Glycemic response and fiber content of some foods.

The proposed mechanisms for the action of dietary fiber (DF) on the glycemic response suggest a nonlinear relationship between glycemic index (GI) and DF content. This relationship was analyzed by using the newly reported total DF (TDF) values, assuming a nonlinear regression curve. The empirical equation obtained was Y = 19.9X-0.322. Similar regression curves were also obtained for soluble DF (SDF) and insoluble DF (IDF). The two regression curves indicated that the correlation coefficient between observed GI and GI calculated from the IDF regression curve (-0.781) was virtually identical to that for SDF (-0.780), but a given content (eg, 5% vs available carbohydrate) of SDF gave a lower GI (39) than did IDF (48). This stronger dependency of GI on SDF suggests a major function of SDF in the TDF hypoglycemic effect. From the regression curve of GI vs TDF, we propose a supplemental GI to predict the glycemic response to foods with no published GI.

Quinolone antimicrobial agents substituted with morpholines at the 7-position. Syntheses and structure-activity relationships.

A series of novel 7-substituted 1-cyclopropyl-6,8-difluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acids have been prepared and tested for antibacterial activities and for convulsive activities in combination with nonsteroidal antiinflammatory drug. Structure-activity relationships revealed that 7-(2-(aminomethyl)morpholino) derivative 28 had a better Gram-positive activity than the reference quinolones, such as ciprofloxacin, norfloxacin, and ofloxacin. Its Gram-negative activity was equipotent with those of norfloxacin and ofloxacin but was inferior to that of ciprofloxacin. In mouse systemic infection models, 28 showed an excellent therapeutic efficacy which might result from the potent antibacterial activity and suitable physicochemical properties. Convulsive activities of 7-morpholino derivatives in combination with nonsteroidal antiinflammatory drug fenbufen or its metabolite biphenylacetic acid markedly diminished as compared to those of 7-piperazino derivatives in the electrophysiological, biochemical, and behavioral experiments. These results suggest that 28 (Y-26611) is a novel quinolone with reduced neurotoxic excitatory adverse reaction.

Antagonists of GABA responses, studied using internally perfused frog dorsal root ganglion neurons.

Responses of frog dorsal root ganglion neurons to GABA were studied under conditions of internal perfusion. Conductances to Na, Ca and K were pharmacologically blocked, C1 concentrations were maintained equal on both sides of the membrane and a small holding potential was used. Under these conditions GABA-induced C1 currents could be studied in isolation without shifts in EC1 occurring after GABA application. GABA currents were blocked by a variety of agents. The blockade by bicuculline and Zn was competitive, while that to penicillin was competitive at low concentrations (6 x 10(-5) M) and non-competitive at high concentrations (3 x 10(-4) M). Picrotoxin was non-competitive at all concentrations studied. The time course of the GABA-induced currents was changed in the presence of antagonists, including those that were competitive. These actions appear to be due to a change in the rates of receptor desensitization rather than shifts in EC1. Pretreatment with antagonists increased the degree of inhibition only for picrotoxin as compared to simultaneous application of GABA plus antagonist. The voltage dependence of the GABA response was altered by penicillin but not by other antagonists. GABA responses on frog dorsal root ganglion cell were also depressed by a variety of other metal ions (Cd, Ni, Cu, Co, Mn) and other drugs (strychnine, curare, 4-acetamide, 4'-isothiocyano-stilbene-2,2'-dilsulfonic acid disodium salt, 4,4'-diisothiocyano-stilbene-2,2'-dilsulfonic acid disodium salt trihydrate, bemegride and folic acid). In this preparation bicuculline and the heavy metal ions appear to block at or very near to the agonist binding site, while penicillin probably blocks the ion channel. The non-competitive action of picrotoxin appears not to be channel blockade, but to be due to a slowly equilibrating action at a site different from either the agonist binding site or the channel.

Blockade of 5-HT3 receptor-mediated currents in dissociated frog sensory neurones by benzoxazine derivative, Y-25130.

