Telomere length and mitochondrial DNA copy number in bipolar disorder: identification of a subgroup of young individuals with accelerated cellular aging.

The 10-15-years decrease in life expectancy observed in individuals with bipolar disorder (BD) has been linked to the concept of accelerated cellular aging. Telomere length (TL) and mitochondrial DNA copy number (mtDNAcn) have been proposed as markers of cellular aging and comparisons between individuals with BD and healthy controls (HC) sometimes led to conflicting results. Previous studies had moderate sample sizes and studies combining these two markers into a single analysis are scarce. Using quantitative polymerase chain reaction, we measured both TL and mtDNAcn in DNA (peripheral blood) in a sample of 130 individuals with BD and 78 HC. Regression analyses, receiver operating characteristic (ROC), and clustering analyses were performed. We observed significantly lower TL and mtDNAcn in individuals with BD as compared to HC (respective decrease of 26.5 and 35.8%). ROC analyses showed that TL and mtDNAcn highly discriminated groups (AUC = 0.904 for TL and AUC = 0.931 for mtDNAcn). In the whole population, clustering analyses identified a group of young individuals (age around 36 years), with accelerated cellular aging (both shorter TL and lower mtDNAcn), which consisted mostly of individuals with BD (85.5%). The subgroup of patients with young age but accelerated aging was not characterized by specific clinical variables related to the course of BD or childhood maltreatment. However, patients in this subgroup were more frequently treated with anticonvulsants. Further characterization of this subgroup is required to better understand the molecular mechanisms and the risk factors of accelerated cellular aging in BD.

Adeno-associated virus may play a protective role against human papillomavirus-induced cervical lesions independent of HIV serostatus.

This study investigated the prevalence of adeno-associated virus (AAV) and human papillomavirus (HPV) DNA in cervical samples of HIV-seropositive and -seronegative women attending a clinic in south-eastern Brazil. Both viruses were investigated by polymerase chain reaction (PCR) and cytological exams were performed. AAV was typed by PCR and restriction fragment length polymorphism analysis. AAV prevalence was 19.7% (56/284), with 18.7% (21/112) and 20.3% (35/172) in HIV-positive and -negative women, respectively. AAV type 2 was the single virus type detected. AAV was detected with higher frequency in HPV-infected women (P < 0.05) as was HPV in HIV-positive women (P < 0.05). The AAV-HPV co-infected women showed a lower rate of atypical squamous cells of undetermined significance or cervical intraepithelial neoplasia development compared with those infected only with HPV. The prevalence of AAV2 confirms this type as the most common in human samples. This is the first report examining AAV in cervical samples of HIV-infected women and indicates that HIV infection does not appear to influence AAV prevalence or AAV-HPV co-infection.

Hydration, sporoderm breaking and germination of Cupressus arizonica pollen.

In vitro and in vivo rehydration and germination in Cupressus arizonica pollen were examined using light and scanning electron microscopy. Shed pollen has 12.6% water content, which reduced to 8.2% after dispersal, and this latter pollen survived for some months at room temperature and for years at -10 degrees C. Rehydration requires breaking of the sporoderm walls and depends on the composition and pH of the rehydration medium. Acidity restrains the breakage, while alkalinity promotes it. Pollen division follows exine shedding and requires the persistence of the mucilaginous layer; hence, pH values countering these outcomes prevent division. Division results in a large and a small cell separated by a callosic wall. A pollen tube develops from the innermost intine of the large cell, which is callosic, and extends into the mucilaginous middle intine. The percentage germination never exceeded 17% in all tested media. In vivo, pollen rehydrates and casts off the exine in the micropylar drop. Drop withdrawal brings pollen to the apical nucellar cells that degenerate in the meantime, and it leaves a deposit on the surface of the micropylar canal. After contaction of the nucellar cells, the pollen flattens and its mucilaginous layer shrinks and disappears. This occurs simultaneously with sealing of the micropylar canal. During this time, pollen divides asymmetrically without the callosic wall, and the larger cell develops a tube in the interface with the nucellus. Only some pollen grains accomplish adhesion to the nucellus and germinate. The in vitro and in vivo developmental stages are discussed.

