Epigenetic regulation of 5α reductase-1 underlies adaptive plasticity of reproductive function and pubertal timing.

BACKGROUND: Women facing increased energetic demands in childhood commonly have altered adult ovarian activity and shorter reproductive lifespan, possibly comprising a strategy to optimize reproductive success. Here, we sought to understand the mechanisms of early-life programming of reproductive function, by integrating analysis of reproductive tissues in an appropriate mouse model with methylation analysis of proxy tissue DNA in a well-characterized population of Bangladeshi migrants in the UK. Bangladeshi women whose childhood was in Bangladesh were found to have later pubertal onset and lower age-matched ovarian reserve than Bangladeshi women who grew-up in England. Subsequently, we aimed to explore the potential relevance to the altered reproductive phenotype of one of the genes that emerged from the screens. RESULTS: Of the genes associated with differential methylation in the Bangladeshi women whose childhood was in Bangladesh as compared to Bangladeshi women who grew up in the UK, 13 correlated with altered expression of the orthologous gene in the mouse model ovaries. These mice had delayed pubertal onset and a smaller ovarian reserve compared to controls. The most relevant of these genes for reproductive function appeared to be SRD5A1, which encodes the steroidogenic enzyme 5α reductase-1. SRD5A1 was more methylated at the same transcriptional enhancer in mice ovaries as in the women's buccal DNA, and its expression was lower in the hypothalamus of the mice as well, suggesting a possible role in the central control of reproduction. The expression of Kiss1 and Gnrh was also lower in these mice compared to controls, and inhibition of 5α reductase-1 reduced Kiss1 and Gnrh mRNA levels and blocked GnRH release in GnRH neuronal cell cultures. Crucially, we show that inhibition of this enzyme in female mice in vivo delayed pubertal onset. CONCLUSIONS: SRD5A1/5α reductase-1 responds epigenetically to the environment and its downregulation appears to alter the reproductive phenotype. These findings help to explain diversity in reproductive characteristics and how they are shaped by early-life environment and reveal novel pathways that might be targeted to mitigate health issues caused by life-history trade-offs.

Management of XLP-1 and ITK deficiency: The challenges posed by PID with an unpredictable spectrum of disease manifestations.

The incorporation of next generation sequencing into routine immunological practice has enabled the identification of novel inborn errors of disease, helped define new categories of immune deficiency and extended the clinical spectrum associated with many long-recognised diseases. The family of EBV (Epstein Barr Virus)-sensitive primary immune deficiencies is one such group and in this paper we describe three families: two with X-linked lymphoproliferative disease type-1 (XLP-1) and one with deficiency of Interleukin-2 Inducible T-cell Kinase (ITK). Both diseases have a wide range of clinical manifestations and are united by an exquisite predisposition to EBV, dysgammaglobulinemia, hemophagocytic lymphohistiocytosis, and lymphoma. We detail our approach to diagnosis, treatment, and risk stratification in these diseases where both clinicians and patients must grapple with constant uncertainty.

Characterization of the response of in vitro cultured Myrtus communis L. plants to high concentrations of NaCl.

Effect of salt stress was examined in in vitro shoot cultures of Myrtus communis L. a species of the Mediterranean maquis. To determine the effects of high salt concentrations on myrtle plantlets and contribute toward understanding the mechanisms adopted from this species to counteract soil salinity, in vitro rooted shoots were transferred to a liquid culture medium containing 0, 125 or 250 mM NaCl for 30 days. After 15 and 30 days of in vitro culture, shoot and root growth, chlorosis and necrosis extension, chlorophylls, carotenoids, proline, arginine, cysteine and total sugars content, as well as guaiacol peroxidase (G-POD, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11) activities were determined. In treated plants shoot and root growth, as well as chlorophyll content, significantly decreased, while carotenoids content was not affected by the NaCl treatment. Among osmolytes, proline did not significantly increase, arginine and cysteine decreased, while total sugars were found to be higher in the treated plants than in the control. Enhancement of G-POD and APX activities was positively related to increasing salt concentrations in the culture media, regardless of the exposure time. Salt-treated plants did not show significant changes in lipid peroxidation or DNA fragmentation after 30 days salt treatment, regardless of the NaCl concentrations applied. The results represent a contribution towards understanding the mechanisms adopted by this species to high salinity.