1. The effect of Y-25130, ((+-)-N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4-dih ydr o- 2H-1,4-benzoxazine-8-carboxamide hydrochloride), a high affinity 5-hydroxytryptamine3 (5-HT3) receptor ligand, was examined on the 5-HT-induced response in dissociated frog dorsal root ganglion (DRG) neurones by use of the extremely rapid concentration-jump ('concentration-clamp') and the conventional whole-cell patch-clamp techniques. 2. 5-HT induced a rapid transient inward current associated with an increase in membrane conductance at a holding potential of -70 mV. The current amplitude increased sigmoidally as 5-HT concentration increased. The half-maximum value (Ka) and the Hill coefficient estimated from the concentration-response curve were 1.7 x 10(-5) M and 1.7, respectively. 3. The current-voltage (I-V) relationship of 5-HT-induced current (I5-HT) showed inward rectification at potentials more positive than -40 mV. The reversal potential (E5-HT) was -11 mV. The E5-HT value was unaffected by total replacement of intracellular K+ by Cs+, indicating that the 5-HT-gated channels might be large cation channels. 4. Both the activation and inactivation phases of I5-HT were single exponentials. The time constants of activation and inactivation (tau a and tau i) decreased with increasing 5-HT concentration. 5. The 5-HT response was mimicked by a selective 5-HT3 receptor agonist, 2-methyl-5-HT, but the maximum response induced was approximately 25% that of 5-HT. The 5-HT response was reversibly antagonized by the 5-HT3 receptor antagonists, ICS 205-930, metoclopramide and Y-25130, but not by a 5-HTIA receptor antagonist, spiperone, and a 5-HT2 receptor antagonist, ketanserin. The half-inhibition concentrations (IC50) were 4.9 x 10-10 M for Y-25130, 4.8 x 10-10 M for ICS 205-930 and 8.6 x 10-9 M for metoclopramide.6. Y-25130 (5 x 10-10 M) caused a rightward shift of the concentration-response curve for 5-HT while decreasing the maximum response.7. The results suggest that Y-25130 is a potent antagonist of the 5-HT3 receptor-channel complex.

Non-competitive inhibition of GABAA responses by a new class of quinolones and non-steroidal anti-inflammatories in dissociated frog sensory neurones.

1. The interaction of a new class of quinolone antimicrobials (new quinolones) and non-steroidal anti-inflammatory agents (NSAIDs) with the GABAA receptor-Cl- channel complex was investigated in frog sensory neurones by use of the internal perfusion and 'concentration clamp' techniques. 2. The new quinolones and the NSAIDs (both 10(-6)-10(-5) M) had little effect on the GABA-induced chloride current (ICI) when applied separately. At a concentration of 10(-4) M the new quinolones, and to a lesser degree the NSAIDs, produced some suppression of the GABA response. 3. The co-administration of new quinolones and some NSAIDs (10(-6)-10(-14) M) resulted in a marked suppression of the GABA response. The size of this inhibition was dependent on the concentration of either the new quinolone or the NSAID tested. The inhibitory potency of new quinolones in combination with 4-biphenylacetic acid (BPAA) was in rank order norfloxacin (NFLX) much greater than enoxacin (ENX) greater than ciprofloxancin (CPFX) much greater than ofloxacin (OFLX), and that of NSAIDs in combination with ENX was BPAA much greater than indomethacin = ketoprofen greater than naproxen greater than ibuprofen greater than pranoprofen. Diclofenac, piroxicam and acetaminophen did not affect GABA responses in the presence of ENX. 4. In the presence of ENX or BPAA, there was a small shift to the right of the concentration-response curve for GABA without any effect on the maximum response. However, the co-administration of these drugs suppressed the maximum of the GABA concentration-response curve, indicating a non-competitive inhibition, for which no voltage-dependency was observed.5. Simultaneous administration of ENX and BPAA also suppressed pentobarbitone (PB)-gated Icl. On the other hand, both PB and phenobarbitone reversed the inhibition of GABA-induced Ic, by coadministration of ENX and BPAA.6. The effect on GABAA responses of co-administration of new quinolones and NSAIDs was not via an interaction with benzodiazepine receptors coupled to the GABAA receptor, since this effect was not reversed by Rol5-1788 or diazepam.7. It is concluded that the co-administration of new quinolones and some of the NSAIDs inhibit GABAergic transmission, and could result in convulsions.

Effects of benzodiazepines and non-benzodiazepine compounds on the GABA-induced response in frog isolated sensory neurones.

