnetty korkeimman hallinto-oikeuden ratkaisukäytäntöä sekä vero-oikeudellista kirjallisuutta. Tutkielmassa selvitettyjen seikkojen perusteella voidaan todeta, että yksiselitteisen tulkintasuosituksen antaminen tutkimusongelman ratkaisemiseksi on haastavaa, kos-ka jokaisen yrityksen tarpeet kiinteistön omistamiseen vaihtelevat liiketoiminnan olo-suhteiden mukaisesti. Toisaalta konsernirakenteen synnyttämistä emoyhtiön ja kiin-teistöyhtiön välille voidaan perustella esimerkiksi osinkotulon verotehokkaalla kana-voimiselle kiinteistöyhtiön tasolta emoyhtiölle sekä konsernin efektiivisen veroasteen pienentämisellä konserniyhtiöiden välisen lainan avulla. Lisäksi kiinteistöyhtiön luovu-tustilanteessa verotuksen tulolähdejaon merkitys korostuu, kun ratkaistaan voidaanko myyntiin soveltaa elinkeinoverolain mukaista käyttöomaisuusosakkeiden luovutuksen verovapaussäännöksiä, ja toisaalta, miten mahdollisia kiinteistöyhtiön myynnistä johtuvia luovutustappioita käsitellään emoyhtiön tuloverotuksen näkökulmasta.

The combination of atorvastatin and ethanol is not more hepatotoxic to rats than the administration of each drug alone

Animal studies and premarketing clinical trials have revealed hepatotoxicity of statins, primarily minor elevations in serum alanine aminotransferase levels. The combined chronic use of medicines and eventual ethanol abuse are common and may present a synergistic action regarding liver injury. Our objective was to study the effect of the chronic use of atorvastatin associated with acute ethanol administration on the liver in a rat model. One group of rats was treated daily for 5 days a week for 2 months with 0.8 mg/kg atorvastatin by gavage. At the end of the treatment the livers were perfused with 72 mM ethanol for 60 min. Control groups (at least 4 animals in each group) consisted of a group of 2-month-old male Wistar EPM-1 rats exposed to 10% ethanol (v/v) ad libitum replacing water for 2 months, followed by perfusion of the liver with 61 nM atorvastatin for 60 min, and a group of animals without chronic ethanol treatment whose livers were perfused with atorvastatin and/or ethanol. The combination of atorvastatin with ethanol did not increase the release of injury marker enzymes (alanine aminotransferase, aspartate aminotransferase, and lactic dehydrogenase) from the liver and no change in liver function markers (bromosulfophthalein clearance, and oxygen consumption) was observed. Our results suggest that the combination of atorvastatin with ethanol is not more hepatotoxic than the separate use of each substance.

The cytotoxicity of methacryloxylethyl cetyl ammonium chloride, a cationic antibacterial monomer, is related to oxidative stress and the intrinsic mitochondrial apoptotic pathway

Antibacterial monomers incorporated in dentin bonding systems may have toxic effects on the pulp. Thus, the cytotoxicity of antibacterial monomers and its underlying mechanisms must be elucidated to improve the safety of antibacterial monomer application. The influence of an antibacterial monomer, methacryloxylethyl cetyl ammonium chloride (DMAE-CB), on the vitality of L929 mouse fibroblasts was tested using MTT assay. Cell cycle progression was studied using flow cytometry. Production of intracellular reactive oxygen species (ROS) after DMAE-CB treatment was measured using 2,7-dichlorodihydrofluorescein diacetate staining and flow cytometry analysis. Loss of mitochondrial membrane potential, disturbance of Bcl-2 and Bax expression, as well as release of cytochrome C were also measured using flow cytometry analysis or Western blot to explore the possible involvement of the mitochondrial-related apoptotic pathway. DMAE-CB elicited cell death in a dose-dependent manner and more than 50% of cells were killed after treatment with 30 µM of the monomer. Both necrosis and apoptosis were observed. DMAE-CB also induced G1- and G2-phase arrest. Increased levels of intracellular ROS were observed after 1 h and this overproduction was further enhanced by 6-h treatment with the monomer. DMAE-CB may cause apoptosis by disturbing the expression of Bcl-2 and Bax, reducing the mitochondrial potential and inducing release of cytochrome C. Taken together, these findings suggest that the toxicity of the antibacterial monomer DMAE-CB is associated with ROS production, mitochondrial dysfunction, cell cycle disturbance, and cell apoptosis/necrosis.

