Tris–sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61

The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris–sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.     

Influence of the selective oestrogen receptor modulator (raloxifene hydrochloride) on IL-6, TNF-alpha, TGF-beta1 and bone turnover markers in the treatment of postmenopausal osteoporosis

BACKGROUND: Osteoporosis that is encountered frequently in postmenopausal women, may cause an increased incidence of vertebral and iliac fractures that are associated with excess morbidity. Raloxifene hydrochloride, a selective oestrogen receptor modulator, has been shown to increase bone mineral density and decrease biochemical markers of bone turnover in postmenopausal women, without stimulatory effects on breast or uterus. Levels of proinflammatory cytokines, including IL-6, and TNF-alpha and TGF-beta1 which are important cytokines involved in remodeling, have been evaluated previously in in vitro studies of osteoporosis. However, there seems to be a paucity of in vivo research concerned with changes in these cytokines in osteoporosis. OBJECTIVE: In this study, we evaluated the effects of raloxifene (Evista); Lilly Pharmaceutical Co. USA, 60 mg/day) on biochemical bone turnover markers, serum parathyroid hormone, and 25-OH vitamin D, as well as the serum levels of IL-6, TNF-alpha and TGF-beta1, in 22 postmenopausal, osteoporotic women before and after 12 weeks of raloxifene treatment. METHODS: Well-matched, postmenopausal, non-osteoporotic control subjects were also enrolled in the study. Serum levels of all the parameters were measured in postmenopausal, osteoporotic women at baseline and end of the study. RESULTS: It was found that serum osteocalcin and parathyroid hormone, and urine deoxypyridinoline levels decreased to normal levels with treatment. Serum 25-OH vitamin D levels after treatment in the patient group were higher than those in the control group. Serum IL-6, TNF-alpha and TGF-beta1 levels did not change significantly with treatment. However, serum levels of IL-6 and TGF-beta1 in the patient group after treatment, decreased to levels lower than those found in the control group. Serum TNF-alpha levels in the patient group before and after treatment, were lower than those in the control group. CONCLUSION: Raloxifene treatment reduces bone turnover biochemical markers, parathyroid hormone and induces 25-OH vitamin D in postmenopausal women. Moreover, it also affects some serum cytokine levels in the postmenopausal period.     

Molecular ecology and phylogeography of the bent-wing bat complex (Miniopterus schreibersii) (Chiroptera: Vespertilionidae) in Asia Minor and adjacent regions

In this study we investigate population genetic structure and phylogeography of the bent-wing bat complex ( Miniopterus schreibersii ) in Asia Minor and adjacent regions. PCR amplification and sequencing of the first hypervariable domain of the mitochondrial control region were used to obtain the genetic data. Morphometric differentiation between lineages was analysed by comparing forearm lengths. We found two reciprocally monophyletic lineages within the M . schreibersii complex, identified as M.   s. schreibersii and M. s . pallidus . Distributions of the lineages were allopatric with a U-shaped suture zone passing through Central Anatolia. The suture zone separated coastal regions occupied by M.   s. schreibersii from inland, higher altitude regions occupied by M.   s. pallidus . The lineages showed a considerable sequence divergence of c . 9%, accompanied by a corresponding difference in forearm length. The presence of the genetically distinct lineages, with allopatric distribution and corresponding morphometric differences, probably reflects their long isolation during the ice-age in the Balkans and the Caspian/Caucasus refugia, followed subsequently by expansion into different habitats. Based on the present data, the lineages can be recognized as evolutionary significant units.     

Gem1 and ERMES do not directly affect phosphatidylserine transport from ER to mitochondria or mitochondrial inheritance

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.     

A quantitative comparison of mechanical blood damage parameters in rotary ventricular assist devices: shear stress, exposure time and hemolysis index

Ventricular assist devices (VADs) have already helped many patients with heart failure but have the potential to assist more patients if current problems with blood damage (hemolysis, platelet activation, thrombosis and emboli, and destruction of the von Willebrand factor (vWf)) can be eliminated. A step towards this goal is better understanding of the relationships between shear stress, exposure time, and blood damage and, from there, the development of numerical models for the different types of blood damage to enable the design of improved VADs. In this study, computational fluid dynamics (CFD) was used to calculate the hemodynamics in three clinical VADs and two investigational VADs and the shear stress, residence time, and hemolysis were investigated. A new scalar transport model for hemolysis was developed. The results were compared with in vitro measurements of the pressure head in each VAD and the hemolysis index in two VADs. A comparative analysis of the blood damage related fluid dynamic parameters and hemolysis index was performed among the VADs. Compared to the centrifugal VADs, the axial VADs had: higher mean scalar shear stress (sss); a wider range of sss, with larger maxima and larger percentage volumes at both low and high sss; and longer residence times at very high sss. The hemolysis predictions were in agreement with the experiments and showed that the axial VADs had a higher hemolysis index. The increased hemolysis in axial VADs compared to centrifugal VADs is a direct result of their higher shear stresses and longer residence times. Since platelet activation and destruction of the vWf also require high shear stresses, the flow conditions inside axial VADs are likely to result in more of these types of blood damage compared with centrifugal VADs.     

