Elemental diet modulates the growth of Clostridium difficile in the gut flora.

BACKGROUND AND AIMS: Tube feeding is regarded as a risk factor for Clostridium difficile-associated diarrhoea. Recently, we reported that C. difficile toxin was frequently found in patients receiving an elemental diet. The present study was conducted to clarify whether elemental diets are associated with the growth of C. difficile in the gut flora. METHODS: C. difficile was cultured for 72 h in various concentrations of elemental diet containing 3% thioglycollate, and the growth rate or activity of C. difficile was evaluated by Gram stain or by measuring optical density at 560 nm. Faecal samples from 10 healthy adults were cultured in elemental diet + 3% thioglycollate. RNA was extracted from faeces with glass powder, which can eliminate PCR inhibitors, and mRNA of C. difficile toxin B was measured by reverse transcription PCR. RESULTS: Maximum OD560 value during culture in thioglycollate-containing elemental diet was 2.4 times higher than that in thioglycollate alone (P = 0.0163). Viability of C. difficile was decreased in thioglycollate but not in thioglycollate-containing elemental diet. Toxin B mRNA was detected in five faecal samples (50%) before culture and in all samples after culture. CONCLUSIONS: Our results suggest that an elemental diet can modulate the growth of C. difficile in the gut flora.

Mutations to Gly2370, Gly2373 or Gly2375 in malignant hyperthermia domain 2 decrease caffeine and cresol sensitivity of the rabbit skeletal-muscle Ca2+-release channel (ryanodine receptor isoform 1).

Mutations G2370A, G2372A, G2373A, G2375A, Y3937A, S3938A, G3939A and K3940A were made in two potential ATP-binding motifs (amino acids 2370-2375 and 3937-3940) in the Ca(2+)-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [(3)H]ryanodine binding by Ca(2+), caffeine and ATP (adenosine 5'-[beta,gamma-methylene]triphosphate, AMP-PCP) was used as an assay for channel opening, since ryanodine binds only to open channels. Caffeine-sensitivity of channel opening was also assayed by caffeine-induced Ca(2+) release in HEK-293 cells expressing wild-type and mutant channels. Equilibrium [(3)H]ryanodine-binding properties and EC(50) values for Ca(2+) activation of high-affinity [(3)H]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of 1 mM AMP-PCP, Ca(2+)-activation curves were shifted to higher affinity and maximal binding was increased to a similar extent for wild-type RyR1 and mutants. ATP sensitivity of channel opening was also similar for wild-type and mutants. These observations apparently rule out sequences 2370-2375 and 3937-3940 as ATP-binding motifs. Caffeine or 4-chloro-m-cresol sensitivity, however, was decreased in mutants G2370A, G2373A and G2375A, whereas the other mutants retained normal sensitivity. Amino acids 2370-2375 lie within a sequence (amino acids 2163-2458) in which some eight RyR1 mutations have been associated with malignant hyperthermia and shown to be hypersensitive to caffeine and 4-chloro-m-cresol activation. By contrast, mutants G2370A, G2373A and G2375A are hyposensitive to caffeine and 4-chloro-m-cresol. Thus amino acids 2163-2458 form a regulatory domain (malignant hyperthermia regulatory domain 2) that regulates caffeine and 4-chloro-m-cresol sensitivity of RyR1.

Adaptive changes in dynamic properties of human disparity-induced vergence.

PURPOSE: Vergence eye movements undergo adaptive recalibration in response to a training stimulus in which the initial disparity is changed just after vergence begins (the double-step paradigm). In the present study the changes in the dynamic properties of convergence, speed and acceleration, were examined by using this double-step paradigm, before and after adaptation. METHODS: Four normal subjects participated. Three-dimensional visual stimuli were provided by a head-mounted display with two liquid crystal diode (LCD) panels. To induce adaptation, a double step of disparity was used: an initial step from distances of 2 to 1 m was followed by a second step to distances of 0.7 m ("increasing paradigm") or 1.4 m ("decreasing paradigm") after a constant period of 0.2 seconds. The dynamic properties of vergence were compared before and after 30 minutes of training with these paradigms. RESULTS: Peak velocity of convergence became significantly greater (increasing paradigm) or smaller (decreasing paradigm) after 30 minutes' training. Changes in the dynamic properties of convergence were also obvious in phase-plane (velocity versus position) and main sequence (peak velocity versus amplitude) plots. Further analysis revealed that adaptive increases in vergence velocity were accomplished by an increase in the duration of the acceleration period, whereas adaptive decreases were induced by a decrease in the maximum value of acceleration. CONCLUSIONS: The pattern of change in the dynamic characteristics of vergence after adaptation was similar to that of saccades and the initiation of pursuit eye movements, suggesting common neural mechanisms for adaptive changes in the open-loop control of eye movements.

