Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca^2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With ^D63NS100B as an exception (^D63NK_D = 50 ± 9 μM), Ca^2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (^D61NK_D = 412 ± 67 μM, ^D65NK_D = 968 ± 171 μM, and ^E72AK_D = 471 ± 133 μM), when compared to wild-type protein (^WTK_D = 56 ± 9 μM). For the TRTK-12 complexes, the Ca^2 +-binding affinity to wild type (^WT + TRTKK_D = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (^{D61N + TRTK}K_D = 29 ± 1.2 μM, ^{D63N + TRTK}K_D = 10 ± 2.2 μM, ^{D65N + TRTK}K_D = 73 ± 4.4 μM, and ^{E72A + TRTK}K_D = 18 ± 3.7 μM). In addition, R_ex, as measured using relaxation dispersion for side‐chain ¹⁵N resonances of Asn63 (^D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, ^D61NS100B, ^D63NS100B, and ^D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca^2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) ^D63NS100B showed no change in Ca^2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca^2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca^2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.     

Alterations in ryanodine receptors and related proteins in heart failure

Sarcoplasmic reticulum (SR) Ca^2 + release plays an essential role in mediating cardiac myocyte contraction. Depolarization of the plasma membrane results in influx of Ca^2 + through l-type Ca^2 + channels (LTCCs) that in turn triggers efflux of Ca^2 + from the SR through ryanodine receptor type-2 channels (RyR2). This process known as Ca^2 +-induced Ca^2 +release (CICR) occurs within the dyadic region, where the adjacent transverse (T)-tubules and SR membranes allow RyR2 clusters to release SR Ca^2 + following Ca^2 + influx through adjacent LTCCs. SR Ca^2 + released during systole binds to troponin-C and initiates actin–myosin cross-bridging, leading to muscle contraction. During diastole, the cytosolic Ca^2 + concentration is restored by the resequestration of Ca^2 + into the SR by SR/ER Ca^2 +-ATPase (SERCA2a) and by the extrusion of Ca^2 + via the Na⁺/Ca^2 +-exchanger (NCX1). This whole process, entitled excitation–contraction (EC) coupling, is highly coordinated and determines the force of contraction, providing a link between the electrical and mechanical activities of cardiac muscle. In response to heart failure (HF), the heart undergoes maladaptive changes that result in depressed intracellular Ca^2 + cycling and decreased SR Ca^2 + concentrations. As a result, the amplitude of CICR is reduced resulting in less force production during EC coupling. In this review, we discuss the specific proteins that alter the regulation of Ca^2 + during HF. In particular, we will focus on defects in RyR2-mediated SR Ca^2 + release. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.     

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO₂ assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100 μmol photons m⁻² s⁻¹ light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100 μmol photons m⁻² s⁻¹ light condition. Under 15 μmol photons m⁻² s⁻¹ light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803.     

In vitro production of bovine embryos in medium supplemented with a serum replacer: Effects on blastocyst development,cryotolerance and survival to term

In this study, we evaluated a serum replacer (SR; Knockout SR^®, Invitrogen) in our in vitro culture systems. We hypothesized that SR would benefit bovine embryo development, since SR supported survival of embryonic stem cells (which originate from embryos). Experiment 1 compared oocyte maturation with SR versus fetal bovine serum (FBS). Following fertilization, blastocyst development was lower for oocytes matured with SR (21.5 versus 34.1, P < 0.05). Experiment 2 evaluated SR for culturing embryos. Following fertilization, embryos were cultured for 3 days in KSOM, and then assigned to treatments: (1) KSOM static culture (KNM); (2) fresh KSOM (KD3); (3) KSOM + SR or (4) KSOM + FBS and cultured to Day 7 (fertilization = Day 0). Blastocyst development in FBS or SR was higher than either KNM or KD3 (48.2, 47.2, 32.7, and 35.5, respectively, P < 0.05). Experiment 3 evaluated cryosurvival of embryos cultured in the same manner as Experiment 2. On Day 7, embryos were vitrified and upon warming, embryos cultured in SR had greater 24 h survival rates (70.6%) than all other treatments (P < 0.05). Finally, Experiment 4 evaluated effects of SR on pregnancy rate and development to term. Culture in SR was not detrimental to pregnancy or calving rates (50 and 50%, respectively), and SR calves had normal birth weights (mean = 38.8 kg ± 1.5). In conclusion, the use of SR for maturation of oocytes was not beneficial; however, SR enhanced embryo culture by improving development in vitro, cryotolerance and survival, effectively replacing serum in culture.     

