Immunochemical comparison between an oxacillin-hydrolyzing penicillinase of Aeromonas hydrophila and those mediated by R plasmids.

Antiserum against an oxacillin-hydrolyzing penicillinase of Aeromonas hydrophila did not show immunochemical cross-reaction with four oxacillin-hydrolyzing penicillinases mediated by R plasmids.     

Studies on {partial}-amino levulinic acid deydratase in radish cotyledons during chloroplast development

The subcellular localization and biosynthetic site of 8-aminolevulinic acid dehydratase [EC 4.2.1.24 [EC] , ALAD] were investigatedin relation to chloroplast development in radish cotyledons. ALAD was mainly located in the chloroplasts and cytoplasm. Mostof the ALAD in the chloroplasts was readily released by hypotonicshock. The enzyme was also found in the proplastids of etiolatedcotyledons. The normal increase in the activity of ALAD in the chloroplastsas well as the cytoplasm was inhibited by cycloheximide butunaffected by D-threo chloramphenicol and kanamycin during thegreening of radish cotyledons. We concluded that the ALAD inboth the cytoplasm and chloroplasts was synthesized on the cytoplasmic80S-ribosomes. This suggests that the ALAD formed on the 80S-ribosomesmight be incorporated into chloroplasts during their development. When etiolated radish seedlings were illuminated, ALAD in boththe cytoplasm and chloroplasts increased up to the point ofthe full development of the chloroplasts, and thereafter itdecreased. (Received August 20, 1975; )     

Plasminogen with type-I mutation is polymorphic in the Japanese population

Summary A functionally inactive plasminogen (PLG) variant designated as PLG M5 is polymorphic in the Japanese population and has a feature common to PLG with type-I mutation that has a codon 601 missense mutation in exon 15 (GCT for AlaACT for Thr). This study was conducted to clarify whether the type-I mutation of PLG is present in PLG M5 and polymorphic in the Japanese population. Direct sequencing of the amplified DNA from the PLG gene in a heterozygote for PLG M5 revealed that the sequence of the exon 15 in the gene for PLG M5 is identical with that in the PLG gene with type-I mutation. In addition, the amplified DNA from the PLG gene in 12 heterozyotes for PLG M5 reacted with the probe for the type-I mutation in dot blot hybridization with an allele-specific oligonucleotide probe. The heterozygote for PLG with type-I mutation was found in 2.2% of 360 unrelated healthy subjects. These data indicate that the type-I mutation of PLG is present in PLG M5 and polymorphic in the Japanese population. The data also suggest that the PLG M5 is identical with PLG Tochigi and Kagoshima.     

Calcium-induced formation of chlorophyll b and light-harvesting chlorophyll complex in cucumber cotyledons in the dark

Dark-grown cucumber seedlings were exposed to intermittent light (2 min light and 98 min dark) and then cotyledons were incubated with 50 mM CaCl₂ in the dark. Chlorophyll (Chl) a was selectively accumulated under intermittent light and Chl b was accumulated during the subsequent dark incubation with CaCl₂. The change in chlorophyll-protein complexes during Chl b accumulation induced by CaCl₂ in the dark was investigated by SDS-polyacrylamide gel electrophoresis. Chlorophyll-protein complex I and free chlorophyll were major chlorophyll-containing bands of the cotyledons intermittently illuminated 10 times. When these cotyledons were incubated with CaCl₂ in the dark, the light-harvesting Chl complex was formed. When the number of intermittent illumination periods was extended to 55, small amounts of Chl b and light-harvesting Chl complex were recognized at the end of intermittent light treatment, and these two pigments were further increased during the subsequent incubation of the cotyledons with CaCl₂ in the dark compared to water controls.     

Calpain activation in shear-induced platelet aggregation

Fluid shear stress has been known to activate platelet reaction such as aggregation, but the exact mechanism of shear-induced platelet aggregation (SIPA) has not been fully understood. Calpain, an intracellular calcium-activated cysteine protease, is abundant in platelets and is considered to be activated and involved in the proteolytic processes during platelet activation. A possible activation of calpain in SIPA was investigated, employing a newly developed aggregometer and specific monoclonal antibodies to detect activation of calpain. When a shear stress gradient varying between 6 and 108 dyn/cm² was applied to platelets, activation of μ-calpain was observed only in high-shear-stressed platelets, resulting in the proteolysis of talin. At 1 min after the onset of constant high shear stress of 108 dyn/cm², μ-calpain activation and proteolysis of talin were detected and increased in a time-dependent manner. Constant shear stress more than 50 dyn/cm², applied for 5 min, caused μ-calpain activation and proteolysis of talin, which were increased in a shear-force-dependent manner. Calpeptin, a calpain-specific peptide antagonist, caused the complete inhibition of both μ-calpain activation and proteolysis of talin, while SIPA profiles with calpeptin showed almost no change compared to those without calpeptin. These results suggest the possibility of calpain involvement in late phases of shear-induced platelet activation such as cytoskeletal reorganization. J. Cell. Biochem. 66:54–64, 1997. © 1997 Wiley-Liss, Inc.     

Structural changes of alpha2- and ovomacroglobulins

The plasma ₂-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f ₀ of human 2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f ₀ and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as ₂-macroglobulin.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Alternate fast and slow stepping of a heterodimeric kinesin molecule

A conventional kinesin molecule travels continuously along a microtubule in discrete 8-nm steps. This processive movement is generally explained by models in which the two identical heads of a kinesin move in a 'hand-over-hand' manner. Here, we show that a single heterodimeric kinesin molecule (in which one of the two heads is mutated in a nucleotide-binding site) exhibits fast and slow (with the dwell time at least 10 times longer than that of the fast step) 8-nm steps alternately, presumably corresponding to the displacement by the wild-type and mutant heads, respectively. Our results provide the first direct evidence for models in which the roles of the two heads alternate every 8-nm step.     

Aging-associated B7-DC+ B cells enhance anti-tumor immunity via Th1 and Th17 induction

Because most patients with cancer are aged and because immunological functions are altered during aging, it is important to account for aging-associated immunological alterations in the design of new cancer immunotherapies. We thus compared immune populations in young and aged mice and found that B7-DC(+) (PD-L2/CD273) B cells, a minor population in young mice, were significantly increased in aged mice. Induction of both Th1 and Th17 cells was significantly augmented by B7-DC(+) B cells from aged mice, and this effect was blocked with anti-B7-DC antibodies in vitro and in vivo. Moreover, retardation of tumor growth in aged mice was largely B7-DC dependent. Tumor growth in young mice was significantly inhibited by immunization with B7-DC(+) B cells from aged mice owing to increased induction of tumor antigen-specific cytotoxic T lymphocytes. These data indicate that B7-DC(+) B cells could play an important role in aging-associated cancer immunopathology as well as in other aging-associated diseases and further suggest that B7-DC(+) B cells have potential for future cancer immunotherapy.