Empirical Selection of Informative Microsatellite Markers within Co-ancestry Pig Populations Is Required for Improving the Individual Assignment Efficiency.

The Lanyu is a miniature pig breed indigenous to Lanyu Island, Taiwan. It is distantly related to Asian and European pig breeds. It has been inbred to generate two breeds and crossed with Landrace and Duroc to produce two hybrids for laboratory use. Selecting sets of informative genetic markers to track the genetic qualities of laboratory animals and stud stock is an important function of genetic databases. For more than two decades, Lanyu derived breeds of common ancestry and crossbreeds have been used to examine the effectiveness of genetic marker selection and optimal approaches for individual assignment. In this paper, these pigs and the following breeds: Berkshire, Duroc, Landrace and Yorkshire, Meishan and Taoyuan, TLRI Black Pig No. 1, and Kaohsiung Animal Propagation Station Black pig are studied to build a genetic reference database. Nineteen microsatellite markers (loci) provide information on genetic variation and differentiation among studied breeds. High differentiation index (FST) and Cavalli-Sforza chord distances give genetic differentiation among breeds, including Lanyu's inbred populations. Inbreeding values (FIS) show that Lanyu and its derived inbred breeds have significant loss of heterozygosity. Individual assignment testing of 352 animals was done with different numbers of microsatellite markers in this study. The testing assigned 99% of the animals successfully into their correct reference populations based on 9 to 14 markers ranking D-scores, allelic number, expected heterozygosity (HE) or FST, respectively. All miss-assigned individuals came from close lineage Lanyu breeds. To improve individual assignment among close lineage breeds, microsatellite markers selected from Lanyu populations with high polymorphic, heterozygosity, FST and D-scores were used. Only 6 to 8 markers ranking HE, FST or allelic number were required to obtain 99% assignment accuracy. This result suggests empirical examination of assignment-error rates is required if discernible levels of co-ancestry exist. In the reference group, optimum assignment accuracy was achievable achieved through a combination of different markers by ranking the heterozygosity, FST and allelic number of close lineage populations.

Complexity in biological signaling systems.

Biological signaling pathways interact with one another to form complex networks. Complexity arises from the large number of components, many with isoforms that have partially overlapping functions; from the connections among components; and from the spatial relationship between components. The origins of the complex behavior of signaling networks and analytical approaches to deal with the emergent complexity are discussed here.

Resolution of a signal transfer region from a general binding domain in gbeta for stimulation of phospholipase C-beta2.

Signaling by guanine nucleotide-binding proteins (G proteins) involves sequential protein-protein interactions. G protein-betagamma subunit (Gbetagamma) interactions with phospholipase C-beta2 (PLC-beta2) were studied to determine if all Gbeta contacts are required for signaling. A peptide encoding Gbeta amino acid residues 86 to 105 stimulated PLC-beta2. Six residues (96 to 101) within this sequence could transfer signals and thus constitute a core signal transfer region. Another peptide, encoding Gbeta amino acid residues 115 to 135, did not substantially stimulate PLC-beta2 by itself but inhibited Gbetagamma stimulation, indicating that residues 115 to 135 constitute a general binding domain. Resolution of signal transfer regions from general binding domains indicates that all protein-protein contacts are not required for signal transfer and that it may be feasible to synthesize agonists and antagonists that regulate intracellular signal flow.

Molecular basis for interactions of G protein betagamma subunits with effectors.

Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.

Comparison of elderly- and young-onset rheumatoid arthritis in an Asian cohort.

OBJECTIVE: To describe the demographic characteristics, clinical features, functional status and quality of life of elderly-onset (EORA) and young-onset (YORA) rheumatoid arthritis (RA) patients in an Asian cohort. METHODS: We studied all RA patients in our prospective disease registry, utilizing baseline data. EORA was defined as disease onset at 60 years or older. We collected data from January 2001 to December 2012. RESULTS: There were 1206 patients in our cohort, of which 178 (14.8%) had EORA, with a mean age of onset of 66.7 ± 5.6 years. There were more males in the EORA than YORA group (23.0% vs. 14.7%, P = 0.005). EORA patients were diagnosed sooner after symptom onset and had a higher number of comorbidities (median 2 [inter-quartile range 1-3] vs. 1 (0-2), P < 0.001). They were less likely to be rheumatoid factor positive, had higher erythrocyte sedimentation rate values and lower hemoglobin concentrations. There was no significant difference in joint counts, Disease Activity Score of 28 joints activity score and prevalence of radiographic erosions. Though EORA patients had worse Health Assessment Questionnaire scores and poorer functional status than YORA ones, they had lower pain scores and higher scores in the general health and mental component summary of the Short Form-36. EORA patients received significantly lower numbers of disease-modifying anti-rheumatic drugs. CONCLUSIONS: EORA and YORA patients had different demographic characteristics. Although they had similar disease activities, EORA patients received less intensive treatment. EORA patients had a higher number of RA-related co-morbidities and poorer physical functioning but they coped better emotionally and mentally.