1. The effects of benzodiazepines and non-benzodiazepine compounds on the gamma-aminobutyric acid (GABA)-induced chloride current (ICl) were studied in frog isolated sensory neurones by use of a concentration-jump (termed 'concentration-clamp') technique, under single-electrode voltage-clamp conditions. The drugs used were classified into four categories as follows: full benzodiazepine receptor agonists (diazepam, clonazepam, nitrazepam, midazolam, clotiazepam and etizolam), partial agonists (CL 218,872, Ro 16-6028, Ro 17-1812 and Ro 23-0364), inverse agonists (Ro 15-3505, FG 7142 and beta-CCE) and a benzodiazepine receptor antagonist, Ro 15-1788 (flumazenil). 2. All full agonists at concentrations of 3 x 10(-6) M or less increased dose-dependently the peak amplitude of ICl elicited by 3 x 10(-6) M GABA to twice to three times larger than the control. However, no further augmentation of the GABA response was observed at concentrations of 1 x 10(-5) M or higher. Partial agonists also showed a dose-dependent augmentation of the GABA response at concentrations ranging from 3 x 10(-8) M to 3 x 10(-5) M, but their efficacies of augmentation of the GABA response were only about half or less of those of full agonists. Of the inverse agonists, beta-CCE had a unique dose-dependent effect on the GABA response. Beta-CCE reduced dose-dependently the GABA response at concentrations of less than 3 x 10(-6) M, but augmented it at concentrations of 3 x 10(-5) M and 6 x 10(-5) M. The inverse agonists reduced dose-dependently the GABA response. The benzodiazepine antagonist, flumazenil, slightly augmented the GABA response at concentrations between 3 x 10 7M and 3 x 10 5 M. 3. These results show clear differences in the effects on the GABA response between these four categories of compounds known to affect the benzodiazepine recognition site of the GABA/ benzodiazepine receptor-chloride channel complex. Our experimental system of frog isolated sensory neurones and a 'concentration-clamp' technique appears to be useful for evaluating efficacy of compounds on responses mediated by the GABA/benzodiazepine receptor-chloride channel complex.

Differential properties of type I and type II benzodiazepine receptors in mammalian CNS neurones.

1. The effects of benzodiazepine receptor (BZR) partial agonists, Y-23684 and CL218,872, were compared with its full agonist, diazepam, on gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) in acutely dissociated rat cerebral cortex (CTX), cerebellar Purkinje (CPJ) and spinal ventral horn (SVH) neurones, by the whole-cell mode patch-clamp technique. 2. The GABA-induced responses were essentially the same in both SVH and CPJ neurones, but the KD value of the GABA response in CTX neurone was lower than those in the other two brain regions. 3. Enhancement of the GABA response by the two partial agonists was about one-third of that by diazepam in the SVH neurones (where type II subtype of BZR, BZ2, is predominant), whereas these partial agonists potentiated the GABA response as much as diazepam in CPJ neurones (where the type I subtype of BZR, BZ1, is predominant). In CTX neurones where both type I and II variants are expressed, the augmentation ratio of the GABA response by diazepam was between the values in CPJ and SVH neurones. 4. In concentration-response relationships of BZR partial agonists, the threshold concentrations, KD values and maximal augmentation ratio of the GABA response were similar in all CTX, CPJ and SVH neurones. Also, in all preparations, the threshold concentration and KD values of diazepam action were 10 fold less than those induced by partial agonists. 5. All BZR agonists shifted the concentration-response relationship for GABA to the left without changing the maximum current amplitude, indicating that activation of both BZ1 and BZ2 increase the affinity of the GABAA receptor for GABA. 6. The results are important in clarifying the mechanism of anxiety and might explain the anxioselectivity of BZR partial agonists.

Capacity for epithelial differentiation in synovial sarcoma: analysis of a new human cell line.