REGγ is a strong candidate for the regulation of cell cycle, proliferation and the invasion by poorly differentiated thyroid carcinoma cells

REGγ is a proteasome activator that facilitates the degradation of small peptides. Abnormally high expression of REGγ has been observed in thyroid carcinomas. The purpose of the present study was to explore the role of REGγ in poorly differentiated thyroid carcinoma (PDTC). For this purpose, small interfering RNA (siRNA) was introduced to down-regulate the level of REGγ in the PDTC cell line SW579. Down-regulation of REGγ at the mRNA and protein levels was confirmed by RT-PCR and Western blot analyses. FACS analysis revealed cell cycle arrest at the G1/S transition, the MTT assay showed inhibition of cell proliferation, and the Transwell assay showed restricted cell invasion. Furthermore, the expression of the p21 protein was increased, the expression of proliferating cell nuclear antigen (PCNA) protein decreased, and the expression of the p27 protein was unchanged as shown by Western blot analyses. REGγ plays a critical role in the cell cycle, proliferation and invasion of SW579 cells. The alteration of p21 and PCNA proteins related to the down-regulation of REGγ suggests that p21 and PCNA participate in the process of REGγ regulation of cell cycle progression and cell proliferation. Thus, targeting REGγ has a therapeutic potential in the management of PDTC patients.

Diastolic function is associated with quality of life and exercise capacity in stable heart failure patients with reduced ejection fraction

Exercise capacity and quality of life (QOL) are important outcome predictors in patients with systolic heart failure (HF), independent of left ventricular (LV) ejection fraction (LVEF). LV diastolic function has been shown to be a better predictor of aerobic exercise capacity in patients with systolic dysfunction and a New York Heart Association (NYHA) classification ≥II. We hypothesized that the currently used index of diastolic function E/e' is associated with exercise capacity and QOL, even in optimally treated HF patients with reduced LVEF. This prospective study included 44 consecutive patients aged 55±11 years (27 men and 17 women), with LVEF<0.50 and NYHA functional class I-III, receiving optimal pharmacological treatment and in a stable clinical condition, as shown by the absence of dyspnea exacerbation for at least 3 months. All patients had conventional transthoracic echocardiography and answered the Minnesota Living with HF Questionnaire, followed by the 6-min walk test (6MWT). In a multivariable model with 6MWT as the dependent variable, age and E/e' explained 27% of the walked distance in 6MWT (P=0.002; multivariate regression analysis). No association was found between walk distance and LVEF or mitral annulus systolic velocity. Only normalized left atrium volume, a sensitive index of diastolic function, was associated with decreased QOL. Despite the small number of patients included, this study offers evidence that diastolic function is associated with physical capacity and QOL and should be considered along with ejection fraction in patients with compensated systolic HF.

DNA methylation regulates expression of VEGF-C, and S-adenosylmethionine is effective for VEGF-C methylation and for inhibiting cancer growth

DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growthin vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.

Fat gain with physical detraining is correlated with increased glucose transport and oxidation in periepididymal white adipose tissue in rats

As it is a common observation that obesity tends to occur after discontinuation of exercise, we investigated how white adipocytes isolated from the periepididymal fat of animals with interrupted physical training transport and oxidize glucose, and whether these adaptations support the weight regain seen after 4 weeks of physical detraining. Male Wistar rats (45 days old, weighing 200 g) were divided into two groups (n=10): group D (detrained), trained for 8 weeks and detrained for 4 weeks; and group S (sedentary). The physical exercise was carried out on a treadmill for 60 min/day, 5 days/week for 8 weeks, at 50-60% of the maximum running capacity. After the training protocol, adipocytes isolated from the periepididymal adipose tissue were submitted to glucose uptake and oxidation tests. Adipocytes from detrained animals increased their glucose uptake capacity by 18.5% compared with those from sedentary animals (P<0.05). The same cells also showed a greater glucose oxidation capacity in response to insulin stimulation (34.55%) compared with those from the S group (P<0.05). We hypothesize that, owing to the more intense glucose entrance into adipose cells from detrained rats, more substrate became available for triacylglycerol synthesis. Furthermore, this increased glucose oxidation rate allowed an increase in energy supply for triacylglycerol synthesis. Thus, physical detraining might play a role as a possible obesogenic factor for increasing glucose uptake and oxidation by adipocytes.