Actin accelerates plasmin generation by tissue plasminogen activator.

Actin has been found to bind to plasmin's kringle regions, thereby inhibiting its enzymatic activity in a noncompetitive manner. We, therefore, examined its effect upon the conversion of plasminogen to plasmin by tissue plasminogen activator. Actin stimulated plasmin generation from both Glu- and Lys-plasminogen, lowering the Km for activation of Glu-plasminogen into the low micromolar range. Accelerated plasmin generation did not occur in the presence of epsilon-amino caproic acid or if actin was exposed to acetic anhydride, an agent known to acetylate lysine residues. Actin binds to tissue plasminogen activator (t-Pa) (Kd = 0.55 microM), at least partially via lysine-binding sites. Actin's stimulation of plasmin generation from Glu-plasminogen was inhibited by the addition of aprotinin and was restored by the substitution of plasmin-treated actin, indicating the operation of a plasmin-dependent positive feedback mechanism. Native actin binds to Lys-plasminogen, and promotes its conversion to plasmin even in the presence of aprotinin, indicating that plasmin's cleavage of either actin or plasminogen leads to further plasmin generation. Plasmin-treated actin binds Glu-plasminogen and t-PA simultaneously, thereby raising the local concentration of t-PA and plasminogen. Together, but not separately, actin and t-PA prolong the thrombin time of plasma through the generation of plasmin and fibrinogen degradation products. Actin-stimulated plasmin generation may be responsible for some of the changes found in peripheral blood following tissue injury and sepsis.     

Epitope mapping of four monoclonal antibodies recognizing the hexose core domain of Salmonella lipopolysaccharide.

Four murine monoclonal antibodies reactive with distinctive regions of the hexose core domain of Salmonella lipopolysaccharide (LPS) were generated and their epitope specificities were delineated. MAST 56 (IgG1) and MAST 50 (IgG3) antibodies elicited by immunizations with Salmonella typhimurium Rb1 and Rb2 mutants, reacted selectively in enzyme immunoassay with the LPS from rough mutants. In contrast, MATy 1 (IgM) and MATy 2 (IgG2b) antibodies raised by an attenuated Salmonella typhi 620 Ty strain were reactive with LPS from both smooth and rough Salmonellae. Immunoblotting analysis showed that MATy 1 distinguished only the bottom bands (naked LPS core) among the heterogeneous LPS populations, whereas MATy 2 gave a ladder pattern (reactive with both naked and O-chain-substituted LPS cores). Differential binding specificities of MATy 1 and MATy 2 antibodies to the naked and capped LPS cores were further analyzed utilizing S. typhimurium polysaccharide fractions with different O-chain:core ratios which were obtained after separation by Sephacryl S-200 chromatography. Steric effects on the antibody reactivity by the bulky O-polysaccharide chain were detected. The use of chemically defined native and synthetic saccharides as inhibitors, in combination with the conformation of the Salmonella core oligosaccharide, permitted the definition of antigenic determinants carried in the core domain recognized by each antibody: (i) the branches I and VIII are essential for MATy 1 recognition, (ii) the backbone III-IV-V for MATy 2, (iii) the backbone II-III-IV-V for MAST 56, and (iv) the backbone plus the branch III-IV-V-VIII for MAST 50. (formula; see text)     

The disaccharide L-alpha-D-heptose1-->7-L-alpha-D-heptose1-->of the inner core domain of Salmonella lipopolysaccharide is accessible to antibody and is the epitope of a broadly reactive monoclonal antibody.

We generated a panel of mAb containing at least one specificity against each of the known chemotypes of the Salmonella LPS core domain and used them to investigate the accessibility of core determinants in smooth LPS. Most of the mAb were reactive with at the most three chemotypes of the core as determined by enzyme immunoassay and failed to bind smooth LPS or any of the complete cores of E. coli. One mAb, MASC1-MM3 (MM3), reacted with six different Salmonella core chemotypes, the R2 core of Escherichia coli and a variety of smooth LPS. This mAb reacted equally well with live and heat-killed bacteria. It bound to 123 of 126 clinical isolates of Salmonella and 11 of 73 E. coli strains in a dot-immunoblot assay. Typical ladder-like patterns of bands were observed after immunoblotting of this mAb against electrophoretically resolved smooth LPS from the five major serogroups of Salmonella species (A, B, C1, D1, and E). MM3 had no reactivity with BSA conjugates of O-Ag polysaccharides from the above serogroups confirming specificity for a core epitope. Polysaccharides derived from or synthetic saccharides representative of the various chemotypes of Salmonella LPS core were tested as competitive inhibitors of the binding of MM3 to LPS. The results led to a conclusion that MM3 recognizes the structure, L-alpha-D-Heptose1-->7-L-alpha-D-Heptose1-->disaccharide present as a branch in the Ra, Rb1, Rb2, Rb3 and Rc but lacking in the Rd1, Rd2, and Re chemotypes of the Salmonella LPS core. This disaccharide seems free and accessible on the basis of the previously calculated conformations of the Salmonella (Ra) and E. coli complete cores (R1, R2, R3, R4, and K12). It therefore defines or contains an epitope within the inner core subdomain of Salmonella LPS that is accessible to antibody in long-chained LPS and in intact bacteria with complete LPS.     

