Yoji Suzuki

Specific cleavage of secretory leukoprotease inhibitor by neutrophil elastase and saliva

In an attempt to explore the process of naturally occurring secretory leukoprotease inhibitor (SLPI) fragmentation, the cleavage profile of SLPI, which had been prepared by recombinant techniques, was investigated biochemically. Restricted fragments of SLPI were detected using SDS-PAGE after treatment with human neutrophil elastase (NE) or normal saliva and sequenced at their cleavage sites. Among these restricted fragments, two species of nearly half-length SLPIs that contained the C-terminal domain, (Arg58-Ala107)SLPI and (Arg59-Ala107)SLPI, were detected. They were both as active at inhibiting NE as the parent SLPI. These results suggest that functional SLPI derivatives may be generated physiologically in the respiratory tract under inflammatory and healthy conditions.

Pharmacological activity of the C-terminal and N-terminal domains of secretory leukoprotease inhibitor in vitro

1 In order to characterize the physiological functions of the domain structure of secretory leukoprotease inhibitor (SLPI), the biological capacities of half‐length SLPIs, (Serl‐Pro54)SLPI and (Asn55‐Ala107)SLPI, were investigated and compared with those of full‐length SLPI. 2 The activities of these inhibitors against several serine proteases were determined using synthetic chromogenic substrates. The inhibitory capacity of the C‐terminal domain, (Asn55‐Ala107)SLPI, was as strong as that of full‐length SLPI against human neutrophil elastase (NE), cathepsin G and chymotrypsin. It possessed less trypsin inhibitory activity than intact SLPI. For the N‐terminal domain of SLPI, (Serl‐Pro54)SLPI, no inhibitory activity could be detected against the serine proteases tested in this study. 3 The inhibitory activity of (Asn55‐Ala107)SLPI against the proteolysis of the natural substrates elastin and collagen by NE was comparable with that of full‐SLPI (elastin, IC50 = 907±31 nM for SLPI, 767±33nM for (Asn55‐Ala107)SLPI; collagen, IC50 = 862 ± 36 nM for SLPI, 727 ±47 nM for (Asn55‐Ala107)SLPI). 4 The binding affinities of full‐ and half‐length SLPIs for heparin were measured by affinity column chromatography. Full‐length SLPI showed high affinity for heparin while the binding capacities of both half‐length SLPIs were lower. (Concentration of NaCl for elution, 0.45 m for SLPI, 0.24 m for (Serl‐Pro54)SLPI, 0.27 m for (Asn55‐Ala107)SLPI). 5 The effects of full‐SLPI and (Asn55‐Ala107)SLPI on blood coagulation were measured using the activated partial thromboplastin time (APTT). Full‐length SLPI prolonged clotting time dose‐dependently (1.25, 2.5 and 5.0 um), whereas (Asn55‐Ala107)SLPI had no effect even at the highest concentration. 6 In conclusion, the C‐terminal domain of SLPI is a promising candidate for the treatment of inflammatory diseases in which participation of neutrophil proteases has been suggested.

A new culturing method for production of filamentous hemagglutinin of Bordetella pertussis

Abstract The addition of cyclodextrins, especially heptakis (2,6- O -dimethyl)β-cyclodextrin (MeβCD), to synthetic medium, such as Stainer-Scholte medium (D.W. Stainer and M.J. Scholte, J. Gen. Microbiol. 63: 211–220) greatly stimulated the production of filamentous hemagglutinin (F-HA) by Bordetella pertussis Tohama phase I under shaking conditions. Purified F-HA from this culturing system was demonstrated immunochemically and morphologically to be identical with that from static culture.

Factors Involved in Beta-Lactam Antibiotic Resistance in Pseudomonas aeruginosa

A temperature-sensitive mutant, strain SS, which is assumed to be devoid of β-lactamase activity and deficient in a permeability barrier to antibiotics, was isolated from a β-lactamase-less mutant, strain L-2, derived from Pseudomonas aeruginosa IFO 3080. By comparing the β-lactam antibiotic susceptibility between strains IFO 3080, L-2, and SS, the involvement of both β-lactamase and a permeability barrier in determining the β-lactam antibiotic resistance was estimated.

Protective effects of human gamma globulin preparations against experimental aerosol infections of mice with Bordetella pertussis

The protective effects of three types of pooled human gamma-globulin preparations (intact: GG, S-sulfonated: GGS, and pepsin-treated: GGP) for intravenous use against experimental aerosol infection of mice with Bordetella pertussis have been evaluated. The gamma-globulin preparations GG and GGS showed significant protective activity whereas GGP did not, as evaluated by survival numbers, body weight gains and suppression of leucocytosis. GG and GGS but not GGP also possessed neutralizing activity against leucocytosis-promoting and histamine-sensitizing activities of pertussis toxin (PT). Evaluation of protective activities of GG, GGS and GGP prepared from rabbit gamma-globulin, highly immunized with PT and not containing any anti-F-HA (filamentous or agglutinin antibodies, demonstrated that anti-PT-GG and anti-PT-GGS but not anti-PT-GGP protected against experimental infection by B. pertussis. These studies showed that the protective activity was correlated with anti-PT titre but that the Fc portion of the gamma-globulin molecule is necessary for actual protection against whooping cough by B. pertussis.