High doses of atrazine do not disrupt activity and expression of aromatase in female gonads of juvenile goldfish (Carassius auratus L.).

Juveniles female goldfish were exposed to atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) at high doses, 100 and 1000 microg l(-1) during 56 days in order to evaluate the potential action of the herbicide as an endocrine disruptor. Plasma concentration of estradiol (E2) and 11-ketotestosterone (11-KT) as well as activity and expression of aromatase in the gonads were evaluated. These parameters were completed with morphological measures such as gonadosomatic index (GSI) and histological analyses of gonads. Morphological parameters at both 100 and 1000 microg l(-1) did not show any significant differences with the control groups. Correlated to the pathway hypothesized, no time-, dose-related effects were detected on the aromatase activity and the expression in the gonads of juvenile female goldfish. The same conclusion was attributed regarding the circulating E2 where no perceptible variation was detected. Nevertheless, a hormonal imbalance was detected for plasma concentration of the sex steroid 11-KT of fish exposed to 1000 microg l(-1) after 56 days exposure. In these particular experimental conditions, we failed to demonstrate an effect of atrazine through the induction of aromatase and hormonal imbalance associated.

Interactions between saporin, a ribosome-inactivating protein, and DNA: a study by atomic force microscopy.

Saporins are enzymes belonging to the PNAG class (polynucleotide: adenosine glycosidase), plant enzymes commonly known as ribosome-inactivating proteins (RIP), as a result of their property of irreversibly damaging eukaryotic ribosomes. Direct imaging with tapping-mode atomic force microscopy (AFM) has been used to study pGEM-4Z plasmid DNA binding to the saporin-SO6 (isoform from Saponaria officinalis seeds). Saporin wrapped the plasmidic DNA, and distribution of the enzyme molecules along the DNA chain was markedly variable; plasmid digested with saporin-SO6 appeared fragmented or topologically modified. The supercoiled DNA strands were cleaved, giving rise to a linearized form and to relaxed forms. Electrophoretic analysis of the effect of standard preparations of saporin-SO6 on pGEM-4S confirmed the presence of DNA strand-cleaving activity.

Prevalence of human cytomegalovirus infection in pregnant and non-pregnant women.

OBJECTIVES: To determine human cytomegalovirus (HCMV) cervical reactivation in both pregnant and non-pregnant women and to ascertain whether or not it occurs in conjunction with hematogenic dissemination. METHODS: Clinical specimens were obtained from 40 pregnant and 62 non-pregnant women attended at the Ambulatory of the Gynecology-Obstetrics Unit of the Federal University of Espírito Santo (UFES) in Southeastern Brazil. Specimens under investigation were blood samples submitted to seroprevalence determination, antigenemia assay, HCMV-DNA detection, and vaginal secretion, submitted to HCMV-DNA detection. RESULTS: Viral seroprevalence was found in 98% of the women investigated, two of whom were found to be IgM positive, while no difference could be determined between pregnant and non-pregnant women. Antigenemia assay was negative in all cases. HCMV gB gene amplification was found in 5.1 and 8.5% of WBCs and in 10 and 14.5% of vaginal secretion from pregnant and non-pregnant women, respectively. CONCLUSION: The high seroprevalence observed is in accordance with previous Brazilian surveys. Antigenemia assay was unable to detect the occurrence of active infection in the immunocompetent women studied, most likely because it either occurred in a viral load undetectable by this assay or did not occur at all. Although the highest incidence of positivity was observed by gene amplification both in WBCs and secretion from non-pregnant than in pregnant women, the rate of viral detection was statistically similar for both groups.

Cytomegalovirus in human abortion in Espírito Santo, Brazil.

