Intracellular localisation of platelet-activating factor during mammalian embryo development in vitro: a comparison of cattle, mouse and human.

Platelet-activating factor (PAF) is a well-known marker for embryo quality and viability. For the first time, we describe an intracellular localisation of PAF in oocytes and embryos of cattle, mice and humans. We showed that PAF is represented in the nucleus, a signal that was lost upon nuclear envelope breakdown. This process was confirmed by treating the embryos with nocodazole, a spindle-disrupting agent that, as such, arrests the embryo in mitosis, and by microinjecting a PAF-specific antibody in bovine MII oocytes. The latter resulted in the absence of nuclear PAF in the pronuclei of the zygote and reduced further developmental potential. Previous research indicates that PAF is released and taken up from the culture medium by preimplantation embryos invitro, in which bovine serum albumin (BSA) serves as a crucial carrier molecule. In the present study we demonstrated that nuclear PAF does not originate from an extracellular source because embryos cultured in polyvinylpyrrolidone or BSA showed similar levels of PAF in their nuclei. Instead, our experiments indicate that cytosolic phospholipase A2 (cPLA2) is likely to be involved in the intracellular production of PAF, because treatment with arachidonyl trifluoromethyl ketone (AACOCF3), a specific cPLA2 inhibitor, clearly lowered PAF levels in the nuclei of bovine embryos.

A de novo loss-of-function DYNC1H1 mutation in a patient with parkinsonian features and a favourable response to levodopa.

Graphical summary of 'A de novo loss-of-function DYNC1H1 mutation in a patient with parkinsonian features and a favourable response to levodopa' by Szczaluba et al..

Platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3) is required for the formation of the meiotic spindle during in vitro oocyte maturation.

Platelet-activating factor (PAF) is a well-described autocrine growth factor involved in several reproductive processes and is tightly regulated by its hydrolysing enzyme, PAF acetylhydrolase 1B (PAFAH1B). This intracellular enzyme consists of three subunits: one regulatory, 1B1, and two catalytic, 1B2 and 1B3. PAFAH1B3 has remained uncharacterised until now. Here, we report that PAFAH1B3 is present during the different stages of the first meiotic division in bovine, murine and human oocytes. In these species, the PAFAH1B3 subunit was clearly present in the germinal vesicle, while at metaphase I and II, it localised primarily at the meiotic spindle structure. In cattle, manipulation of the microtubules of the spindle by nocodazole, taxol or cryopreservation revealed a close association with PAFAH1B3. On the other hand, disruption of the enzyme activity either by P11, a selective inhibitor of PAFAH1B3, or by PAFAH1B3 antibody microinjection, caused arrest at the MI stage with defective spindle morphology and consequent failure of first polar body extrusion. In conclusion, our results show that one of the catalytic subunits of PAFAH1B, namely PAFAH1B3, is present in bovine, murine and human oocytes and that it plays a functional role in spindle formation and meiotic progression during bovine oocyte maturation.

Regioselective hydroxylation of cholecalciferol, cholesterol and other sterol derivatives by steroid C25 dehydrogenase.

Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D3 used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6-9 % (w/v) 2-hydroxypropyl-ß-cyclodextrin as a solubilizer and 1.25-5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8-15 mM K3[Fe(CN)6] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min-1 mg-1), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH2 groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.

Adiponectin affects uterine steroidogenesis during early pregnancy and the oestrous cycle: An in vitro study.