Highly efficient bioconversion of glucose into fructose diphosphate with fed-batch-grown Saccharomyces cerevisiae cells

Summary One of the methods commonly used for manufacturing fructose 1,6-diphosphate is based on the enzymatic phosphorylation of glucose with inorganic phosphate using permeabilized brewer's yeast cells. Our results demonstrate that a substantial improvement in the yield of bioconversion can be achieved using fed-batch-grown Saccharomyces cerevisiae cells. Under an appropriate glucose and phosphate to cell ratio the efficiency of bioconversion reaches 70% of the theoretical value. Offprint requests to: C. Compagno     

Biospecific adsorption of hepatic DT-diaphorase on immobilized dicoumarol. II. Purification of mitochondrial and microsomal DT-diaphorase from 3-methylcholanthrene-treated rats

Liver mitochondrial and microsomal DT-diaphorase have been purified from 3-methylcholanthrene-treated rats. A 1150-fold and 3500-fold purification of mitochondrial and microsomal DT-diaphorase, respectively, is achieved after solubilization of the membranes with deoxycholate followed by affinity chromatography on azodicoumarol Sepharose 6B and subsequent gel filtration on Sephadex G-100. From this purification procedure, 65–70% of mitochondrial DT-diaphorase is recovered and the purified enzyme has a specific activity comparable to that of cytosolic DT-diaphorase; i.e., 50.4 kat/kg protein. Microsomal DT-diaphorase is obtained with a yield of 45% and a specific activity of 15.5 kat/kg protein.Purified mitochondrial DT-diaphorase exhibits an absorption spectrum characteristic of a flavoprotein and very similar to that of the cytosolic enzyme. Purification of both mitochondrial and microsomal DT-diaphorase results in fractions enriched in a polypeptide with a molecular weight of 28,000 which comigrates with purified cytosolic DT-diaphorase on SDS-polyacrylamide gel electrophoresis. Employing antiserum raised against cytosolic DT-diaphorase, immunological identity between DT-diaphorase isolated from the three cell fractions is observed with both the Ouchterlony immunodiffusion technique and fused rocket immunoelectrophoresis. The latter method also reveals that DT-diaphorase isolated from mitochondria and microsomes contains several antigenic forms identical to those observed in purified cytosolic DT-diaphorase. Furthermore, this antiserum inhibits DT-diaphorase to about the same extent whether the enzyme is isolated from mitochondria, microsomes, or cytosol. In addition, this antiserum efficiently inhibits membrane-bound microsomal DT-diaphorase.     

Responses of calves to a simulated altitude of 5000 m

Six calves were exposed in succession 12 days at 400 m altitude (control), 12 days at a simulated altitude of 5000 m in a low pressure chamber (experimental), and 14 days at 400 m altitude (recovery). Exposure to 5000 m produced the following changes: intake of feed and water decreased by 47 and 35% respectively, and body weight gain ceased. Rectal temperature rose by 0.4°C. Heart rate increased by 65%. Respiratory rate and blood pH increased moderately. There was an S-shaped rise of haematocrit (from 33 to 45%), which was paralleled by blood viscosity. Plasma viscosity showed a sharp, but transient rise. Short term measurements made during ascent to and descent from 5000 m altitude, both lasting for four hours, showed that some of the changes developed rapidly.     

Constituents of human muscle in isometric fatigue.

Three subjects performed five successive isometric contractions to fatigue; the tension in any one experiment was constant at tensions varying from 20 to 80% of the maximal voluntary contraction (MVC). The interval between contractions was held constant at 11 min. Muscle biopsy specimens were obtained at the start of the experiment, after the first, fourth, and fifth, and before the second and fifth of the successive contractions. The concentrations of ATP, CP, glycogen, and lactate were measured in each sample of muscle. Changes in ATP and glycogen were insufficient to be held accountable for the development of isometric fatigue. Changes in CP and lactate were large after fatigue at intermediate tensions, but those of CP were considered unlikely to be responsible for the fatigue. At tensions of 30-50% MVC the increase in lactate could be responsible for fatigue either directly or by indirect changes in pH; at higher and lower tensions the possibility that lactate is directly implicated in the development of fatigue seems remote.     

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.