Ancient DNA analyses reveal high mitochondrial DNA sequence diversity and parallel morphological evolution of late pleistocene cave bears

Cave bears (Ursus spelaeus) existed in Europe and western Asiauntil the end of the last glaciation some 10,000 years ago.To investigate the genetic diversity, population history, andrelationship among different cave bear populations, we havedetermined mitochondrial DNA sequences from 12 cave bears thatrange in age from about 26,500 to at least 49,000 years andoriginate from nine caves. The samples include one individualfrom the type specimen population, as well as two small-sizedhigh-Alpine bears. The results show that about 49,000 yearsago, the mtDNA diversity among cave bears was about 1.8-foldlower than the current species-wide diversity of brown bears(Ursus arctos). However, the current brown bear mtDNA gene poolconsists of three clades, and cave bear mtDNA diversity is similarto the diversity observed within each of these clades. The resultsalso show that geographically separated populations of the high-Alpinecave bear form were polyphyletic with respect to their mtDNA.This suggests that small size may have been an ancestral traitin cave bears and that large size evolved at least twice independently.     

Effect of Cd(2+) and Hg(2+) on the activity of Na(+)/K(+)-ATPase and Mg(2+)-ATPase adsorbed on polystyrene microtiter plates.

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4 degrees C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na(+)/K(+)-ATPase and Mg(2+)-ATPase, retained 70-80% activity after the adsorption. In addition, adsorption stabilizes Na(+)/K(+)-ATPase and Mg(2+)-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na(+)/K(+)-ATPase and Mg(2+)-ATPase and their sensitivity to and mechanism of Cd(2+)- or Hg(2+)-induced inhibition. The only exception is the "high affinity" Mg(2+)-ATPase moiety, whose affinity for ATP and sensitivity toward Cd(2+) were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Chemical Composition and Antimicrobial Activity of the Essential Oil of Stachys officinalis (L.) Trevis. (Lamiaceae)

Characterization by GC‐FID and GC/MS analyses of the Stachys officinalis (L.) Trevis . essential oil obtained by hydrodistillation of the aerial parts allowed the identification of 190 components that represented 97.9% of the total oil content. The main constituents identified were germacrene D (19.9%), β‐caryophyllene (14.1%), and α‐humulene (7.5%). Terpenoids were by far predominant (89.4%), with sesquiterpene hydrocarbons (69.1%) and oxygenated sesquiterpenes (14.8%) being the most abundant compounds detected in the oil. Based on the present and previously published results, multivariate statistical comparison of the chemical composition of the essential oils was performed within the species. Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) of the data on the volatile profiles of S. officinalis taxa revealed no pronounced differences among the samples originated from the Balkan Peninsula. Additionally, the oil was screened for in vitro antibacterial and antifungal activity using the broth microdilution assay. The oil's best antimicrobial activities were obtained against the mold Aspergillus niger (minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of 2.5 and 5.0 mg/ml, resp.) and the yeast Candida albicans (MIC and MFC of 5.0 mg/ml).     

The role of IgG avidity determination in diagnosis of Epstein-Barr virus infection in immunocompetent and immunocompromised patients

There is a high degree of variability in the serologic response to Epstein-Barr virus (EBV) infection, especially in viral capsid antigen (VCA)-IgM antibodies. Therefore, additional tests are needed to confirm primary infection. We evaluated the value of IgG avidity determination in diagnosis of EBV infection in immunocompetent and immunocompromised patients. A total of 236 serum samples from immunocompetent patients with symptoms suggestive of EBV infection were tested for the presence of VCA-IgM/IgG antibodies and IgG avidity. Using IgG avidity, acute primary infection was confirmed in 56.7% of the immunocompetent patients with positive and in 1.8% of patients with negative VCA-IgM. Recent primary infection was documented in 8.9% of the IgM positive and 3.5% of the IgM negative patients. In patients with indeterminate serology (equivocal IgM), 6.7% were classified by avidity index (AI) as acute primary infection, 10.0% as post-acute and 83.3% as past infection cases. Concerning the 32 immunocompromised patients, recent primary infection was documented in 3 of the 14 IgM positive patients. High AI was detected in 11 of these patients, indicating an IgM response due to reactivation. Determination of IgG avidity in combination with classical serologic markers seems to be a reliable method to confirm primary infection both in immunocompetent and immunocompromised patients. It may be especially useful to differentiate cases of primary infection in patients with undetectable VCA-IgM antibodies or indeterminate routine EBV serology.     

