In vivo and in vitro interaction of high and low molecular weight single-chain urokinase-type plasminogen activator with rat liver cells.

The plasma clearance and the interaction of high (HMW) and low (LMW) molecular weight single-chain urokinase-type plasminogen activator (scu-PA) with rat liver cells was determined. 125I-Labeled HMW- and LMW-scu-PA were rapidly cleared from plasma with a half-life of 0.45 min and a maximal liver uptake of 55% of the injected dose. Liver uptake of scu-PA was mediated by parenchymal cells. Excess of unlabeled HMW-scu-PA reduced the liver uptake of 125I-HMW-scu-PA strongly. In vivo liver degradation of scu-PA was reduced by inhibitors of the lysosomal pathway. A high affinity binding site (Kd 45 nM, Bmax 1.7 pmol/mg cell protein) for both HMW- and LMW-scu-PA was determined on isolated parenchymal liver cells. Cross-competition binding studies showed that LMW- and HMW-scu-PA bind to the same site. Tissue-type plasminogen activator, mannose- or galactose-terminated glycoproteins did not affect the scu-PA binding to parenchymal liver cells. It is concluded that LMW- and HMW-scu-PA are taken up in the liver by a common, newly identified recognition site on parenchymal liver cells and are subsequently degraded in the lysosomes. It is suggested that this site is important for the regulation of the turnover of scu-PA.     

IL-2 and a contact-mediated signal provided by TCR alpha beta + or TCR gamma delta + CD4+ T cells induce polyclonal Ig production by committed human B cells. Enhancement by IL-5, specific inhibition of IgA synthesis by IL-4.

To study the role of T cells in T-B cell interactions resulting in isotype production, autologous purified human splenic B and T cells were cocultured in the presence of IL-2 and Con A. Under these conditions high amounts of IgM, IgG, and IgA were secreted. B cell help was provided by autologous CD4+ T cells whereas autologous CD8+ T cells were ineffective. Moreover, CD8+ T cells suppressed Ig production when added to B cells cocultured with CD4+ T cells. Autologous CD4+ T cells could be replaced by allogeneic activated TCR gamma delta,CD4+ or TCR alpha beta,CD4+ T cell clones with nonrelevant specificities, indicating that the TCR is not involved in these T-B cell interactions. In contrast, resting CD4+ T cell clones, activated CD8+, or TCR gamma delta,CD4-,CD8- T cell clones failed to induce IL-2-dependent Ig synthesis. CD4+ T-B cell interaction required cell-cell contact. Separation of the CD4+ T and B cells by semiporous membranes or replacement of the CD4+ T cells by their culture supernatants did not result in Ig synthesis. However, intact activated TCR alpha beta or TCR gamma delta,CD4+ T cell clones could be replaced by plasma membrane preparations of these cells. Ig synthesis was blocked by mAb against class II MHC and CD4. These data indicate that in addition to CD4 and class II MHC Ag a membrane-associated determinant expressed on both TCR alpha beta or TCR gamma delta,CD4+ T cells after activation is required for productive T-B cell interactions resulting in Ig synthesis. Ig production was also blocked by mAb against IL-2 and the IL-2R molecules Tac and p75 but not by anti-IL-4 or anti-IL-5 mAb. The CD4+ T cell clones and IL-2 stimulated surface IgM-IgG+ and IgM-IgA+, but not IgM+IgG- or IgM+IgA- B cells to secrete IgG and IgA, respectively, indicating that they induced a selective expansion of IgG- and IgA-committed B cells rather than isotype switching in Ig noncommitted B cells. Induction of Ig production by CD4+ T cell clones and IL-2 was modulated by other cytokines. IL-5 and transforming growth factor-beta enhanced, or blocked, respectively, the production of all isotypes in a dose-dependent fashion. Interestingly, IL-4 specifically blocked IgA production in this culture system, indicating that IL-4 inhibits only antibody production by IgA-committed B cells.     

Biogenesis of outer membrane protein PhoE of Escherichia coli. Evidence for multiple SecB-binding sites in the mature portion of the PhoE protein.

