Neck circumference as a potential marker of metabolic syndrome among college students / Circunferência do pescoço como possível marcador para síndrome metabólica em universitários / La circunferencia del cuello como posible indicador del síndrome metabólico en universitarios

OBJECTIVE: to relate neck circumference with metabolic syndrome and its criteria among college students. METHOD: cross-sectional study conducted with 702 college students in Fortaleza, CE, Brazil from September 2010 to June 2011. Socio-demographic data, waist circumference and neck circumference were collected together with blood pressure, fasting blood sugar, triglyceride levels, and HDL-C. RESULTS: 1.7% of the studied sample presented metabolic syndrome. Of these, 58.3% presented altered neck circumference (p<0.006). As neck circumference decreases, pressure levels improve (p<0.001). Additionally, college students with high fasting blood sugar (p=0.003) and high triglyceride levels (p<0.001) presented higher values of neck circumference. CONCLUSION: neck circumference is a potential predictive marker in the detection of metabolic syndrome and its components among college students. .

OBJETIVO: relacionar a circunferência do pescoço com a síndrome metabólica e seus critérios em universitários. MÉTODO: estudo transversal, realizado com 702 universitários de Fortaleza, CE, Brasil, no período de setembro de 2010 a junho de 2011. Coletaram-se dados sociodemográficos, circunferência da cintura, circunferência do pescoço, níveis de pressão arterial e glicemia plasmática de jejum, triglicerídeos e lipoproteína de alta densidade. RESULTADOS: 1,7% da amostra investigada tinha a síndrome metabólica. Desses, 58,3% apresentaram circunferência do pescoço alterada (p<0,006). Na medida em que decresce a circunferência do pescoço, os valores pressóricos dos universitários melhoram (p<0,001). Também, observou-se que universitários com valores de glicemia de jejum plasmática (p=0,003) e triglicerídeos (p<0,001) elevados apresentaram maiores valores de circunferência do pescoço. CONCLUSÃO: a circunferência do pescoço mostrou-se um possível marcador preditivo para detecção da síndrome metabólica e seus componentes em universitários. .

OBJETIVO: relacionar la circunferencia del cuello con el síndrome metabólico y sus criterios en universitarios. MÉTODO: estudio transversal realizado con 702 universitarios de Fortaleza-CE, Brasil, en el período de septiembre de 2010 a junio de 2011. Se recolectaron datos sociodemográficos, circunferencia de la cintura, circunferencia del cuello, niveles de presión arterial y glucemia plasmática de ayuno, triglicéridos y HDL-C. RESULTADOS: 1,7% de la muestra investigada tenían el síndrome metabólico. De estos, 58,3% presentaron circunferencia del cuello alterada (p<0,006). A medida que decrece la circunferencia del cuello mejoran los valores de la presión de los universitarios (p<0,001). También, se observó que los universitarios con valores de glucemia de ayuno plasmática (p=0,003) y triglicéridos (p<0,001) elevados presentaron mayores valores de circunferencia del cuello. CONCLUSIÓN: la circunferencia del cuello se mostró un posible indicador de predicción para la detección del síndrome metabólico y sus componentes, en universitarios. .

Quantitative in Vivo Detection of Brain Cell Death after Hypoxia Ischemia Using the Lipid Peak at 1.3 ppm of Proton Magnetic Resonance Spectroscopy in Neonatal Rats

This study was performed to determine the accuracy of proton magnetic spectroscopy (1H-MRS) lipid peak as a noninvasive tool for quantitative in vivo detection of brain cell death. Seven day-old Sprague Dawley rats were subjected to 8% oxygen following a unilateral carotid artery ligation. For treatment, cycloheximide was given immediately after hypoxic ischemia (HI). Lipid peak was measured using 1H-MRS at 24 hr after HI, and then brains were harvested for fluorocytometric analyses with annexin V/propidium iodide (PI) and fluorescent probe JC-1, and for adenosine-5'-triphosphate (ATP) and lactate. Increased lipid peak at 1.3 ppm measured with 1H-MRS, apoptotic and necrotic cells, and loss of mitochondrial membrane potential (DeltaPsi) at 24 hr after HI were significantly improved with cycloheximide treatment. Significantly reduced brain ATP and increased lactate levels observed at 24 hr after HI showed a tendency to improve without statistical significance with cycloheximide treatment. Lipid peak at 1.3 ppm showed significant positive correlation with both apoptotic and necrotic cells and loss of DeltaPsi, and negative correlation with normal live cells. Lipid peak at 1.3 ppm measured by 1H-MRS might be a sensitive and reliable diagnostic tool for quantitative in vivo detection of brain cell death after HI.