Engineering tissue with BioMEMS

In summary, microfluidic-BioMEMS platforms are increasingly contributing to tissue engineering in many different ways. First, the accurate control of the cell environment in settings suitable for cell screening and with imaging compatibility is greatly advancing our ability to optimize cell sources for a variety of tissue-engineering applications. Second, the microfluidic technology is ideal for the formation of perfusable networks, either to study their stability and maturation or to use these networks as templates for engineering vascularized tissues. Third, the approaches based on microfluidic and BioMEMS devices enable engineering and the study of minimally functional modules of complex tissues, such as liver sinusoid, kidney nephron, and lung bronchiole. This brief article highlighted some of the unique advantages of this elegant technology using representative examples of tissue-engineering research. We focused on some of the universal needs of the area of tissue engineering: tissue vascularization, faithful recapitulation in vitro of functional units of our tissues and organs, and predictable selection and differentiation of stem cells that are being addressed using the power and versatility of microfluidic-BioMEMS platforms.     

Enhanced prereceptor glucocorticoid metabolism and lipogenesis impair insulin signaling in the liver of fructose-fed rats

Overconsumption of fructose, as a highly lipogenic sugar, may profoundly affect hepatic metabolism and has been associated with many components of the metabolic syndrome, particularly with insulin resistance and Type 2 diabetes. In this study, we proposed that high fructose diet may enhance lipogenesis and decrease insulin sensitivity in the liver through dysregulation of glucocorticoid signaling. Therefore, we examined the effects of long-term consumption of 10% fructose solution on triglyceridemia, liver histology and intracellular corticosterone level, as well as on 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and hexose-6-phosphate dehydrogenase (H6PDH) mRNA and protein levels in the rat liver. Glucocorticoid action was assessed by glucocorticoid receptor (GR) expression and intracellular redistribution. We also analyzed the expression of enzymes involved in gluconeogenesis and lipogenesis, phosphoenolpyruvate carboxykinase (PEPCK) and lipin-1. The results have shown that fructose-rich diet led to increase in 11βHSD1 and H6PDH protein levels, while hepatic corticosterone concentration remained unchanged. Concomitantly, GR was increasingly accumulated in the cytoplasm, whereas its nuclear level was unchanged and accompanied by diminished PEPCK mRNA level. Elevation of lipin-1 in the liver microsomes suggested that fructose diet led to an increase in lipogenesis and consequently to hypertriglyceridemia. The observed increase of insulin receptor supstrate-1 phosphorylation on Ser³⁰⁷ represents a hallmark of impaired insulin signaling in the liver of fructose-fed rat and probably is a consequence of the alterations in 11βHSD1 and lipin-1 levels. Overall, our findings suggest that fructose-rich diet may perturb hepatic prereceptor glucocorticoid metabolism and lipogenesis, resulting in hypertriglyceridemia and attenuated hepatic insulin sensitivity.     

Cholecystokinin-producing (I) cells of intestinal mucosa in dexamethasone-treated rats

The aim of this study was to investigate the morphological, immunohistochemical and ultrastructural changes of cholecystokinin-producing (I) cells of gastrointestinal mucosa in dexamethasone-treated rats (D). After 12-daily intraperitoneal administration of 2mg/kg dexamethasone, rats developed diabetes similar to human diabetes mellitus type 2. The mean diameter of the duodenum was significantly decreased due to significant reduction of volume fraction and profile area of lamina propria. There was a decrease in volume fraction and number of cholecystokinin (CCK)-producing cells per mm(2) of mucosa, as well as their numerical density, but without statistical significance. Also, dexamethasone induced appearance of hyperactive duodenal I-cells with small number of granules and dilated endoplasmic reticulum. In conclusion, the present study showed that morphological changes in duodenum cholecystokinin-producing (I) cells occurred in diabetic rats, in a manner which, suggests compensatory effort of CCK cells in diabetic condition.     

Kinetics of Tissue Iron in Experimental Autoimmune Encephalomyelitis in Rats

To elucidate the role of iron in the pathomechanisms of autoimmune CNS disorders, we estimated the tissue concentrations of Fe²⁺ in the brain, spinal cord, and liver in the chronic relapsing form of experimental autoimmune encephalomyelitis (EAE). The disease was induced in Dark Agouti (DA) strain of rats, by subcutaneous injection of bovine brain homogenate in complete Freund's adjuvant (CFA). Control rats consisted of unsensitized rats and of rats treated with CFA or saline. The data obtained by clinical assessment and by inductively coupled plasma spectrometry have shown that the attacks of disease (on the 12th and 22nd post-immunization day) were followed by high accumulation of iron in the liver. Additionally, during the second attack of disease, the decreased concentration of Fe²⁺ was found in cervical spinal cord. The data point to regulatory effects of iron and hepatic trace elements regulating mechanisms in the pathogenesis of EAE.     

Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.)

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1 mM ammonium (NH₄⁺) as the sole source of nitrogen. Growth of NH₄⁺-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH₄⁺ medium with 25 mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH₄⁺ induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH₄⁺ as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20 mM) or Gln (10 mM) in combination with NH₄⁺ (1 mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.