Reproduction in females is an energetically demanding process. We assumed that adiponectin (ADPN), known for its role in energy balance maintenance, is also engaged in the regulation of uterine steroidogenesis in the pig. We determined the impact of ADPN alone or in combination with insulin (INS) on testosterone (T), estrone (E1) and estradiol (E2) secretion by porcine endometrium and myometrium, uterine expression of CYP17A1 and CYP19A3 genes, and endometrial abundance of P450C17 and P450AROM proteins during the peri-implantation period and the oestrous cycle, using radioimmunoassay, qPCR, and Western Blot, respectively. During pregnancy, in the endometrial explants from days 10-11, ADPN decreased CYP17A1 gene expression, P450C17 protein abundance and T secretion, whereas increased E1 secretion. On days 12-13 of pregnancy, ADPN decreased CYP17A1 and CYP19A3 expression, P450C17 and P450AROM protein abundance and E1 secretion, but stimulated T secretion. On days 15-16 of pregnancy, ADPN decreased P450C17 protein accumulation but enhanced CYP19A3 expression and E1 secretion. On days 27-28 of pregnancy, ADPN increased CYP17A1 and CYP19A3 mRNA content and T secretion in this tissue and decreased P450C17 content. ADPN effect on myometrial explants was dependent on stage of gestation or oestrous cycle. Moreover, INS treatment modulated basal and ADPN-affected steroidogenic enzymes gene and protein expression and steroids secretion. The results obtained indicate that ADPN may affect processes required for successful implantation such as steroidogenesis. ADPN and INS were also shown to modulate each other action, which indicates that the proper course of uterine steroidogenesis may be dependent on both hormones' interaction.

Alcohol and tobacco, and the risk of cancers of the upper aerodigestive tract in Latin America: a case-control study.

BACKGROUND: Cancers of the upper aerodigestive tract (UADT; including oral cavity, pharynx, larynx and oesophagus) have high incidence rates all over the world, and they are especially frequent in some parts of Latin America. However, the data on the role of the major risk factors in these areas are still limited. METHODS: We have evaluated the role of alcohol and tobacco consumption, based on 2,252 upper aerodigestive squamous-cell carcinoma cases and 1,707 controls from seven centres in Brazil, Argentina, and Cuba. RESULTS: We show that alcohol drinkers have a risk of UADT cancers that is up to five times higher than that of never-drinkers. A very strong effect of aperitifs and spirits as compared to other alcohol types was observed, with the ORs reaching 12.76 (CI 5.37-30.32) for oesophagus. Tobacco smokers were up to six times more likely to develop aerodigestive cancers than never-smokers, with the ORs reaching 11.14 (7.72-16.08) among current smokers for hypopharynx and larynx cancer. There was a trend for a decrease in risk after quitting alcohol drinking or tobacco smoking for all sites. The interactive effect of alcohol and tobacco was more than multiplicative. In this study, 65% of all UADT cases were attributable to a combined effect of alcohol and tobacco use. CONCLUSIONS: In this largest study on UADT cancer in Latin America, we have shown for the first time that a prevailing majority of UADT cancer cases is due to a combined effect of alcohol and tobacco use and could be prevented by quitting the use of either of these two agents.

Individual-oocyte transcriptomic analysis shows that genotoxic chemotherapy depletes human primordial follicle reserve in vivo by triggering proapoptotic pathways without growth activation.

Gonadotoxic chemotherapeutics, such as cyclophosphamide, can cause early menopause and infertility in women. Earlier histological studies showed ovarian reserve depletion via severe DNA damage and apoptosis, but others suggested activation of PI3K/PTEN/Akt pathway and follicle 'burn-out' as a cause. Using a human ovarian xenograft model, we performed single-cell RNA-sequencing on laser-captured individual primordial follicle oocytes 12 h after a single cyclophosphamide injection to determine the mechanisms of acute follicle loss after gonadotoxic chemotherapy. RNA-sequencing showed 190 differentially expressed genes between the cyclophosphamide- and vehicle-exposed oocytes. Ingenuity Pathway Analysis predicted a significant decrease in the expression of anti-apoptotic pro-Akt PECAM1 (p = 2.13E-09), IKBKE (p = 0.0001), and ANGPT1 (p = 0.003), and reduced activation of PI3K/PTEN/Akt after cyclophosphamide. The qRT-PCR and immunostaining confirmed that in primordial follicle oocytes, cyclophosphamide did not change the expressions of Akt (p = 0.9), rpS6 (p = 0.3), Foxo3a (p = 0.12) and anti-apoptotic Bcl2 (p = 0.17), nor affect their phosphorylation status. There was significantly increased DNA damage by γH2AX (p = 0.0002) and apoptosis by active-caspase-3 (p = 0.0001) staining in the primordial follicles and no change in the growing follicles 12 h after chemotherapy. These data support that the mechanism of acute follicle loss by cyclophosphamide is via apoptosis, rather than growth activation of primordial follicle oocytes in the human ovary.

