Diversification of lupine Bradyrhizobium strains: evidence from nodulation gene trees

Bradyrhizobium strains isolated in Europe from Genisteae and serradella legumes form a distinct lineage, designated clade II, on nodulation gene trees. Clade II bradyrhizobia appear to prevail also in the soils of Western Australia and South Africa following probably accidental introduction with seeds of their lupine and serradella hosts. Given this potential for dispersal, we investigated Bradyrhizobium isolates originating from a range of native New World lupines, based on phylogenetic analyses of nodulation (nodA, nodZ, noeI) and housekeeping (atpD, dnaK, glnII, recA) genes. The housekeeping gene trees revealed considerable diversity among lupine bradyrhizobia, with most isolates placed in the Bradyrhizobium japonicum lineage, while some European strains were closely related to Bradyrhizobium canariense. The nodA gene tree resolved seven strongly supported groups (clades I to VII) that correlated with strain geographical origins and to some extent with major Lupinus clades. All European strains were placed in clade II, whereas only a minority of New World strains was placed in this clade. This work, as well as our previous studies, suggests that clade II diversified predominately in the Old World, possibly in the Mediterranean. Most New World isolates formed subclade III.2, nested in a large "pantropical" clade III, which appears to be New World in origin, although it also includes strains originating from nonlupine legumes. Trees generated using nodZ and noeI gene sequences accorded well with the nodA tree, but evidence is presented that the noeI gene may not be required for nodulation of lupine and that loss of this gene is occurring.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.     

Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, is considered as the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Here we report the 1.95Å structure of the complex formed by the DNA-binding domains (DBDs) the EcR and the Usp, bound to the natural pseudopalindromic response element. Comparison of the structure with that obtained previously, using an idealized response element, shows how the EcRDBD, which has been previously reported to possess extraordinary flexibility, accommodates DNA-induced structural changes. Part of the C-terminal extension (CTE) of the EcRDBD folds into an α-helix whose location in the minor groove does not match any of the locations previously observed for nuclear receptors. Mutational analyses suggest that the α-helix is a component of EcR-box, a novel element indispensable for DNA-binding and located within the nuclear receptor CTE. This element seems to be a general feature of all known EcRs.     

Diagnostic difficulties in adrenal incidentaloma--analysis of 125 cases

INTRODUCTION: Therapeutic approach to incidentaloma, in spite of existing algorithms, is not always obvious due to diagnostic difficulties. The aim of the study was to assess the validity of the initial diagnoses of incidentaloma which determined the qualification for the operation. MATERIAL AND METHODS: 125 patients hospitalised in the Endocrinology Dept. of the Medical University of Bialystok in the years 2003-2005 and in the Endocrinology dept. of Voivodeship Hospital of Bialystok. The patients were clinically and hormonally examined (metanephrines in daily urine collection, daily cortisol rhythm, short dexamethasone test, aldosterone, and renin plasma activity, Na, K levels in the serum) as well as computer tomography of the adrenal glands were performed. RESULTS: 42 patients were qualified for adrenalectomy. Adenoma was confirmed in 25 patients (in 7 subclinical Cushing syndrome was diagnosed, in 2 Conn disease, in 16 inactive changes), phaeochromocytoma in 6 patients, cysts in 3, lipoma in 2, carcinoma in one, in 4 patients metastases (in 2 of kidney carcinoma, in 1 of malignant melanoma and in 1 of planocellular carcinoma) and in one oncocytoma. Metanephrines urine measurements showed 33% of false positive results. CONCLUSIONS: Qualification for adrenalectomy requires an assessment of tumor's enlargement, its tissue density, morphology and growth dynamics. To reduce the percentage of false positive results of metanephrine measurement there is to eliminate an influence of some drugs, victuals, beverages and nicotine and eventually to carry out additional tests. The decision as to proceeding with adrenal incidentaloma should be individualized based on clinical symptoms, hormonal tests and tumor morphology.     

Osteoprotegerin--does it play a protective role in the pathogenesis of bone loss in obese perimenopausal women?

INTRODUCTION: Assessment of serum osteoprotegerin (OPG) concentrations in obese patients in comparison to healthy controls and evaluation of a possible correlation between OPG and other markers of bone turnover or calcitropic hormones. MATERIAL AND METHODS: 50 obese perimenopausal women without concomitant diseases (BMI 36.7 +/- 4.1 kg/m(2), mean age 50.4 +/- 4.9 yrs). The control group consisted of 19 healthy women (BMI 24.2 +/- 2.1 kg/m(2); mean age 53.8 +/- 5.1 yrs). In all patients serum concentration of OPG, C telopeptide of type I collagen containing the crosslinking site (CTX), osteocalcin, parathormone (PTH) and vitamin D (25-OH-D(3)) was assessed. Dual energy x-ray absorptiometry (the DXA method) of the lumbar spine and femoral neck was performed using a Lunar DPXL to measure bone marrow density (BMD). RESULTS: In obese perimenopausal women serum OPG, osteocalcin and 25-OH-D(3) levels were significantly lower, and the serum PTH level was significantly higher in comparison to healthy controls. A significantly positive correlation was found between serum OPG level and age in both obese and control subjects. CONCLUSION: The serum OPG level in obese perimenopausal women is significantly lower in comparison to healthy controls and does not correlate significantly with biochemical markers of bone turnover, calcitropic hormones and BMD. It probably cannot play a protective role in the pathogenesis of bone loss in obese perimenopausal women.     