AIM: To analyse the capacity for epithelial differentiation in synovial sarcoma using a new human cell line. METHODS: A new human cell line, KU-SS-1, was established from a monophasic, spindle cell type of synovial sarcoma by grafting those cells on to severe combined immunodeficient (SCID) mice and then transferring them to in vitro culture systems. The KU-SS-1 cells were characterised by light and electron microscopy, and by immunohistochemical, flow cytometric, and cytogenetic analysis. RESULTS: Primary tumour and cultured cells at passage 20 showed a positive reaction for vimentin, which is a mesenchymal marker. After 40 passages, subcultured cells were injected into SCID mice to induce further tumours. These advanced subcultured cells and the tumour cells that they induced were positive for cytokeratin, an epithelial marker, and exhibited epithelial ultrastructural features such as intermediate junctions. Furthermore, two colour immunofluorescent analysis for proliferating nuclear cell antigen (PCNA) and intermediate filaments showed that a large number of PCNA expressing cells were positive for vimentin, and that part of this fraction also expressed cytokeratin. The existence of cells with reactivity for these three markers indicated that, in this cell line, a fraction with high proliferating capacity had both mesenchymal and epithelial markers. In addition, cytogenetically, this cell line expressed the SYT-SSX chimaeric transcript as a result of the t(X;18) (p11;q11) translocation. CONCLUSIONS: A human synovial sarcoma cell line was established and stably maintained in cell culture for more than 70 passages. In addition, this cell line showed epithelial differentiation, which supports the hypothesis that synovial sarcoma is a carcinosarcoma like tumour with true epithelial differentiation. This cell line will be a useful tool for investigating the nature of this tumour and will contribute to clinical studies.

Augmentation of GABA-induced chloride current in frog sensory neurons by diazepam.

The effect of diazepam (DZP) on the GABA-induced macroscopic and microscopic Cl- current was investigated in isolated frog sensory neurons using both 'concentration-clamp' and patch-clamp techniques. At concentration range between 10(-9) and 10(-4) M, DZP itself evoked no response but potentiated time- and dose-dependently the subthreshold GABA responses, though at high DZP concentrations beyond 10(-5) M the potentiation ratio decreased. The potentiation effect was long-lasting and desensitized slowly over the course of several 10 minutes after washing-out of DZP. DZP potentiated GABA response without shifting the GABA reversal potential. The entire GABA dose-response curve was shifted in a parallel manner to the left by adding DZP without changing cooperatively: the Hill slope was 2.0. The potentiation of GABA response by DZP did not depend on either inward or outward direction of the Cl- current but slightly on the membrane potential. The time constants of activation of desensitization of GABA-gated Cl- current consisted of fast and slow components, respectively. The slow components were concentration-dependent, and significantly changed in the presence of DZP, while DZP had little effects on fast components. In the 'inside-out' configuration, the addition of DZP activated GABA-receptor ionophore complexes under subthreshold without changing the single Cl- channel conductance. It is concluded that DZP may act at a site to modulate GABA binding, in which DZP increases GABA binding affinity and also affects the kinetics of GABA-gated Cl- channels, indicating that DZP has dual action on the GABA-induced responses.

Restricted distribution of Streptococcus milleri carbohydrate type antigens amongst other viridans streptococci.

The distribution of oral Streptococcus milleri carbohydrate type antigens in other viridans streptococcus species was examined. Rantz-Randall extracts of cells of the test strains grown in broth containing glucose were allowed to react with typing or grouping antisera for S. milleri serotypes a-k, or Lancefield groups A-G and K. Of 93 strains comprising more than 12 streptococcal species that included S. mutans and S. sanguis complexes, only 15 S. salivarius strains and one S. mitis strain were immunologically related to S. milleri serotype f. Unlike S. milleri strains, S. salivarius type f strains belonged to Lancefield group K, whereas the S. mitis strain was closely related to S. milleri serotype f but did not react with any of the Lancefield grouping antisera tested. Results suggest that oral S. milleri strains can be distinguished serologically from other oral viridans streptococci and that the typing antisera used in our researches might differentiate S. milleri isolates from the mouth from those associated with systemic purulent infections.

Serological variation in oral Streptococcus milleri.

Serological variation in 71 oral isolates and three reference strains of Streptococcus milleri was examined. Antisera were raised by immunising rabbits with cells of 10 selected strains, followed by absorption of non-specific antibodies. Double diffusion of the typing sera and the Rantz and Randall extracts of the strains in agar gel demonstrated that 70 strains were divided into 10 serotypes (a-j) on the basis of cell-surface carbohydrate antigens. Only four strains were untypable. The typing scheme proposed depends on type antigens other than the Lancefield group antigens A, C, F, G and others, although strains belonging to the serotypes a, c and f strictly corresponded to those of the groups A, C and F respectively. Close correlation between the present serotyping scheme and the previously proposed biotyping scheme for S. milleri was demonstrated. Distribution of these strains in dental plaque obtained from young adults was also investigated.