Molecular Identification of Schizophyllum commune as a Cause of Allergic Fungal Sinusitis

Schizophyllum commune, a basidiomycetous fungus, rarely causes disease in humans. We report a rare case of allergic fungal sinusitis caused by S. commune in a 14-yr-old girl. The patient presented with nasal obstruction and a purulent nasal discharge. Materials obtained during endoscopic surgery of the frontal recess revealed allergic mucin and a few fungal hyphae. A potato dextrose agar (PDA) culture from the allergic mucin yielded a rapidly growing white woolly mold. Although no distinctive features including hyphae bearing spicules or a clamp connection were present, the case isolate disclosed compatible mycological features including growth at 37degrees C, susceptibility to cycloheximide, and production of a tart and disagreeable smell. S. commune was confirmed by sequence analysis of the internal transcribed spacer region and D1/D2 regions of the 26S ribosomal DNA. We believe this is the first report of allergic fungal sinusitis caused by S. commune in Korea. Moreover, this report highlights the value of gene sequencing as an identification tool for non-sporulating isolates of S. commune.

The methyl ester of rosuvastatin elicited an endothelium-independent and 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent relaxant effect in rat aorta

The relaxant effect of the methyl ester of rosuvastatin was evaluated on aortic rings from male Wistar rats (250-300 g, 6 rats for each experimental group) with and without endothelium precontracted with 1.0 µM phenylephrine. The methyl ester presented a slightly greater potency than rosuvastatin in relaxing aortic rings, with log IC50 values of -6.88 and -6.07 M, respectively. Unlike rosuvastatin, the effect of its methyl ester was endothelium-independent. Pretreatment with 10 µM indomethacin did not inhibit, and pretreatment with 1 mM mevalonate only modestly inhibited the relaxant effect of the methyl ester. Nω-nitro-L-arginine methyl ester (L-NAME, 10 µM), the selective nitric oxide-2 (NO-2) inhibitor 1400 W (10 µM), tetraethylammonium (TEA, 10 mM), and cycloheximide (10 µM) partially inhibited the relaxant effect of the methyl ester on endothelium-denuded aortic rings. However, the combination of TEA plus either L-NAME or cycloheximide completely inhibited the relaxant effect. Inducible NO synthase (NOS-2) was only present in endothelium-denuded aortic rings, as demonstrated by immunoblot with methyl ester-treated rings. In conclusion, whereas rosuvastatin was associated with a relaxant effect dependent on endothelium and hydroxymethylglutaryl coenzyme A reductase in rat aorta, the methyl ester of rosuvastatin exhibited an endothelium-independent and only slightly hydroxymethylglutaryl coenzyme A reductase-dependent relaxant effect. Both NO produced by NOS-2 and K+ channels are involved in the relaxant effect of the methyl ester of rosuvastatin.

Biomasa fúngica y bacteriana como indicadoras del secuestro de C en suelos de sabanas sustituidos por pinares en Uverito, Venezuela

