Comparison of bone marrow extracellular matrices.

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.     

Random Amplified Polymorphic DNA Variation among and within Selected Ixora (Rubiaceae) Populations and Mutants

Random amplified polymorphic DNA (RAPD) analysis was used tostudy variation among and within selectedIxora (Rubiaceae) populationsand mutants. Six populations of I. congesta yielded identicalbanding patterns suggesting genetic uniformity of this species.However, six populations of I. coccinea varieties (three red-flowered,two yellow-flowered and one red-flowered wild-type) exhibitedinfraspecific differences in RAPD profiles. Small and largeleaves of an atavistic mutant cultivar of I. coccinea were alsosubjected to RAPD analysis. An extra band was amplified in thelarge leaves that was absent in small leaves, suggesting thatthe phenotypic alteration in this taxon is due to genetic mutationrather than epigenetic changes. Similarly, an extra band wasdetected in the white sectors of I. Variegated compared to thegreen sectors, suggesting that the shoot apical meristems ofthis cultivar exist as a genetic chimera. DNA gel blot hybridizationwas performed to confirm the specificities of selected bands.Our study indicates that differences among individuals of variouspopulations and mutants may be detected using RAPD markers.Copyright 1999 Annals of Botany Company Ixora L., variegated variety, RAPD fingerprinting, DNA gel blot, intraspecific genetic similarity, atavistic mutant.     

Cytochrome P450 2E1-derived reactive oxygen species mediate paracrine stimulation of collagen I protein synthesis by hepatic stellate cells.

To evaluate possible fibrogenic effects of CYP2E1-dependent generation of reactive oxygen species, a model was developed using co-cultures of HepG2 cells, which do (E47 cells) or do not (C34 cells) express cytochrome P450 2E1 (CYP2E1) with stellate cells. There was an increase in intra- and extracellular H(2)O(2), lipid peroxidation, and collagen type I protein in stellate cells co-cultured with E47 cells compared with stellate cells alone or co-cultured with C34 cells. The increase in collagen was prevented by antioxidants and a CYP2E1 inhibitor. CYP3A4 did not mimic the stimulatory effects found with CYP2E1. Collagen mRNA levels remained unchanged, and pulse-chase analysis indicated similar half-lives of collagen I protein between both co-cultures. However, collagen protein synthesis was increased in E47 co-culture. Hepatocytes from pyrazole-treated rats (with high levels of CYP2E1) induced collagen protein in primary stellate cells, and antioxidants and CYP2E1 inhibitors blocked this effect. These results suggest that increased translation of collagen mRNA by CYP2E1-derived reactive oxygen species is responsible for the increase in collagen protein produced by the E47 co-culture. These co-culture models may be useful for understanding the impact of CYP2E1-derived ROS on stellate cell function and activation.     

The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

Potassium depletion selectively inhibits sustained diacylglycerol formation from phosphatidylinositol in angiotensin II-stimulated, cultured vascular smooth muscle cells

Potassium depletion decreases blood pressure in vivo and blunts the pressor response to angiotensin II (ang II) without down-regulating the receptor. In cultured rat aortic smooth muscle cells, the ang II-induced signaling sequence is biphasic with rapid hydrolysis of the polyphosphoinositides producing an early (15 s) diacylglycerol (DG) peak and a transient rise in inositol trisphosphate (IP3) and more delayed phosphatidylinositol (PI) hydrolysis resulting in sustained DG formation (peak at 5 min). Exposure of intact vascular smooth muscle cells to low potassium growth medium for 24 h or acutely potassium-depleting cells with nigericin causes selective, marked inhibition of late DG formation (5-min peak inhibited by 60 +/- 8% and 84 +/- 7%, respectively). The early cell response, namely polyphosphoinositide hydrolysis, inositol bis- and trisphosphate production and the 15-s DG peak, is not affected. Analysis of 125I-ang II-binding data reveals no significant differences in either receptor number or binding affinity (Kd) in potassium-depleted cells. Together with its marked inhibitory effect on sustained ang II-induced DG formation, acute potassium depletion effectively blocks internalization of 125I-ang II: there is no significant internalization of the ligand after 5 min at 37 degrees C versus 64 +/- 7% internalization in control cells. Thus, potassium depletion does not alter ang II binding or initial membrane signaling in rat aortic smooth muscle but blocks ligand internalization and selectively and markedly inhibits the development of direct PI hydrolysis and sustained diacylglycerol formation. These findings suggest a role for ligand-receptor processing in generating the sustained cell response and potentially explain the lower blood pressure and decreased pressor response to ang II seen in hypokalemic states in vivo. Furthermore, the ability of K+ depletion to alter secondary signal generation may provide insight into the mechanisms underlying the K+ dependence of a variety of cell functions.     

