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.     

Comprehensive family practice clerkship in a minority institution

A comprehensive family practice clerkship program at Howard University College of Medicine has been conducted since 1970. This institution is one of three predominantly black institutions offering a family practice program. The senior clerkship is mandatory and at least 20 to 25 percent of each class elect to participate in a four-to six- week family practice preceptorship. As a result of the clerkship''s success, over 50 percent of the program''s graduates actively practice in primary medical manpower shortage or medically underserved areas.     

Analysis of pressure waves as a mean of diagnosing vascular obstructions

A feasibility study was made to examine whether pressure measurements can be used to diagnose vascular obstructions in blood vessels. Distortion of a pressure wave due to an obstruction in an elastic tube was investigated theoretically and experimentally. Linear theory and the method of characteristics were employed in developing mathematical expressions for the distortion of the pressure wave. The quality of the models developed was examined by performing experiments on a latex tube with rigid obstructions. A nonlinear model using the method of characteristics was in good agreement with the experiment data for obstructions with any severity, while a linear model was applicable to small obstructions. The nonlinear model is proposed as a mathematical model for the detection of vascular obstructions by analysing pressure waves.     

Experimental infection of pigs with aTaenia species from Korea: parasitological and serological aspects

Belgian Landrace piglets were experimentally infected with eggs of aTaenia sp. of Korean origin. At autopsy, metacestodes were present only in the livers. The proportion of degenerated metacestodes increased from 12%–39% at 5 weeks to 94%–100% at 10 weeks after infection. A sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies raised against the excretory-secretory products ofT. saginata metacestodes detected circulating antigen in the sera of the pigs at 1 week post-infection. A good correlation was found between the presence of viable metacestodes and the detection of circulating antigen; the latter disappeared as the metacestodes died off. However, the antibodies were detected only after 3 weeks of infection and onwards until the necropsy of the pigs.Financial support for this study was provided by the Institute for Scientific Research in Industries and Agriculture (IWONL, Brussels)     

Pathogenesis and Autointerference in a Virus Disease of Crabs

The course of an infection apparently caused by a filterable virus of the blood of the shore crab. Carcinus maenas, has been experimentally studied in this host both at Roscoff, France. where it was originally found, and at Woods Hole, Mass., where the local species was also found susceptible. Although a portion of the infected animals die with the symptoms of inadequate blood clotting, recovery of this function occurred promptly in about two-thirds of the animals. Half of the animals that recovered this function did so within 4 to 6 days. Recovery was not accompanied by disappearance of the virus from the whole blood. In tests done as late as 40 days after recovery, virus was still present. Autointerference was demonstrated after acute infection. It was found in the serum of animals with manifest disease on all days tested and in whole blood of animals taken more than 2 days after the clotting defect appeared. It was not demonstrable within the whole blood within the first 2 days of disease. The role of this in the recovery phenomenon is discussed.     

Adult-onset type II citrullinemia and idiopathic neonatal hepatitis caused by citrin deficiency: involvement of the aspartate glutamate carrier for urea synthesis and maintenance of the urea cycle

Citrin is a mitochondrial aspartate glutamate carrier primarily expressed in the liver, heart, and kidney. We found that adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin. In this report, we describe the frequency of SLC25A13 mutations, the roles of citrin as a member of the urea cycle and as a member of the malate-aspartate shuttle, the relationship between its functions and symptoms of citrin deficiency, and therapeutic issues.     

Modulation of large conductance calcium-activated K+ channel by membrane-delimited protein kinase and phosphatase activities

Large conductance Ca²⁺-activated K⁺ channel was identified and studied in excised inside-out membrane patches of freshly dispersed smooth muscle cells from rabbit gastric antrum. The current-voltage relationship of the single channel was linear from -80 to +80 mV of pipette voltage in which single channel conductance was 249±17.8 pS (n=19) in symmetrical concentration of K⁺ (145mM) across the patch. Activity of the channel (NPo) depended not only on cytoplasmic calcium concentration but also on membrane potential. MgATP increased NPo in a dose-dependent manner and Mg²⁺ was prerequisite for the effect. Okadaic acid (l00nM), inhibitor of protein phosphatases, increased NPo further in the presence of MgATP. Therefore, it would be concluded that activity of the calcium-activated K⁺ channel in gastric smooth muscle cells was controlled by phosphorylation state of the channel protein and the state is further modulated by membrane-delimited protein kinase and protein phosphatase activities.     

Differences and relationships between the physical properties and the microscopic structure of human femoral,tibial and fibular cortical bone

Coefficients of correlation between certain physical properties and the histological components of the break area were calculated on an IBM 7090 computer for 56 femoral, 79 tibial and 37 fibular specimens of embalmed cortical bone of standardized size and shape. Strong positive correlations (0.01–0.02 significance level) were found between tensile strength and the percentage of interstitial lamellae in the break area; between hardness and the number of osteons/mm²; and between hardness and the percentage of osteons in the break area. Equally high negative correlations were found between tensile strength and percentage of osteons in the break area; between shearing strength and average area/osteon remnant; between elastic modulus and percentage of spaces in the break area; and an even higher correlation (0.001) between hardness and percentage of spaces in the break area. Negative correlations (at slightly more than 0.05 significance level) were found between shearing strength and modulus and average area/osteon. Osteons tend to reduce the tensile strength and elastic modulus of bone while interstitial lamellae tend to increase them. The probable reason is the relatively greater amount of cement lines, which are sites of weakness where failure can occur, in Haversian bone as compared with lamellar bone. The predominant orientation of collagen fibers and the amount and distribution of calcium may also be involved. These factors are now being investigated.     

Mechanism of slowing of mucociliary transport induced by SO2 exposure

Responses of nasal mucociliary transport mechanisms to exposure to 6 ppm SO₂ were studied in chickens in vivo. This model takes advantage of the natural cleft palate which exposes the mucociliated base of the nasal septum. Exposure to 6 ppm SO₂ decreased the mucociliary transport rate along the base of the nasal septum. The minimum force required to move an iron particle along this area of mucous membrane by use of a magnetic field in vivo increased significantly after SO₂ exposure, while the minimum force required to move an iron particle on a pool of mucus collected from the same chicken and tested in vitro showed no change after SO₂ exposure. The elastic recoil distance of mucus was measured both in vivo and in vitro. The in vivo recoil distance decreased significantly after SO₂ exposure, while SO₂ exposure did not change recoil distance in vitro. It is proposed that exposure of chickens to SO₂ results in the formation of multiple points of adhesion of strands of mucus between the acinar gland cells and the emergent extracellular mucus or adhesion of a mucous blanket to the cilia, causing mucociliary transport to be retarded or static.