Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.     

Clonorchis sinensis: molecular cloning and localization of myosin regulatory light chain

One cDNA clone was purified from an adult Clonorchis sinensis cDNA library, and its deduced polypeptide sequence was found to be homologous with myosin regulatory light chain (MRLC) of invertebrates and vertebrates. Two amino-acid residues, Thr and Ser, were conserved at the phosphorylation sites that regulate the function of MRLCs. Recombinant C. sinensis MRLC (rCsMRLC) protein was produced and purified from Escherichia coli, and mouse anti-CsMRLC immune sera recognized a protein of molecular weight 24 kDa from a soluble protein preparation of C. sinensis. The CsMRLC protein was immunohistochemically localized to the muscle fibers of the subtegumental muscle layer and to the muscles of oral and ventral suckers. However, the rCsMRLC protein proved to be less useful antigen for the serodiagnosis of human clonorchiasis.The nucleotide sequence reported herein was submitted to GenBank and assigned accession number AY519356.     

Effects of topography and composition of titanium surface oxides on osteoblast responses

To investigate the roles of composition and characteristics of titanium surface oxides in cellular behaviour of osteoblasts, the surface oxides of titanium were modified in composition and topography by anodic oxidation in two kinds of electrolytes, (a) 0.2 M H(3)PO(4), and (b) 0.03 M calcium glycerophosphate (Ca-GP) and 0.15 M calcium acetate (CA), respectively. Phosphorus (P: ca.10at%) or both calcium (Ca: 1-6at%) and phosphorus (P: 3-6at%) were incorporated into the anodized surfaces in the form of phosphate and calcium phosphate. Surface roughness was slightly decreased or enhanced (R(a) in the range of 0.1-0.5 microm) on the anodized surfaces. The geometry of the micro-pores in the anodized surfaces varied with diameters up to 0.5 microm in 0.2 M H(3)PO(4) and to 2 microm in 0.03 M Ca-GP and 0.15 M CA, depending on voltages and electrolyte. Contact angles of all the anodic oxides were in the range of 60-90 degrees. Cell culture experiments demonstrated absence of cytotoxicity and an increase of osteoblast adhesion and proliferation by the anodic oxides. Cells on the surfaces with micro-pores showed an irregular and polygonal growth and more lamellipodia, while osteoblasts on the titanium surface used as a control or on anodic oxides formed at low voltages showed many thick stress fibres and intense focal contacts. Alkaline phosphatase (ALP) activity of the cells did not show any correlation with surface characteristics of anodic oxides.     

Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells

Toll-like receptors (TLR) are sentinel receptors capable of recognizing pathogen-associated molecule patterns (PAMP) such as lipopolysaccharide (LPS) and CpG-containing oligonucleotides (CpG ODN). TLR2 and TLR4 are major receptors for Gram-positive and Gram-negative bacterial cell wall components, respectively. TLR9 is necessary for CpG signalling. LPS or CpG ODN can activate immature dendritic cells (DC) and induce DC maturation characterized by production of cytokines, up-regulation of co-stimulatory molecules, and increased ability to activate T cells. However, little is known regarding the regulation of TLR gene expression in mouse DC. In this study, we investigated the regulation of TLR2, TLR4 and TLR9 gene expression by LPS in murine immature DC. TLR2, TLR4 and TLR9 mRNA were up-regulated following LPS stimulation. The up-regulation of TLR9 expression coincided with significantly increased production of tumour necrosis factor-alpha induced by LPS plus CpG ODN. While inhibition of extracellular signal-related kinase and NF-kappaB activation suppressed the up-regulation of the expression of TLR2, TLR4 and TLR9 mRNA, inhibition of p38 kinase prevented the up-regulation of TLR2 and TLR4 mRNA expression but enhanced the up-regulation of TLR9 expression. These results demonstrated that TLR2, TLR4 and TLR9 gene expression was differently regulated by LPS in mouse immature DC. Up-regulation of TLR2, TLR4 and TLR9 expression by LPS might promote the overall responses of DC to bacteria and help to explain the synergy between LPS and other bacterial products in the induction of cytokine production.     

Frozen-section services by telepathology: Experience of 100 cases in the San-in District, Japan

The early experience is reported here of the use of Intra-operative frozen-section service by telepathology using the Integrated Service Digital Network (ISDN), a commercially available system that is being connected between the Department of Pathology of Tottori University and Matsue City Hospital, a distance of 30 km. The transfer rate is currently 64kbit/s. The frozen-section service was conducted for a total of 117 tissue specimens (organs) from 100 patients between August 1993 and May 1995. The average time taken for examination of each specimen of frozen section was 13min, ranging between 2 and 42min. The average number of transmitted Images was 6.2. Six cases necessitated more than 11 transmitted Images to make a diagnosis, while 13 cases could be diagnosed from two images only. Correct and permissible diagnoses were obtained in 109 (93.2%) out of 117 specimens when comparing the telepathology diagnosis with that of direct microscopy. Improper or misdiag-nosis was made for eight cases (specimens), which were misinterpreted as papillary carcinoma in Basedow's disease, adenoma and hyperplasia in two pheochromocytomas, solid-tubular carcinoma in phyilodes tumor, mastopathy in invasive carcinoma, metastatic carcinoma in astrocytoma, follicular lymphoma in reactive hyperplasia, and lymphadenitis in follicular lymphoma. in retrospect, diagnosis of these cases should have been deferred. From the results, it was concluded that the Intraoperatlve frozen-section service by telepathology may be a worthwhile substitute for hospitals with limited accessibility to local pathology service, in spite of pitfalls in some cases. Well prepared, high-quality frozen sections, sufficient verbal communication with surgeons, and a rather conservative attitude on the part of a well-trained pathologist seem to be the essential Ingredients for reaching an accurate decision when using telepathology.     