Amino sugars: new inhibitors of zeaxanthin epoxidase, a violaxanthin cycle enzyme

The effect of three sugars and their amino derivatives on violaxanthin cycle enzymes activity was investigated in duckweed (Lemna trisulca), a model water-plant. No effect of sugars and amino sugars on violaxanthin de-epoxidase was observed independent of incubation time; however, epoxidation of zeaxanthin to violaxanthin was inhibited. The minimum amino sugar concentrations causing maximum inhibition of zeaxanthin epoxidation have been estimated. Amino sugars but not sugars caused more than a 50% inhibition of zeaxanthin epoxidation in duckweed after a 24h incubation when applied at a concentration of 0.5%. Incubation with amino sugars under a 6d photoperiod enhanced the inhibitory effect. Zeaxanthin epoxidation was completely inhibited under such conditions, whereas only a minor inhibitory effect was observed in sugar treated plants. The strong amino sugar inhibition of zeaxanthin epoxidase activity represents additional evidence for the creation of an unstable carotenoid carbocation in the molecular mechanism of epoxidation.     

Neuropeptide FF (NPFF) reduces the expression of morphine- but not of ethanol-induced conditioned place preference in rats

Neuropeptide FF (NPFF) has been described as an anti-opioid peptide. It plays a role in opioid antinociception, dependence and tolerance. Previous study has indicated that 1DMe ([D-Tyr(1), (NMe)Phe(3)]NPFF), a stable analog of NPFF, inhibits acquisition of the rewarding effect of morphine but not of ethanol in mice. The rewarding effects of these drugs were measured in the unbiased paradigm of conditioned place preference (CPP). The present study examines the influence of NPFF on the expression of morphine- and ethanol-induced CPP in the biased procedure in rats. Our experiments showed that NPFF, given intracerebroventricularly (i.c.v.) at the doses of 5, 10 and 20 nmol, inhibited the expression of morphine-induced CPP. NPFF gave itself, neither induced place preference nor aversion, although a tendency to aversive effect was seen at the highest dose of 20 nmol. NPFF did not indicate fear behavior in the elevated plus maze test, and did not disturb locomotor activity of rats. However, NPFF was unable to inhibit the expression of ethanol-induced CPP. Probably this effect is due to the fact that ethanol reward is a more complex process and apart from the role of opioids, there are other neurotransmitters also involved in this mechanism. These results suggest that NPFF is involved in the expression of morphine reward. Moreover, our study supports an anti-opioid character of this peptide.     

Cytochemical and immunocytochemical studies of the localization of histones and protamine-type proteins in spermatids of Chara vulgaris and Chara tomentosa

Spermiogenesis in Chara algae, which has been divided into 10 phases (sp I-X), is similar to spermiogenesis in animals. The most important process during spermiogenesis in animals is remodeling of chromatin leading to "sleeping genome", being the result the exchange of histone proteins into protamine-like proteins. Cytochemical studies showed in both Chara species (C. vulgaris, C. tomentosa) that at spI-IV phases only histones were present, at spV-VIII phases--the amount of nuclear protamine-type proteins progressively increased and that of histones decreased while at spIX-X only pro-tamine-type proteins were present. This was also confirmed with capillar electrophoresis. In order to localize more precisely both histones and protamines the immunocytochemical studies with the use of anti-protamine antibodies (protamine-type proteins were obtained from C. tomentosa antheridia) and anti-histone H3 antibodies, have been carried out. More specific immunocytochemical studies confirmed cytochemical results including the exchange of histones into protamine-type during spermiogenesis (spV-VIII) in both Chara species. At phase V spermiogenesis these strong strand-like anti-protamine signals were observed in cytoplasm which might suggest that protamine synthesis took place in ER.     

Apoptosis in ovarian cells in postmenopausal women

Apoptosis is a natural process which accompanies human ovary from the moment of birth until old age. While it is a well-known process at the reproductive age, it still needs to be thoroughly examined when referring to the postmenopausal age. The study involved 30 postmenopausal women who had their ovaries removed by laparotomy due to nonneoplastic diseases of the uterus. The women were divided into 3 groups depending on the time that had passed since the last menstruation. Group A consisted of women who had their last menstruation no more than 5 years earlier. In group B menopause occurred 5 to 10 years earlier. Group C was composed of patients who had the last menstruation over 10 years earlier. In all the patients concentrations of follitropin (FSH) and estradiol (E2) in blood plasma were measured. Ovarian tissue was obtained during surgery. For morphological studies, ovaries were fixed in Bouin's solution and 4% formalin and embedded in paraffin. Morphological analysis was carried out after hematoxylin-eosin (H-E) staining. For histochemical detection of apoptotic cells (in situ localization of fragment DNA), the TUNEL method was used. The expression of caspase-3 positive cells was determined immunohistochemically in paraffin-embedded specimens. Comparing to groups A and B, the ovaries in group C contained small number of corpora albicantia located in the medullary part as well as thinned blood vessels and few lymphatic vessels and nerves. In contrast to group A where the number of TUNEL-positive cells was high and caspase-3 expression was observed, no TUNEL-positive nuclei and caspase-3 expression were found in the examined ovaries of group C women.