Regulation of serotonin transporter gene expression in human glial cells by growth factors.

The aims of this study were to identify monoamine transporters expressed in human glial cells, and to examine the regulation of their expression by stress-related growth factors. The expression of serotonin transporter mRNA was detected by reverse transcriptase-polymerase chain reaction in normal human astrocytes, whereas the dopamine transporter (DAT) and the norepinephrine transporter (NET) were not detected. The cDNA sequence of the "glial" serotonin transporter in astrocytes was consistent with that reported for the "neuronal" serotonin transporter (SERT). Moreover, we also demonstrated SERT expression in glial fibrillary acidic protein-positive cells by immunocytochemical staining in normal human astrocytes. Serotonin transporter gene expression was also detected in glioma-derived cell lines (A172, KG-1-C and KGK). Addition of basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) for 2 days increased serotonin transporter gene expression in astrocytes and JAR (human choriocarcinoma cell line). Basic fibroblast growth factor, but not epidermal growth factor, increased specific [3H]serotonin uptake in astrocytes in a time (1-4 days)- and concentration (20-100 ng/ml)-dependent manner. The expression of genes for basic fibroblast growth factor and epidermal growth factor receptors was detected in astrocytes. These findings suggest that the expression of the serotonin transporter in human glial cells is positively regulated by basic fibroblast growth factor.

An optimal condition of bronchial cell proliferation stimulated by insulin-like growth factor-I.

BACKGROUND: It is known that growth hormones such as insulin-like growth factor-I (IGF-I) and several kinds of cytokines are involved in the regeneration process of injured epithelial cells in chronic respiratory inflammatory diseases such as bronchial asthma. Repetitive degeneration/regeneration processes of the airway epithelial layer is supposed to be responsible for the remodeling and irreversible organic changes of the airway in bronchial asthma. The purpose of this study is to establish a simple and reliable in vitro method for studying airway epithelial cell growth and proliferation using IGF-I. METHOD: By altering the number of cultured epithelial cells (strain NCI-H(292)), culture duration before stimulation with IGF-I, concentration of IGF-I, and duration of IGF-I stimulation, the optimum conditions for epithelial cell growth was determined. The epithelial cell growth was evaluated using [methyl-(3)H]thymidine uptake. RESULT: Among various culture conditions, the epithelial cells cultured at 1 x 10(3) cells/well for 24 h followed by 24 h of stimulation by 10(-8) M of IGF-I showed the highest growth. CONCLUSION: The method for the evaluation of epithelial cell growth established in this study requires a small number of cells and has no complicated procedure. This simple model enables us to investigate the effect of various substances on bronchial epithelial cell growth in the presence of IGF-I.

Whole-blood flow-cytometric analysis of induction of adhesion molecules expression on eosinophils stimulated by phorbol-12-myristate-13-acetate/ionomycin.

BACKGROUND: Activation of eosinophils is closely associated with the pathology of allergic inflammatory disease, especially bronchial asthma. We recently investigated the activation of eosinophils by applying whole blood to a flow cytometer. We measured here beta1 and beta2 integrin on eosinophils stimulated by phorbol-12-myristate-13-acetate (PMA)/ionomycin to evaluate eosinophil activation in vitro using whole blood. METHODS: Heparinized whole blood was diluted with the same volume of RPMI 1640, then cells were incubated in the presence or absence of PMA and ionomycin for 45 min at 37 degrees C. After hemolyzation with lysing solution, flow-cytometric findings for CR3, LFA1-alpha, LFA1-beta and VLA-4 expression on eosinophils were examined. RESULTS: Mean fluorescent intensity (MFI) of CR3 and LFA1-beta stimulated by PMA and ionomycin was significantly higher than that of the unstimulated control. MFI of LFA1-alpha showed no significant difference from the unstimulated control. On the other hand, MFI of VLA-4 tended to decrease. CONCLUSIONS: Our method to distinguish eosinophils from various cell groups in whole blood is simple and time-saving, similar to conditions in vivo and may allow intensive investigation of eosinophils in clinical laboratories as well as in research laboratories. We are currently investigating the influence of different kinds of stimulations, regulation factors or agents on eosinophils using this method.