Effect of CDP-choline on age-dependent modifications of energy- and glutamate-linked enzyme activities in synaptic and non-synaptic mitochondria from rat cerebral cortex

The effect of aging and CDP-choline treatment (20 mg kg⁻¹ body weight i.p. for 28 days) on the maximal rates (V_max) of representative mitochondrial enzyme activities related to Krebs’ cycle (citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic “light” and “heavy” mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months.During aging, enzyme activities vary in a complex way respect to the type of mitochondria, i.e. non-synaptic and intra-synaptic. This micro-heterogeneity is an important factor, because energy-related mitochondrial enzyme catalytic properties cause metabolic modifications of physiopathological significance in cerebral tissue in vivo, also discriminating pre- and post-synaptic sites of action for drugs and affecting tissue responsiveness to noxious stimuli.Results show that CDP-choline in vivo treatment enhances cerebral energy metabolism selectively at 18 months, specifically modifying enzyme catalytic activities in non-synaptic and intra-synaptic “light” mitochondrial sub-populations. This confirms that the observed changes in enzyme catalytic activities during aging reflect the bioenergetic state at each single age and the corresponding energy requirements, further proving that in vivo drug treatment is able to interfere with the neuronal energy metabolism.     

Influence of organic systems on milk fatty acid profile and CLA in goats

The effect of organic system on the fatty acid profile of milk and CLA content was evaluated using 30 pregnant pluriparous goats, divided into two homogeneous groups (S and O) of 15 goats each. Group S was housed in a stable and received alfalfa hay as forage, while group O was raised according EC Regulation 834/2007 and led to pasture. After the kids weaning, goats were milked twice a day for 5 months. Daily milk yield was recorded and, monthly, representative milk samples from the two daily milkings were analysed for chemical and fatty acid profile. Average milk yield did not differ statistically between the groups. The goats of the O group had significantly higher fat content in milk than those of group S (65.9 g/day vs. 54.3 g/day, P < 0.01). Among milk fatty acids, organic system significantly affected the percentages of C18:1 c9, C18:1 t11, linoleic acid, alpha-linolenic acid, monounsaturated fatty acid and polyunsaturated fatty acid. Organic system highly influenced the c9 t11 conjugated linoleic acid (CLA) (0.810 g/100 g of fat vs. 0.542 g/100 g of fat, for groups O and S, respectively, P < 0.01), t10 c12 CLA (0.041 g/100 g of fat vs. 0.024 g/100 g of fat, for groups O and S, respectively, P < 0.01) and ∑CLA (0.87 g/100 g of fat vs. 0.58 g/100 g of fat for groups O and S, respectively, P < 0.01) concentrations of milk.     

Rhizobium indicum sp. nov., isolated from root nodules of pea (Pisum sativum) cultivated in the Indian trans-Himalayas