Fungal and bacterial biomass as indicators of soil C sequestration in savannas soils substituted by pine plantations. A transformation of any natural ecosystem to an agricultural or forest system leads to an important soil modification, not only in the total carbon pool, but also in the carbon associated to the microbial biomass. This way, carbon quantification on soil quality is important for the determination of impacts of agricultural practices and land use changes. The aim of this study was to the determine, through the selective inhibition technique, the fungal and bacterial biomass, and fungal-to-bacterial ratio (F:B) in pine plantations (Pinus caribaea var. hondurensis), to establish if these parameters are sensible indicators of changes in the carbon content in Uverito soils (Venezuela). Furthermore, the inhibitor additivity ratio (IAR) and total combined inhibition (TCI) were carried out to determine if the antibiotics caused non-target inhibition. The quantification of fungal and bacterial biomass was carried out by using of cyloheximide as fungal inhibitor, and streptomycin and chloranphenicol as specific bacterial inhibitors. This research evidences that this land use change exerted a significant effect on soil microbial biomass, and shows that in pine plantations there is a dominance of the fungal component, in contrast to the native savanna, in which the bacterial biomass dominates. The substitution of native savanna by pine plantation in Uverito promotes a major soil carbon sequestration. The values of the inhibitor additivity ratio (IAR) as for native savanna as pine system, were both>1.0. The total combined inhibition (TCI) was smaller in the pine systems, from which it is possible to infer that a high proportion of microbial biomass was affected by the combination of the inhibitors. Rev. Biol. Trop. 58 (3): 977-989. Epub 2010 September 01.

Cualquier transformación de un ecosistema natural a un sistema agrícola o forestal conduce a una modificación importante no sólo del pool del carbono total, sino también del carbono asociado con la biomasa microbiana. Su cuantificación es importante en la determinación del impacto de las prácticas agrícolas y el cambio de uso de la tierra sobre la calidad del suelo. El objetivo de este estudio fue determinar, a través del método de inhibición selectiva, la biomasa fúngica y bacteriana y la relación (H:B) en suelos de sabana nativa sustituidos por pinares (Pinus caribaea var. hondurensis), para establecer si éstos parámetros son indicadores sensibles de cambios en el contenido de carbono en suelos de Uverito, Venezuela. La relación de aditividad del inhibidor (RAI) y la inhibición total por efecto combinado del inhibidor (ITC) se llevaron a cabo para determinar, si los inhibidores microbianos tuvieron actividad sobre otros organismos para los cuales éstos no estaban destinados. La cuantificación de la biomasa fúngica y bacteriana se llevó a cabo mediante el uso de la cycloheximida como inhibidor fúngico, y la estreptomicina y el cloranfenicol como inhibidores bacterianos. Esta investigación evidencia que este cambio de uso de la tierra ejerció un efecto significativo sobre la biomasa microbiana del suelo, y muestra que en el sistema de pinares existe una dominancia del componente fúngico, en contraste con la sabana nativa, en la cual domina la biomasa bacteriana. La sustitución de la sabana nativa por plantaciones de pino en Uverito, promueve un mayor secuestro del carbono en el suelo. Los valores de la relación de aditividad del inhibidor (RAI) tanto para la sabana nativa como para el sistema de pinares, resultaron ambos >1.0. La inhibición total combinada (ITC) resultó menor en el sistema de pinares; a partir de lo cual, es posible inferir que una elevada proporción de la biomasa microbiana fue afectada por la combinación de los inhibidores.

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor gamma

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor gamma (PPAR gamma) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPAR gamma activity or knockdown of PPAR gamma expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPAR gamma through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPAR gamma siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress- induced gene expression by suppressing translation via activation of PPAR gamma in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPAR gamma.

Neuroprotective Effect of Cycloheximide on Hypoxic-Ischemic Brain Injury in Neonatal Rats

This study was done to determine the neuroprotective effect of cycloheximide on neonatal hypoxic-ischemic brain injury. Seven day-old newborn rat pups were subjected to 90 min of 8% oxygen following a unilateral carotid artery ligation. The extent of cerebral infarction was evaluated at 1 and 4 week of recovery. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluoresceinated annexin V and propidium iodide. Brain infarction area was significantly increased at 4 week compared to 1 week after hypoxia-ischemia in the control group. With cycloheximide treatment, the number of TUNEL positive cells in the ipsilateral cerebral cortex at 48 hr and peri-infarct area at 1 and 4 week of recovery was significantly reduced, both apoptotic and necrotic cells by flow cytometry 48 hr after the injury were significantly reduced, and the extent of cerebral infarction at 1 and 4 week of recovery was also significantly attenuated compared to the hypoxia-ischemia control group. In summary, our data suggest that apoptosis plays an important role in the development of delayed infarction, and inhibition of apoptosis with cycloheximide significantly reduces the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia.

