Regulation of brain capillary endothelial thrombomodulin mRNA expression.

BACKGROUND AND PURPOSE: Endothelial cells regulate hemostasis in part via expression of thrombomodulin, a potent anticoagulant protein. The purpose of this study was to analyze brain capillary endothelial cell expression of thrombomodulin mRNA. METHODS: Bovine brain capillary endothelial cells were grown in a blood-brain barrier model in which endothelial cells form capillary-like structures. In situ hybridization and polymerase chain reaction (PCR) were used to examine thrombomodulin expression. Endothelial cells were then cocultured with astrocytes. We examined both coculture and monoculture preparations for gamma-glutamyl transpeptidase (GGTP), a marker of the blood-brain barrier. We then used quantitative-competitive PCR to compare thrombomodulin expression in endothelial monocultures and astrocyte-endothelial cocultures after 1 and 7 days of culture. RESULTS: Both in situ hybridization and PCR studies demonstrated thrombomodulin mRNA expression by endothelial cells. During 1 week of astrocyte-endothelial coculture, there was (1) progressive association of astrocytes with capillary-like structures and (2) expression of GGTP; endothelial monocultures did not express GGTP. There was no significant difference in thrombomodulin mRNA expression for cocultures versus monocultures after 1 day. After 1 week, however, astrocyte-endothelial cocultures had markedly decreased thrombomodulin mRNA compared with monocultures (9 +/- 2 versus 189 +/- 62 pg/mL; P < .025). This thrombomodulin mRNA decrease thus occurred when elements of the blood-brain barrier phenotype were demonstrable, ie, when astrocyte association with capillary-like structures was maximal and when GGTP was expressed in cocultures. CONCLUSIONS: These findings indicate astrocyte regulation of thrombomodulin mRNA expression in vitro and suggest an important role for the blood-brain barrier in the regulation of thrombomodulin.

Cryopreserved callus: a source of protoplasts for rice transformation.

We cryopreserved whole rice calli (Oryza sativa L cv Taipei 309) to investigate the ability of the surviving cells to regenerate plants and yield protoplasts competent for genetic transformation. Four out of six callus lines cryopreserved after four months in culture contained small sectors able to continue cell division and subsequently regenerate fertile plants. Both cryopreservation efficiency and regeneration ability decreased when using eight month old cultures. High yields of protoplasts were obtained from different cryopreserved callus lines. Protoplasts were transfected with chimeric genes consisting of the maize ubiquitin 1 promoter, first exon and first intron fused to the coding region of either the GUS or BAR marker genes. Levels of transient gene expression from both marker genes were similar to those previously obtained using protoplasts derived from callus that had not been frozen. Stable transformants were selected by their resistance to Bialaphos and could be identified with the pH indicator chlorophenol red. Southern blot analysis confirmed the integration of the BAR gene into the rice genome. Therefore, cryopreservation does not affect the ability of rice cells to integrate and express foreign genes.

Hematogenous factors and prediction of delayed ischemic deficit after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Delayed ischemic deficits contribute to the high morbidity and mortality rates associated with subarachnoid hemorrhage. We evaluated the potential usefulness of measuring coagulation and hemorheological variables and cardiolipin antibodies for prediction of delayed ischemic deficit after subarachnoid hemorrhage. METHODS: Consecutive patients with subarachnoid hemorrhage were studied. Coagulation and hemorheological variables and cardiolipin antibodies were measured on admission, within 7 days of subarachnoid hemorrhage. A subset of patients was studied on admission and at two subsequent occasions. RESULTS: Sixty-nine patients were studied. Sixty-one of these were without clinical manifestations of vasospasm at admission, and 16 developed delayed ischemic deficit during their hospitalization. None of the laboratory variables measured were significantly different between patients with or without later development of delayed ischemic deficit. Elevation of the fibrin fragment D-dimer was found in the group of eight patients admitted with ischemic symptoms and in 49% (34 of 69) of all patients, but this was not associated with delayed ischemic deficit. Sixteen patients were studied on three occasions; this group showed a significant decrease in hematocrit, an increased white blood cell count, and no change in fibrinogen concentration. Fibrin D-dimer levels rose significantly after surgery (from 5.01 +/- 0.69 to 5.53 +/- 0.58 ln-ng/ml, p less than 0.025) and after onset of delayed ischemic deficit (from 4.71 +/- 0.64 to 5.84 +/- 0.34 ln-ng/ml, p less than 0.01). CONCLUSIONS: Hemostatic measurements, hemorheological variables, and cardiolipin immunoreactivity did not predict delayed ischemic deficit in this population.