Assessment of hospital disaster plans for conventional mass casualty incidents following terrorist explosions using a live exercise based upon the real data of actual patients.

PURPOSE: The National Committee for Hospital Preparedness for Conventional Mass Casualty Incidents and the Hospital Preparedness Division of the Home Front Command are in charge of preparing live exercises held yearly in public hospitals in Israel. Our experience is that live exercises are limited in their ability to test clinical decision making and its influence upon incident management. A live exercise was designed upon real patient data and tested in several public hospitals. The aim of the manuscript is to describe the impact of this new format on clinical decision making in large-scale live exercises. METHODS: A database of histories, physical examination findings, laboratory results and imaging results for 420 patients treated following terrorist explosions was created using information derived from actual patient encounters. Similar information for 100 patients treated following motor vehicle accidents was also collected. Information from the database was used to create victim profiles used during the course of exercises held in eight public hospitals with 60-800-bed capacities. RESULTS: Before implementing the new injury tags, no conclusions could be made concerning the quality of clinical decision making. Conducting the exercise using the new format helped identify deficiencies in the hospital disaster plan in triage, emergency department management and in the proper utilisation of resources such as radiology, operating rooms and the secondary transfer of patients. CONCLUSION: Previous knowledge of patient diagnoses and resource needs allow the identification and quantification of deficiencies and problems identified in clinical decision making, resource utilisation and incident management.

New approach for the removal of metal ions from water: adsorption onto aquatic plants and microwave reaction for the fabrication of nanometals.

We adsorb heavy metal ions such as Ag(+), Pb(2+), and Ru(3+) onto an aquatic plant and convert the adsorbed ions to the corresponding nanometallic particles by the polyol reaction carried out in a microwave oven.

[What is the role of compulsory ECT therapy today?].

Electro-convulsive therapy (ECT) has been effective for years, but it arouses opposition among patients and especially in the general public. ECT treatment is limited and compared to other medical treatment it is considered exceptional by the law, regulations and treatment personnel. A question arises as to the position of therapists regarding compulsory ECT treatment. A questionnaire was sent on this subject to all the units utilizing ECT in Israel. Opinions ranged from complete negation of compulsory ECT, to regarding such treatment as possible in cases when the patient is compulsorily hospitalized and/or when the patient's guardian supports this treatment. The authors' opinion is that the Law of Patients' Rights regarding special treatment when the patient is in extreme danger must be followed. The law requires that three physicians agree to the treatment, and compulsory treatment is no longer applicable when the danger passes. ECT treatment is important and imperative in certain conditions, especially conditions endangering patients' lives. In these conditions the law provides the authority to physicians to make decisions regarding treatment.

Projection structure and oligomeric properties of a bacterial core protein translocase.

The major route for protein export or membrane integration in bacteria occurs via the Sec-dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein-conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 A projection structure was calculated using electron cryo-microscopy. The structure exhibits P12(1) symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

The involvement of polyphenols and peroxidase activities in heavy-metal accumulation by epidermal glands of the waterlily (Nymphaeaceae).

Co-localization of polyphenols and peroxidase activity was demonstrated in epidermal glands of the waterlily (Nymphaea) by histochemistry. Total phenols, tannins and peroxidase activity were determined quantitatively in plant extracts. Polyphenols were partially identified and were found to consist mainly of hydrolyzable tannins, gallic and tannic acid derivatives. Nymphaea polyphenols were shown to chelate Cr, Hg, and Pb in vitro, and Cd-binding by polymerized polyphenols was demonstrated in leaves exposed to Cd in vivo. Both polyphenols and peroxidases were found at very high constitutive levels, which were not induced or altered by external conditions, such as light and heavy-metal stress. It is suggested that the polymerization of polyphenols by peroxidases, enhanced after heavy-metal uptake and detoxification, is responsible for the binding of heavy metals in Nymphaea epidermal glands.

