[Studies on the step-wise extraction of BCG cell surface components and immunological properties of the extracts].
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Biomedical subjects
Publications and source records attributed to T Shimono.
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Addition of a lauroyl, stearoyl or docosanoyl group to the primary hydroxy group at the C-6 position of N-acetylmuramyl-L-alanyl-D-isoglutamine gave lipophilic derivatives that had definite adjuvancies in induction of delayed-type hypersensitivity and enhancement of antibody production against a test protein antigen, ovalbumin, when administered to guinea pigs as liposomes, that is without mineral oil. When administered as mineral oil-in-water emulsion, including Ribitype emulsions, rather than as water-in-mineral oil emulsions, N-acetylmuramyl-L-alanyl-D-isoglutamine and its 6-O-acyl derivatives showed only weak immunoadjuvancies.
1. In lightly nembutalized cats, effects of high frequency (60-100/sec) repetitive stimulation of the midbrain reticular formation (RF) were tested upon the thalamo-cortical (T-C) neurones which project from the anterior ventral (VA) nucleus of the thalamus and its vicinities to the parietal association cortex and convey impulses responsible for cortical recruiting and spindling-like responses. 2. Tonic maintained or rhythmic grouped firings of the T-C neurones recorded as extracellular units with microelectrode in the thalamus were in the majority suppressed by high frequency RF stimulation during and often for a short time after the stimulation, and they showed later tonic discharges for many seconds or minutes. Intracellular recording from the T-C neurones revealed hyperpolarizing potential changes corresponding to the suppression on high frequency RF stimulation. Field potential analysis in the VA nucleus indicated that the hyperpolarization is ascribed at least in part to IPSPs elicited in the T-C neurones. Responses in some other types of thalamic neurones to the RF stimulation were exemplified to be compared and related with those of the T-C neurones. 3. Desynchronization of the parietal electrocorticogram coincided with the suppression of the T-C neurones and lasted for the time of the later tonic discharges, which contrasted with the rhythmic grouped discharges of the T-C neurones in association with recruiting and spindling-like responses of the cortex. Relations between the thalamo-cortical recruiting system and the ascending reticular activating system were discussed.
(1) Functional commencements of the neuronal elements in the cerebellar cortex of young rats were studied electrophysiologically by means of laminar field potential analyses in the cortex on stimulation of the cerebellar surface (Loc) and the white matter (WM). (2) The antidromic action potential of Purkinje cells on the WM stimulation was observed at one day after birth. The climbing fiber excitation of Purkinje cells on the stimulation was noted at 3 days after birth. (3) The mossy fiber-granule cell synapses were found to function at 10 days after birth and the Golgi cell inhibition of granule cells could be proved at the same time. The excitatory action of parallel fibers and the inhibitory action of basket-stellate cells on Purkinje cells appeared simultaneously at about 12 days after birth. The transverse distribution across the cerebellar folium of the basket-stellate cell inhibitory action on Purkinje cells was found to be narrow up to 60 days after birth. (4) These results concerning the dates of commencement of excitatory and inhibitory synaptic actions in the cerebellar cortex were compared with those of synaptogenesis studied morphologically, and some implications of the dates in the functional development of the cerebellar cortex were discussed.
Cell walls of Mycobacterium smegmatis were able to produce much more severe arthritis in rats than the delipidated cells, whereas cell envelope and cell membrane fractions were unable to produce the disease. The lysozyme-solubilized product was able to produce mild disease with only 30% of incidence with an optimum dose, whereas the higher and the lower doses did not produce the disease. The rats immunized with cell envelope, cell membrane fraction and nonarthritogenic doses of lysozyme-solubilized product were protected against the subsequent homologous and heterologous challenge of delipidated cells. It was discussed that this preventative effect can be the result of antigenic competition between the arthritogenic and nonarthritogenic components of M. smegmatis. On the other hand, all the fractions separated here were able to serve as an immunoadjuvant in terms of inducing delayed hypersensitivity to ovalbumin in guinea pigs.
Bayol F preparations from different commercial sources were incorporated into water-in-oil emulsions containing mycobacterial fractions and a protein antigen. It was found that an adjuvant effect was obtained with some mineral oils and a toxic immunosuppressive effect with others.
A total of 14 different N-acetylmuramyl-peptides or -amino acids with or without configurations inherent to bacterial cell wall peptidoglycans were synthesized and their pyrogenicities on intravenous injection into rabbits were tested. N-Acetylmuramyl-peptides, and especially N-acetylmuramyl-L-alanyl-D-isoglutamine and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-lysine, which were previously shown to be adjuvant-active in both induction of delayed-type hypersensitivity and stimulation of increased serum antibody levels to ovalbumin in guinea pigs, exhibited distinct pyrogenicity at as low dose as 16 mug per rabbit. However, none of the adjuvant-inactive analogues or diastereomers of the above N-acetylmuramyl-dipeptide or related compounds caused any significant febrile response in rabbits, even at a dose of 250 mug per animal.