TP53 and EGFR mutations in combination with lifestyle risk factors in tumours of the upper aerodigestive tract from South America.

Cancers of the upper aerodigestive tract [(UADT): oral cavity, pharynx, larynx and oesophagus] have high incidence rates in some parts of South America. Alterations in the TP53 gene are common in these cancers. In our study, we have estimated the prevalence and patterns of TP53 mutations (exons 4-10) in 236 UADT tumours from South America in relation to lifestyle risk factors, such as tobacco smoking and alcohol drinking. Moreover, we have conducted a pilot study of EGFR mutations (exons 18-21) in 45 tumours from the same population. TP53 mutation prevalence was high: 59% of tumours were found to carry mutant TP53. We found an association between TP53 mutations and tobacco smoking and alcohol drinking. The mutation rate increased from 38% in never-smokers to 66% in current smokers (P-value for trend = 0.09). G:C>T:A transversions were found only in smokers (15%). Alcohol drinkers carried more G:C>A:T transitions (P = 0.08). Non-exposed individuals were more probable to carry G:C>A:T transitions at CpG sites (P = 0.01 for never-smokers and P < 0.001 for never-drinkers). EGFR mutations were found in 4% of cases. Inactivation of TP53 by mutations is a crucial molecular event in the UADT carcinogenesis and it is closely related to exposure to lifestyle risk factors. EGFR mutations do not appear to be a common event in UADT carcinogenesis in this population.

Drinking of maté and the risk of cancers of the upper aerodigestive tract in Latin America: a case-control study.

Cancers of the upper aerodigestive tract (UADT: oral cavity, oropharynx, hypopharynx, larynx, esophagus) have high incidence rates all over the world and they are especially frequent in some parts of Latin America. In this study, we have evaluated the role of the consumption of maté, a hot herb-based beverage, based on 1168 UADT squamous-cell carcinoma cases and 1,026 frequency-matched controls enrolled from four centers in Brazil and Argentina. The effect of maté drinking on the risk of head-and-neck cancers was borderline significant. A significant effect was observed only for cancer of the esophagus (OR 3.81 (95% CI 1.75-8.30)). While duration of maté drinking was associated with the risk of all UADT cancers, the association with cumulative maté consumption was restricted to esophageal cancer (p-value of linear trend 0.006). The analyses of temperature at which maté was drunk were not conclusive. The increased risk associated with maté drinking was more evident in never-smokers and never-alcohol drinkers than in other individuals. Our study strengthens the evidence of an association between maté drinking and esophageal cancer; the hypothesis of an association with other UADT cancers remains to be clarified.

Sepiapterin reductase deficiency in a 2-year-old girl with incomplete response to treatment during short-term follow-up.

Sepiapterin reductase (SR) catalyses the last step in the tetrahydrobiopterin biosynthesis pathway; it converts 6-pyruvoyl-tetrahydropterin (6-PTP) to BH(4) in an NADPH-dependent reaction. SR deficiency is a very rare autosomal recessive disorder with normal phenylalanine (Phe) concentration in blood and diagnostic abnormalities are detected in CSF. We present a 16-month-old girl with SR deficiency. From the newborn period she presented with an adaptation regulatory disorder. At the age of 3 months, abnormal eye movements with dystonic signs and at 4.5 months psychomotor retardation were noticed. Since that time axial hypotonia with limb spasticity (or rather delayed reflex development), gastro-oesophageal reflux and fatigue at the end of the day has been observed. Brain MRI was normal; EEG was without epileptiform discharges. Analysis of biogenic amine metabolites in CSF at the age of 16 months showed very low HVA and 5-HIAA concentrations. Analysis of CSF pterins revealed strongly elevated dihydrobiopterin (BH(2)), slightly elevated neopterin and elevated sepiapterin levels. Plasma and CSF amino acids concentrations were normal. A phenylalanine loading test showed increased Phe after 1 h, 2 h and 4 h and very high Phe/Tyr ratios. SR deficiency was confirmed in fibroblasts and a novel homozygous g.1330C>G (p.N127K) SPR mutation was identified. On L-dopa and then additionally 5-hydroxytryptophan, the girl showed slow but remarkable progress in motor and intellectual ability. Now, at the age of 3 years, she is able to sit; expressive speech is delayed (to 1 1/2 years), passive speech is well developed. Her visual-motor skills, eye-hand coordination and social development correspond to the age of 2 1/2 years.

