The Purification of a GroEL-Like Stress Protein from Aerobically Adapted Campylobacter jejuni

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion-exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star-shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N-terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross-reactive with the antiserum for the 60-kDa HSP of Yersinia enterocolitica. Production of the 62-kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62-kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.     

Chromatin arrangements in intact interphase nuclei examined by laser confocal microscopy

Using a laser confocal microscope, chromatin arrangements in intact interphase nuclei were investigated in four plant species. Chromosomes in these plants have specific segments that can be stained with the fluorescent dye chromomycin A₃ (CMA). We stained centromeres inHordeum vulgare, sub-telomeric regions inSecale cereale, satellites inChrysanthemum multicore, and the satellites and the short arms of chromosomes with satellites inHemerocallis middendorfii. The following points were shown: (1) In mitotic interphase nuclei, the centromere and the telomeres of both arms touched the nuclear membrane and had evident polarity. Some CMA-bodies in sub-telomeric regions do not contact the nuclear membrane. (2) Differentiated nuclei had a non-random construction. Polarity of chromosomes is maintained, however, the chromosomes are far apart from the nuclear membrane. (3) Associations in sub-telomeric regions in the interphase nuclei ofSecale cereale were probably due to the association of heterochromatic regions with identical repeated sequences rather than telomere associlations. (4) In interphase nuclei ofChrysanthemum multicore, satellites fused during interphase.     

N-2′-Acetoxybenzoyl derivatives of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose

N-2′-Acetoxybenzoyl (aspirin) derivatives (degree of substitution 0·35–1·00) of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose were prepared by methods that gave yields in the range 65–86%. The salicylate of chitosan was isolated with a 98% yeild. Aspirin or salicylic acid was released much more slowly from N-(2′-acetoxybenzoyl)-chitosan than from the salicylate of chitosan, and much faster at 37°C in 0·1 m NaOH solution than in 2% aqueous acetic acid solution. Salicylic acid was isolated from the dialysate (0·1 m NaOH solution) of N-(2′-acetoxybenzoyl)-chitosan.     

Whole-cell-mount cytochemistry by the colloidal gold labeling method

Summary Binding, redistribution, and endocytosis of colloidal gold (CG)-labeled concanavalin A (ConA) were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mouse peritoneal macrophages were cultured on Formvar-coated platinum grids. Either fixed or unfixed cells were labeled by the indirect ConA-CG labeling method. Specimens were critical-pointdried and observed by TEM and SEM in the same region. Surface-bound ConA-CG was easily seen by SEM. Stereomicroscopic observation by TEM clearly showed the threedimensional distribution of ConA on the cell surface as well as in the cytoplasmic vesicles and vacuoles. In the prefixed cells, CG was distributed randomly on the cell surface. When unfixed cells were labeled at 0° C, a similar binding pattern was observed, although the density of bound CG was decreased. When cells labeled with ConA-CG at 0° C were further incubated at 37° C, redistribution and endocytosis of the label were seen. Endocytosed CG in the cytoplasmic vesicles and vacuoles was clearly seen by TEM. In addition, three-dimensional location and relationship with other organelles were easily observed. Combined TEM and SEM observation of CG-labeled whole-cell-mount specimens is a useful method to study the dynamics of cellbound ligands.This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan, and the Japan Private School Promotion Foundation     

Monoacylcadaverines in the blood of schizophrenic patients

Concentrations of cadaverine, monoacetylcadaverine and monopropionylcadaverine in the blood of schizophrenic and nonschizophrenic subjects were measured. Two groups, one from the U.S.A. the other from Japan, were tested. Monoacetylcadaverine and monopropionylcadaverine were found elevated in the blood of some schizophrenic patients in comparison with those in controls in each group. Their increase could be caused by a reduced monoamine oxidase activity or by an increased acylation in schizophrenic patients.     

The effects of N-substitution of chitosan and the physical form of the products on the rate of hydrolysis by chitinase from Streptomyces griseus

N-Formyl, N-chloroacetyl, N-glycyl, N-isobutyryl, and N-pentanoyl derivatives of chitosan have been prepared. N-Acetylchitosan was the derivative most susceptible to chitinase from Streptomyces griseus and lysozyme from chicken egg-white, but the susceptibility was not restrictive. The relative rates of hydrolysis by chitinase with respect to R in the RCONH group were CH₃ > CH₃CH₂ > H > CH₃CH₂CH₂ > (CH₃)₂CH > NH₂CH₂ > ClCH₂. Neither enzyme hydrolysed chitosan or its N-methylene, N-benzylidene, N-benzoyl, N-nicotinyl, and N-fatty acyl (C₅C₁₈) derivatives, and lysozyme did not hydrolyse N-butyrylchitosan. N-Acetylhexanoyl-chitosans, which had d.s. ratios of ~0.7: ~0.3 and ~0.3; ~0.7, were hydrolysed at ~0.75 and ~0.04 of the rate of N-acetylchitosan (powder) by chitinase. O-Acylation of N-acylchitosans caused a decrease in the rates of hydrolysis by chitinase. N-Acetylchitosan gels were hydrolysed at 8–13 times the rate for crab-shell chitin. These results indicate that not only N- and O-substituents but also the physical form of the substrates influence the rates of hydrolysis by these enzymes.     

Aminoacylase pellets.

Aminoacylase was immobilized on the mycelium pellets of Aspergillus ochraceus by using albumin and glutaraldehyde. No difference in the optimum pH was observed between native aminoacylase and aminoacylase pellets. The aminoacylase pellets were stable in pH 4-8 but they were unstable in alkaline conditions. The aminoacylase pellets were more stable against heavy metal ions and inhibitors than native aminoacylase. However, the degree of the activation of aminoacylase with cobalt ion decreased with the immobilization. It was suggested that most of aminoacylase was covalently coupled to the mycelium with glutaraldehyde.     

Resolution of anomeric 2-amino-2-deoxy-d-glycofuranosides and -glycopyranosides by cation-exchange chromatography

Methyl 4,6-O-benzylidene-2-deoxy-α-D-ribo-hexopyranoside (1) is converted into methyl 3,4-di-O-benzoyl-6-bromo-2,6-dideoxy-α-D-ribo-hexopyranoside (3) via the 3-O-benzoyl derivative (2) of 1 by subsequent treatment with N-bromosuccinimide. Compound 3 is the key intermediate in high-yielding, preparative syntheses of the title dideoxy sugars, which are constituents of many antibiotics. Dehydrohalogenation of 3 affords the 5,6-unsaturated glycoside 7. which undergoes stereospecific reduction by hydrogen with net inversion at C-5 to give methyl 3,4-di-O-benzoyl-2,6-dideoxy-β-L-lyxo-hexopyranoside (8), whereas reductive dehalogenation of 3 provides the corresponding D-ribo derivative 4. The unprotected glycosides 9 (L-lyxo) and 5 (D-ribo) are readily obtained by catalytic transesterification, and mild, acid hydrolysis gives the crystalline title sugars 10 (L-lyxo) and 6 (D-ribo) in 45 and 57% overall yield from 1 without the necessity of chromatographic purification at any of the steps.