Use of radioactive—Labeled ABA for rapid determination of leaf abscission periods in cotton seedlings

A simple and rapid method is described for determination of cotton leaf abscission periods using radioactive-labeled ABA in cotton seedlings. A step-by-step protocol for cotton aged 5–6 days is provided.     

Time-course expression of metallothioneins and tissue metals in chronic relapsing form of experimental autoimmune encephalomyelitis

To elucidate the role of metallothioneins (MTs) in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MTs I+II and the tissue concentrations of Zn2+ and Cu2+ in the brain, spinal cord (SC) and in the liver during the periods of attacks and remissions in chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE). Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control rats were treated with CFA. The data, obtained by clinical assessment, immunohistochemistry and inductivity coupled plasma spectrometry, have shown that during the first attack (on the 12th day) MTs I+II were markedly upregulated in subarachnoid regions and perivascular space on astrocytes, microglia and on spinal neurons. Simultaneously, the concentrations of zinc in the SC and zinc and copper in the liver have found to be increased. During the second attack (on the 22nd day) a new overexpression of MTs was found in the cerebellum, in sulcus hippocampi, in spinal neurons and particularly in hepatocytes around the central vein. Concomitantly, in the brain and SC the concentration of copper increased. The data point to a neuroprotective role of MTs and to an important regulatory role of essential metals and hepatic MTs in the pathogenesis of CR-EAE.     

ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice

ELOVL2 is a member of the mammalian microsomal ELOVL fatty acid enzyme family, involved in the elongation of very long-chain fatty acids including PUFAs required for various cellular functions in mammals. Here, we used ELOVL2-ablated (Elovl2(-/-)) mice to show that the PUFAs with 24-30 carbon atoms of the ω-6 family in testis are indispensable for normal sperm formation and fertility in male mice. The lack of Elovl2 was associated with a complete arrest of spermatogenesis, with seminiferous tubules displaying only spermatogonia and primary spermatocytes without further germinal cells. Furthermore, based on acyl-CoA profiling, heterozygous Elovl2(+/-) male mice exhibited haploinsufficiency, with reduced levels of C28:5 and C30:5n-6 PUFAs, which gave rise to impaired formation and function of haploid spermatides. These new insights reveal a novel mechanism involving ELOVL2-derived PUFAs in mammals and previously unrecognized roles for C28 and C30 n-6 PUFAs in male fertility. In accordance with the function suggested for ELOVL2, the Elovl2(-/-) mice show distorted levels of serum C20 and C22 PUFAs from both the n-3 and the n-6 series. However, dietary supplementation with C22:6n-3 could not restore male fertility to Elovl2(+/-) mice, suggesting that the changes in n-6 fatty acid composition seen in the testis of the Elovl2(+/-) mice, cannot be compensated by increased C22:6n-3 content.     

Corneal thickness in pseudoexfoliative glaucoma

Measurements of central cornea thickness (CCT) have a very important value in glaucoma patients; if the central cornea is thinner than it suggests, then the intraocular pressure is falsely low. This study compares the central cornea thickness between patients with pseudoexfoliative glaucoma, open angle glaucoma, angle closure glaucoma and control group. This study included 34 patients with pseudoexfoliative glaucoma, 31 patient with open angle laucoma, 28 patients with angle closure laucoma and 36 normal subjects in a control group. Patients in all groups and also normal subjects in control group had no other corneal disorders, no history of trauma, corneal surgery and were not patients with contacts lens use. Patients with pseudoexfoliative glaucoma and also patients with open angle glaucoma had significantly lower values of central cornea thickness compared with normal subjects in control group. Tomey EM 3000 is a non contact specular microscope which was used to measure central corneal thickness in this study. Pachymetry is an important method for diagnoses of glaucoma and for examination of the intraocular pressure in glaucoma patients, because values of the central corneal thickness affect the exact intraocular pressure readings.     

Biomechanical properties of bones from rats treated with sevelamer

Sevelamer hydrochloride is used for ten years in patients on dialysis as a phosphate binder. We have previously shown that oral application of sevelamer prevents the bone loss and increases the bone volume in ovariectomized rats. In this study we further analysed the biomechanical properties of bones from rats treated with sevelamer utilizing a threepoint bending test to determine the mechanical properties of the cortical bone of the mid-shaft femur, while the indentation test was used to determine the mechanical properties of cancellous bone in the marrow cavity of the distal femoral metaphysis. Parameters analyzed included: maximum load (F(u)), stiffness (S), energy absorbed (W), toughness (T) and ultimate strength (sigma). The intrinsic properties, stress, elastic modulus and toughness were determined from measured maximum load, strains, stiffness, energy absorbed, outer and inner diameters, and calculated bone cross-sectional moment of inertia. Sevelamer was given to rats for 25 weeks with a content of 3% of sevelamer in a standard diet, starting immediately following ovariectomy (OVX). Animals were divided to the following groups: (1) Sham; (2) Sham + sevelamer 3%; (3) OVX; (4) OVX + sevelamer 3%. Our results showed that sevelamer particularly influenced the rat trabecular bone by increasing the maximum load for 26.2%, energy absorbed for 24.2% and the ultimate strength for 26.2% in sham animals treated with sevelamer 3%, as compared to sham rats. Sevelamer 3% in OVX rats also increased the maximum load for 71.4%, stiffness for 70.7%, energy absorbed for 55.9% and the ultimate strength for 71.3% as compared to OVX controls. In the three bending test sevelamer had a very little effect on preventing loss of bone strenght in the cortical bone. These results collectively suggest that sevelamer improves bone biomechanical properties, mainly affecting trabecular bone quality in both normal and ovariectomized rats.     