1. Responses evoked in the cerebellar cortex by stimulation of the parietal association cortex (rostral portions of the middle suprasylvian gyrus) were recorded and analysed in cats, and were compared with those by stimulation of the motor cortex (anterior sigmoid gyrus). 2. The parietal stimulation elicited early mossy fibre and late climbing fibre responses in the cerebellar cortex. The mossy fibre responses appeared at a latency of 2.0--2.5 msec and predominantly in the lateral (hemispherical) part of the contralateral cerebellum (mainly crus I, crus II and paramedian lobules). Cutting of the inferior cerebellar peduncle produced little or no influence upon the mossy fibre responses, which suggests that the mossy fibre responses are mediated chiefly by the pontine nuclei. 3. The climbing fibre responses were recorded at a latency of 17--19 msec and markedly in the contralateral intermediate and medial parts of IV--VI lobules. The responses were easily sppressed by anaesthesia and depended on the conditions of experimental animals. The unstable appearance of the responses and their longer latencies than those of the climbing fibre responses due to stimulation of the motor cortex imply indirect pathways from the parietal association cortex to the inferior olive. 4. The predominant projection of the parietal-induced mossy fibre responses to the lateral part of the cerebellum was compared with the mossy fibre projection from the motor cortex and was discussed as an important component in the cerebrocerebellar loops.
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A variety of N-acetylmuramyl-peptides (or -amino acids) were prepared by condensation of benzyl N-acetyl-4, 6-O-brnzylidene-alpha-muramide with various peptide (or amino acid) benzyl esters by the dicyclohexylcarbodiimide--N-hydroxysuccinimide or ethylchlorocarbonate--N-methylmorpholine method and removal of the protecting groups by hyderogenolysis. N-Acetylmuramyl-L-alanyl-D-isoglutamine was identified as the minimum structural entity essential for the immunoadjuvant activities characteristic of bacterial cell walls. Consequently N-acetylmuramyl-L-alanine was not adjuvant active. The tetrapeptide portion of adjuvant-active N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-lysyl-D-alanine proved to be inert, at least in induction of delayed-type hypersensitivity. The possible adjuvant activities of various analogues or diastereomers of the above N-acetylmuramyl-dipeptide and related compounds were studied. N-Acetylmuramyl-L-alanyl-D-glutamic acid exhibited weak, but definite adjuvancy, but none of the others, including N-acetylmuramyl-L-alanyl-L-isoglutamine, N-acetylmuramyl-L-alanyl-D-glutamine and N-acetylmuramyl-L-alanyl-D-isoasparagine, had any adjuvant activity. This clearly indicated the importance of the configuration of the glutamic acid residue or its amides, i.e. the presence of the D-isoglutamine residue in the N-acetylmuramyl-dipeptide, for manifestation of adjuvant activities in stimulation of both antibody-mediated and cell-mediated immune responses. Neither N-acetylmuramyl-D-isoglutamine nor N-acetylmuramyl-D-alanine had any adjuvancy.
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The cell walls from all 21 species of gram-positive bacteria examined, except lysozyme-susceptible Micrococcus lysodeikticus (NCTC 2665) and lysozyme-resistant Staphylococcus epidermidis (ATCC 155), were found to be definitely adjuvant-active in both stimulation of increased serum antibody levels and induction of delayed-type hypersensitivity to ovalbumin when administered to guinea-pigs as water-in-oil emulsions. Using various cell wall lytic enzymes, the immunoadjuvant principles were solubilized with full retention of the adjuvant activities from walls of Staphylococcus aureus (Copenhagen), Streptococcus pyogens (group A, type 6; S43/100), Streptococcus salivarius (IFO 3350), Streptococcus faecalis (IFO 12580), Streptococcus mutans (BHT), Lactobacillus plantarum (ATCC 8014), Bacillus megaterium (IFO 12068), Corynebacterium diphtheriae (Park-Williams No. 8), Mycobacterium smegmatis, and Actinomyces viscous (ATCC 15987). Evidence was obtained that the non-peptidoglycan portion of the cell walls is not essential for manifestation of immunoadjuvancy.
Attempts were made to isolate and identify the unit chemical structure essential for manifestation of the immunoadjuvant activities characteristic of bacterial cell walls. The N-acetylmuramyl-peptide subunit monomers, Nalpha-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-Nepsilon-(glycylglycyl)-L-lysyl-D-alanine from the cell walls of Staphylococcus aureus (FDA 209P) and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid and/or N-acetylmuramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine from those of Lactobacillus plantarum (ATCC 8014), were shown to be unit chemical entities with definite adjuvant activity both in stimulation of antibody production and in induction of delayed-type hypersensitivity to ovalbumin when administered to guinea-pigs as water-in-oil emulsions.
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