Efficient in vivo translocation of the precursor of Escherichia coli outer membrane protein PhoE across the inner membrane is shown to depend on SecB protein. A set of mutants, carrying internal deletions in the phoE gene, was used to locate a possible SecB-binding site and/or a site that makes the protein dependent on SecB for export. Except for two small mutant PhoE proteins, the in vivo and in vitro translocation of all mutant proteins was more efficient in the presence of SecB. The interaction of SecB protein with wild-type and mutant PhoE proteins, synthesized in vitro, was further studied in co-immunoprecipitation experiments with anti-SecB protein serum. The efficiencies of co-immunoprecipitation of precursor and mature PhoE were very similar, indicating the absence of a SecB-binding site in the signal sequence. Moreover, all mutant proteins with deletions in the mature moiety of the PhoE protein were co-immunoprecipitated in these assays, albeit mostly with reduced efficiency. Taken together, these results indicate the existence of multiple SecB-binding sites in the mature portion of the PhoE protein.     

International Commission for Protection Against Environmental Mutagens and Carcinogens. A method for combining and comparing short-term genotoxicity test data: preface. A report from ICPEMC Committee 1.

The ability to repair 'mis-instructive', O6-methylguanine, and 'non-instructive', AP sites, DNA lesions in Fischer 344 rat livers at various ages was determined. Different behaviours were observed. While the AP-endodesoxyribonuclease enzymes displayed a high constant level throughout the animals' lifetime, the O6-methylguanine-DNA methyltransferase activity presented a stepwise modulation (DNA normalisation of results): the O6-MT activity significantly increased within the first month of animal life and enhanced again after 6 months reaching a maximum plateau in the 12-18-month-old animals. Thereafter a net significant decrease of O6-MT enzyme was detected in the 24-month-old group. While the repair of the widely formed AP sites appeared uniformly efficient like 'house keeping' functions, the removal of the rare precancerous O6-methylguanine is age-dependent indicating a decreased protection of the youngest and oldest animals against this 'mis-instructive' damage. However, any extrapolation of the age-associated cancer risk needs further assessment.     

Mutagenic potency of heterocyclic amines towards Salmonella typhimurium; possible causes of variability in the results observed.

The potent food mutagens and carcinogens 2-amino-3-methylimidazol[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MEIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are probably the most active bacterial mutagens so far discovered. Important discrepancies were found, however, in the specific mutagenicities published for these compounds. This paper analyzes a number of experimental factors that could explain these differences: purity of the compounds, stability under the experimental conditions employed, solvents used, bacterial toxicity, testing procedure, amount and age of the S9 fraction, dose-effect relationships, day-to-day variability, origin of the compounds investigated or of the bacterial strain and age of the strain culture used. None of these factors was found to play a critical role, when the other experimental conditions were strictly standardized. The in-house testing procedure used probably explains the interlaboratory variations observed.     

Plant size,geitonogamy and seed set in Ipomopsis aggregata

Summary We used powdered fluorescent dyes to estimate receipt of self vs. outcross pollen in the self-incompatible species Ipomopsis aggregata (Polemoniaceae). Flowers on small and large plants received equal amounts of outcross pollen, whereas flowers on large plants received more self pollen, so the proportion of self pollen delivered through geitonogamy increased with plant size. In natural populations emasculation of all flowers on a plant raised average seed set per flower from 5.19 to 6.99 and also raised fruit set, though not significantly. From these results one expects a negative correlation between plant size and seeds per flower. The opposite trend was observed in a sample of plants in the field, suggesting that deleterious effects of geitonogamy on female fecundity in large plants can be overruled by other factors such as size-related fruit or seed abortion. Results are discussed in relation to the evolution of gynodioecy.     

