The possibility of obtaining intergeneric hybrids via White Kołuda (Anser anser L.) goose insemination with fresh and frozen-thawed Canada goose (Branta canadensis L.) gander semen

The objective of the present experiments was to produce the intergeneric hybrids of domesticated and wild goose via artificial insemination with fresh and frozen-thawed semen. The experiments were carried out during two successive goose reproductive seasons, on eight five-year-old Canada Goose (Branta canadensis L.) males used as semen donors and 16 two-year-old White Ko?uda geese designated to fertility tests. Pooled semen was collected twice a week by the dorso-abdominal massage. In freshly collected semen, ejaculate volume, color, consistency, degree of fecal or blood contamination, spermatozoa concentration, motility, and morphology were evaluated. Part of the semen collected in the first year of the experiment (Experiment 1) was used for geese insemination with fresh semen, while the remainder was frozen. In Experiment 2 all samples were subjected exclusively to freezing procedure. Geese were inseminated once a week with fresh semen in a dose of 80 μl or 160 μl, and twice a week with frozen-thawed semen in a dose of 80 μl (160 μl per wk) or 100 μl (200 μl per wk). Eggs were set weekly and incubated up to hatching.The volume of ejaculates varied from 0.100 to 0.470 ml; spermatozoa concentration from 140 to 310 million ml⁻¹; progressive movement was observed in 40 to 60% of spermatozoa; the percentage of total live spermatozoa ranged from 69.3 to 92.0%, the highest percentage (34.0-68.3) was represented by live normal spermatozoa and those with bulb-head (13.3-41.0). Cryopreservation caused a decrease in percentage of motile cells to 30%; total live spermatozoa contribution by 27.2%p, including those live normal by 15.9%p (in relation to the fresh semen), bulb-head spermatozoa by 10.9%p, and increase (by 5.9%p) in number of spermatozoa with other deformations. Goose insemination 1×/week with fresh semen containing about 10.3 million live normal spermatozoa resulted in 66.7% of fertile eggs and with dose higher by 2.8 million spermatozoa (on average) the fertility increased by 20.9%p (up to 87.6% on average). Hatchability from set and fertile eggs was 55.9% and 83.9% vs. 66.3% and 75.6%, respectively. After twice a week insemination with frozen-thawed semen containing about 10.2 million live normal cells 58.2% eggs were fertile; hatchability from set eggs was 42.8% and from fertile eggs 71.7%, while insemination dose increase by 2.7 million spermatozoa per week caused a fertilization increase by 3.8%p (62.0% on average), this increase was not statistically significant, but hatchability from the fertile eggs (95.4%), was significantly (P < 0.05) higher.The use of AI with fresh semen in the creation of intergeneric hybrids of Canada goose males and White Ko?uda females allows a high level of egg fertility to be obtained. Furthermore, one limitation which is the short reproductive season of the Canada goose may be overcome by the use of cryopreserved semen.     

Brain levels of arginine-vasotocin and isotocin in dominant and subordinate males of a cichlid fish

The nonapeptides arginine-vasotocin (AVT) and isotocin (IT), which are the teleost homologues of arginine-vasopressin and oxytocin in mammals, have well established peripheral effects on osmoregulation and stress response, and central effects on social behavior. However, all studies that have looked so far into the relationship between these nonapeptides and social behavior have used indirect measures of AVT/IT activity (i.e. immunohistochemistry of AVT/IT immunoreactive neurons, or AVT/IT or their receptors mRNA expression with in situ hybridization or qPCR) and therefore direct measures of peptide levels in relation to social behavior are still lacking. Here we use a recently developed high-performance liquid chromatography analysis with fluorescence detection (HPLC-FL) method to quantify the levels of both AVT and IT in macro-dissected brain areas [i.e. olfactory bulbs, telencephalon, diencephalon, optic tectum, cerebellum, and hindbrain (= rhombencephalon minus cerebellum)] and pituitary of dominant and subordinate male cichlid fish (Oreochromis mossambicus). The pituitary shows higher levels of both peptides than any of the brain macroareas, and the olfactory bulbs have the highest AVT among all brain areas. Except for IT in the telencephalon there is a lack of correlations between central levels and pituitary peptide levels, suggesting an independent control of hypophysial and CNS nonapeptide secretion. There were also no correlations between AVT and IT levels either for each brain region or for the pituitary gland, suggesting a decoupled activity of the AVT and IT systems at the CNS level. Subordinate AVT pituitary levels are significantly higher than those of dominants, and dominant hindbrain IT levels are significantly higher than those of subordinates, suggesting a potential involvement of AVT in social stress in subordinate fish and of IT in the regulation of dominant behavior at the level of the hindbrain. Since in this species dominant males use urine to communicate social status and since AVT is known to have an antidiuretic effect, we have also investigated the effect of social status on urine storage. As predicted, dominant males stored significantly more urine than subordinates. Given these results we suggest that AVT/IT play a key role in orchestrating social phenotypes, acting both as central neuromodulators that promote behavioral plasticity and as peripheral hormones that promote integrated physiological changes.     

