Resistin as a new player in the regulation of porcine corpus luteum luteolysis: in vitro effect on proliferation/viability, apoptosis and autophagy.

The formation and luteolysis of the corpus luteum (CL) is strictly controlled by many factors. Imbalance between proliferation and apoptosis processes leads to deficiency of the luteal phase and infertility. Our previous study showed resistin expression in porcine luteal cells and an inhibitory effect on progesterone synthesis. Thus, the aim of the present study was to examine the in vitro effect of resistin on the proliferation/viability, apoptosis and autophagy of porcine luteal cells as well as the involvement of mitogen-activated kinase (MAP3/1), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) in these processes. Porcine luteal cells were incubated with resistin (0.1-10 ng/mL) for 24-72 h and viability was assessed using the alamarBlue or 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the time-dependent effect of resistin on mRNA and protein expression of proliferating cell nuclear antigen (PCNA), caspase 3, BCL2-like protein 4 (BAX), B-cell lymphoma 2 (BCL2), beclin1, microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal-associated membrane protein 1 (LAMP1) was measured by real-time polymerase chain reaction (PCR) and immunoblotting, respectively. We found that resistin enhanced luteal cell viability with no effect on caspase 3 mRNA and protein, increased the BAX/BCL2 mRNA and protein ratio and significantly stimulated the initiation of autophagy, which promotes the maintenance of CL function rather than its regression. Additionally, using pharmacological inhibitors of MAP3/1 (PD98059), AKT (LY294002) and STAT3 (AG490), we observed that the effect of resistin was reversed to the control level in viability and, by influence, MAP3/1 and STAT3 in autophagy. Taken together, our results suggest that resistin, in addition to its well-known effect on granulosa cell function has direct influence on CL luteolysis and the formation and maintenance of luteal cell function.

Cadmium mobility in a circular food-to-waste-to-food system and the use of a cultivated mushroom (Agaricus subrufescens) as a remediation agent.

Cadmium (Cd) and its mobility and toxicity in the food chain receives considerable public and scientific attention. Levels in soil have been increasing, partly caused by mineral fertiliser use. In many areas in the EU, intake of Cd is not far from the maximum tolerable level. Now, urban and more circular approaches to food production are being promoted, in which waste materials substitute growing media and fertilizers. Can this make the situation worse, or is it, on the contrary, possible to extract Cd from the cycle using practical microbial methods? Cd concentration, availability and accumulation in materials and crops in a closed greenhouse system was investigated first. Secondly, we tested if fungal remediation was able to remove a significant amount of Cd. A sequential extraction analysis showed how anaerobic digestion and composting changed chemical Cd availability. Cd levels in the system were comparable to agricultural soils. The vegetable crops did not accumulate Cd. However, the culture of the edible mushroom Agaricus subrufescens in the greenhouse had a strong effect on Cd mobility: It extracted 80% of the Cd from the growing medium into the mushrooms, mostly at the start of fruiting. These early Cd enriched mushrooms can be treated as special waste, while the later harvest complied with EU Cd limits for foodstuffs and can potentially be consumed. Thus, growing this mushroom species in an integrated waste-to-crop system can be a cost-efficient way to remediate Cd from the food chain.

Obesity and obesogenic growth are both highly heritable and modified by diet in a nonhuman primate model, the African green monkey (Chlorocebus aethiops sabaeus).

OBJECTIVE: In humans, the ontogeny of obesity throughout the life course and the genetics underlying it has been historically difficult to study. We compared, in a non-human primate model, the lifelong growth trajectories of obese and non-obese adults to assess the heritability of and map potential genomic regions implicated in growth and obesity. STUDY POPULATION: A total of 905 African green monkeys, or vervets (Chlorocebus aethiops sabaeus) (472 females, 433 males) from a pedigreed captive colony. METHODS: We measured fasted body weight (BW), crown-to-rump length (CRL), body-mass index (BMI) and waist circumference (WC) from 2000 to 2015. We used a longitudinal clustering algorithm to detect obesogenic growth, and logistic growth curves implemented in nonlinear mixed effects models to estimate three growth parameters. We used maximum likelihood variance decomposition methods to estimate the genetic contributions to obesity-related traits and growth parameters, including a test for the effects of a calorie-restricted dietary intervention. We used multipoint linkage analysis to map implicated genomic regions. RESULTS: All measurements were significantly influenced by sex, and with the exception of WC, also influenced by maternal and post-natal diet. Chronic obesity outcomes were significantly associated with a pattern of extended growth duration with slow growth rates for BW. After accounting for environmental influences, all measurements were found to have a significant genetic component to variability. Linkage analysis revealed several regions suggested to be linked to obesity-related traits that are also implicated in human obesity and metabolic disorders. CONCLUSIONS: As in humans, growth patterns in vervets have a significant impact on adult obesity and are largely under genetic control with some evidence for maternal and dietary programming. These results largely mirror findings from human research, but reflect shorter developmental periods, suggesting that the vervet offers a strong genetic model for elucidating the ontogeny of human obesity.