A gymnosperm extensin contains the serine-tetrahydroxyproline motif

The extensin family is a diverse group of hydroxyproline-rich glycoproteins located in the cell wall and characterized by repetitive peptide motifs glycosylated to various degrees. The origin of this diversity and its relationship to function led us earlier to compare extensins of the two major groups of angiosperms from which we concluded that the highly glycosylated Ser-Hyp₄ motif was characteristic of advanced herbaceous dicots, occurring rarely or not at all in a representative graminaceous monocot (Zea mays) and a chenopod (Beta vulgaris) representative of primitive dicots. Because these results could arise either from loss or acquisition of a characteristic feature, we chose a typical gymnosperm representing seed-bearing plants more primitive than the angiosperms. Thus, salt eluates of Douglas fir (Pseudotsuga menziesii) cell suspension cultures yielded two monomeric extensins differing in size and composition. The larger extensin reported earlier lacked the Ser-Hyp₄ motif, was rich in proline and hydroxyproline, and contained peptide motifs similar to the dicot repetitive proline-rich proteins. The smaller extensin monomer reported here (Superose-6 peak 2 [SP2]) was compositionally similar to typical dicot extensins such as tomato P1, mainly consisting of Hyp, Thr, Ser, Pro, Val, Tyr, Lys, His, abundant arabinose, and a small but significant galactose content. A chymotryptic peptide map (on Hamilton PRP-1) of anhydrous hydrogen fluoride-deglycosylated SP2 yielded eight peptides sequenced after further purification on a high-resolution fast-sizing column (polyhydroxyethyl aspartamide; Poly LC). Significantly, two of the eight peptides contained the Ser-Hyp₄ motif, consistent both with the SP2 amino acid composition as well as the presence of hydroxyproline tetraarabinoside as a small (4% of total Hyp) component of the hydroxyproline arabinoside profile; thus, hydroxyproline tetraarabinoside corroborates the presence of Ser-Hyp₄, in agreement with our earlier observation that Hyp contiguity and Hyp glycosylation are positively correlated. Interestingly, other peptide sequences indicate that SP2 contains motifs such as Ser-Hyp₃-Thr-Hyp-Tyr, Ser-Hyp₄-Lys, and (Ala-Hyp)_n repeats that are related to and typify dicot extensins P1, P3, and arabinogalactan proteins, respectively. Overall, these peptide sequences confirm our previous prediction that Ser-Hyp₄ is indeed an ancient motif and also strongly support our suggestion that the extensins comprise an extraordinarily diverse, but nevertheless phylogenetically related, family of cell wall hydroxyproline-rich glycoproteins.     

Sensitivity to in vitro lipid peroxidation in liver and brain of aged rats.

Lipid peroxidation in rat liver and brain has been studied to see if it increases with old age. No significant differences in the level of endogenous, nonstimulated lipid peroxidation (TBA-RS) is found between 9 month-old (mature adults) and 28 month-old animals in liver or cerebral cortex. Liver homogenates subjected in vitro to an oxidative stress (ascorbate-Fe++), show a clearly slower peroxidation rate in old than in young animals. On the other hand, the in vitro peroxidation rate of cerebral homogenates was similar in young and old animals. The in vitro peroxidation rate was much higher in brain than in liver tissue. These results do not support the view that old rats liver and brain are more susceptible to free radical oxidative damage than those of young ones.     

Effect of brain serotonin on the arterial pressure and plasma renin in normal and hypertensive rats]

The effects of changes in brain serotonin content after injections of p-chlorophenylalanine (p-CPA), L-5-hydroxytryptophan (L-5HTP) and 5-6-dihydroxytryptamine (5-6DHT) on the mean arterial pressure (MAP), plasma renin activity (PRA) and peripheral levels of atrial natriuretic peptide (ANP) have been studied in normal and hypertensive (2K:1C model) male Wistar rats. The p-CPA (250 mg/kg) and L-5HTP (200 mg/kg) were injected i.p., while 5-6 DHT (15 micrograms/animal in 10 mu/animal vehicle) was injected into lateral brain ventricles. The effects were studied 24 h after the p-CPA injection, 2 h after L-5HTP and 10 or 20 days after 5-6DHT administration. The fall in brain serotonin produced by p-CPA and 5-6DHT did not modify the MAP values in the normal and hypertensive rat model, whereas the increase induced after L-5HTP injection only caused a slight decrease in arterial pressure in normotensive animals. The ARP experimented remarkable rises in the normal and hypertensive rats, these values increasing after L-5HTP and falling after p-CPA and 5-6 DHT injections. Similar changes are detected in the normal group after administration of these substances related to serotoninergic brain activity. The ANP levels rose after renal artery constriction, and they are not affected by the above mentioned substances. Only p-CPA and 5-6DHT reduced a low decrease in the ANP levels 10 days after their administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by pentoxifylline of procoagulant activity produced by endotoxin-active monocytes]

When appropriately stimulated, monocytes are able to initiate blood coagulation through the membrane expression of tissue factor. This procoagulant activity is thought to play a role in activating coagulation in response to inflammatory stimuli in vivo. We found that pentoxifylline, a methylxanthine derivative already reported to regulate some monocyte functions, inhibits the procoagulant activity developed by monocytes in vitro in response to endotoxin. This effect was accompanied by an early increase in intracellular levels of cyclic AMP and was mimicked by compounds that induce an increase in cyclic AMP levels. These results suggest that the suppressive effect of pentoxifylline occurs at least in part via an increase in intracellular cyclic AMP levels.