The efficacy of a topical gel with triester glycerol oxide in denture-related mucosal injuries

doi: 10.1111/j.1741‐2358.2011.00549.x
The efficacy of a topical gel with triester glycerol oxide in denture‐related mucosal injuries Objective: The efficacy of a topical gel with triester glycerol oxide for treating ulcerations related to new complete dentures was studied and compared with a placebo gel. Materials and methods: Fifty edentulous patients were enrolled in this double‐blind clinical study. All subjects received new maxillary and mandibular complete dentures and were recalled 1, 3 and 7 days after denture placement. Twenty‐five subjects received topical gel with triester glycerol oxide (TGO), and 25 subjects received topical gel without TGO. At each visit, pain and discomfort with the new dentures was assessed, and at the last visit, healing of ulcerations was also graded by a clinician. Subjects also graded the gel’s taste, smell and ease of use. Results: Specifically, there was no statistically significant difference in ulcer healing (p > 0.05), ratings of smell, taste and ease of use (p > 0.05) or pain and discomfort scores (p > 0.05). However, within‐group differences were observed in pain and discomfort scores between follow‐up sessions (p = 0.01 for all comparisons). Conclusion: Clinically, TGO gel is not effective in the treatment of mucosal injuries occurring following placement of complete dentures.     

High microsatellite and mitochondrial diversity in Anatolian native horse breeds shows Anatolia as a genetic conduit between Europe and Asia

The horse has been a food source, but more importantly, it has been a means for transport. Its domestication was one of the crucial steps in the history of human civilization. Despite the archaeological and molecular studies carried out on the history of horse domestication, which would contribute to conservation of the breeds, the details of the domestication of horses still remain to be resolved. We employed 21 microsatellite loci and mitochondrial control region partial sequences to analyse genetic variability within and among four Anatolian native horse breeds, Ayvac?k Pony, Malakan Horse, H?n?s Horse and Canik Horse, as well as samples from indigenous horses of unknown breed ancestry. The aims of the study were twofold: first, to produce data from the prehistorically and historically important land bridge, Anatolia, in order to assess its role in horse domestication and second, to analyse the data from a conservation perspective to help the ministry improve conservation and management strategies regarding native horse breeds. Even though the microsatellite data revealed a high allelic diversity, 98% of the genetic variation partitioned within groups. Genetic structure did not correlate with a breed or geographic origin. High diversity was also detected in mtDNA control region sequence analysis. Frequencies of two haplogroups (HC and HF) revealed a cline between Asia and Europe, suggesting Anatolia as a probable connection route between the two continents. This first detailed genetic study on Anatolian horse breeds revealed high diversity among horse mtDNA haplogroups in Anatolia and suggested Anatolia’s role as a conduit between the two continents. The study also provides an important basis for conservation practices in Turkey.     

Heterogeneity of ATP-sensitive K+ Channels in Cardiac Myocytes: ENRICHMENT AT THE INTERCALATED DISK*

Ventricular ATP-sensitive potassium (K_ATP) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative K_ATP channel-associated proteins. We investigated whether the association of K_ATP channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co-immunoprecipitation experiments confirmed physical interaction between K_ATP channels and PKP2 and plakoglobin in rat heart. Immunolocalization experiments demonstrated that K_ATP channel subunits (Kir6.2 and SUR2A) are expressed at a higher density at the intercalated disk in mouse and rat hearts, where they co-localized with PKP2 and plakoglobin. Super-resolution microscopy demonstrate that K_ATP channels are clustered within nanometer distances from junctional proteins. The local K_ATP channel density, recorded in excised inside-out patches, was larger at the cell end when compared with local currents recorded from the cell center. The K_ATP channel unitary conductance, block by MgATP and activation by MgADP, did not differ between these two locations. Whole cell K_ATP channel current density (activated by metabolic inhibition) was ∼40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of K_ATP channels was absent in the PKP2 deficient mice, but the K_ATP channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of K_ATP channel distribution within a cardiac myocyte. The higher K_ATP channel density at the intercalated disk implies a possible role at the intercellular junctions during cardiac ischemia.     

Agrobacterium-mediated transformation of Arabis gunnisoniana

An efficient method for adventitious shoot regeneration for Arabis drummondii and a transformation protocol for A. gunnisoniana from hypocotyl explants are described. Hypocotyl explants from 7-day-old aseptically grown seedlings were cultured on MS medium containing plant growth regulators (6-benzylaminopurine, 1-phelyl-3- (1,2,3-thiadiazol-5-yl) urea, -naphthaleneacetic acid and 2,4-dichlorophenoxy-acetic acid). After 4 weeks in culture, high frequency of adventitious shoot regeneration was observed. Regenerated shoots were rooted on half-strength MS basal medium supplemented 1% (w/v) sucrose, with or without NAA. This protocol was then used to produce transformed Arabis gunnisoniana plants. A. gunnisoniana hypocotyl explants were co-cultivated with Agrobacterium tumefaciens strain GV3101 harbouring pBJ40. Transgenic shoots were selected on MS 21 medium supplemented with 50 mg l kanamycin. PCR analysis verified the presence of the nptII gene in the plant DNA isolated from kanamycin resistant shoots.