Porcine high molecular weight kininogen: its purification and properties as a thiol proteinase inhibitor as compared to human high molecular weight kininogen

1. High mol. wt kininogen (HMW kininogen) was purified to a homogeneous state from porcine plasma. 2. The protein exhibited a strong inhibitory activity for thiol proteinases such as ficin, papain and calpain I, whereas it did not inhibit serine proteinases, trypsin and chymotrypsin. 3. The mol. wt, isoelectric point, amino acid and carbohydrate compositions, stabilities to temperature and pH, kinetic constants, and immunological properties of the porcine HMW kininogen were determined and compared with those of human HMW kininogen.     

Correlation between the virulence of streptococci for mice and their IgG FcR activity in in vivo experiments

The immunological analysis of 24 spontaneous Strr, Rifr and Kanr mutants of streptococcal strain IP, highly virulent for mice and capable of binding polyclonal human IgG (IgG FcR+), was made. The characteristic feature of all these mutants was decreased virulence, restored after their passage in vivo. 23 mutants were capable of binding polyclonal IgG; one Strr mutant had no such capacity, but acquired it, together with an increase in virulence, after its passage in vivo. When stored in meat-peptone agar without antibiotics, 5 out of 10 Strr mutants lost their capacity for binding polyclonal human IgG. After passage in vivo they regained this property simultaneously with virulence.     

Microflora of the upper respiratory tract in young children under normal conditions and in respiratory tract diseases. Microbial count of the upper respiratory tract in healthy children and in children with pneumonia

The results of the inoculation of material taken from the anterior section of the nasal cavity and from the pharyngeal mucosa of 50 healthy young children and 298 acute pneumonia patients were analyzed. 23 microbial species were isolated. In the samples taken from the anterior section of the nasal cavity, monocultures were detected in 86 samples and 54 variants of associations including 2-4 species, in 139 samples. In the samples taken from the pharynx, monocultures were detected in 59 samples and 180 variants of associations including 2-6 species, in 282 samples. Differences in the contamination of the nasal cavity and the pharynx in healthy children and in pneumonia patients were revealed. These differences were manifested in the structure of the microflora (monocultures, associations, their composition), the assortment of microbial species and their concentration. In young children with pneumonia the microflora of the upper respiratory tract was found to reflect the severity of acute pneumonia and the intensity of the pathological process in the lungs (uncomplicated, pyodestructive pneumonia, pyodestructive pneumonia with fatal termination, acute purulent pleurisy).     

Changes in the properties of adenylate cyclase from guinea pig lungs in tuberculosis

Changes in the properties of adenylate cyclase from the lungs of tuberculotic guinea pigs were revealed. The number of beta-adrenergic receptors in the lungs was found to be reduced by 30% at the second and by 70% at the third stage of the disease. The degree and the value of Ka for adenylate cyclase activation by isoproterenol remained thereby unchanged. The basal activity of adenylate cyclase was increased by 20% against the control level at the second stage and decreased by 20% at the third stage of the disease. At these periods, the stimulating effects of guanylyl imidodiphosphate, NaF and forskolin on lung adenylate cyclase were diminished. The experimental results point to the significant role of the enzymes of cAMP metabolism and reflect the course of the tuberculosis process in experimental animals.