CCR3 mRNA expression in bronchial epithelial cells and various cells in allergic inflammation.

BACKGROUND: RANTES and eotaxin are important chemokines involved in the activation and migration of eosinophils and are considered to play a major role in allergic inflammation. METHODS: In this study, we used RT-PCR to investigate the kinds of cells that express mRNA for CCR3, a common receptor of these chemokines, and eotaxin, a ligand for CCR3. RESULTS: CCR3 mRNA was expressed in eosinophils, peripheral mononuclear cells, an eosinophilic cell line (EoL-1), a bronchial epithelial cell line (NCI-H(292)), human endothelial cells and nasal washings from patients with allergic rhinitis. CONCLUSION: These results suggest that the CCR3-eotaxin system plays an important role in generating inflammation, since these substances are expressed not only in cells implicated in activation or migration of eosinophils but also in various other cells involved in allergic inflammation.

Effect of eotaxin on the generationof reactive oxygen species from eosinophil cell line, YY-1.

BACKGROUND: The CC chemokine eotaxin is a selective chemoattractant for eosinophils. Eosinophils have been considered to be the major effector cells in allergic inflammation, since not only eosinophil-specific granule proteins but also reactive oxygen species (ROS) from eosinophils may cause the damage to the cells or tissue of the mucosal epithelium. In this study, we examined the effect of eotaxin on ROS from an eosinophil cell line, YY-1. METHODS: ROS in luminol-dependent reaction were examined. Calcium ionophore A23187 were added to the mixture of YY-1 cells with luminol, and then ROS were determined. RESULTS: Eotaxin primed the production of ROS from YY-1 cells. ROS from untreated YY-1 cells evoked with calcium ionophore A23187 in luminol-dependent chemiluminescence gave a maximal value of 1,928 +/- 223 intensity counts (IC; mean +/- SE, n = 4) and an integral value of 17.04 +/- 1. 51 IC (x10(-4)), while eosinophils that were treated with eotaxin gave a maximal value of 2,264 +/- 86 IC (10 nM), 2,691 +/- 124 IC (100 nM) and an integral value of 21.22 +/- 0.67 IC (x10(-4); 10 nM), 26.20 +/- 1.41 IC (x10(-4); 100 nM). CONCLUSION: Eotaxin might play important roles in the pathogenesis of allergic inflammation through eosinophil activation by priming of eosinophil oxidative metabolism as well as involvement in selective eosinophil chemotaxis.

Postnatal development of NK1, NK2, and NK3 neurokinin receptors expression in the rat retina.

The biological effects of tachykinins are mediated by three distinct receptors, the neurokinin 1 receptor (NK1-R), NK2-R, and NK3-R. There is no information available concerning the development of these receptors in the retina. In the present study, we investigated the localization of tachykinin receptors, using antisera directed against NK1-R, NK2-R, and NK3-R in the adult and developing rat retinas. Numerous NK1-R immunoreactive (NK1-R IR) cells were already observed in the proximal part of the neuroblastic layer in the retina at postnatal day 5 (P5). The distribution and intensity of NK1-R IR cells and processes in the inner nuclear layer (INL) and inner plexiform layer (IPL) at P10 were similar to those of adult retina. Most NK1-R IR cells located in the proximal part of INL, which were morphologically amacrine cells. In the contrast to the early expression of NK1-R IR cells, no NK3-R IR structures existed in the neuronal elements of the retina until P10. NK3-R IR processes were first detected in the outer plexiform layer (OPL) at P10. At P15, NK3-R IR somata were slightly stained in the distal and middle parts of the INL, and NK3-R IR processes were present in the OPL and the upper part of the IPL. During P15-P30, the number of NK3-R IR somata located in the INL remarkably increased. These NK3-R IR cells were morphologically bipolar and amacrine cells. This study provides differential cellular distribution of NK1-R IR cells and NK3-R IR cells in the INL of the rat retina. Our findings suggest that NK1-R and NK3-R are involved in different visual circuits and retinal maturation, and NK3-R may play previously unknown important roles in the visual processes of the rat.

