William Sun

THE RON/STK RECEPTOR TYROSINE KINASE IS ESSENTIAL FOR PERI-IMPLANTATION DEVELOPMENT IN THE MOUSE

The Ron/STK receptor tyrosine kinase is a member of the c-Met family of receptors and is activated by hepatocyte growth factor-like protein (HGFL). Ron activation results in a variety of cellular responses in vitro, such as activation of macrophages, proliferation, migration, and invasion, suggesting a broad biologic role in vivo. Nevertheless, HGFL-deficient mice grow to adulthood with few appreciable phenotypic abnormalities. We report here that in striking contrast to the loss of its only known ligand, complete loss of Ron leads to early embryonic death. Embryos that are devoid of Ron (Ron-/-) are viable through the blastocyst stage of development but fail to survive past the peri-implantation period. In situ hybridization analysis demonstrates that Ron is expressed in the trophectoderm at embryonic day (E) 3.5 and is maintained in extraembryonic tissue through E7.5, compatible with an essential function at this stage of development. Hemizygous mice (Ron+/-) grow to adulthood; however, these mice are highly susceptible to endotoxic shock and appear to be compromised in their ability to downregulate nitric oxide production. These results demonstrate a novel role for Ron in early mouse development and suggest that Ron plays a limiting role in the inflammatory response.

Genetic elimination of prothrombin in adult mice is not compatible with survival and results in spontaneous hemorrhagic events in both heart and brain

Mice carrying a conditional prothrombin knockout allele (fII(lox)) were established to develop an experimental setting for exploring the importance of thrombin in the maintenance of vascular integrity, the inflammatory response, and disease processes in adult animals. In the absence of Cre-mediated recombination, homozygous fII(lox/lox) mice or compound heterozygous mice carrying one fII(lox) allele and one constitutive-null allele were viable. Young adults exhibited neither spontaneous bleeding events nor diminished reproductive success. However, the induction of Cre recombinase in fII(lox) mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fII(lox) allele within the liver, the loss of circulating prothrombin, and profound derangements in coagulation function. Consistent with the notion that thrombin regulates coagulation and inflammatory pathways, an additional early consequence of reducing prothrombin was impaired antimicrobial function in mice challenged with Staphylococcus aureus peritonitis. However, life expectancy in unchallenged adults genetically depleted of prothrombin was very short ( approximately 5-7 days). The loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, particularly in the heart and brain. Unlike the constitutive loss of either clotting or platelet function alone, the conditional loss of prothrombin is uniformly not compatible with maintenance of hemostasis or long-term survival.

Using the lymph fistula rat model to study the potentiation of GIP secretion by the ingestion of fat and glucose.

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin produced in the K cells of the intestine and secreted into the circulating blood following ingestion of carbohydrate- and fat-containing meals. GIP contributes to the regulation of postprandial insulin secretion and is essential for normal glucose tolerance. We have established a method of assaying GIP in response to nutrients using the intestinal lymph fistula model. Administration of Ensure, a mixed-nutrient liquid meal, stimulated a significant increase in intestinal lymphatic GIP levels that were approximately threefold those of portal plasma. Following the meal, lymph GIP peaked at 60 min (P < 0.001) and remained elevated for 4 h. Intraduodenal infusions of isocaloric and isovolumetric lipid emulsions or glucose polymer induced lymph GIP concentrations that were four and seven times the basal levels, respectively. The combination of glucose plus lipid caused an even greater increase of lymph GIP than either nutrient alone. In summary, these findings demonstrated that intestinal lymph contains high concentrations of GIP that respond to both enteral carbohydrate and fat absorption. The change in lymphatic GIP concentration is greater than the change observed in the portal blood. These studies allow the detection of GIP levels at which they exert their local physiological actions. The combination of glucose and lipid has a potentiating effect in the stimulation of GIP secretion. We conclude from these studies that the lymph fistula rat is a novel approach to study in vivo GIP secretion in response to nutrient feeding in conscious rats.

Characterization of the mouse Ron/Stk receptor tyrosine kinase gene.

In an effort to understand the mechanisms governing the regulation of the mouse Ron receptor gene, a mouse genomic library was screened and overlapping clones coding for the Ron gene and flanking DNA were identified. Continuous DNA sequence was obtained for approximately 16.4 kilobases. The gene, from the initiator methionine to the polyadenylation site, is contained within 13 244 basepairs and contains 19 exons. Primer extension analyses were performed to determine the transcription start site of the mouse Ron transcript. Multiple transcription start sites were found which also appear to be used in transfected reporter constructs containing Ron 5′ flanking DNA. To determine the location of sites which may be critical for the function of the Ron gene promoter, a series of chimeric genes containing serial deletions of the Ron gene promoter fused to the coding sequences for the chloramphenicol acetyltransferase gene were constructed. Transient transfection analyses of these hybrid genes into various cell lines demonstrated that two regions of the Ron gene promoter, encompassing nucleotides −585 to −465 and from −465 to −285, are important for expression of this transcript in CMT-93 cells. Further analysis of the Ron promoter utilizing gel mobility shift analyses suggests that regions encompassing nucleotides −585 to −508 and nucleotides −375 to −285 appear to bind specific proteins which may be involved in the negative and positive regulation, respectively, of the mouse Ron gene.

Prothrombin Carora: hypoprothrombinaemia caused by substitution of Tyr‐44 by Cys

Two members of a family from Carora, Venezuela, were found to have prothrombin activity levels at 4% of normal and undetectable antigen levels. All exons of the prothrombin gene from the proband were sequenced and a mutation at nucleotide 1305 was identified that would result in the substitution of Cys for Tyr at residue 44. Residue 44 is present in the aromatic stack region of the protein. Substitution of a Cys in this region would result in an abnormal folding of the protein which could be the cause for the observed lack of secretion of the abnormal prothrombin.