Efficiency of a novel "Food to waste to food" system including anaerobic digestion of food waste and cultivation of vegetables on digestate in a bubble-insulated greenhouse.

At urban locations certain challenges are concentrated: organic waste production, the need for waste treatment, energy demand, food demand, the need for circular economy and limited area for food production. Based on these factors the project presented here developed a novel technological approach for processing organic waste into new food. In this system, organic waste is converted into biogas and digester residue. The digester residue is being used successfully as a stand-alone fertilizer as well as main substrate component for vegetables and mushrooms for the first time - a "digeponics" system - in a closed new low energy greenhouse system with dynamic soap bubble insulation. Biogas production provides energy for the process and CO2 for the greenhouse. With very limited land use highly efficient resource recycling was established at pilot scale. In the research project it was proven that a low energy dynamic bubble insulated greenhouse can be operated continuously with 80% energy demand reduction compared to conventional greenhouses. Commercial crop yields were achieved based on fertilization with digestate; in individual cases they were even higher than the control yields of vegetables such as tomatoes, cucumber and lettuce among others. For the first time an efficient direct use of digestate as substrate and fertilizer has been developed and demonstrated.

Increased formation of autophagosomes in ectromelia virus-infected primary culture of murine bone marrow-derived macrophages.

Induction of autophagy by ectromelia virus (ECTV) in primary cultures of bone marrow-derived macrophages (BMDMs) was investigated. The results showed that ECTV infection of BMDMs resulted in increased formation of autophagosomes, increased level of LC3-II protein present in aggregates and extensive cytoplasmic vacuolization. These data indicate an increased autophagic activity in BMDMs during ECTV infection.

In vivo induction of autophagy in splenocytes of C57BL/6 and BALB/c mice infected with ectromelia orthopoxvirus.

Autophagy is a self-degradation process of cellular components. It plays both antiviral and pro-viral roles in the life cycle of different viruses and the pathogenesis of different viral diseases. In this study, we evaluated autophagy induction in splenocytes of ectromelia virus (ECTV)-resistant C57BL/6 and ECTV-susceptible BALB/c mice during infection with the Moscow strain of the ectromelia virus (ECTV-MOS). Autophagy was analyzed using the Western blot method by assessing type II microtubule-associated protein 1 (MAP1) light chain 3 (LC3) and Beclin 1 expression levels relative to beta-actin. Results indicated an increased ratio of LC3-II to beta-actin in splenocytes of C57BL/6 mice only at 7 day post infection (d.p.i.) compared to uninfected animals. LC3-II/beta-actin and Beclin 1/beta-actin ratios in splenocytes of BALB/c mice increased at 5 d.p.i. and remained high until day 14 and 7 p.i., respectively. We confirmed the formation of autophagosome structures in the spleen of BALB/c mice by transmission electron microscopy (TEM). Moreover, autophagy accompanied necrosis in the splenocytes of infected animals. Results suggest that ECTV-MOS induced autophagy, especially in the spleen of the susceptible mouse strain, may support viral replication and promote cell necrosis.