The incorporation of iodine in thyroid hormone may stem from its role as a prehistoric signal of ecologic opportunity: an evolutionary perspective and implications for modern diseases

To optimize fitness under conditions of varying Darwinian opportunity, organisms demonstrate tremendous plasticity in their life-history strategies based on their perception of available resources. Higher-energy environments generally promote more aggressive life-history strategies, such as faster growth, larger adult size, greater genetic variation, shorter lifespan, larger brood sizes, and offspring ratio skewed towards the larger-sized gender. While numerous mechanisms regulate life-history plasticity including genetic imprinting, methylation, and growth factors, evidence suggests that thyroid hormone plays a central role. Given the pivotal adaptive role of thyroid hormone, the teleology of its dependence on dietary iodine for production remains unexplained. We hypothesize that iodine may have emerged as a substrate for production of thyroid hormone in prehistoric ecosystems because the former represented a reliable proxy for ecologic potential that enabled the latter to modulate growth, reproduction, metabolic rate, and lifespan. Such a scenario may have existed in early marine ecosystems where ocean-surface vegetation, which concentrates iodine for its antimicrobial and antioxidant properties, formed the basis of the food chain. Teleologic parallels can be drawn to the food-chain accumulation of antimicrobials that also exhibit antioxidant properties and promote adult size, brood size, and offspring quality by modulating central hormonal axes. As each higher species in the food chain tunes its life-history strategy based on iodine intake, the coupling of this functional role of iodine with its value as a resource signal to the next member of the food-chain may promote runaway evolution. Whereas predators in prehistoric ecosystems successfully tuned their life-history strategy using iodine as a major input, the strategy may prove maladaptive in modern humans for whom the pattern of iodine intake is decoupled from resource availability. Iodine acquired through sodium iodide supplementation may independently contribute to some biologic dysfunctions currently attributed to sodium.     

涎腺癌肉瘤临床及病理分析

目的：探讨涎腺癌肉瘤的临床病理学特点及其鉴别诊断。方法：对3例涎腺癌肉瘤患者的临床资料进行回顾性研究并复习相关文献，对全部病例的组织学标本重新进行镜下观察。结果：涎腺癌肉瘤临床表现常为迅速增大的颜面部肿物并伴疼豢。光匀下组织学观察常可见肉瘤和癌两种成分并存，癌多为鳞状细胞癌和腺癌，肉瘤以骨或软骨肉瘤为主。结论：涎腺癌肉瘤的临床特点与涎腺其他恶性肿瘤较难区别，但涎腺癌肉瘤的恶性程度极高。     

Single nucleotide variation analysis in 65 candidate genes for CNS disorders in a representative sample of the European population

The detailed investigation of variation in functionally important regions of the human genome is expected to promote understanding of genetically complex diseases. We resequenced 65 candidate genes for CNS disorders in an average of 85 European individuals. The minor allele frequency (MAF), an indicator of weak purifying selection, was lowest in radical amino acid alterations, whereas similar MAF was observed for synonymous variants and conservative amino acid alterations. In noncoding sequences, variants located in CpG islands tended to have a lower MAF than those outside CpG islands. The transition/transversion ratio was increased among both synonymous and conservative variants compared with noncoding variants. Conversely, the transition/transversion ratio was lowest among radical amino acid alterations. Furthermore, among nonsynonymous variants, transversions displayed lower MAF than did transitions. This suggests that transversions are associated with functionally important amino acid alterations. By comparing our data with public SNP databases, we found that variants with lower allele frequency are underrepresented in these databases. Therefore, radical variants obtain distinctively lower database coverage. However, those variants appear to be under weak purifying selection and thus could play a role in the etiology of genetically complex diseases.     

Neurons in the rostral cingulate motor area monitor multiple phases of visuomotor behavior with modest parametric selectivity

We examined the cellular activity in the rostral cingulate motor area (CMAr) with respect to multiple behavioral factors that ranged from the retrieval and processing of associative visual signals to the planning and execution of instructed actions. We analyzed the neuronal activity in monkeys while they performed a behavioral task in which 2 visual instruction cues were given successively with an intervening delay. One cue instructed the location of the target to be reached; the other cue instructed which arm was to be used. After a second delay, the monkey received a motor-set cue to be prepared to initiate the motor task in accordance with instructions. Finally, after a GO signal, the monkey reached for the instructed target with the instructed arm. We found that the activity of neurons in the CMAr changed profoundly throughout the behavioral task, which suggested that the CMAr participated in each of the behavioral processing steps. However, the neuronal activity was only modestly selective for the spatial location of the visual signal. We also found that selectivity for the instructional information delivered with the signals (target location and arm use) was modest. Furthermore, during the motor-set and movement periods, few CMAr neurons exhibited selectivity for such motor parameters as the location of the target or the arm to be used. The abundance and robustness of the neuronal activity within the CMAr that reflected each step of the behavioral task and the modest selectivity of the same cells for sensorimotor parameters are strikingly different from the preponderance of selectivity that we have observed in other frontal areas. Based on these results, we propose that the CMAr participates in monitoring individual behavioral events to keep track of the progress of required behavioral tasks. On the other hand, CMAr activity during motor planning may reflect the emergence of a general intention for action.     

Two dinucleotide repeat polymorphisms at the D8S1442 and D8S1443 loci

Two polymorphic dinucleotide (CA) repeat clones were isolated from cosmids, cCI8-1121 and cCI8-1199, mapped to chromosome 8p11.2-p12.