Three strains of rhizobia isolated from effective root nodules of pea (Pisum sativum L.) collected from the Indian trans-Himalayas were characterized using 16S rRNA, atpD and recA genes. Phylogeny of the 16S rRNA genes revealed that the newly isolated strains were members of the genus Rhizobium with ≥99.9% sequence similarity to the members within the “Rhizobium leguminosarum” group. Phylogenetic analyses based on the concatenated sequences of atpD and recA gene, and 92 core genes extracted from the genome sequences indicated that strains JKLM 12A2^T and JKLM 13E are grouped as a separate clade closely related to R. laguerreae FB206^T. In contrast, the strain JKLM 19E was placed with “R. hidalgonense” FH14^T. Whole-genome average nucleotide identity (ANI) values were 97.6% within strains JKLM 12A2^T and JKLM 13E, and less than 94% with closely related species. The digital DNA-DNA hybridization (dDDH) values were 81.45 within the two strains and less than 54.8% to closely related species. The major cellular fatty acids were C_18:1w7c in summed feature 8, C_{14:0 3OH}/C_{16:1 iso I} in summed feature 2, and C_18:0. The DNA G + C content of JKLM 12A2^T and JKLM 13E was 60.8 mol%. The data on genomic, chemotaxonomic, and phenotypic characteristics indicates that the strains JKLM 12A2^T and JKLM 13E represent a novel species, Rhizobium indicum sp. nov. The type strain is JKLM 12A2^T (= MCC 3961^T = KACC 21380^T = JCM 33658^T). However, the strain JKLM 19E represents a member of “R. hidalgonense” and the symbiovar viciae.     

Survivability of indigenous microflora and a Salmonella typhimurium marker strain in poultry mash treated with buffered propionic acid

Buffered propionic acid (BPA) was evaluated as a potential treatment for the elimination of Salmonella spp. in poultry mash. A primary poultry isolate marker strain of Salmonella typhimurium was added as either a broth or in a dry chalk carrier form to poultry mash containing soybean meal as a protein supplement. The mash was supplemented with buffered propionic acid at 2, 4, 6, 8, 10, 20, 30, 50 and 100 g kg⁻¹ diet and samples were enumerated for indigenous aerobic bacteria, fungi and the S. typhimurium marker strain. Total indigenous aerobic bacteria and fungal populations were generally decreased by addition of more than 20 g BPA kg⁻¹, but an addition of 100 g BPA kg⁻¹ mash was usually required to achieve reductions of approximately 90% of indigenous aerobic bacteria and 99% of indigenous fungi. After 7 days of storage, 8 g BPA kg⁻¹ mash also reduced S. typhimurium populations by more than 90% in mash inoculated via chalk, while at least 50 g BPA kg⁻¹ mash was required to provide the same level of reduction in mash inoculated with a liquid culture of S. typhimurium. Although BPA does not appear to be an overly effective antimicrobial agent with respect to indigenous aerobic bacterial populations in animal feed, higher concentrations may have the potential for reducing fungal and Salmonella spp. contamination in poultry mash.     

Imaging performance of phase-contrast breast computed tomography with synchrotron radiation and a CdTe photon-counting detector

PurposeWithin the SYRMA-CT collaboration based at the ELETTRA synchrotron radiation (SR) facility the authors investigated the imaging performance of the phase-contrast computed tomography (CT) system dedicated to monochromatic in vivo 3D imaging of the female breast, for breast cancer diagnosis.MethodsTest objects were imaged at 38 keV using monochromatic SR and a high-resolution CdTe photon-counting detector. Signal and noise performance were evaluated using modulation transfer function (MTF) and noise power spectrum. The analysis was performed on the images obtained with the application of a phase retrieval algorithm as well as on those obtained without phase retrieval. The contrast to noise ratio (CNR) and the capability of detecting test microcalcification clusters and soft masses were investigated.ResultsFor a voxel size of (60 μm)³, images without phase retrieval showed higher spatial resolution (6.7 mm⁻¹ at 10% MTF) than corresponding images with phase retrieval (2.5 mm⁻¹). Phase retrieval produced a reduction of the noise level and an increase of the CNR by more than one order of magnitude, compared to raw phase-contrast images. Microcalcifications with a diameter down to 130 μm could be detected in both types of images.ConclusionsThe investigation on test objects indicates that breast CT with a monochromatic SR source is technically feasible in terms of spatial resolution, image noise and contrast, for in vivo 3D imaging with a dose comparable to that of two-view mammography. Images obtained with the phase retrieval algorithm showed the best performance in the trade-off between spatial resolution and image noise.     