A genetic evaluation of seed dispersal in the neotropical tree Jacaranda copaia (Bignoniaceae).

Seed dispersal is a critical but poorly understood life-history stage of plants. Here we use a genetic approach to describe seed dispersal patterns accurately in a natural population of the Neotropical tree species Jacaranda copaia (Bignoniaceae). We used microsatellite genotypes from maternally derived tissue on the diaspore to identify which individual of all possible adult trees in the population was the true source of a given seed collected after it dispersed. Wind-dispersed seeds were captured in two different years in a large array of seed traps in an 84-ha mapped area of tropical forest on Barro Colorado Island, Panama. We were particularly interested in the proportion of seeds that traveled long distances and whether there was evidence for direct dispersal into gaps, which are required for successful recruitment of this pioneer tree species. Maximum likelihood procedures were used to fit single- and multiple-component dispersal kernels to the distance data. Mixture models, with separate distributions near and far, best fit the observed dispersal distances, albeit with considerable uncertainty in the tail. We discuss the results in light of different mechanisms responsible for separate distributions near the adult source and in the tail of the curve.

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)

p21ras supports the survival of chick embryonic motor neurones.

Various trophic factors present in muscle extract can promote the survival of cultured motor neurones. However, little is known about the signal transduction pathways used in these cells. The proto-oncogene product p21ras has been shown to play an important role in proliferative and differentiative signalling pathways. We report here that cytoplasmic introduction of its oncogenic form, p21ras(G12V), fully supports the in vitro survival of chick embryonic motor neurones. The proto-oncogenic form of p21ras also showed a dose-dependent survival effect, while a C-terminally truncated counterpart of p21ras(G12V) was ineffective. These results suggest an involvement of p21ras in signal transduction pathways leading to motor neurone survival and may be of relevance for the development of therapeutic strategies for motor neurone disease.

Neuropeptide Y receptor gene regulation in mouse adrenocortical Y-1 cells.

The mouse adrenocortical Y-1 cell line expresses a high level of neuropeptide Y1 receptor (NPY-Y1). Moreover the receptor density can be up-regulated by dexamethasone or down-regulated by cAMP. To determine whether such regulation occurs at the level of gene expression, Y1 receptor mRNA was measured using a reverse transcriptase-competitive PCR method. Dexamethasone treatment increased Y1 mRNA in Y-1 cells, whereas the cAMP and ACTH decreased it. We also observed that the amount of Y1 receptor RNA was unaffected by phorbol 12-myristate 13-acetate, a protein kinase C stimulator, but was abolished in a cell line expressing apolipoprotein E (apoE). The results indicated that NPY-Y1 receptor mRNA in Y-1 cells is highly regulated by several intracellular messengers. The role of apoE in such regulation is of particular interest in view of evidence that the isoform of the molecule is highly correlated to the age of onset of Alzheimer's disease. The effect observed in the Y-1 cell line which expresses apoE may implicate a possible role of this protein in the process of neuronal death that occurred in the Alzheimer's disease.

A proposed bovine neuropeptide Y (NPY) receptor cDNA clone, or its human homologue, confers neither NPY binding sites nor NPY responsiveness on transfected cells.

Receptors with seven transmembrane domains (7TM) constitute a large family of structurally and functionally related proteins which respond to various types of ligands. We describe here the cloning and expression of a human 7TM receptor, denoted hFB22 (human Fetal Brain 22), which is the homologue (92% amino acid identity) of a bovine receptor (LCR1) reported by others to bind neuropeptide Y (NPY) with a pharmacological profile of the Y3 receptor subtype. However, upon expression in COS1 (confirmed by Northern analysis), COS7 or CHO-K1 cells, the hFB22 receptor did not confer specific 125I-Bolton-Hunter-NPY, 3H-propionyl-NPY or 125I-peptide YY (PYY) binding sites, in either intact cells or in membrane preparations. Similarly, cells transfected with the corresponding bovine clone (LCR1) did not show specific NPY/PYY binding exceeding that resulting from endogenous binding sites; mock-transfected COS7 cells, used frequently for heterologous expression of receptors, were found to have endogenous specific 125I-NPY binding sites (Bmax = 112 fmol/mg protein; Kd = 0.25 nM). Moreover, the hFB22 transfected cells, when compared to control transfected cells, did not display de novo NPY- or PYY-induced second messenger responses, i.e., (1) inhibition of forskolin-stimulated cAMP accumulation or (2) 45Ca2+ influx. The presence of hFB22 mRNA was detected in several human neuroblastoma cell lines, none of which was found to express Y3-like NPY binding sites. hFB22 displays 39% amino acid sequence identity (in the transmembrane regions) to the human interleukin-8 receptor, and 32-36% amino acid identity to the human receptors of angiotensin II, bradykinin, and n-formylpeptide, but only 23% amino acid identity to the previously described human NPY/PYY receptor of the Y1 receptor subtype. Our results show that hFB22 and LCR1 do not encode NPY receptors, and their true ligand(s) remains to be identified.