Standard and quantitative analysis of cyclin E threshold by cyclin E/DNA multiparameter flow cytometry / 华中科技大学学报（医学）（英德文版）

The threshold of cyclin E expression at G1/S boundary is a characteristic feature of cell cycle progressing. In this study, we tried to develop a quantitative approach to analyze cyclin E threshold by multiparameter flow cytometry. The expression of cyclin E in exponentially growing MOLT-4 cells was detected under different photomultiplier tube (PMT) voltages by cyclin E/DNA multiparameter flow cytometry. Additionally, cyclin E was detected in cells which were treated with caffeine and cycloheximide (CHX) under the same PMT voltage. Moreover, the expression of cyclin E in MOLT-4 cells was compared with that in JURKAT cells. Cyclin E threshold was quantified by formula B2/AxC (A, B, C indicates the minimum, threshold, and maximum of cyclin E fluorescence intensity, respectively). Results showed that in MOLT-4 cells, cyclin E threshold calculated by formula B2/AxC was invariable under different PMT settings. It was decreased in cells treated with caffeine and remained changeless in cells treated with cycloheximide. Cyclin E threshold in JURKAT cells was much lower than that in MOLT-4 cells. It was suggested that Formula B2/AxC we firstly set up could be used to analyze cyclin E expression threshold quantitatively.

15-Deoxy-delta12,14-PGJ2inhibits IL-6-induced Stat3 phosphorylation in lymphocytes

15-deoxy-delta12,14-PGJ2(15d-PGJ2) is a natural ligand that activates the peroxisome proliferators-activated receptor (PPAR) gamma, a member of nuclear receptor family implicated in regulation of lipid metabolism and adipocyte differentiation. Recent studies have shown that 15d-PGJ2 is the potent anti-inflammatory agent functioning via PPARgamma-dependent and -independent mechanisms. Most postulated mechanisms for anti-inflammatory action of PPARgamma agonists are involved in inhibiting NF-kappaB signaling pathway. We examined the possibility that IL-6 signaling via the Jak-Stat pathway is modulated by 15d-PGJ2 in lymphocytes and also examined whether the inhibition of IL-6 signaling is dependent of PPARgamma. 15d-PGJ2 blocked IL-6 induced Stat1 and Stat3 activation in primary human lymphocytes, Jurkat cells and immortalized rheumatoid arthritis B cells. Inhibition of IL-6 signaling was induced rapidly within 15 min after treatment of 15d-PGJ2. Other PPARgamma-agonists, such as troglitazone and ciglitazone, did not inhibit IL-6 signaling, indicating that 15d-PGJ2 affect the IL-6-induced Jak-Stat signaling pathway via PPARgamma-independent mechanism. Although cycloheximide reversed 15d-PGJ2-mediated inhibition of Stat3 activation, actinomycin D had no effect on 15d-PGJ2-mediated inhibition of IL-6 signaling, indicating that inhibition of IL-6 signaling occur independent of de novo gene expression. These results show that 15d-PGJ2 specifically inhibit Jak-Stat signaling pathway in lymphocytes, and suggest that 15d-PGJ2 may regulate inflammatory reactions through the modulation of different signaling pathway other than NF-kappaB in lymphocytes.

Protein mediated cholesterol absorption in locusts Schistocerca gregaria (Forskal) and Locusta migratoria (Linn).