Adipohormones as prognostric markers in patients with nonalcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) represents one of the most common liver diseases. It is strongly associated with obesity and insulin resistance and is thought to be part of the metabolic syndrome. NASH can progress to cirrhosis and liver failure. Adipohormones, synthesized in adipose tissue, are involved in the pathophysiology of many acute and chronic liver diseases. The aim of this study was to evaluate the plasma concentrations of adiponectin, resistin, leptin, TNF-alpha and Il-6 in patients with NASH, as well as their correlation with the pathologic parameters. Serum concentration of leptin, adiponectin, resistin, insulin, TNF-alpha, IL-6 were measured with ELISA method. Liver biopsies were obtained from 18 (age 42.55+/-21 years) patients. NASH has been classified according to Dixon score. The control group was represented by 16 non-obese subjects. Mean serum concentration of adiponectin in patients with NASH was significantly lower than in healthy subjects (4.87+/-1.96 vs. 8.33+/-4.56 ng/ml; p<0.05). Mean serum levels of TNF-alpha in patients with NASH were significantly higher than in controls (34.2+/-19.7 vs. 20.7+/-15.5 ng/ml; p<0.05). In patients with more advanced inflammation (grade 2-3) and fibrosis (stage 2) in pathology, serum concentration of leptin was significantly higher than in patients with steatosis and less advanced inflammation (grade 1) and fibrosis (stage 1) (median 8.94 vs. 16.2 ng/ml; p<0.05). No significant differences of serum concentration of others adipohormones between these two groups of patients were stated. Moreover, we observed the correlation in serum levels (examined group vs controls) between: resistin and TNF-alpha (r = 0.62; p<0.05), adiponectin and IL-6 (r = -0.60; p<0.05) and leptin and insulin (r = -0.51; p<0.05). In conclusion, based on our study we speculate that changes of adipohormones levels may be markers of NASH and the serum level of leptin can be associated with more advanced form of NASH.

A narrow and highly significant linkage signal for severe bipolar disorder in the chromosome 5q33 region in Latin American pedigrees.

We previously reported linkage of bipolar disorder to 5q33-q34 in families from two closely related population isolates, the Central Valley of Costa Rica (CVCR) and Antioquia, Colombia (CO). Here we present follow up results from fine-scale mapping in large CVCR and CO families segregating severe bipolar disorder, BP-I, and in 343 population trios/duos from CVCR and CO. Employing densely spaced SNPs to fine map the prior linkage peak region increases linkage evidence and clarifies the position of the putative BP-I locus. We performed two-point linkage analysis with 1134 SNPs in an approximately 9 Mb region between markers D5S410 and D5S422. Combining pedigrees from CVCR and CO yields a LOD score of 4.9 at SNP rs10035961. Two other SNPs (rs7721142 and rs1422795) within the same 94 kb region also displayed LOD scores greater than 4. This linkage peak coincides with our prior microsatellite results and suggests a narrowed BP-I susceptibility regions in these families. To investigate if the locus implicated in the familial form of BP-I also contributes to disease risk in the population, we followed up the family results with association analysis in duo and trio samples, obtaining signals within 2 Mb of the peak linkage signal in the pedigrees; rs12523547 and rs267015 (P = 0.00004 and 0.00016, respectively) in the CO sample and rs244960 in the CVCR sample and the combined sample, with P = 0.00032 and 0.00016, respectively. It remains unclear whether these association results reflect the same locus contributing to BP susceptibility within the extended pedigrees.

What influences the level of oxidative stress as measured by 8-hydroxy-2'-deoxyguanosine in patients on hemodialysis?

Oxidative stress is at play in the progression of chronic renal failure (CRF) and in the genesis of atherosclerosis. The aim of the present study was to evaluate the factors that might influence the oxidative-antioxidative balance in patients on hemodialysis. The study group consisted of 71 hemodialysis patients due to CRF. Sixteen healthy subjects constituted a control group. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), C-reactive protein (CRP), and the blood lipid profile were measured in both groups. The results showed significantly higher mean levels of both 8-OHdG and CRP in the hemodialysis patients compared with that in the control subjects. The highest level of 8-OHdG was found in the subgroups of the patients with CRF primarily caused by diabetes (16.4 ng/ml) and with hypertensive nephropathy (15.8 ng/ml). More than a 2.5-fold higher level of 8-OHdG in the hemodialysis patients compared with the control subjects points to the presence of intensive oxidative stress in the patients.

Induction of insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells by cells of trophoblast origin.