Human cortical areas activated in relation to vergence eye movements-a PET study.

Human cortical areas activated in relation to vergence eye movements were determined using positron emission tomography. Binocular disparity-driven visual stimuli were presented using a head-mounted display. Eye movements were monitored continuously by an infrared limbus tracker. A combination of a bar and a cross was used as the target. In the vergence task, subjects were instructed to follow an approaching bar, while ignoring a stationary cross. Activation in relation to vergence eye movement was discriminated from activation in relation to motion vision by using the ignore-bar task as the control. In the ignore-bar task, subjects were instructed to fixate on a stationary cross, while ignoring an approaching bar. The fixation task was used as the basic control for both the vergence and the ignore-bar tasks. Areas of activation in relation to vergence eye movements were found in the bilateral temporooccipital junction, the left inferior parietal lobule, and the right fusiform gyrus by comparing regional cerebral flow between the vergence and ignore-bar tasks and by the conjunctive analyses of vergence-vs-ignore comparison with vergence-vs-fixation comparison.

Further characterization of the type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm.

We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RyR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RyR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (Po), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity with Po approximately 1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RyR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RyR3 between [3H]ryanodine binding and channel activity.

[Present status of unsealed radioisotope therapy in Japan based on the nation-wide questionnaire].

In Japan, clinical application of unsealed radioisotopes is strictly regulated. Especially in the field of therapy, we are allowed to use only Na131I at present. Under such circumstances, the present status of therapeutic nuclear medicine in Japan was surveyed by means of a nation-wide questionnaire, conducted in 193 hospitals. Then, 113 hospitals replied to such questionnaire (recovery rate: 58.5%), and it was found that in 77 hospitals, radioisotope therapy is being performed for Graves' disease and/or thyroid cancer. The questionnaire covered the following points: for Graves' disease the basic strategy of 131I therapy, its indications, absorbed doses planned to be given, whether the therapy had been conducted on outpatient basis or in-patient basis, method of thyroid weight estimation, interval of administration in case of multiple doses, number of patients treated per year (1996) etc., and for thyroid cancer--strategy for thyroid remnant, the doses to be given, the maximum doses permitted by the authorities in each hospital both per day and per year, handling of highly contaminated urine in each hospital, interval of administration in case of multiple doses, number of patients treated per year (1996) etc. Also questioned were dissatisfaction with the present regulation by the authorities and/or requests for the better daily work, if any. Based on the above questionnaire, the present status of unsealed radioisotope therapy in Japan was investigated.

RANTES augments eosinophil lucigenin-dependent chemiluminescence.

BACKGROUND: RANTES (regulated on activation, normal T expressed and secreted) has been shown to possess chemotactic activity for eosinophils. Eosinophils have been considered to play a key role in the allergic inflammation through the release of inflammatory molecules such as radical oxygen products. Thus, in this study, we examined the effect of RANTES on radical oxygen products from eosinophils. METHODS: Eosinophils were isolated from heparinized venous blood of patients with bronchial asthma by the modified CD16-negative depletion method. Radical oxygen products were examined in terms of lucigenin-dependent chemiluminescence. To a mixture of 50 microl of eosinophils (2x10(6)/ml) and 50 microl of lucigenin (5x10(-4)M), 50 microl of calcium ionophore A23187 (final concentration 10(-5)M) was added, and radical oxygen products were determined for 600 s. RESULTS: RANTES treatment resulted in the enhancement of peak value (0.64+/-0.23 RLU) and integrated value (119.08+/-20.52 RLU) as compared to untreated cells (0.15+/-0.03 RLU, 29.48+/-8.92 RLU, respectively). CONCLUSIONS We could conclude that RANTES might play an important role in the pathogenesis of allergic inflammation through involvement in selective eosinophil infiltration and eosinophil activation by augmentation of eosinophil oxidative metabolism.

Involvement of beta1 and beta2 integrin stimulation in RNA synthesis in an eosinophilic cell line (EoL-1).