Diversity of Selected Lupinus angustifolius L. Genotypes at the Phenotypic and DNA Level with Respect to Microscopic Seed Coat Structure and Thickness

The paper investigates seed coat characteristics (as a percentage of overall seed diameter) in Lupinus angustifolius L., a potential forage crop. In the study ten L. angustifolius genotypes, including three Polish cultivars, two Australian cultivars, three mutants originated from cv. ‘Emir’, and one Belarusian and one Australian breeding line were evaluated. The highest seed coat percentage was recorded in cultivars ‘Sonet’ and ‘Emir’. The lowest seed coat thickness percentage (below 20%) was noted for breeding lines 11257-19, LAG24 and cultivar ‘Zeus’ (17.87%, 18.91% 19.60%, respectively). Despite having low seed weight, the Australian line no. 11257-19 was characterized by a desirable proportion of seed coat to the weight of seeds. In general, estimation of the correlation coefficient indicated a tendency that larger seeds had thinner coats. Scanning Electron Microscopy images showed low variation of seed coat sculpture and the top of seeds covered with a cuticle. Most of the studied genotypes were characterized by a cristatepapillate seed coat surface, formed by elongated polygonal cells. Only breeding line no. 11267-19 had a different shape of the cells building the surface layer of the coat. In order to illustrate genetic diversity among the genotypes tested, 24 ISSR primers were used. They generated a total of 161 polymorphic amplification products in 10 evaluated narrow-leaved lupin genotypes.     

ERK1/2 is dephosphorylated by a novel phosphatase--CacyBP/SIP

Recently, we have reported that the CacyBP/SIP protein binds ERK1/2 (Kilanczyk et al., BBRC, 2009). In this work we show that CacyBP/SIP exhibits a phosphatase activity toward ERK1/2 kinases while its E217K mutant does not. The K_m and V_max values established for a standard phosphatase substrate, p-NPP, are 16.9 ± 3.6 mM and 4.3 ± 0.4 μmol/min, respectively. The CacyBP/SIP phosphatase activity is decreased by okadaic acid (IC₅₀ = 45 nM). Our experimental results are supported by a theoretical analysis which revealed important sequence similarities between CacyBP/SIP and the phosphatase-like proteins as well as certain MAP kinase phosphatases.     

The Mechanism of Calcium-Induced Inhibition of Muscle Fructose 1,6-bisphosphatase and Destabilization of Glyconeogenic Complex

The mechanism by which calcium inhibits the activity of muscle fructose 1,6-bisphosphatase (FBPase) and destabilizes its interaction with aldolase, regulating glycogen synthesis from non-carbohydrates in skeletal muscle is poorly understood. In the current paper, we demonstrate evidence that Ca²⁺ affects conformation of the catalytic loop 52–72 of muscle FBPase and inhibits its activity by competing with activatory divalent cations, e.g. Mg²⁺ and Zn²⁺. We also propose the molecular mechanism of Ca²⁺-induced destabilization of the aldolase–FBPase interaction, showing that aldolase associates with FBPase in its active form, i.e. with loop 52–72 in the engaged conformation, while Ca²⁺ stabilizes the disengaged-like form of the loop.     

Essential Oils,Silver Nanoparticles and Propolis as Alternative Agents Against Fluconazole Resistant Candida albicans,Candida glabrata and Candida krusei Clinical Isolates

Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO₂ against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.     

Oxidative stress-dependent p66Shc phosphorylation in skin fibroblasts of children with mitochondrial disorders

p66Shc, the growth factor adaptor protein, can have a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress. We investigated relationships between the extent of p66Shc phosphorylation at Ser36, mitochondrial dysfunctions and an antioxidant defense reactions in fibroblasts derived from five patients with various mitochondrial disorders (two with mitochondrial DNA mutations and three with methylglutaconic aciduria and genetic defects localized, most probably, in nuclear genes). We found that in all these fibroblasts, the extent of p66Shc phosphorylation at Ser36 was significantly increased. This correlated with a substantially decreased level of mitochondrial superoxide dismutase (SOD2) in these cells. This suggest that SOD2 is under control of the Ser36 phosphorylation status of p66Shc protein. As a consequence, an intracellular oxidative stress and accumulation of damages caused by oxygen free radicals are observed in the cells.     

Neutrophils as a Source of Chitinases and Chitinase-Like Proteins in Type 2 Diabetes

Purpose

The pathophysiological role of human chitinases and chitinase-like proteins (CLPs) is not fully understood. We aimed to determine the levels of neutrophil-derived chitotriosidase (CHIT1), acidic mammalian chitinase (AMCase) and chitinase 3-like protein 1 (YKL-40) in patients with type 2 diabetes (T2D) and verify their association with metabolic and clinical conditions of these patients.

Methods

Neutrophils were obtained from the whole blood by gradient density centrifugation from 94 T2D patients and 40 control subjects. The activities of CHIT1 and AMCase as well as leukocyte elastase (LE) were measured fluorometrically and concentration of YKL-40 immunoenzymatically. Also, routine laboratory parameters in serum/plasma were determined by standard methods.

Results

The levels of all three examined proteins were about 2-times higher in diabetic patients in comparison to control subjects. They were significantly correlated with the activity of LE and increased progressively across tertiles of LE activity. Moreover, the activities of CHIT1 and AMCase were significantly correlated with each other. Metabolic compensation of diabetes did not influence the levels of these proteins. In the subgroup of patients with inflammatory evidence only YKL-40 concentration was significantly higher compared to those without inflammation. The highest levels of all three proteins were observed in patients with macroangiopathies. Insulin therapy was associated with lower levels of examined proteins.

Conclusions

We revealed that neutrophils may be an important source of the increased levels of chitinases and CLPs in T2D, and these proteins may participate in inflammatory mechanisms in the course of the disease and consequent development of diabetic angiopathies.     

Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.