Biological changes of Enterococcus faecalis in the viable but nonculturable state.

Enterococcus faecalis may enter a viable but nonculturable (VBNC) state under adverse conditions. E. faecalis, the major bacterial species present in failed root canal treatments, is thought to survive after endodontic treatment by entering a VBNC state. In this study, we characterized the VBNC state of E. faecalis. We designed 3 different protocols to successfully induce the VBNC state. Approximately one-third of bacteria entered a VBNC state after 15-30 days, and all remained viable for at least 2 months. The morphology, glycometabolism, and adhesion capabilities of VBNC cells differed from those of E. faecalis during the exponential growth phase. Specifically, VBNC E. faecalis cells could not decompose lactose, D-mannitol, or D-sorbitol, although they were able to metabolize sucrose. Transmission electron microscopy showed that the morphology of the VBNC E. faecalis cells changed significantly; the cytoplasmic matrix was unevenly condensed and the overall morphology of the cells became irregular, but the cell membranes remained intact. Although the adhesion ability of the bacteria decreased, VBNC E. faecalis could still adhere to collagen fiber type I and tooth dentine. The persistence of this adhesion ability may be important in the virulence of VBNC E. faecalis.

Tensile ductility and necking of metallic glass.

Metallic glasses have a very high strength, hardness and elastic limit. However, they rarely show tensile ductility at room temperature and are considered quasi-brittle materials. Although these amorphous metals are capable of shear flow, severe plastic instability sets in at the onset of plastic deformation, which seems to be exclusively localized in extremely narrow shear bands approximately 10 nm in thickness. Using in situ tensile tests in a transmission electron microscope, we demonstrate radically different deformation behaviour for monolithic metallic-glass samples with dimensions of the order of 100 nm. Large tensile ductility in the range of 23-45% was observed, including significant uniform elongation and extensive necking or stable growth of the shear offset. This large plasticity in small-volume metallic-glass samples did not result from the branching/deflection of shear bands or nanocrystallization. These observations suggest that metallic glasses can plastically deform in a manner similar to their crystalline counterparts, via homogeneous and inhomogeneous flow without catastrophic failure. The sample-size effect discovered has implications for the application of metallic glasses in thin films and micro-devices, as well as for understanding the fundamental mechanical response of amorphous metals.

The role of COOH-terminal and acidic domains in the activity and stability of human insulin receptor protein tyrosine kinase studied by purified deletion mutants of the beta subunit domain.

We have previously expressed the human insulin receptor beta subunit domain containing transmembrane and cytoplasmic domains (IRTMTPK) in insect cells, and showed that the purified IRTMTPK was highly active (Li, S. L., Yan, P.-F., Pax, I. B., and Fujita-Yamaguchi, Y. (1992) Biochemistry 31, 12455-12462). To investigate the role of COOH-terminal and acidic domains of the insulin receptor kinase, we have expressed deletion mutants IRTMTPK delta CT (delta 76 amino acids) and IRTMTPK delta Acid (delta 19 amino acids). Both enzymes were purified by a one-step method using the same immunoaffinity column as used for IRTMTPK. While Km and Vmax for prephosphorylated IRTMTPK and delta Acid mutant enzyme determined using poly(Glu, Tyr)(4:1) were similar, catalytic efficiency of the delta CT mutant enzyme was significantly lower than those of IRTMTPK and delta Acid mutant enzyme as judged by Km and Vmax. Experiments for thermostability and susceptibility to proteases revealed that Tm of delta CT mutant enzyme was 3.5 degrees C lower than that of IRTMTPK enzyme (= 33.3 degrees C) and that delta CT mutant enzyme was digested by either trypsin or Lys-C into a 28,000 core domain much faster than IRTMTPK. Activation of delta CT mutant enzyme by polylysine was less significant than that of IRTMTPK and delta Acid mutant enzyme, approximately 4-versus approximately 17-fold. These studies suggested that the COOH-terminal domain plays important roles in both catalytic efficiency and stability of the insulin receptor kinase, and that the acidic domain by itself is not responsible for kinase activation by polylysine.

Human insulin receptor beta-subunit transmembrane/cytoplasmic domain expressed in a baculovirus expression system: purification, characterization, and polylysine effects on the protein tyrosine kinase activity.

We have expressed, purified, and characterized the insulin receptor protein tyrosine kinase (PTK) retaining the transmembrane and downstream domains. The proteins expressed in insect cells using a baculovirus expression system were identified as membrane-bound by immunofluorescence staining and biochemical characterization. One-step purification by immunoaffinity chromatography from Triton X-100 cell extracts resulted in a approximately 360-fold increase in the specific kinase activity with a yield of approximately 50%. An appMr = approximately 60,000 protein was the major component identified by both silver staining of the purified enzyme and immunostaining of the crude extracts after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Using nondenaturing conditions, the molecular weight was estimated to be approximately 250,000 and approximately 500,000 by glycerol gradient centrifugation and gel permeation chromatography, respectively, suggesting that oligomers of the beta-subunit domains such as tetramers and octamers are formed. The basal PTK activity of this enzyme was much higher than those of previously reported soluble-form insulin receptor PTKs expressed in insect cells or the native receptor. Km and Vmax for two substrates, src-related peptide and poly(Glu, Tyr) (4:1), were 2.4 mM and 2.5 mumol min-1 mg-1 and 0.26 mM and 1.2 mumol min-1 mg-1, respectively. Specific activities measured under two previously reported conditions using histone H2B as a substrate were 100 or 135 nmol min-1 mg-1, in contrast to those of soluble PTKs which were reported to be 20 or 70 nmol min-1 mg-1, respectively. The purified enzyme was autophosphorylated at Tyr residues. Autophosphorylation activated the enzyme approximately 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Restriction endonucleases from three strains of Haemophilus influenzae.

The restriction endonucleases, Hin P1 I1), Hin S1 I and Hin S2 I are isolated from three strains of Haemophilus influenzae respectively. By polymin P treatment, ammonium sulphate, precipitation and column chromatography on phosphocellulose and on heparin-Sepharose Hin P1 I is partially purified. No contaminating deoxyribonuclease activities have been detected in this purified enzyme preparation. The fact that the digestion patterns of Hin P2 I and Hha I on phage lambda, plasmids ColE1 an pBR 322 DNAs are identical that they are isoschizomers but theri splitting sites are different. The banding patterns of Hin S1 I and Hin S2 I are also the same as that of Hha I.