Heat shock protein-27 attenuates foam cell formation and atherogenesis by down-regulating scavenger receptor-A expression via NF-κB signaling

Previously, we showed an inverse correlation between HSP27 serum levels and experimental atherogenesis in ApoE^?/? mice that over-express HSP27 and speculated that the apparent binding of HSP27 to scavenger receptor-A (SR-A) was of mechanistic importance in attenuating foam cell formation. However, the nature and importance of the interplay between HSP27 and SR-A in atheroprotection remained unclear. Treatment of THP-1 macrophages with recombinant HSP27 (rHSP27) inhibited acLDL binding (? 34%; p < 0.005) and uptake (? 38%, p < 0.05). rHSP27 reduced SR-A mRNA (? 39%, p = 0.02), total protein (? 56%, p = 0.01) and cell surface (? 53%, p < 0.001) expression. The reduction in SR-A expression by rHSP27 was associated with a 4-fold increase in nuclear factor-kappa B (NF-κB) signaling (p < 0.001 versus control), while an inhibitor of NF-κB signaling, BAY11-7082, attenuated the negative effects of rHSP27 on both SR-A expression and lipid uptake. To determine if SR-A is required for HSP27 mediated atheroprotection in vivo, ApoE^?/? and ApoE^?/? SR-A^?/? mice fed with a high fat diet were treated for 3 weeks with rHSP25. Compared to controls, rHSP25 therapy reduced aortic en face and aortic sinus atherosclerotic lesion size in ApoE^?/? mice by 39% and 36% (p < 0.05), respectively, but not in ApoE^?/?SR-A^?/? mice. In conclusion, rHSP27 diminishes SR-A expression, resulting in attenuated foam cell formation in vitro. Regulation of SR-A by HSP27 may involve the participation of NF-κB signaling. Lastly, SR-A is required for HSP27-mediated atheroprotection in vivo.     

Novel insights into cyclooxygenases,linoleate diol synthases,and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs

Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O₂. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n ? 6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n ? 6 and n ? 9 fatty acids and by analysis of D-KIE. COX-1 oxidized C₂₀ and C₁₈ fatty acids in the following order of rates: 20:2n ? 6 > 20:1n ? 6 > 20:3n ? 9 > 20:1n ? 9 and 18:3n ? 3 ≥ 18:2n ? 6 > 18:1n ? 6. 18:2n ? 6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n ? 6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n ? 6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-²H]18:2n ? 6 with similar D-KIE (~ 53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n ? 6 by COX-1 and COX-2 took place with a D-KIE of 3–5 as probed by incubations of [11,11-²H₂]- and [11S-²H]18:2n ? 6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n ? 6 by COX-1 and 18:1n ? 9 by 8R-DOX were both accompanied by large D-KIE (> 20).     

Alteration of plankton communities and biogeochemical cycles by harmful Cochlodinium polykrikoides (Dinophyceae) blooms

Cochlodinium polykrikoides is a globally distributed, ichthyotoxic, bloom-forming dinoflagellate. Blooms of C. polykrikoides manifest themselves as large (many km²) and distinct patches with cell densities exceeding 10³ ml⁻¹ while water adjacent to these patches can have low cell densities (<100 cells ml⁻¹). While the effect of these blooms on fish and shellfish is well-known, their impacts on microbial communities and biogeochemical cycles are poorly understood. Here, we investigated plankton communities and the cycling of carbon, nitrogen, and B-vitamins within blooms of C. polykrikoides and compared them to areas in close proximity (<100 m) with low C. polykrikoides densities. Within blooms, C. polykrikoides represented more than 90% of microplankton (>20 μm) cells, and there were significantly more heterotrophic bacteria and picoeukaryotic phytoplankton but fewer Synechococcus. Terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA genes revealed significant differences in community composition between bloom and non-bloom samples. Inside the bloom patches, concentrations of vitamin B₁₂ were significantly lower while concentrations of dissolved oxygen were significantly higher. Carbon fixation and nitrogen uptake rates were up to ten times higher within C. polykrikoides bloom patches. Ammonium was a more important source of nitrogen, relative to nitrate and urea, for microplankton within bloom patches compared to non-bloom communities. While uptake rates of vitamin B₁ were similar in bloom and non-bloom samples, vitamin B₁₂ was taken up at rates five-fold higher (>100 pmol⁻¹ L⁻¹ d⁻¹) in bloom samples, resulting in turn-over times of hours during blooms. This high vitamin demand likely led to the vitamin B₁₂ limitation of C. polykrikoides observed during nutrient amendment experiments conducted with bloom water. Collectively, this study revealed that C. polykrikoides blooms fundamentally change microbial communities and accelerate the cycling of carbon, some nutrients, and vitamin B₁₂.     