Absorption and transport of 3H cholesterol from the midgut to hemolymph and other tissues was studied in the locusts Schistocerca gregaria and Locusta migratoria. S. gregaria are able to absorb dietary cholesterol in the midgut and release into the hemolymph in vivo and into the incubation medium in virto. Certain proteins of midgut origin are involved in the absorption and release of cholesterol. The proteins designated as cholesterol binding proteins (CBP's) were fractionated by gel filtration chromatography using Sepharose CL-6B-200 column. Presence of a protein and its binding with cholesterol is confirmed by TCA precipitation after subsequent incubation of midgut in the incubation medium. Cholesterol binding with the proteins was also confirmed in native polyacrylamide gel electrophoresis. Biosynthesis of this protein takes place in the midgut which is inhibited by a protein synthesis inhibitor, cycloheximide. It also inhibits absorption and release of cholesterol from the midgut. The cholesterol binding activity was associated with a peak containing proteins ranging from molecular weights of 17-32 kDa in SDS-PAGE gels. Treatment of midgut with cycloheximide resulted in reduced cholesterol binding activity. Dilipidation of mucin and transport in presence of bile salts yielded a higher cholesterol binding activity. Although the absorption and release of cholesterol was observed in the hemolymph of both sexes, the ovary exhibited higher cholesterol binding as compared to testis.

Vibrio vulnificus cytolysin induces hyperadhesiveness of pulmonary endothelial cells for neutrophils through endothelial P-selectin: a mechanism for pulmonary damage by Vibrio vulnificus cytolysin

Vibrio vulnificus cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung. To elucidate the mechanism we investigated whether the cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils. The cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentrationand time-dependent manner. The increase of adhesiveness occurred within several minutes after the cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide. Furthermore, flow cytometric analyses showed that cytolysin enhanced the level of P-selectin on CPAE cell surface. Therefore, these results suggest that the cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface.

Vibrio vulnificus cytolysin induces hyperadhesiveness of pulmonary endothelial cells for neutrophils through endothelial P-selectin: a mechanism for pulmonary damage by Vibrio vulnificus cytolysin

Vibrio vulnificus cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung. To elucidate the mechanism we investigated whether the cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils. The cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentrationand time-dependent manner. The increase of adhesiveness occurred within several minutes after the cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide. Furthermore, flow cytometric analyses showed that cytolysin enhanced the level of P-selectin on CPAE cell surface. Therefore, these results suggest that the cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface.

Insulin alone can lead to a withdrawal of meiotic arrest in the carp oocyte.

Meiotic arrest of oocyte in an Indian carp, Labeo rohita Ham. has been found for the first time to be withdrawn by insulin only. Addition of insulin to oocytes in vitro caused germinal vesicle breakdown (GVBD), one of the first visual markers to determine initiation of the final maturational process. Under the influence of insulin the germinal vesicle (GV) of the oocyte migrated towards the animal pole, reached the micropyle and then dissolved (GVBD). By using different concentrations of insulin i.e., 0.063, 0.63, 6.3 and 12.6 mM, optimum amount required was found to be 6.3 mM. Induction of GVBD by insulin could be blocked by cycloheximide (Chx), a translation inhibitor, while actinomycin D (AcD) had no effect suggesting non-involvement of transcriptional activity in this process. Addition of the maturation-inducing steroid 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) stimulated (P<0.01) GVBD of carp oocytes and its combination with insulin showed an additive effect. Gonadotropin (GtH) caused GVBD but its effect was greatly augmented by insulin. Our results demonstrate that not only can insulin alone induce GVBD in carp oocytes, but it also augments the stimulatory effect of DHP or IGF-I or GtH on GVBD. This information will be important in hormonal manipulation during induced breeding of carp.

Effect of actinomycin D and cycloheximide on ischemic preconditioning-induced delayed cardioprotective effect in rats.

The present study was designed to investigate the effect of actinomycin D, a transcription inhibitor, and cycloheximide, a translation inhibitor, on the delayed cardioprotective effect of ischemic preconditioning. Left thoracotomy was performed in anaesthetized rats at 4th/5th intercostal space and polypropylene suture (5-0) was employed to occlude left common coronary artery. Ischemic preconditioning was produced by four episodes of 5 min of coronary artery occlusion followed by 5 min of reperfusion and thoracic cavity was sutured. Left thoracotomy was performed again after 24 hr of ischemic preconditioning and left coronary artery was occluded for 30 min followed by reperfusion for 120 min. Area at risk and infarct size was estimated by patent blue and TTC staining respectively. Total left ventricular RNA was isolated and estimated quantitatively. Ischemic preconditioning, 24 hr after its induction, produced significant decrease in myocardial infarct size occurred as a result of sustained ischemia and reperfusion but produced no marked effect on ventricular RNA content. Actinomycin D and cycloheximide only, in high dose, markedly attenuated ischemic preconditioning induced decrease in myocardial infarct size. However, no such effect was noted with low dose of cycloheximide. The results suggest that delayed cardioprotective effect of ischemic preconditioning may be mediated through denovo synthesis of protein(s) which is regulated both at transcriptional and translational level.

