Understanding down-regulation of photosynthesis under water stress: future prospects and searching for physiological tools for irrigation management

Photosynthetic down-regulation and/or inhibition under water stress conditions are determinants for plant growth, survival and yield in drought-prone areas. Current knowledge about the sequence of metabolic events that leads to complete inhibition of photosynthesis under severe water stress is reviewed. An analysis of published data reveals that a key regulatory role for Rubisco in photosynthesis is improbable under water stress conditions. By contrast, the little data available for other Calvin cycle enzymes suggest the possibility of a key regulatory role for some enzymes involved in the regeneration of RuBP. There are insufficient data to determine the role of photophosphorylation. Several important gaps in our knowledge of this field are highlighted. The most important is the remarkable scarcity of data about the regulation/inhibition of photosynthetic enzymes other than Rubisco under water stress. Consequently, new experiments are urgently needed to improve our current understanding of photosynthetic down-regulation under water stress. A second gap is the lack of knowledge of photosynthetic recovery after irrigation of plants which have been subjected to different stages of water stress. This knowledge is necessary in order to match physiological down-regulation by water stress with controlled irrigation programmes.     

Prognostic biomarkers for survival in mucosal melanoma

Mucosal melanoma (MM) is a rare subtype of melanoma with an aggressive clinical course. In cutaneous melanoma (CM), the absence of pigmentation and presence of NRAS/KRAS mutations are biomarkers indicating an aggressive clinical course with shorter overall survival. Similar data for MM are missing. We present the real-world outcome data in a cohort of genotyped MM patients and assessed the prognostic relevance of pigmentation- and NRAS/KRAS mutation status. We correlated pathological reports and clinical data with overall survival of patients with MM. Furthermore, we performed clinically integrated molecular genotyping and analyzed real world treatment regimens for covariates associated with clinical outcome. We identified 39 patients with available clinical and molecular data. Patients with amelanotic MM had a significantly shorter overall survival (p = .003). In addition, the presence of a NRAS or KRAS mutation was significantly associated with poor overall survival (NRAS or KRAS p = .024). Currently, it is unknown if the same prognostic relevance for the lack of pigmentation and RAS mutations in CM, exists in MM. Here we analyzed a cohort of MM for outcome measures and determined that two known prognostic biomarkers for CM are in fact novel prognosticators for MM.     

Histochemical localization of 3 beta-hydroxysteroid dehydrogenase in the testes of chicken-pheasant hybrids.

Synopsis Histochemical studies on the activity of 3-hydroxysteroid dehydrogenase (3-HSD) in the testes of adult chicken-pheasant hybrids and domestic fowl of similar age were carried out using dehydroepiandrosterone as the substrate. The reaction for 3-HSD was positive in the interstitial tissue and negative within seminiferous tubules of domestic fowl. In chicken-pheasant hybrids, a strong positive reaction for 3-HSD was observed in the interstitial tissue and within the seminiferous tubules suggesting that, in hybrid testes, both Leydig cells and cells of seminiferous tubules may be capable of steroid biosynthesis. Since the plasma testosterone levels in these birds were found to be extremely low, it is hypothesized that either the chicken-pheasant hybrids do not release testosterone in sufficient amounts in the circulation or the type of steroid produced by the 3HSD-positive cells may be different from testosterone that is required for the maintenance of normal fertility and the development of secondary sexual characteristics.     

STUDY ON THE PROLIFERATIVE STATE OF HAEMOPOIETIC STEM CELLS (CFU)

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUJ in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rate of endogenous CFU_S (endo-CFU,) and exogenous CFU_S (exo-CFU_s) are identical. After irradiation (650 R) the surviving endo-CFU_s begin to proliferate immediately. By contrast exo-CFU, transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosine 3′,5′-monophosphate shortly after marrow cell graft, triggers the transplanted CFU_S into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFU, become to be increasingly sensitive to hydroxyurea.     

Recent advances in the development of steroid sulphatase inhibitors

Inhibition of steroid sulphatase is now an important target for the development of new drugs for the treatment of women with endocrine-dependent breast tumours. The first potent sulphatase inhibitor identified, oestrone-3-O-sulphamate (EMATE) proved, unexpectedly, to be oestrogenic. A number of strategies have therefore been adopted to design and synthesize a non-oestrogenic inhibitor. For this, a number of modifications have been made to the A and D rings of the oestrone nucleus. 2 Methoxyoestrone-3-O-sulphamate, while having similar in vitro and in vivo sulphatase inhibitory potency to that of EMATE, was devoid of oestrogenic activity when tested at 2 mg/kg in an ovariectomised rat uterine weight gain assay. 17-Deoxyoestrone-3-O-sulphamate was also a potent steroid sulphatase inhibitor and while it was devoid of oestrogenic activity when tested at 0.1 mg/kg, did stimulate uterine growth at 1.0 mg/kg. As an alternative approach to the use of steroid-based inhibitors a number of single ring, bicyclic non-fused ring, and two fused ring sulphamate analogues were designed, synthesized and tested for their ability to inhibit steroid sulphatase activity. In general, although the single ring and bicyclic non-fused ring sulphamate analogues could inhibit sulphatase activity, they were considerably less potent than EMATE. The mono- and bis-sulphamate derivatives of 5,7-dihydroxyisoflavone were relatively potent, inhibiting in vivo steroid sulphatase activity by 62 and 81% respectively at a single oral dose of 10 mg/kg. A study of the structure-activity relationship of a series of coumarin-based sulphamates has led to the development of a number of potent non-steroidal inhibitors, one of which has a similar potency to that of EMATE. The identification of potent steroid- and non-steroid-based sulphatase inhibitors will enable the therapeutic value of this therapy to be examined in the near future.     