BACKGROUND: Despite the established implication of human cytomegalovirus (HCMV) in congenital infection, there are still conflicting reports regarding the association of HCMV with spontaneous abortion. Viral antigens and nucleic acid were already described in tissues from abortions cases, but did not indicate HCMV pathogenical role. OBJECTIVES: (1) To access viral seroprevalence (total and IgM antibodies) in pregnant, non-pregnant and in women in abortion process, (2) to evaluate if antigenemia assay can detect active infection in these populations, (3) to detect viral DNA in peripheral leukocytes, and (4) in abortion tissues. STUDY DESIGN: Blood samples from 95 patients in abortion process and from two control groups (40 pregnant and 60 non-pregnant women) were obtained for determination of viral seroprevalence, for detection of antigen and viral DNA by PCR from peripheral leukocytes. Specimens obtained from 88 patients in abortion process, spontaneous or induced, were submitted to gB gene amplification (PCR and nested-PCR). RESULTS: Viral seroprevalence were found in 97.3 with 2.5% of IgM positive cases. Antigenemia assay were negative in all cases, however, viral nucleic acid were found in 6.3 and in 6.0% of the patients in abortion and in control groups, respectively. Nucleic acid in conception tissue was present in 6.6%. CONCLUSION: This high seroprevalence observed is according to previous surveys in Brazil. If active infection due to viral reactivation occurred during the abortion process, it cannot be accessed by antigenemia or anti-IgM assays. Nucleic acid found by PCR in peripheral blood cells enriched with polymorphonuclear cells (PMN) corresponds to viral circulation in immunocompetent person, as similar results were found for the three groups. Although viral DNA had been found in 6.6% from abortion tissues, this result does not support HCMV as a major abortion-related factor as we could not found any correlation between abortion and active HCMV infection.

Effects of atrazine on osmoregulation in the Chinese mitten crab, Eriocheir sinensis.

This study integrates results from acute contamination with atrazine of isolated perfused gills, and from in vivo chronic contamination of euryhaline Chinese mitten crabs, Eriocheir sinensis, acclimated to freshwater. Atrazine 1 mg/l in contact with the basolateral membrane (IN) increases the transepithelial potential difference (TEP) from -20.8 +/- 4.9 to -29.7 +/- 3.8 mV in isolated perfused posterior gills (P < 0.01). This effect is only partially explained by a modification of Na(+) and Cl(-) active influxes. No TEP modification is detected when atrazine is added (OUT) indicating that molecular mechanisms located on the basolateral membrane are likely to be the only ones affected. Another explanation would be that cuticular barrier prevents atrazine penetration into the gill. Haemolymph osmolarity, Na(+) and Cl(-) concentrations of crabs living in freshwater contaminated with atrazine 1 mg/l during 14 days are not significantly modified. We conclude that although atrazine can disturb osmoregulatory mechanisms of isolated gills, this pollutant would be of minor importance in affecting osmoregulatory capacities of the Chinese mitten crab in natural conditions.

Nuclear damage induced by liposomes containing fitc-labelled saporin on human melanoma cells in vitro.

Ribosome-inactivating proteins are enzymes of plant origin which de-adenilate the major ribosomal RNA, making it unable to bind the elongation factor and thus arresting protein synthesis. Recently the N-glycosidase activity of these enzymes has been extended also to deoxyribonucleotides substrates. In the present study we report the successful entrapment of the type 1 ribosome-inactivating protein saporin, covalently labelled with fluorescein isothiocyanate (FITC) into L-alpha lecitin/cholesterol liposomes and describe its delivery to human melanoma cells in vitro. The fluorescein reacted toxin maintained its enzymatic activity, although to a reduced extent; its interaction with liposomes resulted in the entry of the protein through the lipid bilayers. The resulting vesicles are carriers that can deliver the toxin inside cells; as a consequence the cytotoxic effects of the encapsulated enzyme were evident at a concentration two order of magnitude lower than that of the native one. In particular the nuclear damage, as revealed by micronuclei formation, was evident within 44 hr. The intracellular dynamics of the enzyme, as analyzed by confocal microscopy, point to an endocytic pathway of vesicles entry.

Antiproliferative effect and apoptotic response in vitro of human melanoma cells to liposomes containing the ribosome-inactivating protein luffin.