Serological and biological characteristics of "Streptococcus milleri" isolates from systemic purulent infections.

Ninety-one Streptococcus milleri strains isolated from various systemic purulent lesions of 68 patients were examined by physiological and serological tests. Most strains formed a smooth colony (66 strains), did not form spontaneous aggregation of cells in BHI broth culture (79), were non-beta-haemolytic (alpha-35 or non-41), and belonged to biotype Ia (49) or Ib (34) and to API taxa S. milleri I (41) or II (38). Almost all of the beta-haemolytic strains as well as two-fifths of the non-beta-haemolytic belonged to API taxon I; strains of API taxa II and III were non-beta-haemolytic and non-haemolytic, respectively. Two-fifths (38) of the isolates belonged to one of eight serotypes, a-g and k, and more than half (47) to Lancefield groups A, C, F or G, the most frequent being type b (19) and group F (33). Fifteen strains carried simultaneously type a/group A, b/C, c/C, e/G, f/F or k/G antigens. Nineteen were neither typable nor groupable. All the 38 serotypable isolates were non-beta-haemolytic and not members of API taxon III, and were serologically and physiologically similar to oral S. milleri. The isolates from various infected sites--sputum, thorax, abdomen, urogenitalia, skin, eye and dental--exhibited distinct combinations of biological and serological properties. These results suggest that serotyping, haemolytic properties and API taxon, and their combinations, would be useful methods to trace oral S. milleri in systemic infections.

Characterization of the K+ current mediated by 5-HT1A receptor in the acutely dissociated rat dorsal raphe neurons.

The action of 5-hydroxytryptamine (5-HT) via the 5-HT1A receptor on dissociated rat dorsal raphe neurons was characterized under the whole-cell mode by using the nystatin-perforated patch-clamp technique. Under voltage-clamp conditions, 5-HT induced an inwardly rectifying K+ current (I5-HT) in a concentration-dependent manner. I5-HT was mimicked by 8-OH-DPAT and buspirone, which are both 5-HT1A receptor agonists. I5-HT was reversibly blocked by such 5-HT1A receptor antagonists as (S)-UH-301 a 5-HT4 receptor antagonist. I5-HT was antagonized concentration-dependently by such K+ channel blockers as quinine, Ba2+ and 4-aminopyridine but was relatively insensitive to both CS+ and tetraethylammonium. When the neurons were loaded with guanosine 5'-O-3-thiotriphosphate through a patch pipette, the K+ current induced by 5-HT became irreversible. N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, irreversibly blocked I5-HT. The intracellular perfusion with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a Ca2+ chelator, or neomycine, a phospholipase C inhibitor, never significantly affected the 5-HT-induced response. 12-Myristate 13-acetate diester (PMA), a protein kinase C (PKC) activator, had only a weak inhibitory effect on I5-HT, and staurosporine, a PKC inhibitor, failed to significantly occlude I5-HT. Therefore, the K+ conductance activated via the 5-HT1a receptor of dorsal raphe neurons was thus characterized by the sensitivity to such K+ channel blockers as quinine, Ba2+ and 4-aminopyridine. Moreover, G protein which is NEM-sensitive and can couple to the 5-HT1A receptor, is thus considered to activate the inwardly rectifying K+ conductance without being mediated by such second messengers as Ca2+ and PKC.

Influences of external Ca2+ on the GABA-induced chloride current and the efficacy of diazepam in internally perfused frog sensory neurons.

The effects of extracellular Ca2+ on the gamma-aminobutyric acid (GABA)-induced Cl- current and the efficacy of diazepam in the facilitation of GABA response were studied in frog isolated sensory neurons, using a 'concentration clamp' technique which combines a suction pipette (internal perfusion and voltage clamp) and a rapid drug application system. When nominal Ca2+-free external solution was changed to the solution containing 2 mM Ca2+, the response elicited by 1 x 10(-5) M GABA was reduced by about 40% of the control obtained in nominal Ca2+-free solution. The dose-response curve for GABA was shifted to the right without affecting the maximum response. It can be suggested that the application of external Ca2+ modulates the affinity of the GABA receptor to its agonist, GABA. Diazepam at the concentration of 3 x 10(-6) M shifted the dose-response curve for GABA to the left without changing the maximum response with or without external Ca2+. However, the augmentatory action of diazepam on the GABA response was reduced in the presence of external Ca2+. Possible mechanisms for inhibitory action of external Ca2+ on the GABA-gated response and the reduced efficacy of diazepam are discussed.