Accumulation of heavy metals in epidermal glands of the waterlily (Nymphaeaceae).

This study investigates the anatomical aspects of heavy-metal accumulation in the waterlily (Nymphaea 'Aurora', Nymphaeaceae). Epidermal glands were identified by light microscopy on the abaxial side of the leaf laminae and on the epidermis of the rhizome; glandular trichomes were observed in the petiole epidermis. Glands were not observed in the roots. Accumulation of heavy metals in these glands was monitored using a scanning electron microscope equipped for energy-dispersive spectroscopy. Further experiments showed maximal cadmium and calcium accumulation in the mature leaf lamina in daylight, and this accumulation was inhibited by the herbicide 3-(3',4'-dichlorophenyl)-1,1-dimethylurea. These results suggest that, in Nymphaea, heavy metals are accumulated primarily in association with glands found in plant organs that have direct contact with water or mud. Deposition and storage of heavy metals by these glands may represent a stage in the sequestration and detoxification of the metals. Our results raise the possibility of utilizing waterlilies for the removal of heavy metals from polluted environments.

Phenols and phenol oxidases are involved in cadmium accumulation in the water plants Nymphoides peltata (Menyanthaceae) and Nymphaeae (Nymphaeaceae).

This comparative study investigates the mechanism of cadmium accumulation in the semiaquatic plant Nymphoides peltata (Menyanthaceae) and the aquatic plant Nymphaea (Nymphaeaceae). It was conducted as part of an ongoing study of the use of water plants for phytoremediation. Epidermal structures, known as hydropotes, are located on the abaxial epidermis of the leaf laminae of Nymphoides peltata and are shown to contain phenols, peroxidase and polyphenol oxidase activities. When plants are subjected to 50 mg/l of cadmium in the growth medium, these hydropotes accumulate cadmium. Cadmium-induced increases in phenols, peroxidase and polyphenol oxidase activities were determined in plant extracts. Cadmium binding by polymerized phenols was demonstrated in vivo. In comparison with Nymphaeae epidermal glands, N. peltata hydropotes are larger, open, and create bigger crystal, the latter principally composed of calcium and, proportionally, less cadmium. Although both plants showed similar levels of cadmium accumulation, N. peltata was sensitive while Nymphaeae was resistant to this cadmium level. It is suggested that in these water plants the main mechanism for cadmium accumulation is based on the trapping of cadmium crystals by polymerized phenols in specialized epidermal structures and this is due to peroxidase and polyphenol oxidase activities. Nymphaeae, with greater peroxidase activity and more polyphenols, is more resistant to this heavy metal than N. peltata.

The transduction of the signal for grape bud dormancy breaking induced by hydrogen cyanamide may involve the SNF-like protein kinase GDBRPK.

Alterations in gene expression during early stages of dormancy release in grapevine buds were analyzed to facilitate the identification of gene products that may mediate the signal transduction of a dormancy-release signal, or derepression of meristematic activity. In the present report we describe the identification of GDBRPK, a transcript for an SNF-like protein kinase that is up-regulated upon chemical induction of dormancy release by hydrogen cyanamide (HC). Since SNF and SNF-like protein kinases are known as sensors of stress signals, we hypothesize that GDBRPK may be involved in the perception of a stress signal induced by HC. We also describe a simultaneous and remarkable induction of both PDC and ADH transcripts that was observed shortly after HC application, and was of a transient nature. These data may imply that HC application leads to a transient respiratory stress, which likely results in a temporary increase in the AMP/ATP ratio. Since AMP is known as a stress signal that is sensed by SNF-like kinases, we suggest that the SNF-like GDBRPK could serve as the sensor of this signal.

Photostabilization of the herbicide norflurazon by using organoclays.