The present study describes the liposome-mediated delivery of the type 1 ribosome-inactivating protein luffin to human melanoma cells in vitro. Luffin from Luffa cylindrica seeds has been successfully incorporated into lecithin/cholesterol and lecithin/cholesterol/dicetylphosphate negatively charged liposomes. The exposure of melanoma cells to the two types of liposomes resulted in the inhibition of protein synthesis and cell growth; apoptotic cell death was verified by means of TUNEL reaction and quantitation of cytosolic oligonucleosome-bound DNA. The toxicity of encapsulated luffin varied with the lipid composition of the vesicles; the strongest effect was observed with lecithin/cholesterol liposomes. These results identify liposome-incorporated luffin as a possible alternative to immunotoxins for the treatment of human melanoma in situ.

The plant ribosome inactivating protein saporin induces micronucleus formation in peripheral human lymphocytes in vitro.

Saporin belongs to the family of plant enzymes known as ribosome inactivating proteins (RIPs) for their property to depurinate the major rRNA, thus leading to inactivation of ribosomes. In this work we analyzed the genotoxic effects of saporin, purified from root cultures of Saponaria officinalis, by evaluating micronucleus formation and by the quantitative determination of cytosolic histone-associated DNA fragments. Saporin induces micronuclei formation in cultured human lymphocytes in a dose dependent manner; treated lymphocytes show a decrease in cell viability and a concomitant increase in the apoptotic response evidenced by the appearance of cytosolic oligonucleosomes. On the other hand saporin treatment failed to induce sister-chromatid exchange (SCE) at any of the doses tested.

Differential response of human melanoma and Ehrlich ascites cells in vitro to the ribosome-inactivating protein luffin.

The cytotoxicity and inhibitory effect on proliferation of the type 1 ribosome-inactivating protein luffin purified from the seeds of Luffa aegyptiaca were investigated both in human metastatic melanoma cells and in murine Ehrlich ascites tumour cells. Results indicate that luffin from the seeds of Luffa aegyptiaca is cytotoxic to the cell lines tested, with approximately 10 times greater potency in Ehrlich cells. Luffin was found to induce an increase in cytosolic oligonucleosome-bound DNA in both melanoma and Ehrlich ascites tumour cells, the level of DNA fragmentation in the former cell line being higher than in the latter. Experiments with melanoma cells indicate that an increase in cytosolic nucleosomes could be supportive of apoptosis as the type of cell death induced by luffin.

A simple method for the purification of type 1 RIPs.

We describe a straightforward and simple method for obtaining pure and active preparations of type 1 ribosome inactivating proteins (RIPs). The very high isoelectric point values, characteristic of these proteins, allow this purification in a single chromatographic step.

Saporin production from in vitro cultures of the soapwort Saponaria officinalis L.

We report here the successful establishment of callus, cell and root cultures from explants of in-vitro-grown plantlets of the soapwort Saponaria officinalis L. The production of saporin in the different tissue systems was evaluated by determining the capability of crude extracts to inactivate protein synthesis and by Western blotting analysis. Protein synthesis inhibition varied in callus and derived cell suspensions and in cultured roots, the latter, in particular, showing the lowest specific activity. The ribosome-inactivating principle from root cultures was purified to homogeneity by cation exchange chromatography.

RolB expression pattern in the early stages of carrot somatic embryogenesis.

The pattern of expression of the rolB gene, derived from the T-DNA of the plant pathogen Agrobacterium rhizogenes, has been investigated during the early stages of somatic embryo formation in suspension cultures of carrot (Daucus carota L.). The reporter gene GUS (Escherichia coli beta-glucuronidase), under transcriptional control of full-length rolB promoter region, has been utilized in order to evaluate both qualitative and quantitative variations in the expression pattern. Fluorimetric measurements point to the developmental regulation of the gene, while results from histochemical analysis indicate that the promoter of rolB is firstly activated in the central (core) region of the globular embryos.

Production of ribosome-inactivating protein from hairy root cultures of Luffa cylindrica (L.) Roem.