Comparative study on barbiturates using isolated single neurons: GABA-mimetic action and augmentatory action on GABA response.

The comparisons among barbiturate derivatives in both GABA-mimetic action and augmentatory effect on GABA response were made. Experiments were performed on single neurones isolated from frog dorsal root ganglia, using a concentration clamp technique which combines a suction pipette technique (internal perfusion and voltage-clamp) with a rapid external solution exchange within 2 ms. Barbiturates employed here were secobarbital, pentobarbital, hexobarbital and phenobarbital. All barbiturates at the concentrations over 1 x 10(-4) M evoked the chloride current (ICl) dose-dependently. In the GABA-mimetic action of barbiturates the potency was in the order of secobarbital greater than pentobarbital greater than hexobarbital greater than phenobarbital. All barbiturates at the concentrations over 1 x 10(-6) M (but up to 1 x 10(-3) M) augmented the GABA-induced ICl. Potency order of barbiturates in augmenting GABA response was the same as that in their GABA-mimetic actions when the concentration was 1 x 10(-4) M or less. However at the concentration of 1 x 10(-3) M the efficacies of secobarbital and pentobarbital in augmenting GABA response were greatly reduced while the other two barbiturates produced further augmentation.

Characterization of the currents induced by sigma ligands in NCB20 neuroblastoma cells.

Electrical and pharmacological properties of currents induced by compounds having affinities for putative sigma receptors were investigated with NCB20 cells by use of the whole-cell patch-clamp technique. Antipsychotics and naloxone induced inward currents with a decrease in membrane conductance at a holding potential of -60 mV. The rank order of potency for compounds inducing these currents was bromperidol > haloperidol > mosapramine = clocapramine > carpipramine > chlorpromazine > remoxipride > naloxone. Sulpiride, which does not have affinity for sigma receptors, induced inward currents only slightly. Haloperidol-induced currents were not affected by the pretreatments with 10 microM of sulpiride, dopamine, atropine, N-methyl-D-aspartate, 2-amino-7-phosphonoheptanoic acid, morphine or A23187, 100 nM of ICS 205-930, 100 microM of forskolin, 1 microM of phorbol-12,13-dibutyrate, or 100 ng/ml of cholera or pertussis toxins. The reversal potential of the currents induced by haloperidol, naloxone or remoxipride was dependent on the concentration of external or internal potassium. These results indicate that the currents induced by the tested compounds are due to blockade of tonic, outward potassium currents and suggest that these agents act on putative sigma receptors and that the second messenger systems within the cell are not essential for the coupling between the receptors and the channels.

In vivo tumouricidal effect of T lymphocytes activated by liposomes containing adriamycin on chemically-induced rat malignant fibrous histiocytoma.

The augmentative effect of liposomes containing adriamycin (LADM) in the host-tumour immune mechanism was assessed using 9,10-dimethyl-1,2-benzanthracene induced rat malignant fibrous histiocytoma (MFH). The antitumour effect of LADM was analyzed using a double grafted tumour system in which F344 rats first received simultaneous s.c. inoculations of MFH cells in both right and left flanks and were then injected with 0.2 mg of LADM into the right tumour on Day 10, 12, and 14. The growth of a remote, non-treated tumour was strongly inhibited, and the infiltration of CD8+ or CD4+ cells at the tumour periphery was histologically revealed. This inhibition was not observed in F344 nu/nu athymic rats. Winn neutralizing assay with T cell-rich splenic lymphocytes from each drug-treated MFH-bearing rat showed that complete regression of the tumour at a low effector/target ratio occurred only in the LADM-treated group. Furthermore, the tumouricidal effects were demonstrated in coexistence with CD8+ cells and CD4+ cells by assay with FACS sorting splenic lymphocytes. These results strongly suggest that intratumoural administration of LADM caused the systemic augmentation of the MFH-bearing host immune mechanism, and that the augmented response after LADM treatment was induced by the cytotoxic CD8+ lymphocytes dependent on the CD4+ lymphocytes.

Inter-serotype comparison of polysaccharides produced by extracellular enzymes from Streptococcus mutans.