2-Alkylsulfanyl estrogen derivatives: synthesis of a novel class of multi-targeted anti-tumour agents

A flexible, direct, high yielding synthesis of 2-alkylsulfanyl estrogens from estrone has been developed. 2-Methylsulfanyl estradiol (2-MeSE2) 7 displays a similar anti-proliferative activity to the established 2-methoxyestradiol (2-MeOE2) 1, whilst its 3-O-sulfamate derivative (2-MeSE2MATE) 9 exhibits greatly enhanced anti-proliferative activity, combined with significant inhibition of steroid sulfatase, an enzyme target for the treatment of hormone-dependent tumours.     

Inhibition of steroid sulphatase activity by tricyclic coumarin sulphamates

The identification of the active pharmacophore required for potent inhibition of steroid sulphatase activity, i.e. an aryl-O-sulphamate structure, has led to the synthesis and testing of a large number of 1–4 ring-based inhibitors. 4-Methylcoumarin-7-O-sulphamate (COUMATE) was one of the first non-steroid based inhibitors identified. In an attempt to increase the potency of this class of inhibitor a series of tricyclic COUMATEs (665–6615 COUMATEs) have been synthesised and evaluated. Using placental microsomes as a source of oestrone sulphatase (E1-STS) the size of the third ring of the tricyclic COUMATEs was found to have a marked effect on inhibitor potency. Whereas 665- and 6615-COUMATEs had IC₅₀s of 200 and 370 nM, respectively, the most potent inhibitor in vitro in this series was 6610 COUMATE with an IC₅₀ of 1 nM. Selected inhibitors were tested for their in vivo potency by administration of a single dose (0.1 or 1 mg/kg, p.o.) to female rats. Surprisingly, in vivo 6615 COUMATE proved to be the most active drug, inhibiting rat liver E1-STS activity by 23 and 94% when assayed 24 h after administration of the 0.1 and 1 mg/kg doses. E1-STS activity in brain tissue and white blood cells was also found to be inhibited when selected drugs were tested. These studies have identified a number of tricyclic COUMATEs with therapeutic potential.     

Altered molecular dynamics and antioxidant status in the spermatozoa in testosterone-induced oligospermia in mouse

Though supraphysiological doses testosterone (T) and its derivatives are known to suppress spermatogenesis in mammals by interfering with the hypothalamus-pituitary axis leading to oligozoospermia, no study has been performed to evaluate the integrity of the sperm cells produced by such individuals. In T-induced oligozoospermia in the mouse, the spermatozoa showed suppressed zona-binding ability though the motility and viability remained unchanged. In order to assess whether this decreased zona-binding ability is due to perturbations in the mechanical properties of the sperm membranes, we attempted to examine the molecular dynamics employing a lipophilic spin label (16-doxyl stearate) and a protein-binding label (Mal-Net) in two sets of independent experiments. The results showed that the rotational freedom of lipophilic molecules reduced significantly within the first week of T-treatment. During weeks 1 through 4, the protein rotation was found to be retarded significantly. We observed a sharp increase in the ascorbyl radical associated with the cauda epididymal spermatozoa and epididymal fluid of testosterone-treated mice. Moreover, the glutathione (GSH) content in the spermatozoa and the epididymal fluid increased significantly after testosterone-treatment. Further, there was a elevation in the superoxide dismutase (SOD) activity and suppression in the superoxide anion radical generated by the cauda epididymal spermatozoa of testosterone-treated animals. A change in the mechanical properties of a bilayer could modify both the mechanical properties and the function of incorporated proteins. In many instances, a liquid-crystalline bilayer is necessary for protein function. It is likely that the change in the physical properties of sperm membranes might cause the inhibition of enzymes associated with spermatozoa after T-treatment. The alterations in the sperm membrane structure and the antioxidant potentials of both the spermatozoa and the cauda epididymal fluid could also account for the decrease in the zona-binding index of the spermatozoa in T-treated animals. Thus, this study demonstrates for the first time that supraphysiological doses of testosterone could modify the mechano-dynamic properties of sperm membranes and could perturb the redox status of both spermatozoa and the epididymal fluid.     

A letrozole-based dual aromatase-sulphatase inhibitor with in vivo activity

The role of aromatase inhibitors in the treatment of hormone-dependent breast cancer is well established. However, it is now recognised that steroid sulphatase (STS) inhibitors represent a new form of endocrine therapy. To explore the potential advantage of dual inhibition by a single agent, we recently developed a series of dual aromatase-sulphatase inhibitors (DASIs) based on the aromatase inhibitor YM511. We report here a new structural class of DASI obtained by obtained introducing the pharmacophore for STS inhibition, i.e. a phenol sulphamate ester into another established aromatase inhibitor letrozole. Hence, the bis-sulphamate 9 was synthesised which exhibited IC(50) values of 3044 nM for aromatase and >10 microM for STS in JEG-3 cells. However, at a single oral dose of 10mg/kg, 9 inhibited aromatase and rat liver STS by 60% and 88%, respectively, 24h after administration. A proposed metabolite of 9, carbinol 10, was synthesised. Despite also showing weak STS inhibition in JEG-3 cells, 10 inhibited rat liver STS activity to the same extent as 9 at a single oral dose of 10mg/kg. Thus, the concept of a letrozole-based DASI has been validated and could be further developed and modified for therapeutic exploitation.