Auxin regulated poly(A)polymerase activity in Cicer arietinum epicotyls.

There was a linear increase in poly (A+) polymerase activity in the C. arietinum epicotyls during germination. Six-day-old auxin treated seedlings showed about 3-4 fold stimulation in enzyme activity, accompanied with 3- fold rise in the relative abundance of poly (A+) RNA levels. Actinomycin D, cycloheximide, cordycepin and amino acid analogues caused dramatic decline in poly (A+) polymerase as well as poly (A+) RNA levels. It seems that auxin induced a de novo synthesis of this enzyme.

Regulation of bcl-2 family in hydrogen peroxide-induced apoptosis in human leukemia HL-60 cells

Numerous types of cells have been shown to undergo apoptosis when exposed to oxidant agent such as hydrogen peroxide. In order to understand the functional relationship between the anti- and pro-apoptotic regulatory proteins in the cells under oxidant stress, we have studied the level of expression of apoptosis regulatory proteins, bcl-2 and bax, in human leukemia HL-60 cells. The exposure of HL-60 cells to different concentrations of H2O2 for 6 h resulted in a typical apoptosis of the cells as characterized by flow cytometry, cell cycle analysis, and DNA fragmantation. There was a block in G1 to S transition and apoptotic cells were mainly derived from S and G2 cells. Kinetic study demonstrated that the levels of both bcl-2-mRNA and -protein expression were decreased with the progression of cellular apoptosis whereas the level of bax-mRNA was unchanged but the expressed bax-protein was not detectable. Cycloheximide, a nonspecific translation inhibitor, did not prevent the hydrogen peroxide-mediated apoptosis in HL-60 cells. These results suggest that the regulation of bcl-2, but not of bax are important factor in the oxidative stress-induced apoptosis in HL-60 cells.

Fas-mediated apoptosis and expression of related genes in human malignant hematopoietic cells

Fas transduces apoptotic signals upon cross-linking with the Fas ligand (FasL), which is experimentally replaced by agonistic anti-Fas monoclonal antibodies (mAb). Of eight human malignant hematopoietic cell lines (HL-60, KG-1, THP-1, K562, U937, Jurkat, IM-9, RPMI-8226) examined by flow cytometric analysis, all, except K562, were found to be positive for surface Fas antigen. However, despite surface Fas expression, the agonistic anti-Fas mAb (7C11) induced apoptosis in only three of seven Fas-expressing cell lines (KG-1, Jurkat and IM-9). This Fas-resistance did not correlated with high levels of mRNA either for DcR3, a decoy receptor for FasL, or for FAP-1, a Fas-associated phosphatase that can block the apoptotic function of Fas. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis did not show consistent differences in the expression of Bcl-2 and Bax between Fas-sensitive and Fas-resistant cell lines examined. These findings indicated that the presence or absence of mRNA expression of DcR3, FAP-1, Bcl-2 and Bax did not always correlate with relative sensitivity to Fas-mediated apoptosis. Treatment of cells with cycloheximide converted the phenotype of resistant cell lines from Fas-resistant to Fas-sensitive, and enhanced the sensitivity of Fas-sensitive cell lines. These results suggest that the Fas-resistance is dependent on the presence of labile proteins that determine resistance to Fas-mediated apoptosis and the apoptotic machinery is already in place in Fas-resistant cell lines.