Photostable formulations of the herbicide norflurazon [4-chloro-5-(methylamino)-2-(alpha,alpha, alpha-trifluoro-m-tolyl)pyridazin-3-(2H)-one] were achieved by adsorbing it on pillared clay or on montmorillonite preadsorbed with the organic cation thioflavin T (TFT). Diffuse reflectance Fourier transform infrared spectra showed the existence of strong interactions between the aromatic moieties of preadsorbed TFT and the herbicide, particularly after irradiation. The photostabilization of norflurazon obtained with TFT-clay was mainly due to energy transfer from the herbicide to the organic cation via pi-pi interactions. An additional mechanism is the lower production of radicals from the clay when the clay mineral surface is covered with the organic cation. These radicals are responsible for the enhanced photodegradation observed when norflurazon was irradiated in the presence of untreated montmorillonite.

NADP(+)-isocitrate dehydrogenase gene expression and isozyme activity during citrus fruit development.

The accumulation of citric acid and its decline toward fruit maturation is typical of citrus fruit. We studied NADP(+)-isocitrate dehydrogenase (NADP-IDH), an enzyme involved in citrate metabolism. A cDNA encoding the enzyme was cloned from lemon (Citrus limon) juice sac cells, and is the first-reported NADP-IDH from fruits. Sequence comparisons and phylogenetic analysis indicate that it most probably belongs to a monophyletic clade of plant cytosolic enzymes. The mRNA level in the juice sac cells was induced during lemon fruit growth, and increased by about 15-fold to a peak as the fruit neared maturation. Spectrophotometric assay of the NADP-IDH activity in the pulp during fruit development showed that in young fruit, most of the activity was associated with the mitochondrial preparation and that, as the fruit grew, the activity shifted to the soluble fraction. The two activities could also be distinguished by isozyme gel electrophoresis: while one isozyme was detected in the mitochondrial preparation of young fruit and declined later, the other was induced in the soluble fraction of older fruit and increased as the fruit grew. The increasing activity of NADP-IDH in the soluble fraction throughout fruit development correlated well with the increase in gene expression, which suggests that the soluble activity is regulated by the expression of the cytosolic NADP-IDH gene. The possible role of this form of the enzyme in citric acid catabolism in the pulp is discussed.

Fermentative metabolism in grape berries: isolation and characterization of pyruvate decarboxylase cDNA and analysis of its expression throughout berry development.

The involvement of pyruvate decarboxylase (PDC) in the control of alcohol production during ripening of fruit tissues under aerobic conditions has been only partially studied. Enzymological studies showed a significant increase in PDC activity during the ripening of oranges and pears, concurrently with the induction of ethanol production. In tomato, on the other hand, the induction of ethanol production and ADH gene expression after the onset of ripening was not accompanied by induction of PDC activity. The isolation of PDC cDNA from fruits has not yet been reported, nor has its expression pattern during fruit development. We report here the isolation of a cDNA clone encoding for a grape PDC and the characterization of its expression throughout berry development. The pattern of PDC gene expression throughout berry development, combined with earlier findings on constitutive PDC activity in the berry, may suggest that PDC is not the limiting factor for the production of ethanol in the berry, which is induced only after the onset of berry ripening. Alternatively, the induction of ADH gene expression, which occurs only after the onset of ripening in both tomatoes and grape berries, may serve as a regulator of ethanol production in response to a ripening-related cue.

Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane.

Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.

Characterization of disulfide cross-links between fragments of proteolyzed Na,K-ATPase. Implications for spatial organization of trans-membrane helices.

This study characterizes disulfide cross-links between fragments of a well defined tryptic preparation of Na,K-ATPase, 19-kDa membranes solubilized with C12E10 in conditions preserving an intact complex of fragments and Rb occlusion (Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864). Upon solubilization, cross-links form spontaneously between the beta subunit, 19- and 11.7-kDa fragments of the alpha subunit, containing trans-membrane segments M7-M10 and M1/M2, respectively. Treatment with Cu2+-phenanthroline (CuP) improves efficiency of cross-linking. Sequencing and immunoblot analysis have shown that the cross-linked products consist of a mixture of beta-19 kDa dimers ( approximately 65%) and beta-19 kDa-11.7 kDa trimers ( approximately 35%). The alpha-beta cross-link has been located within the 19-kDa fragment to a 6.5-kDa chymotryptic fragment containing M8, indicating that betaCys44 is cross-linked to either Cys911 or Cys930. In addition, an internal cross-link between M9 and M10, Cys964-Cys983, has been found by sequencing tryptic fragments of the cross-linked product. The M1/M2-M7/M10 cross-link has not been identified directly. However, we propose that Cys983 in M10 is cross-linked either to Cys104 in M1 or internally to Cys964 in M9. Based on this study, cross-linking induced by o-phthalaldehyde (Or, E., Goldshleger, R., and Karlish, S. J. D. (1998) Biochemistry 37, 8197-8207), and information from the literature, we propose an approximate spatial organization of trans-membrane segments of the alpha and beta subunits.

