Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Age-related differences in muscle activity, stride frequency and heart rate response during walking in water.

This study aimed to examine whether walking in water produces age-related differences in muscle activity, stride frequency (SF), and heart rate (HR) response. Surface electromyography (EMG) was used to evaluate muscle activities in six older and six young subjects while they walked in water immersed to the level of the xiphoid process. The trials in water utilized the Flowmill which consists of a treadmill at the base of a water flume. The measurement of maximal voluntary contraction (MVC) of each muscle was made prior to the gait analysis. The %MVCs, which refer to the surface EMG measures, from the gastrocnemius of the older subjects were significantly lower than those of the young subjects, in every experimental condition (P<0.05). In contrast, the %MVCs from the rectus femoris (P<0.05) and the biceps femoris (P<0.001) of older subjects were significantly greater than those of young subjects in every experimental condition. Moreover, the SFs of older subjects were also significantly greater than those of young subjects (P<0.05), while the HR responses of older and young subjects were similar. In conclusion, the older subjects had increased hip musculature activity and decreased ankle plantar flexor activity while walking in water, compared with the young subjects.     

Metabolism of 32-hydroxy-24,25-dihydrolanosterol by purified cytochrome P-45014DM from yeast. Evidence for contribution of the cytochrome to whole process of lanosterol 14 alpha-demethylation

Metabolism of 32-hydroxy-24,25-dihydrolanosterol (lanost-8-ene-3 beta,32-diol), a posturated intermediate of the 14 alpha-demethylation (removal of C-32) of 24,25-dihydrolanosterol (lanost-8-en-3 beta-ol), by a reconstituted system consisting of yeast cytochrome P-450 which catalyzes lanosterol 14 alpha-demethylation (cytochrome P-45014DM) (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660 and Aoyama, Y., Yoshida, Y., and Sato, R. (1984) J. Biol. Chem. 259, 1661-1666) and NADPH-cytochrome P-450 reductase was studied. The reconstituted system converted both 32-hydroxy-24,25-dihydrolanosterol and 24,25-dihydrolanosterol to 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, the 14 alpha-demethylated product of the latter. The metabolism of these compounds was inhibited by a low concentration of ketoconazole which is a potent cytochrome P-45014DM inhibitor. Affinity of cytochrome P-45014DM for 32-hydroxy-24,25-dihydrolanosterol was about 20 times higher than for 24,25-dihydrolanosterol and the cytochrome metabolized the former about 4 times faster than the latter under the experimental conditions. Spectral analysis suggested that the 32-hydroxyl group of 32-hydroxy-24,25-dihydrolanosterol interacted with the heme iron of the oxidized cytochrome and this interaction might support the high affinity of this compound for the cytochrome. These lines of evidence indicate that 32-hydroxy-24,25-dihydrolanosterol is the intermediate of the 14 alpha-demethylation of 24,25-dihydrolanosterol by cytochrome P-45014DM. It is also clear that the cytochrome catalyzes further metabolism of the 32-hydroxylated intermediate to the 14 alpha-demethylated product with higher efficiency than the 32-hydroxylation of the substrate. Cytochrome P-45014DM is thus classified as lanosterol C14-C32 lyase.     

Complete nucleotide sequence and characterization of a cryptic plasmid from Lactobacillus helveticus subsp. jugurti

A small cryptic plasmid, pLJ1, was isolated from Lactobacillus helveticus subsp. jugurti and was cloned into Escherichia coli HB101 by using pBR329 as a vector. Plasmid pLJ1 was 3,292 base pairs long and had single restriction endonuclease sites for PvuII, KpnI, AvaII, Acci, HindIII, and EcoRI. In a maxicell system, pLJ1 produced a protein of about 41 kilodaltons.     

cDNA-directed expression of rat testosterone 7 alpha-hydroxylase using the modified vaccinia virus, T7-RNA-polymerase system and evidence for 6 alpha-hydroxylation and delta 6-testosterone formation

The modified vaccinia virus, T7-RNA-polymerase cDNA-expression system was used to express rat cytochrome P-450a. Various parameters such as host-cell type and density, and duration of infection were tested to optimize the level of expression of cytochrome P-450a enzyme activity. Cytochrome P-450a expressed from the cDNA sequence was exclusively incorporated into the membrane-containing portions of the cell lysates, as expected from its normal association in the liver endoplasmic reticulum. The enzyme displayed a carbon-monoxide-reduced-cytochrome-P-450a difference spectrum with a Soret maximum of 450 nm. Activity measurements revealed that cytochrome P-450a produced three metabolites of testosterone; 7 alpha-hydroxytestosterone and 6 alpha-hydroxytestosterone and delta 6-testosterone at a ratio of about 38:1:1. Under the appropriate conditions, the vaccinia-virus, T7-RNA-polymerase system produces high levels of a single form of cytochrome P-450 in cells that are virtually devoid of endogenous cytochrome P-450. Analysis of the cytochrome P-450 in its natural membrane-bound state, as opposed to artificial-lipid reconstitution studies of purified enzymes, allows accurate and confident measurements of substrate specificities.     

The primary structure of rat ribosomal protein L18a

The amino acid sequence of rat ribosomal protein L18a was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L18a contains 175 amino acids and has a molecular mass of 20,047 Da. Hybridization of the cDNA to digests of rat nuclear DNA and to a preparation of poly(A)+ mRNA suggests that there are 8-11 copies of the L18a gene and that the mRNA for the protein is about 700 nucleotides in length. Rat L18a is related to Schizosaccharomyces pombe L17 and perhaps to Halobacterium marismortui L19.     

A single amino acid substitution converts cytochrome P450(14DM) to an inactive form, cytochrome P450SG1: complete primary structures deduced from cloned DNAS

Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.     

Freeze denaturation of enzymes and its prevention with additives

Freeze inactivation of LDH, MDH, ADH, G-6-PDH, and PK and its prevention with additives such as sodium glutamate and albumin were studied. LDH, MDH, ADH, G-6-PDH, and PK, each lost their activity during frozen storage at -20 degrees C. The speed of the inactivation differed in each. The stability of the enzymes increased with the increase of the enzyme concentration. Sodium glutamate and albumin prevented the freeze inactivation. While the activity of the LDH solution frozen without additives was almost lost during a day of frozen storage, those frozen with either glutamate (0.2 M) or albumin (0.1%) added decreased less quickly. The residual activity after 1 day was 50% the initial prefreeze value for the former and 10% for the latter, respectively. Combined use of glutamate and albumin prevented the inactivation the best and maintained the initial activity almost completely over 6 weeks. The enzymes tested lost some part of their activity when their solutions were diluted by the media. This inactivation was prevented to a significant extent by the addition of sodium glutamate and/or albumin to the diluting media.