OBJECTIVE: To determine the influence of the conceptus on the induction of decidualization in endometrial stromal cells from the baboon. METHODS: For in vivo studies, implantation sites from day 22 of pregnancy in the baboon were analyzed for insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) mRNAs using in situ hybridization. For in vitro studies, Jeg-3 cells or primary cytotrophoblasts isolated from term placenta were cocultured with a monolayer of stromal cells from the baboon endometrium. Cytotrophoblasts were placed either directly on top of the stromal cell monolayer or in transwell cell culture inserts and cultured for 7 days. No exogenous hormones were added. Stromal cells were analyzed for IGFBP-1 mRNA and protein by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. RESULTS: Examination of early implantation sites in the baboon revealed a high expression of IGF-II mRNA in the invading cytotrophoblasts. Conversely, the stromal cells of the endometrium directly adjacent to the cytotrophoblasts expressed IGFBP-1 mRNA. Endometrial stromal cells cocultured with Jeg-3 cells or primary cytotrophoblasts for 7 days expressed IGFBP-1 mRNA and protein. This expression occurred in both the direct coculture system and coculture using the cell culture inserts. Cytotrophoblasts also induced the expression of prolactin in stromal fibroblasts following coculture. CONCLUSIONS: The conceptus is capable of inducing decidualization of endometrial stromal cells. This is shown in both the in vivo and in vitro systems.

Study and analytical application of ion-pair formation in the system fluoxetine-pyrocatechol violet and fluvoxamine-pyrocatechol violet.

Pyrocatechol violet (PCV) reacts in aqueous media with fluoxetine (FLX) and fluvoxamine (FLV) forming coloured ion-association complexes, which are insoluble in water but quantitatively extracted into chloroform-n-butanol mixture. The composition of the compounds, studied by spectrophotometric methods showed that the molar ratio PCV : FLX and PCV : FLV is 1 : 1. The compounds were characterized by UV-VIS, IR and NMR spectrometry. Under optimal experimental conditions fluoxetine and fluvoxamine were determined in the range 1.3-18.0 microg/ml and 2.6-39.1 microg/ml, respectively. The proposed methods have been succesfully applied to the determination of these drugs in pharmaceuticals and natural samples.

Prevalence of BRCA1 founder mutations in western Poland.

The frequency of three BRCA1 founder mutations was examined in a group of Polish breast/ovarian cancer patients and women at increased risk of these cancers based on family history. Among the 15 mutations found (5385-5386insC, 187-188delAG, and 4154delA), eight were detected in women with a low or moderate family history. The frequency of the IVS20+48ins12 variant was also analyzed and its distribution within risk groups argues against its involvement in cancer predisposition.

Allelic imbalance of BRCA1 transcript in the IVS20 12-bp insertion carrier.

One of the unclassified variants of the BRCA1 gene which has drawn considerable attention in recent years is the 12-bp insertion/duplication in intron 20. In this report, we show that a contribution from one chromosome cannot be detected in the BRCA1 transcript of the 12 bp insertion carrier. We also demonstrate here that the single transcript variant we observe by cDNA analysis originates from the same BRCA1 allele that harbours the 12-bp insertion. Hum Mutat 16:371, 2000.

Fibrinogen fractions in the third trimester of pregnancy and in puerperium.

The concentration of fibrinogen, its fractions, and the concentration of C-reactive protein were determined in 45 healthy pregnant women before and after vaginal (27) or caesarean section 18 delivery. The control group consisted of 33 blood donors. In pregnancy, increased concentration of total fibrinogen and its fractions and a normal concentration of C-reactive protein were noted. Three days after vaginal delivery the concentration of total and high molecular weight fibrinogen fraction decreased slightly and the ratio of high to low molecular weight fibrinogen increased. After caesarean section both total and high molecular weight fibrinogen and the ratio of high to low molecular weight fibrinogen increased. The C-reactive protein concentration increased after either type of delivery. The degree of augmentation, however, was ten times as strong after caesarean section. In all women at the end of puerperium the concentrations of the compounds studied returned to normal values. The results suggest that the mechanisms leading to the increase of fibrinogen during pregnancy and after delivery are different. The increase of all fibrinogen fractions in pregnancy may depend first of all on hormones, whereas the increased proportion of high molecular weight to low molecular weight fibrinogen after delivery depends on the acute phase reaction. The degree of this reaction depends on the type of delivery.

High frequency of recurrent mutations in BRCA1 and BRCA2 genes in Polish families with breast and ovarian cancer.

