Effect of aging on bolus kinematics during the pharyngeal phase of swallowing

Swallowing difficulty is a common complaint in the elderly and, although there are data for the biomechanics of liquid swallows, little is known about solid bolus motion, or kinematics, in the elderly. The aims of this study were as follows: 1) to characterize and compare solid and liquid bolus kinematics in the elderly and compare the findings with those in young subjects and 2) to correlate bolus kinematics and dynamics. Concurrent manometric-fluoroscopic techniques were used to study eight young and eight elderly subjects. The subjects performed four swallows each of 0.2-cm-diameter solid barium pellets and 5 ml of liquid barium during sagittal fluoroscopy and six-channel pharyngoesophageal manometry. Images were digitized for analysis of kinematic properties such as velocity and acceleration. Dynamic pressures were recorded and coordinated with kinematic events. Image analysis showed that velocity varied as the pellet passed through the hypopharynx, pharynx, and upper esophageal sphincter. In young subjects, pellet kinematics were characterized by two zones of pellet acceleration: one over the tongue base and another as the pellet passed through the upper esophageal sphincter. Although the elderly showed a similar zone of acceleration over the base of the tongue, the second zone of pellet acceleration was not seen. Decreasing pressure gradients immediately distal to the position of the solid pellet and liquid bolus characterized dynamics for all subjects. This decreasing pressure gradient was significantly larger in elderly than in young subjects. Bolus kinematics and dynamics were significantly altered among elderly compared with young subjects. Among these differences were the absence of hypopharyngeal bolus acceleration and a significant increase in the trans-sphincteric pressure gradient in the elderly.     

Signal transfer from GPCRs to G proteins: role of the G alpha N-terminal region in rhodopsin-transducin coupling

Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Galpha N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R(*)) interaction. The N-terminal fatty acylation of Galpha is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Galpha. In Galphabetagamma, lack of the fatty acid and Galpha truncations up to 33 amino acids had little effect on R(*) binding and R(*)-catalyzed nucleotide exchange, implying that this region is not mandatory for R(*)/Galphabetagamma interaction. However, when the other hydrophobic modification of Galphabetagamma, the Ggamma C-terminal farnesyl moiety, is lacking, R(*) interaction requires the fatty acylated Galpha N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Galphabetagamma with R(*). We propose that in native Galphabetagamma, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R(*). Accordingly, we find that the native fatty acylated Galpha is competent to interact with R(*) even in the absence of Gbetagamma, whereas nonacylated Galpha requires Gbetagamma for interaction. Experiments with N-terminally truncated Galpha subunits suggest that in the second step of the catalytic process, the receptor binds to the alphaN/beta1-loop region of Galpha to reduce nucleotide affinity and to make the Galpha C terminus available for subsequent interaction with R(*).     

Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology

Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase gamma. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.     

Retinol dehydrogenase (RDH12) protects photoreceptors from light-induced degeneration in mice

RDH12 has been suggested to be one of the retinol dehydrogenases (RDH) involved in the vitamin A recycling system (visual cycle) in the eye. Loss of function mutations in the RDH12 gene were recently reported to be associated with autosomal recessive childhood-onset severe retinal dystrophy. Here we show that RDH12 localizes to the photoreceptor inner segments and that deletion of this gene in mice slows the kinetics of all-trans-retinal reduction, delaying dark adaptation. However, accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor apoptosis were also observed in Rdh12(-/-) mice, suggesting that RDH12 plays a unique, nonredundant role in the photoreceptor inner segments to regulate the flow of retinoids in the eye. Thus, severe visual impairments of individuals with null mutations in RDH12 may likely be caused by light damage(1).     

Cytohesin binder and regulator (cybr) is not essential for T- and dendritic-cell activation and differentiation

Cybr (also known as Cytip, CASP, and PSCDBP) is an interleukin-12-induced gene expressed exclusively in hematopoietic cells and tissues that associates with Arf guanine nucleotide exchange factors known as cytohesins. Cybr levels are dynamically regulated during T-cell development in the thymus and upon activation of peripheral T cells. In addition, Cybr is induced in activated dendritic cells and has been reported to regulate dendritic cell (DC)-T-cell adhesion. Here we report the generation and characterization of Cybr-deficient mice. Despite the selective expression in hematopoietic cells, there was no intrinsic defect in T- or B-cell development or function in Cybr-deficient mice. The adoptive transfer of Cybr-deficient DCs showed that they migrated efficiently and stimulated proliferation and cytokine production by T cells in vivo. However, competitive stem cell repopulation experiments showed a defect in the abilities of Cybr-deficient T cells to develop in the presence of wild-type precursors. These data suggest that Cybr is not absolutely required for hematopoietic cell development or function, but stem cells lacking Cybr are at a developmental disadvantage compared to wild-type cells. Collectively, these data demonstrate that despite its selective expression in hematopoietic cells, the role of Cybr is limited or largely redundant. Previous in vitro studies using overexpression or short interfering RNA inhibition of the levels of Cybr protein appear to have overestimated its immunological role.     

Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood

BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSC/HPC) for the reconstitution of the hematopoietic system after clinical transplantation. Cryopreservation of these cells is critical for UCB banking and transplantation as well as for research applications by providing readily available specimens. The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB. METHODS: Cryopreservation of CD34+ HSC/HPC from UCB after mononuclear cell (MNC) preparation was tested in a research-scale setup. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs. In addition, sterility testing of the collected UCB units before further processing was performed. RESULTS: The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5 x 10(7) MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed. Microbial contamination of the collected UCB samples was reduced to a rate of 6.4%. DISCUSSION: Optimized cryopreservation conditions were developed for UCB MNC in respect of the composition of the cryosolution. In addition, our results showed that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.     

Nestin-Cre transgenic mouse line Nes-Cre1 mediates highly efficient Cre/loxP mediated recombination in the nervous system, kidney, and somite-derived tissues

Here we describe the generation of the Nes-Cre1 transgenic mouse line in which Cre recombinase expression is controlled by the rat nestin promoter and intron 2 enhancer. This line has previously been used for conditional loss-of-function studies of various genes in the central nervous system and first branchial arch ectoderm. Here we report the detailed temporal and spatial recombination pattern of Nes-Cre1 using three different reporters of Cre-mediated recombination, ROSA26R (R26R), Z/AP, and Z/EG. Cre/loxP recombination was detected in embryos as early as the head-fold stage. By embryonic day (E)15.5 recombination occurred in virtually all cells of the nervous system and unexpectedly also in somite-derived tissues and kidneys. Tissues with little or no recombination included heart, liver, thymus, and lung. This study suggests that Nes-Cre1-mediated recombination occurs in progenitor cell types present in the neuroectoderm, the developing mesonephros, and the somites.     

鼠类种群动态:食物资源影响力的实验评估

本研究在美国伊利诺斯州中东部测定了中等食物质量(莓系属的牧草)和低食物质量(高杆草牧场)栖息地内橙腹田鼠(Microtusochrogaster)和草原田鼠(M.pennsylvanicus)种群对附加食物的响应。在中等食物质量栖息地内投放附加食物,橙腹田鼠种群的波动幅度高于对照样地,但同类型食物质量栖息地内,种群平均密度和波动模式无显著的差异。仅在低等食物质量的高杆草栖息地内,投放附加食物可导致草原田鼠的种群密度高于对照样地。附加食物不直接影响两种栖息地内橙腹田鼠的成活率或成熟率,在中等食物质量栖息地中投放附加食物,繁殖活跃的成熟雌性个体比例及雄性成体的体重高于对照。附加食物不影响莓系属牧草内草原田鼠,也不能影响该动物在高杆草环境中的存活率。然而,投放附加食物,可缩短低等食物质量栖息地内草原田鼠的成熟期,提高繁殖活跃雌性的比例和迁入个体比例,增加并引起雄性个体体重增加。据此,本研究证明食物资源在橙腹田鼠和草原田鼠种群动态中只起极小的作用。     

The crystal structure of Arabidopsis thaliana allene oxide cyclase: insights into the oxylipin cyclization reaction

We describe the crystallization and structure elucidation of Arabidopsis thaliana allene oxide cyclase 2 (AOC2), a key enzyme in the biosynthesis of jasmonates. In a coupled reaction with allene oxide synthase, AOC2 releases the first cyclic and biologically active metabolite, 12-oxo-phytodienoic acid (OPDA). AOC2 (AT3G25770) folds into an eight-stranded antiparallel beta-barrel with a C-terminal partial helical extension. The protein forms a hydrophobic binding cavity with two distinct polar patches. AOC2 is trimeric in crystals, in vitro and in planta. Based on the observed folding pattern, we assigned AOC2 as a low molecular weight member of the lipocalin family with enzymatic activity in plants. We determined the binding position of the competitive inhibitor vernolic acid (a substrate analog) in the binding pocket. Based on models for bound substrate 12,13-epoxy-9,11,15-octadecatrienoic acid and product OPDA, we propose a reaction scheme that explains the influence of the C15 double bond on reactivity. Reaction is promoted by anchimeric assistance through a conserved Glu residue. The transition state with a pentadienyl carbocation and an oxyanion is stabilized by a strongly bound water molecule and favorable pi-pi interactions with aromatic residues in the cavity. Stereoselectivity results from steric restrictions to the necessary substrate isomerizations imposed by the protein.