In allergic inflammatory disease, especially in bronchial asthma, eosinophils play important roles as essential inflammatory cells. In the accumulation of eosinophils in airway inflammation, eosinophils receive very diverse stimuli. In this study, we investigated the influences of the signal between beta2 integrin/intercellular adhesion molecule-1 (ICAM-1) or beta1 integrin/fibronectin (FN) and RNA synthesis on proteins in eosinophilic cell line-1 (EoL-1), using recombinant soluble ICAM-1 (r-sICAM-1) as a global response of eosinophils. 3H-thymidine incorporation and 3H-uridine incorporation were used as indices for DNA synthesis and RNA synthesis, respectively. In a comparison of the RNA/DNA ratio with various durations of stimulation of 1 microg/ml of r-sICAM-1 and 100 microg/ml of FN, a time-dependent increase was observed, but the increase induced by FN rose slower than that induced by r-sICAM-1. From this result, a beta2 integrin/ICAM-1 signal induced an increase in the RNA/DNA ratio in EoL-1, implying that the signal promotes RNA synthesis, which suggests that various types of protein synthesis, such as the synthesis of various cytokines, are induced by the beta2 integrin/ICAM-1 signal. Similar results were obtained with a beta1 integrin signal using FN, but there was a difference in the time course between beta2 integrin/ICAM-1 and beta1 integrin/FN signals. This experimental method may be useful for understanding these manifestations as a global response of eosinophils.

Whole-blood flow-cytometric analysis of eosinophil EG2 expression as a marker of the pathological conditions of asthma.

UNLABELLED: Using a simple technique detecting the eosinophil fraction in whole-blood flow cytometry, we measured intracellular antigen EG2 (a monoclonal antibody to eosinophil cationic protein) in 56 asthmatic patients (26 during an attack and 30 during an asymptomatic period) and 22 healthy subjects to determine whether EG2 reflects the pathological stages of allergy. METHODS: In brief, preparations of the sample included the following procedures: (1) hemolyzation of heparinized or EDTA-mixed whole blood; (2) fixation of white blood cells with 0.4% parabenzoquinone (PBQ) or paraformaldehyde (PFA); (3) permeabilizing the cell membrane with n-octyl-beta-D-glucopyranoside, and (4) staining of intracellular EG2 antigen with monoclonal EG2 antibody and FITC-labeled secondary antibody. RESULTS: In PBQ-fixed samples, there was a clearer boundary of the eosinophil fraction with a higher yield and purity than in those fixed with PFA. The number of EG2-positive eosinophils was significantly greater in subjects during attacks than in asymptomatic patients. In addition, when compared with normal controls, asthmatic subjects had significantly greater numbers of EG2-positive eosinophils regardless of their current condition. CONCLUSION: Eosinophil intracellular EG2 may indicate the pathological stage of asthma. This simple technique for analysing the properties of eosinophils using whole-blood flow cytometry would save time and labor in laboratories.

[Some considerations on the avoidance of excess body burden in case of high dose 131I treatment: with special reference to the urination frequency].

In case of the treatment of metastatic thyroid cancer with radioiodine (131I), 3.7 GBq (100 mCi) or more is often repeatedly administered to the patient. Therefore, we must be aware of avoiding unnecessary exposure to the radioiodine except for the lesions to be treated. The administered radioiodine is excreted mainly through urinary system, resulting in an accumulation of the urine in the bladder for a certain period, which is highly concentrated with it. Based on this fact, we set one case having a particular pattern of the whole body retention curve. Then, two different modes of urination were considered; mode A indicates every 2 hour-urination x 6 times followed by every 6 hour-urination x 2 times, and mode B indicates every 6 hour-urination x 4 times. Focusing on the different amounts of urine in the bladder upon the different modes of urination, the radiation exposures from the urine to the neighboring organs, such as bladder wall, uterus, ovary and testis, and also to the whole body were calculated. As the results, it was found that the urination mode B would cause radiation exposure from the urine in the bladder twice as much as the urination mode A to the neighboring organs as well as to the whole body. This study will supply arguments for the necessity of frequent urination in the cases receiving radioiodine treatment for metastatic thyroid cancer.

Structural and immunochemical characterization of beta-1,2-linked mannobiosyl phosphate residue in the cell wall mannan of Candida glabrata.

A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-alpha-mannosidase and a beta-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using 13C nuclear magnetic resonance spectroscopy. The results show that the beta-1, 2-linked mannobiosyl residue is esterified to a phosphate group through position C-1 in the alpha-configuration, Manbeta1- 2Manalpha1-HPO3-. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain Manbeta1-2Manalpha1- 2Manalpha1-2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manbeta1- 2Manalpha1-HPO3- in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present and previous findings indicate that serum no. 5 recognizes relatively longer beta-1,2-linked oligomannosyl side-chains, Manbeta1-[2Manbeta1-]n 2Man (n = 1-6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis.