The biochemical and morphological characteristics of polysaccharides synthesized from sucrose by extracellular enzymes from D-glucose-grown Streptococcus mutans representing serotypes a-g were compared. The polysaccharides synthesized by the enzymes from serotypes a, d, and g formed visible aggregates and firmly adhered to glass surfaces, whereas those formed by the enzymes from serotypes b, c, e, and f floated homogeneously and were poorly adherent. The enzymes of serotypes a, d, and g produced large amounts of water-insoluble polysaccharides (IPs, D-glucans), and those of serotypes b, c, e, and f water-soluble polysaccharides (SPs, D-glucans and D- fructans ). As compared with the IPs of serotypes b, c, e, and f, the IPs of serotypes a, d, and g (a) contained a higher proportion of (1----3)-alpha-D-glucosidic linkages and alpha-D-(1----3,6) branch linkages; (b) showed higher susceptibility to (1----3)-alpha-D-glucanase (serotype a excepted) and lower (1----6)-alpha-D-glucanase sensitivity; (c) contained larger amounts of high-molecular-weight fractions; (d) showed higher intrinsic viscosities (serotype b excepted); and (e) had lower S. mutans cell-agglutination activities. On electron-microscope observation, the IPs of all serotypes showed two fibrillar components; a double-stranded fibril, with short, fluffy protrusions extending out of its periphery, and a fine, single-stranded fibril. Thus, the serotypes could be divided into two major groups: a, d, and g; and b, c, e, and f. No similar grouping of serotypes was indicated by the chemical and morphological properties of SPs.

Reduction of the adherence of Streptococcus sobrinus insoluble alpha-D-glucan by endo-(1----3)-alpha-D-glucanase.

Insoluble alpha-D-glucan, previously formed on a glass surface from sucrose by the action of cell-free D-glucosyltransferases of Streptococcus sobrinus OMZ176, was significantly removed by a purified preparation of endo-(1----3)-alpha-D-glucanase (mutanase) from a strain of Pseudomonas sp. Almost complete dissociation of adherent glucan occurred at the highest enzyme concentration (40 mU/mL) tested. Synthesis and de novo adherence on glass of the glucan was markedly inhibited by the presence of mutanase, even at low concentrations (4 mU/mL or less). When compared to native glucan, the mutanase-modified glucan samples (a) contained lower proportion of D-(1----3) linkages; (b) showed lower susceptibility to mutanase and higher susceptibility to (1----6)-alpha-D-glucanase (dextranase); (c) contained larger amounts of low-molecular-weight fractions; (d) had lower intrinsic viscosities; (e) showed higher S. sobrinus cell-agglutinating activities; and (f) consisted of looser entwinement of coalescent single-stranded fibrils (a major component) and shorter double-stranded fibrils (a minor one).

Production and partial characterization of the extracellular polysaccharides from oral Streptococcus salivarius.

The production of polysaccharides from sucrose by extracellular enzymes from oral Streptococcus salivarius isolates and the physico-chemical properties of water-insoluble products (IPs) were investigated. Extracellular enzymes from all the 18 strains tested produced insoluble alpha-D-glucans (IGs) as well as soluble beta-D-fructans, and formed adhering deposits on glass. Generally, the IPs (mostly IGs) of S. salivarius strains differed from the S. sobrinus IPs by (a) containing significant proportions of alpha-D-(1----4)-, in addition to alpha-D-(1----3)- and alpha-D-(1----6)-glucosyl linkages, and much higher proportions of alpha-D-(1----3) than alpha-D-(1----6) linkages, (b) being more susceptible to hydrolysis by mutanase than by dextranase, (c) possessing low or no streptococcal cell-agglutinating ability, and (d) showing weaker adhesion to a glass surface. The degree of the polysaccharide adherence differed greatly among the S. salivarius strains and, therefore, they were divided into three groups of adherence producers; heavy, moderate, and slight. The IPs of the three groups contained, generally in descending order, a higher proportion of higher-molecular-weight fractions, and consisted of higher proportions of IG containing higher proportions of -(1----6)-alpha-D and -(1----4)-alpha-D glucosyl linkages and (1----3,6) branches, but showed higher susceptibility to hydrolysis by mutanase as well as dextranase. Thus, the production and the properties of extracellular insoluble alpha-D-glucans from sucrose differ considerably between oral S. salivarius and cariogenic S. sobrinus.