Monitoring of rat islet allografts with dithizone after induction of donor specific transplant tolerance by intrathymic administration of soluble alloantigens.

Transplantation of whole pancreas or pancreatic islets remains a promising approach to treatment of diabetes mellitus. Since there is no efficient method presently known for in vivo detection of pancreatic islet rejection, we have utilized dithizone [DTZ] to monitor the survival of transplanted islet allografts following the induction of tolerance by a new strategy of deliberate introduction of donor antigens into the adult thymus. In this study, we examined the morphology of islet allografts in vivo and in vitro following pretreatment with intrathymic (IT) inoculation of 2 mg soluble Ag obtained from 3M KCl extracts of resting T-cells with or without ALS immunosuppression in the WF-to-Lewis combination. Fresh isolated rat islets stained pink 3-5 minutes following exposure to medium containing 0.12 mM DTZ solution in DMSO. Intravenous (i.v.) injection of DTZ solution into unmodified recipients of islet allografts that had rejected their grafts showed massive degranulation of islets which did not stain pink with DTZ. This was confirmed by microscopic finding of fibrosis and lymphocytic infiltration. In contrast, i.v. injection of DTZ solution into long-term recipients of islet allografts at 50, 100, and 150 days after transplantation showed viable islet cells which stained crimson red with DTZ and the findings were confirmed with microscopic sections. This study demonstrates that DTZ is an effective means of in vivo and in vitro identification of transplanted pancreatic islets and suggests that this strategy may have potential clinical application in the diagnosis of the pancreatic islet rejection.

[Effect of diphynylhydantoin on the histological appearance of internal organs in cat (preliminary study)]. / Wplyw dwufenylohydantoiny (DPH) na obraz histopatologiczny narzadów wewnetrznych kotów (doniesienie wstepne)

The effects of DPH on internal organs of cats were investigated. The drug (Hydantoinal POLFA) was administered orally in doses 8 to 20 mg/kg daily during from 27 to 560 days. The initial dose was 1.5 mg/kg and it was increased gradually. Consistently occurring changes were found in the liver, kidneys, salivary glands and spleen. In the liver and kidneys the character of changes was mainly degenerative (fatty infiltration of hepatocytes and renal tubular epithelial cells). In the salivary gland the number of mucus-producing cells was increased, in the spleen proliferation of connective tissue and congestion were present.

TP53, EGFR, and KRAS mutations in relation to VHL inactivation and lifestyle risk factors in renal-cell carcinoma from central and eastern Europe.

Renal-cell carcinomas (RCC) are frequent in central and eastern Europe and the reasons remain unclear. Molecular mechanisms, except for VHL, have not been much investigated. We analysed 361 RCCs (334 clear-cell carcinomas) from a multi-centre case-control study for mutations in TP53 (exons 5-9 in the whole series and exons 4 and 10 in a pilot subset of 60 tumours) and a pilot 50 tumours for mutations in EGFR (exons 18-21) or KRAS (codon 12) in relation to VHL status. TP53 mutations were detected in 4% of clear-cell cases, independently of VHL mutations. In non-clear-cell carcinomas, they were detected in 11% of VHL-wild-type tumours and in 0% of tumours with VHL functional mutations. No mutations were found in EGFR or KRAS. We conclude that mutations in TP53, KRAS, or EGFR are not major contributors to the RCC development even in the absence of VHL inactivation. The prevalence of TP53 mutations in relation to VHL status may differ between clear-cell and other renal carcinomas.