Ten-year study on the correlation of clinical and pathohistological diagnosis of dysplastic nevi

The aim of the study was to analyze the clinical prevalence and pathohistological correlation of dysplastic nevi. In the period between 2000 and 2009, in the Outpatient Clinic of Referral Centre for Melanoma of the Ministry of Health and Social Welfare of the Republic of Croatia, 12,344 patients were examined, and 35.07% of them were surgically removed in the same institution. Among the patients, 69.16% had clinically diagnosed melanocytic tumor. Out of them, 28.39% were dysplastic. Dysplastic nevus was pathohistologically diagnosed in 20.02% of pathohistologically diagnosed melanocytic tumors. There was women predominace among patients with clinically diagnosed dysplastic nevi (65.22%). The most frequent localization was the trunk in both sexes, women 78.18%, men 76.75%. The coincidence of clinical and pathohistological diagnosis of dysplastic nevus was 30.70%. The results of this study, based on a large number of patients could be a significant contribution in understanding characteristics of dysplastic nevus, its clinical and pathohistological complexity. We hope that the data will contribute to the creation of general accepted protocols in the diagnostics of dysplastic nevus.     

Ecology and taxonomy of potentially toxic Pseudo-nitzschia species in Lim Bay (north-eastern Adriatic Sea)

The population dynamics of Pseudo-nitzschia in relation to environmental factors was investigated from March 2002 to July 2008 in Lim Bay, in the north-eastern Adriatic Sea. Domoic acid was monitored in the breeding population of Mytilus galloprovincialis from 2005 to 2008. The principal-component analysis of environmental parameters showed that the system is mostly temperature driven. The phytoplankton community was mainly composed of diatoms. Pseudo-nitzschia was the dominant diatom, present in 60% of samples, with a maximum (1.6 × 10⁶ cells L⁻¹) contribution up to 97% of the total diatom abundance. Morphological analysis revealed Pseudo-nitzschia manii and potentially toxic Pseudo-nitzschia pseudodelicatissima, Pseudo-nitzschia pungens, Pseudo-nitzschia fraudulenta and Pseudo-nitzschia calliantha as the dominant species in blooms. Pseudo-nitzschia abundance positively correlated to temperature, phosphate and ammonia in accordance with its maximal abundance in the summer/autumn period when fish farms had a maximum impact on the environment. Domoic acid was detected in M. galloprovincialis in concentrations below regulatory limits, ranging from 0.097 to 0.8721 μg g⁻¹ in five cases from April to October 2005 in Lim Bay, but so far it is not clear which of the species was responsible for DA production. This study is also the first record of P. manii, P pungens and P. fraudulenta species in the Adriatic Sea.     

Sterically congested quinone methides in photodehydration reactions of 4-hydroxybiphenyl derivatives and investigation of their antiproliferative activity

In aqueous media, photochemical excitation (S(1)) of hydroxyadamantyl, diphenylhydroxymethyl, and hydroxypropyl derivatives of 4-phenylphenol 5-9 leads to solvent-assisted deprotonation of the phenol OH, and protonation of the benzyl alcohol coupled with dehydration, that delivers quinone methides (QMs) 14-18. The QMs react with CH(3)OH converting them in high quantum yields to the photosolvolysis products (overall Φ～ 0.1-0.5). QMs were characterized by laser flash photolysis in CH(3)CN-H(2)O and TFE. In TFE, the zwitterionic QM 15 has a lifetime of 250 ns, whereas para QMs 16 and 17 have lifetimes of 500 μs and 1.1 s, respectively. Introduction of the steric hindrance to the parent QM structure (with the adamantyl moiety), or additional stabilization by two phenyl rings, results in an increase of QM lifetimes and selectivity in the reactions with nucleophiles. In vitro studies of the antiproliferative activity of photochemically generated QMs 15-17 were carried out on one human cancer cell line. Irradiation of cells incubated with 7 showed enhanced antiproliferative activity compared to cells that were not irradiated, in accordance with the activity being due to the formation of QM 16.     

Chemical and fatty acid composition of male mouflon (Ovis ammon musimon Pal.) meat

The aim of this research was to determine the effect of age on chemical and fatty acid composition of the male mouflon (Ovis ammon musimon Pal.) meat. The male mouflons included in the study originated from the North Adriatic island Rab, Croatia. Three age groups were included (young, sub-adult, adult). An effect of age on chemical composition was not found. However, higher percentage of total fat, dry matter and ash was determined in the meat of young mouflons. Predominating saturated fatty acids in mouflon meat were myristic, palmitic and stearic acids. Age significantly (p < 0.05) affected only the percentage of palmitic acid being the highest in the meat of young mouflons. Percentage of the predominant monounsaturated fatty acid, oleic acid, did not change significantly with age. A higher (p < 0.05) percentage of the predominant polyunsaturated fatty acid, linoleic acid, was found in the meat of sub-adult mouflons. The meat of the young mouflons had a higher (p < 0.05) percentage of conjugated linoleic acid. The meat of the sub-adult and adult mouflons was found to have a higher (p < 0.05) percentage of total saturated fatty acids. A lower (p < 0.05) ratio of polyunsaturated and saturated fatty acids was found in the meat of young than in the meat of sub-adult mouflons. The ratio of n-6 and n-3 was lower (p < 0.05) in the adult than in the sub-adult mouflons. Higher (p < 0.05) atherogenicity and thrombogenicity indices were found in the young rather than in the adult mouflons. Low fat content (<1 %) and favourable ratios of polyunsaturated, saturated, n-6 and n-3 fatty acids make the mouflon meat a good choice for human diet.