Activity of steroid sulphatase and estrogen sulphotransferase in the boar epididymis during the postpubertal period

The activities of steroid sulphatase (StS) and estrogen sulphotransferase (EST) were determined in the epididymis of 18 boars. The animals were divided into three groups (n = 6) according to age (8, 12 and 16 months). The boars were anesthetized and castrated. The tissue samples of different epididymal parts (caput, corpus and cauda) were taken and homogenized. Activities of StS and EST were assessed using ³H-estrone sulphate (³H-E1S) and free ³H-oestrone (³H-E1) as substrates, respectively. The substrate conversion rates after 60 min of incubation were 51.25% for ³H-E1S and 45.65% for ³H-E1. The activities of both enzymes were significantly higher in the caput epididymis compared to the cauda epididymis (p < 0.05). A significant age-dependent increase of StS and EST activities (p < 0.05) was observed. These results suggest that the availability of estrogens in the boar epididymis may be locally controlled also by StS and EST. The age-dependent increase of StS and EST activities may be related to the process of “biochemical maturation” of the reproductive system during the postpubertal period.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

De novo design of transmembrane helix–helix interactions and measurement of stability in a biological membrane

Membrane proteins regulate a large number of cellular functions, and have great potential as tools for manipulation of biological systems. Developing these tools requires a robust and quantitative understanding of membrane protein folding and interactions within the bilayer. With this in mind, we have designed a series of proteins to probe the net thermodynamic contribution of well-known sequence motifs to transmembrane helix-helix association in a biological membrane. The proteins were designed from first principles (de novo) using current knowledge about membrane insertion and stabilizing interaction motifs. A simple poly-Leu “scaffold” was decorated with individual helix interaction motifs (G-XXX-G, polar residues, heptad repeat) to create transmembrane helix–helix interactions of increasing strength. The GALLEX assay, an in vivo assay for measurement of transmembrane helix self-association, was combined with computational methods to characterize the relative strength and mode of interaction for each sequence. In addition, the apparent free energy contribution (ΔΔG^app) of each motif to transmembrane helix self-association was measured in a biological membrane, results that are the first of their kind for these de novo designed sequences, and suggest that the free energy barrier to overcoming weak association is quite small (< 1.4 kcal mol^− 1) in a natural membrane. By quantifying and rationalizing the contribution of key motifs to transmembrane helix association, our work offers a route to direct the design of novel sequences for use in biotechnology or synthetic biology (e.g. molecular switches) and to predict the effects of sequence modification in known transmembrane domains (for control of cellular processes).     

Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

In this study we evaluated the effects of semen extender supplementation with different concentrations of glutathione (GSH) on buffalo (Bubalus bubalis) bull sperm motility, plasma membrane integrity, viability and DNA integrity as well as in vivo fertility. Semen from three Nili-Ravi buffalo bulls was collected, and qualified semen ejaculates (n = 18) were split into five aliquots for dilution (37 °C; 50 × 10⁶ spermatozoa ml^?1) with experimental tris-citric acid extender containing 0, 0.5, 1.0, 1.5 or 2.0 mM GSH. Extended semen was cooled to 4 °C, equilibrated and filled in French straws. The straws were kept on liquid nitrogen vapors (5 cm above the LN₂ level) for 10 min and plunged in liquid nitrogen for storage. Sperm motility (%), plasma membrane integrity (%), viability (%) and DNA integrity (%) were assessed at 0, 2 and 4 h post-thawing (37 °C). Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0 mM) increased sperm motility, plasma membrane integrity and viability in a dose dependent manner. Sperm DNA integrity was higher (p < 0.05) in all experimental extenders containing GSH when compared to the control extender (0 mM GSH). The in vivo fertility rate of cryopreserved buffalo bull (n = 2) spermatozoa was higher (p < 0.05) in extender containing 2.0 mM GSH compared to that of control. In summary, tris-citric acid extender supplemented with glutathione improved the freezability of buffalo bull spermatozoa in a dose dependant manner. Moreover, the addition of 2.0 mM GSH to the extender enhanced the in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.     