A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores.

The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.

[The preparation and stability of liposome-encapsulated Ara-A].

This paper uses several preparation methods, such as thin-film, reverse-phase evaporation and freeze-thawing methods, and also compares encapsulation percentage of Ara-A in liposome (EN%) of these methods. The best operational conditions of preparing liposome-encapsulated Ara-A are explored. A higher EN%, about 50%, which is ten times that reported in foreign literature, is obtained by using improved freeze-thawing method, and this method is easy to operate and repeatable. At the same time, the physical and chemical stabilities of the liposome-encapsulated Ara-A were observed. The result shows that there are no distinct changes in shape, size distribution of liposome, and EN% as well as Ara-A contained in liposome by means of sterilization at 100 degrees C for 30 minutes. Accelerating test at a constant temperature indicates that liposome-entrapped Ara-A has certain chemical stability.

[Preparation of oral berberine bisulfate liposomes].

The co-precipitate has been made from berberine bisulfate and polyvingyl pyrrolidone (PVP) with the ratio of 1 : 5. The liposomes entrapped berberine bisulfate have been prepared with the ration of drug to lipid 1 : 20 and the ratio of cholesterol to soy phosphatide 2 : 7. The percentage of encapsulation is 48.72%. The test in vivo of small intestinal absorption in rats shows that liposomes can increase the absorption by 4-fold odd compared with free ber-berine bisulfate.

Stimulating ß-cell replication and improving islet graft function by AR231453, A GPR119 agonist.

OBJECTIVE: G protein-coupled receptor 119 (GPR119) is predominantly expressed in ß cells and intestinal L cells. AR231453 is a selective small-molecular GPR119 agonist that enhances glucose-dependent insulin secretion and glucagon-like peptide 1 (GLP-1) release. We investigated whether AR231453 can directly stimulate ß-cell replication and improve islet graft function in diabetic mice. METHODS: A total of 100 syngenic C57BL/6 mouse islets were transplanted under the left kidney of each chemically induced diabetic C57BL/6 mouse. Starting from the day of transplantation, these recipients were given bromodeoxyuridine (BrdU) daily with or without AR231453 at 10 mg/kg/d. Islet graft function was monitored by measuring blood glucose levels. At 4 weeks, left nephrectomy was performed to remove the kidney bearing the islet grafts to determine ß-cell replication in the islet grafts. Insulin and BrdU immunofluorescence staining was performed to detect replicated ß cells. Insulin(+) and BrdU(+) ß cells in islet grafts were counted using a confocal microscope. To determine whether AR231453 increases plasma GLP-1 levels, we collected plasma from AR231453 treated mice at 30 minutes after treatment and measured plasma active GLP-1 by enzyme-linked immunosorbent assay. RESULTS: Although all recipient mice achieved normoglycemia at 28 days with or without treatment, normoglycemia was achieved in significantly fewer days in AR231453-treated mice. The vehicle-treated mice achieved normoglycemia in 16 ± 6 days, while AR231453-treated mice only required only 8 ± 3 days (P < .01). The percentage of insulin(+) and BrdU(+) ß cells in islet grafts was significantly higher in AR231453-treated mice than in vehicle-treated mice. The mean percentage of insulin(+) and BrdU(+) ß cells in islet grafts was 21.5% ± 6.9% in AR231453-treated mice and 5.6% ± 3.7% in vehicle-treated mice (P < .01). The plasma active GLP-1 levels were also significantly higher in AR231453-treated mice than in vehicle-treated mice (P < .05). CONCLUSION: Our data demonstrate that AR231453, a GPR119 agonist, can stimulate ß-cell replication and improve islet graft function.

[The advancement of G protein and coupled signal transduction pathways].

G protein coupled signal transduction system is one major transmembrane signaling pathways in cell. Research of biochemistry and pharmacology in medicine showed that a lot of medicines play their role through G protein coupled signaling pathways. Thus, systematic study on G protein structure and functions had very important effects on new medicine producing. In the process of creature evolution, there are highly conserved amino-acid sequences in G protein. Study on G protein and coupled signal components in plants and insects signaling processes may benefit to determine the mechanism of crop resistance to disease and pest insect and resistant chemicals effect on insect behavior and physiology. This paper reviews the research progress and prospect of practical use in recent studies of G protein and coupled components in animal, insect and plant signaling pathways.