Immunohematologic characteristics of infection-associated cerebral infarction.

We evaluated 50 consecutive patients with acute ischemic stroke to assess the prevalence of systemic infection preceding the neurological event. We analyzed the immunohematologic characteristics of patients with and without signs and/or symptoms of a preceding infectious process. Patients were examined less than or equal to 7 days after cerebral infarction and evaluated for fibrinogen, anticardiolipin antibodies, fibrin D-dimer (a fragment of cross-linked fibrin), plasminogen activator inhibitor-1, and protein S. Of the 50 patients, 17 had symptoms of infection beginning less than or equal to 1 month before the stroke (11 had upper respiratory tract infections, three urinary tract infections, two subacute bacterial endocarditis, and one pneumonia). Compared with patients without infection, patients with infection had significant increases in fibrin D-dimer concentration (5.3 +/- 1.1 versus 4.7 +/- 0.9 log-transformed ng/ml, p less than 0.05) and cardiolipin immunoreactivity, IgG isotype (1.8 +/- 1.3 versus 1.1 +/- 0.9 log-transformed phospholipid units, p less than 0.04), and, when studied less than or equal to 2 days after the stroke, increased fibrinogen levels (459 +/- 126 versus 360 +/- 94 mg/dl, p less than 0.05). In conclusion, infection-associated cerebral infarction is common and is associated with substantial immunohematologic abnormalities.

Regional distribution of thrombomodulin in human brain.

Thrombomodulin, an integral membrane protein with important anticoagulant properties, was evaluated for its distribution in blood vessels of human brain. Adjacent sections from frozen human brain were stained for von Willebrand Factor (an endothelium marker) or thrombomodulin. The number of thrombomodulin-positive blood vessels was expressed as a percentage of all blood vessels in a specific area. The density ratio was compared among cortical and subcortical regions in the human brain. Our results indicated that thrombomodulin density in neocortex, cerebellum, medulla and hippocampus was similar. There was, however, significantly less thrombomodulin in putamen (P less than 0.01), pons (P less than 0.001) and mesencephalon (P less than 0.001) compared to neocortex. The regional distribution of thrombomodulin may prove to be helpful for understanding the development of thrombotic diseases of the brain.

Growth-inhibitory activity of interferon-beta against human colorectal carcinoma cell lines.

Recombinant human interferon beta (rIFN-beta) inhibited in a time- and dose-dependent manner the proliferation of 18/18 human colon carcinoma cell lines in monolayer culture and 8/9 lines in a soft agar assay but had no effect on 4 human fibroblast cell lines. Maximal inhibition of cell proliferation by rIFN-beta required repetitive treatment (every 2 days) with lymphokine (50 units/ml). Furthermore, the inhibitory activity of rIFN-beta was neutralized by polyclonal antibodies against natural IFN-beta. In contrast to rIFN-beta, rIFN-alpha was inactive against all colon cell lines tested, and rIFN-gamma, with the exception of HT-29 cells, was similarly ineffective. These data demonstrate that rIFN-beta is a potent growth inhibitor of colon carcinoma cells in vitro, and suggest that studies on its mechanism of action may lead to a better understanding of the regulation of colon tumor cell proliferation.

Biological, immunological and biochemical characterization of cleaved prolactin generated by lactating mammary gland.

We have previously shown that rat prolactin is proteolytically cleaved in its loop by peripheral tissues of the rat. Of the tissues examined to date, lactating mammary gland exhibits the highest prolactin-cleaving activity. The objective of this study was to characterize cleaved prolactin, biologically, immunologically and chemically. By modifying an established analytical method, we were able to generate large (micrograms) amounts of cleaved rat prolactin from cell fractions of rat mammary gland which could then be assayed for biological and immunological activity relative to intact hormone. The cleaved product showed no significant difference relative to the intact rat prolactin when assayed for its ability to compete with 125I-labelled ovine prolactin for the prolactin receptor and for its ability to stimulate the proliferation of rat Nb2 lymphoma cells. Cleaved rat prolactin, however, did show a 50-60% reduction in activity relative to intact rat prolactin when assayed by radioimmunoassay. Using Edman degradation and partial amino acid analysis, we determined that the second N-terminus of the cleaved rat prolactin begins at amino acid 149. The divergence of biological and immunological activity produced by proteolytic cleavage in the loop of rat prolactin suggests that biological and immunological sites differ in location. The possible physiological implications of a cleaved rat prolactin molecule generated by target tissue with maintained biological activity and reduced immunological activity are discussed.

Proteolytic modification of rat prolactin by subcellular fractions of the lactating rat mammary gland.

The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.