The profitability of vertical mergers between hospitals and physician practices.

We demonstrate that the existence of incentives for vertical mergers between hospitals and physician practices depends upon the relative degree of competitiveness of the two providers' markets. When the degree of competitiveness is comparable, a vertical merger enhances the bargaining position of both merging parties vis-à-vis insurers. In contrast, when one provider's market is much more competitive than the other a vertical merger may reduce the joint profits of the merged entity. Prohibiting the parties from offering their services in conjunction with outside independent entities may restore the profitability of the vertical merger even in this case.

Metal-catalysed cleavage of Na,K-ATPase as a tool for study of structure-function relations.

This paper describes a novel technique for specific cleavage of renal Na/K-ATPase, based on bound transition metal ions. The approach might have application to other P-type pumps or membrane proteins. In one type of experiment, specific cleavages of the alpha subunit have been observed following incubation with ascorbate plus H2O2. Five fragments with intact C-terminals and complementary fragments with intact N-terminals are detectable. The beta subunit is not cleaved. Cleavages depend on the presence of contaminant or added submicromolar concentrations of Fe2+ ions. The results suggest that Fe2+ (or Fe3+) binds with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe2+ (or Fe3+) ion. The rate of cleavage is greatly affected by the conformational state of the protein, E1Na or E2(Rb), respectively. The findings provide information on spatial organization of the protein and suggest that the highly conserved regions of the alpha subunit, within the minor and major cytoplasmic loops, interact in the E2 or E2(Rb) conformations, but move apart in the E1 or E1Na conformations. In a second application of this technique, added Cu2+ ions at micromolar concentrations, have been shown to catalyse specific cleavages of both alpha and beta subunits at the extracellular surface. The experiments provide evidence for trans-membrane topology and proximity between trans-membrane segments M5-M10 within the alpha subunit and for interacting segments of alpha and beta subunits. We discuss the implications of metal-catalysed cleavages for spatial organisation of transmembrane helices of the protein.

Specific cross-links between fragments of proteolyzed Na,K-ATPase induced by o-phthalaldehyde.

We have used o-phthalaldehyde (OPA) to cross-link adjacent fragments of "19 kDa membranes", a tryptic preparation of Na,K-ATPase lacking the ATP site but retaining cation occlusion sites. Treatment with OPA of "19 kDa membranes" or detergent-solubilized membranes containing occluded Rb ions [Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864] yielded cross-linked products of 25 and 31 kDa. Both species contained the 19 kDa fragment of the alpha subunit (transmembrane segments M7-M10). In addition, the 25 kDa product contained the fragment including M5-M6, while the 31 kDa product contained a 16 kDa fragment of the beta subunit. Cross-linking was unaffected by the absence or presence of ligands (Na, Rb, or Mg and ouabain). Cross-linking was largely abolished in thermally inactivated "19 kDa membranes". When proteolytic digestion of the 25 and 31 kDa products was combined with antibody binding, PKA-dependent phosphorylation, and sequencing of fragments, approximate positions of the cross-links were established. In the 25 kDa product, the cross-link was located within the short cytoplasmic segment Asn831-Arg841 of the 19 kDa fragment preceding M7 and within Ala749-Ala770 preceding M5. Thus, M7 and M5 are likely to be in close proximity. In the 31 kDa product, the cross-link was located in the extracellular loop of the alpha subunit between M7 and M8, close to residues which are known to interact with the beta subunit. Functional implications of the interactions between the fragments of the alpha (M5-M6 and M7-M10) and beta subunits are discussed.