Novel gene variants predict serum levels of the cytokines IL-18 and IL-1ra in older adults

Activation of inflammatory pathways measured by serum inflammatory markers such as interleukin-18 (IL-18) and interleukin-1 receptor antagonist (IL-1ra) is strongly associated with the progression of chronic disease states in older adults. Given that these serum cytokine levels are in part a heritable trait, genetic variation may predict increased serum levels. Using the Cardiovascular Health Study and InCHIANTI cohorts, a genome-wide association study was performed to identify genetic variants that influence IL-18 and IL-1ra serum levels among older adults. Multiple linear regression models characterized the association between each SNP and log-transformed cytokine values. Tests for multiple independent signals within statistically significant loci were performed using haplotype analysis and regression models conditional on lead SNP in each region. Multiple SNPs were associated with these cytokines with genome-wide significance, including SNPs in the IL-18-BCO gene region of chromosome 2 for IL-18 (top SNP rs2250417, P = 1.9 × 10^–32) and in the IL-1 gene family region of chromosome 2 for IL-1ra (rs6743376, P = 2.3 × 10^–26). Haplotype tests and conditional linear regression models showed evidence of multiple independent signals in these regions. Serum IL-18 levels were also associated with a region on chromosome 2 containing the NLRC4 gene (rs12989936, P = 2.7 × 10^–19). These data characterize multiple robust genetic signals that influence IL-18 and IL-1ra cytokine production. In particular, the signal for serum IL-18 located on chromosome two is novel and potentially important in inflammasome triggered chronic activation of inflammation in older adults. Replication in independent cohorts is an important next step, as well as molecular studies to better understand the role of NLRC4.     

Synthesis and preclinical characterization of 1-(6′-deoxy-6′-[18F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-6′-[18F]FAZAL) as a positron emission tomography radiotracer to assess tumor hypoxia

Positron emission tomography (PET) using fluorine-18 (¹⁸F)-labeled 2-nitroimidazole radiotracers has proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios. With the aim to develop a compound that is actively transported into cells, 1-(6′-deoxy-6′-[¹⁸F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-[¹⁸F]1), a putative nucleoside transporter substrate, was synthetized by nucleophilic [¹⁸F]fluoride substitution of an acetyl protected labeling precursor with a tosylate leaving group (β-6) in a final radiochemical yield of 12 ± 8% (n = 10, based on [¹⁸F]fluoride starting activity) in a total synthesis time of 60 min with a specific activity at end of synthesis of 218 ± 58 GBq/μmol (n = 10). Both radiolabeling precursor β-6 and unlabeled reference compound β-1 were prepared in multistep syntheses starting from 1,2:5,6-di-O-isopropylidene-α-d-allofuranose. In vitro experiments demonstrated an interaction of β-1 with SLC29A1 and SLC28A1/2/3 nucleoside transporter as well as hypoxia specific retention of β-[¹⁸F]1 in tumor cell lines. In biodistribution studies in healthy mice β-[¹⁸F]1 showed homogenous tissue distribution and excellent metabolic stability, which was unaffected by tissue oxygenation. PET studies in tumor bearing mice showed tumor-to-muscle ratios of 2.13 ± 0.22 (n = 4) at 2 h after administration of β-[¹⁸F]1. In ex vivo autoradiography experiments β-[¹⁸F]1 distribution closely matched staining with the hypoxia marker pimonidazole. In conclusion, β-[¹⁸F]1 shows potential as PET hypoxia radiotracer which merits further investigation.