Expression of mRNA for RANTES in human eosinophils.

RANTES has been considered to play an important role in various immune and allergic disorders since RANTES is a potent chemoattractant for various important inflammatory cells such as eosinophils. Eosinophils, on the other hand, are considered to be the major inflammatory cells in bronchial asthma since eosinophil-specific granule proteins can damage bronchial mucosal cells. The recent demonstration that eosinophils themselves could synthesize some cytokines such as IL-5 indicates the role of these cytokines not only in paracrine but also in autocrine regulation of eosinophil production, differentiation and activation. Therefore, in this study, we examined mRNA for RANTES and release of RANTES by human eosinophils. The present findings revealed that eosinophils obtained from asthmatic patients could express and release RANTES. Taken together, these findings suggest that eosinophils play a crucial role in the pathogenesis, particularly in eosinophil and T lymphocyte recruitment into the inflamed sites in asthma through RANTES production.

Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease.

Malignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle in which a potentially fatal hypermetabolic crisis can be triggered by commonly used anesthetic agents. To date, 17 mutations in the human RYR1 gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have been associated with MH and/or CCD. Although many of these mutations have been linked to MH and/or CCD, with high lod (log of the odds favoring linkage versus nonlinkage) scores, others have been found in single, small families. Independent biochemical evidence for a causal role for these mutations in MH is available for only two mutants. Mutations corresponding to the human MH mutations were made in a full-length rabbit RYR1 cDNA, and wild type and mutant cDNAs were transfected into HEK-293 cells. After about 48 h, intact cells were loaded with the fluorescent Ca2+ indicator, fura-2, and intracellular Ca2+ release, induced by caffeine or halothane, was measured by photometry. Ca2+ release in cells expressing MH or CCD mutant ryanodine receptors was invariably significantly more sensitive to low concentrations of caffeine and halothane than Ca2+ release in cells expressing wild type receptors or receptors mutated in other regions of the molecule. Linear regression analysis showed that there is a strong correlation (r = 0.95, p < 0.001) between caffeine sensitivity of different RYR1 mutants measured by the cellular Ca2+ photometry assay and by the clinical in vitro caffeine halothane contracture test (IVCT). The correlation was weaker, however, for halothane (r = 0.49, p > 0.05). Abnormal sensitivity in the Ca2+ photometry assay provides supporting evidence for a causal role in MH for each of 15 single amino acid mutations in the ryanodine receptor. The study demonstrates the usefulness of the cellular Ca2+ photometry assay in the assessment of the sensitivity to caffeine and halothane of specific ryanodine receptor mutants.

Characterization of beta-1,2-mannosyltransferase in Candida guilliermondii and its utilization in the synthesis of novel oligosaccharides.

A particulate insoluble enzyme fraction containing mannosyltransferases from Candida guilliermondii IFO 10279 strain cells was obtained as the residue after extracting a 105,000 x g pellet of cell homogenate with 1% Triton X-100. Incubation of this fraction with a mannopentaose, Manalpha1-->3(Manalpha1-->6)Manalpha1-->2Manalpha1+ ++-->2Man, in the presence of GDP-mannose and Mn2+ ion at pH 6.0 gave a third type of beta-1,2 linkage-containing mannohexaose, Manbeta1-->2Manalpha1-->3(Manalpha1-->6)Manalpha1++ +-->2Manalpha1-->2Man , the structure of which was identified by means of a sequential NMR assignment. The results of a substrate specificity study indicated that the beta-1,2-mannosyltransferase requires a mannobiosyl unit, Manalpha1--> 3Manalpha1-->, at the nonreducing terminal site. We synthesized novel oligosaccharides using substrates possessing a nonreducing terminal alpha-1,3-linked mannose unit prepared from various yeast mannans. Further incubation of the enzymatically synthesized oligosaccharide with the enzyme fraction gave the following structure, Manbeta1-->2Manbeta1-->2Manalpha1-->3(Manalpha1- ->6)Manalpha1--> 2Manalpha1-->2Man, which has been found to correspond to antigenic factor 9. Incubation of Candida albicans serotype B mannan with the enzyme fraction gave significantly transformed mannan, which contains the third type of beta-1,2-linked mannose units.