Effect of serum and protein synthesis inhibitors on polymorphonuclear neutrophil apoptosis

In this study the programmed cell death [apoptosis] of human neutrophilic granulocytes was investigated in the presence and absence of fetal calf serum [FCS], cycloheximide and actinomycin-D. The results show that when FCS is omitted from cultures, apart from a decrease in viability, the percentage of apoptotic cells and DNA fragmentation increases. Apoptosis is accelerated in serum withdrawal cultures at 6 hours of incubation time. The use of fluorescent dyes and diphenylamine reaction procedures confirm the above results. Treatment of cells with protein synthesis inhibitors, actinomycin-D and cycloheximide promotes apoptosis and produces a typical ladder of internucleosomal cleavage in the cellular chromatin; the extent of fragmentation however, differs

Intraventricular cycloheximide attenuates the restraint-induced long-lasting effect on plus maze exploration

Rats submitted to 2 h of restraint stress show reduced open arm exploration in the elevated plus maze 24 h later. To determine if this effect is dependent on protein synthesis during or after the restraint period, cycloheximide, a protein synthesis inhibitor, was injected into the right cerebral ventricle of male Wistar rats (200-250 g), immediately before (N = 19 animals per group), immediately after (N = 7 animals per group) or 2 h (N = 10 animals per group) following a 2h period of forced restraint. Twenty-four hours later the animals were tested in the elevated plus maze. Non-stressed control groups received saline (SAL, N = 8-9 per group) or cycloheximide (CHX, N = 8-9 per group) and were tested 1 h or 24 h later in the maze. Pre- but not post-stress microinjections of cycloheximide (20 mug in 2 mul) increased exploration in the elevated plus maze (per cent of time spent in open arms, pre-stress injection: SAL = 4.6 ñ 1.2, CHX = 10.7 2.3; number of enclosed arms entries: SAL = 3.6 ñ 0.5, CHX = 5.6 0.4). No drug effect was observed in non-stressed animals. These results suggest that blockade of protein synthesis during the restraint period may attenuate the behavioral consequences of stress.

Kinetics of translation and glycosylation of pituitary glycoproteins

The present study determines the basal kinectics of synthesis of translation (by[(14)C] leucine incorporation. [(14)C] leu-PROT] and of proximal (by [(3)H] mannose incorparation. [(3)H] man-PROT) and distal (by [3H] galactose incorporation, [(3)H] gal-PROT) glycosylation of total adenohypophyseal glycoproteins, by rat pituitary cells in primary culture. In order to obtain more information regarding the role of both septs of glycosylation on the secretory process, the effects of cycloheximide (CH; translation inhibitor) and tunicamycin (TM; glycosylation inhibitor) on the kinetics of synthesis and release of pituitary glycoproteins were also studied. Cells were incubated in medium containing [(14)C] leu plus [(3)H] man or [(14)C] leu plus [(3)H] gal, for various time-intervals (from 0.5 to 5h) in the abscence (control) or presence of different doses od CH (1.0;4.0 or 16.0 mug/ml) or TM (0.5;1.0 or 2.0 mug/ml). The kinetics of synthesis (slope=3488) and release (slope=622) of [(14)C] le-PROT were higler than those of the sugar percursos (slopes: [(3)H] man-PROT=1751 and 526; [(3)H] gal-PROT=1231 and 506). Leucine or mannose-labeled protein was barely detectable in the medium after 2h incubation, whereas galactose-labeled protein had already been released into the incubation medium by 30min. Cycloheximide induced translation blockage and, concomitantly, produced a marked inhibition of [(3)H] man incorporation. On the other hand, TM inhibited the kinetics of synthesis and release of [(3)H] man-PROT without affecting those of [(14)C] leu-PROT. The kinetics of synthesis and release of [(3)H] gal-PROT, although diminished, maintained linearity and increased in function of time, even in the presence of the antibiotics. Thus, the present results on glycoproteins from the pituitary gland are consistent with the previous conclusion for other mammalian glycoproteins that carbohydrate attachment occurs in several steps to molecules destined to be secreted. Addition of mannose (proximal glycosylation) is a co-translational event and that of galactose (distal glycosylation) is post-translational and can be designated as final stages in carbohydrate assembly, occurring close to the time of release. Furthermore, it has been demonstrated that the absence of the carbohydrate side chains of the pituitary glycoprotein does not prevent the intracellular transport of the protein and its export from the cell.