Transformed root lines of Luffa cylindrica (L.) Roem. (Cucurbitaceae) were established by inoculation of in vitro grown plantlets with wild type Agrobacterium rhizogenes strain 1855. Cloned lines of hairy roots were tested for the presence of ribosome-inactivating proteins; crude extracts inhibited protein synthesis in a reaction mixture based on rabbit reticulocyte lysate. Inhibitory activity increased during culture period, reaching a maximum value in the stationary phase. No activity could be detected in the culture medium, nor in extracts from callus and/or suspension cultures. A ribosome-inactivating protein having specific activity of 62,100 U mg protein(-1) and a molecular mass of 26-28,000 Da was purified to homogeneity. The protein showed N-glycosidase activity on rat liver ribosomes. The results demonstrate that hairy root cultures can be successfully utilized for the in vitro production of ribosome-inactivating proteins.

Effectiveness of cascade filtration plasmapheresis in two patients affected by familial hypercholesterolemia.

Hypercholesterolemia has been recognised as a primary risk factor for coronary heart disease. Reduction of plasma levels of total and LDL cholesterol has been shown to decrease coronary atherosclerosis. Plasmapheresis represents an useful non-pharmacological tool to treat severe hypercholesterolemias. We have evaluated the effectiveness of a system of plasmapheresis using a cascade filtration method in two young male subjects (aged 16 and 26 years) with homozygous familial hypercholesterolemia. Both showed severe coronary atherosclerosis as determined by angiography. Procedures were performed at intervals of 7 days in each case. We observed a mean reduction of plasma levels of total cholesterol of 59.5% (range 31.0-75.5%); LDL-cholesterol, 61.6% (range 32.6-77.1%); triglycerides, 48.1%; HDL-cholesterol, 31.1%; apo A-I, 30.8%; and apo B, 57.6%. We also noted a reduction of other parameters, such as fibrinogen (49.9%) and Lp(a) (59.9%). At the end of each procedure about 8 g of cholesterol was removed from the total body pool. A decrease of total proteins (26.9%) and albumin (19.6%) was also observed, but this was completely restored before the next apheresis (1 week). These data show the effectiveness of the removal of LDL in a cascade filtration system, which obtains results not very different from other more selective methods. The lack of selectivity is not much of a problem, since it also reduces other risk factors such as Lp(a) and fibrinogen.

Apo-lipoprotein profile in subjects with extracranial carotid atherosclerosis.

Relationships between plasma lipoproteins and cerebrovascular atherosclerosis are not completely clear. In a group of asymptomatic nondiabetic normolipidemic subjects, plasma lipid and apolipoprotein profiles have been related to extracranial carotid atherosclerotic lesions, as assessed by B-mode ultrasonography and independent relations between lipid and clinical parameters and carotid atherosclerosis have been evaluated. We have found that subjects with atherosclerotic lesions (both intimal thickening or plaque) had TG levels and CHO/HDL-C and LDL-C/HDL-C ratios significantly higher and HDL-C and apo A-I levels significantly lower in comparison with subjects with normal arteries. When patients were divided according to the lesions of carotid arteries subjects with atherosclerotic plaque presented HDL-C and apo A-I levels significantly reduced and TG and apo B levels and CHO/HDL-C and LDL-C/HDL-C ratios significantly increased in comparison with subjects with normal arteries, and HDL-C levels reduced and CHO/HDL-C and LDL-C/HDL-C ratios increased in comparison with subjects with intimal thickening. Patients with intimal thickening and normal subjects differed for HDL-C and TG levels and CHO/HDL-C and LDL-C/HDL-C ratios. At multivariate analysis HDL-C levels (negatively), age, hypertension and cigarette smoking (positively) resulted independently associated with cerebrovascular atherosclerosis. Our data seem to show that, although several lipid and apoprotein abnormalities are able to initiate the atherosclerotic process in extracranial carotid district, probably the presence of low HDL-cholesterol levels is an important condition to determine the further worsening of the lesions.