Germ-line mutations in BRCA1 and BRCA2 genes result in a significantly increased risk of breast and ovarian cancer. Other genes involved in an increased predisposition to breast cancer include the TP53 gene, mutated in Li-Fraumeni syndrome. To estimate the frequency of germ-line mutations in these three genes in Upper Silesia, we have analyzed 47 breast/ovarian cancer families from that region. We found five different disease predisposing mutations in 17 (36%) families. Twelve families (25.5%) carried known BRCA1 mutations (5382insC and C61G), four families (8.5%) carried novel BRCA2 mutations (9631delC and 6886delGAAAA), and one family (2%) harbored novel mutation 1095del8 in the TP53 gene, which is the largest germline deletion in coding sequence of this gene identified thus far. The 5382insC mutation in BRCA1 was found in 11 families and the 9631delC mutation in BRCA2 occurred in three families. These two mutations taken together contribute to 82% of all mutations found in this study, and 30% of the families investigated harbor one of these mutations. The very high frequency of common mutations observed in these families can only be compared to that reported for Ashkenazi Jewish, Icelandic, and Russian high-risk families. This frequency, however, may not be representative for the entire Polish population. The observed distribution of mutations will favor routine pre-screening of predisposed families using a simple and cost-effective test.

Postoperative plasma interleukin-6 in patients with renal cancer correlates with C-reactive protein but not with total fibrinogen or with high molecular weight fibrinogen fraction.

The concentration of fibrinogen (Fb) and its fractions, the levels of interleukin-6 (I1-6), C-reactive protein (CRP), and immunoglobulin G (IgG) were determined in 38 patients operated on because of renal cancer. The increased Fb and I1-6 concentrations were found in approximately one-half of the patients with malignancy. The relations among the high molecular weight (HMW) and two low molecular weight (LMW and LMW') fibrinogen fractions in these patients before surgery did not differ from the corresponding relations in normal subjects. The levels of all (except IgG) compounds studied increased after surgery and the peak of I1-6 was observed on the first postoperative day but that of CRP on the third day. The concentrations of total Fb and of its HMW fraction were the highest also on the third postoperative day and this was in contrast with the decline of low molecular weight fractions at the same time. These variations of estimated variables can be regarded as being relevant to the acute phase response. We have noted a correlation between the maximal concentrations of I1-6 and CRP, but not between the corresponding concentrations of Il-6 and total Fb or HMW Fb; this may suggest that the concentration of Fb is also under the control of a factor other than I1-6.

Novel BRCA1 mutations and more frequent intron-20 alteration found among 236 women from Western Poland.

Three different novel BRCA1 mutations, five independent cases of the same 12 bp insertion-duplication in intron-20 and two novel rare BRCA1 sequence variants were identified among 122 Polish women with positive, in most cases moderate family history of breast and/or ovarian cancer, 80 controls and 34 unselected breast cancer tissue specimens. All mutations and variants were germline. The 4153 delA frameshift mutation, the Tyr105Cys missense mutation and two cases of the alteration in intron-20 were found in the group of healthy women with positive family history. Two other cases of the intronic insertion were found in unselected controls. Their carriers had no family history of breast or ovarian cancer but other cancers occurred in their families. The 1782 Trp/STOP nonsense mutation and one case of the insertion in intron-20 were first found in tissue specimens of breast cancer patient and breast/ovarian cancer patient, respectively. Their carriers also had no family history of breast or ovarian cancer. The distribution of the insertion in intron-20 in analysed groups and results of RT-PCR experiments suggest a less prominent role for this variant considered earlier a splicing mutation. This study shows also, that more population-oriented research is needed, involving women with less profound or even no family history of breast and ovarian cancer, to better understand the role and significance of different BRCA1 variants and mutations.

[Serum C reactive protein as a laboratory indicator of infection in neonates]. / Bialko C reaktywne w surowicy jako laboratoryjny wskaznik zakazenia u noworodków.

CRP was measured in cord blood of 41 neonates born by healthy mothers and those with either risk of infection or its obvious clinical symptoms, and in sera of 122 newborn babies from infection risk group. Results excluded cord blood as a possible specimen for CRP estimation, because of very low values, at lower limit of sensitivity of the immunoturbidimetric method, and showed a correlation of serum CRP and clinical condition and its usefulness in monitoring of anti-bacterial therapy.

[Transferrin and ferritin as laboratory indices of body iron deposits]. / Transferyna i ferrytyna jako laboratoryjne wskazniki stanu zasobów zelaza w organizmie.

The aim of the study was to present the relation between serum parameters connected with iron metabolism; serum iron, transferrin and ferritin. Biochemical data in 126 patients were not compared with microscopic evaluation of erythrocytes. The tendency to decrease of serum transferrin with increased of serum ferritin was found. Significant relations between those proteins and serum iron were observed only in patients with very low ferritin values. It can be concluded that ferritin and transferrin may be considered as independent diagnostic in differential diagnosis of real (latent) and false iron depletion.