Development and validation of a new asthma questionnaire to help achieve a high level of control in school-age children and adolescents.

BACKGROUND: Maintaining good asthma control minimizes the risk of exacerbations and lung function decline and is a primary goal of asthma management. The Japanese Pediatric Asthma Guidelines (JPGL) employs different classification criteria for control status from other guidelines, stressing a higher level of control. Based on JPGL, we previously developed a caregiver-completed questionnaire for assessing and achieving best asthma control in preschoolers. In this study, we aimed to develop a questionnaire for school-age children and adolescents. METHODS: A working questionnaire comprising 14 items for patients and 34 items for caregivers was administered to 362 asthma patients aged 6-15 years and their caregivers. Separately, physicians filled out a questionnaire to determine JPGL-defined control. Logistic regression analysis was performed to construct a model to predict control levels using data from a randomly selected set of completed questionnaires from two-thirds of the subjects. Validation was performed using the remaining questionnaires. RESULTS: A set of 7 questions, encompassing self-assessed control status at the time of the visit and in the past month, and nocturnal/early morning asthma symptoms for patients and frequency of asthma symptoms, dyspnea, rescue beta-agonist use, and asthma hospitalization for caregivers, were selected and the 7-item model showed a good statistical fit with AIC of 110.5. The model has been named the Best Asthma Control Test for School Children and Adolescents (Best ACT-S). Best ACT-S scores differed significantly in the hypothetical direction among the groups of different JPGL-defined control levels, step-up/down treatment decisions, and presence/non-presence of exacerbations in the previous year. CONCLUSIONS: The Best ACT-S is a valid questionnaire for children/adolescents aiming for best asthma control.

Cholinergic sensing of allergen exposure by airway epithelium promotes type 2 immunity in the lungs.

BACKGROUND: Nonneuronal cells, including epithelial cells, can produce acetylcholine (ACh). Muscarinic ACh receptor antagonists are used clinically to treat asthma and other medical conditions; however, knowledge regarding the roles of ACh in type 2 immunity is limited. OBJECTIVE: Our aim was to investigate the roles of epithelial ACh in allergic immune responses. METHODS: Human bronchial epithelial (HBE) cells were cultured with allergen extracts, and their ACh production and IL-33 secretion were studied in vitro. To investigate immune responses in vivo, naive BALB/c mice were treated intranasally with different muscarinic ACh receptor antagonists and then exposed intranasally to allergens. RESULTS: At steady state, HBE cells expressed cellular components necessary for ACh production, including choline acetyltransferase and organic cation transporters. Exposure to allergens caused HBE cells to rapidly release ACh into the extracellular medium. Pharmacologic or small-interfering RNA-based blocking of ACh production or autocrine action through the M3 muscarinic ACh receptors in HBE cells suppressed allergen-induced ATP release, calcium mobilization, and extracellular secretion of IL-33. When naive mice were exposed to allergens, ACh was quickly released into the airway lumen. A series of clinical M3 muscarinic ACh receptor antagonists inhibited allergen-induced IL-33 secretion and innate type 2 immune response in the mouse airways. In a preclinical murine model of asthma, an ACh receptor antagonist suppressed allergen-induced airway inflammation and airway hyperreactivity. CONCLUSIONS: ACh is released quickly by airway epithelial cells on allergen exposure, and it plays an important role in type 2 immunity. The epithelial ACh system can be considered a therapeutic target in allergic airway diseases.

Asynchronous e-learning with technology-enabled and enhanced training for continuing education of nurses: a scoping review.

BACKGROUND: Asynchronous e-learning has become the mainstream choice since the transformation of learning formats by the coronavirus disease-19 pandemic. This scoping review aimed to examine the technologies used in asynchronous e-learning for the continuing education of clinical nurses and their modes of delivery and effectiveness. METHODS: This scoping review covered the period between 2011 and 2023. Six databases were searched for relevant studies following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis extension for Scoping Reviews (PRISMA-ScR) protocol. RESULTS: Sixty articles met the inclusion criteria. There was a noticeable trend toward using diverse technology-enabled and enhanced training (TEET) options after 2017. The enabling technological approaches, such as interactive online modules (25 articles) and video modules (25 articles), are described in the articles. The most commonly used enhancing technologies were scenario-based learning (nine articles), resource access (eight articles), computer simulation or virtual reality (three articles), and gamification (three articles). Among the outcomes, knowledge acquisition was the most commonly examined outcome (41 articles). CONCLUSIONS: Notably, many interactive TEET modules were used in asynchronous e-learning. There were few studies on gamification, computer simulation or virtual reality, and scenario-based learning (techniques to enhance intrinsic motivation further). However, the adoption of asynchronous e-learning with advanced TEET options is anticipated to increase in the future. Therefore, objective outcome measures are required to determine the effects of such learning methods on knowledge acquisition and behavioral changes.

House Dust Mite Subcutaneous Immunotherapy and Lung Function Trajectory in Children and Adolescents with Asthma.

Background: Allergen-specific immunotherapy is currently the only disease-modifying treatment for allergic asthma, and it has been shown to improve control of asthma while reducing both drug use and asthma exacerbations. However, its effects on lung function­especially its long-term effects­remain controversial. We aimed to identify factors associated with a possible beneficial effect of allergen-specific immunotherapy on lung function in asthma by retrospectively evaluating the long-term changes in lung function in children with asthma who received house dust mite subcutaneous immunotherapy (HDM-SCIT). Methods: We enrolled children with asthma who had undergone HDM-SCIT for more than 1 year. Clinical information and lung function measurements were retrieved from the electronic chart system. To characterize the trajectory of lung function change, we performed linear regression analysis to evaluate the maximal expiratory flow at 50% of the forced vital capacity during two periods: before and during HDM-SCIT. Slopes from a least-squares regression line for the two periods, i.e., S1 before HDM-SCIT and S2 during HDM-SCIT, were compared. The subjects were then classified into two groups: an improving group (Group I) defined as S2 − S1 > 0, and a declining group (Group D) defined as S2 − S1 < 0. The clinical factors at the start of HDM-SCIT were compared between the two groups. Results: A total of 16 patients were analyzed. Eight patients were classified into each of Group I and Group D. The mean ages were 10.5 and 11.8 years, and the mean treatment periods were 4.1 and 3.9 years. Group I had a significantly lower blood eosinophil count and a significantly higher HDM-specific IgE level than Group D. Logistic regression showed a strong relationship between those two markers and the lung function trajectory. Conclusion: Control of the blood eosinophil count in highly HDM-sensitized patients may increase the beneficial effect of HDM-SCIT on lung function.

A mouse model of the LEAP study reveals a role for CTLA-4 in preventing peanut allergy induced by environmental peanut exposure.

BACKGROUND: A human study, Learning Early About Peanut Allergy (LEAP), showed that early introduction of peanut products decreases the prevalence of peanut allergy among children. However, the immunologic mechanisms mediating the protective effects of consuming peanut products are not well understood. OBJECTIVE: The objective was to develop a mouse model that simulates the LEAP study and investigate the underlying mechanisms for the study observations. METHODS: Adult naive BALB/c mice were fed a commercial peanut butter product (Skippy) or buffer control and concomitantly exposed to peanut flour through the airway or skin to mimic environmental exposure. The animals were analyzed for anaphylactic reaction and by molecular and immunologic approaches. RESULTS: After exposure to peanut flour through the airway or skin, naive mice developed peanut allergy, as demonstrated by acute and systemic anaphylaxis in response to challenge with peanut extract. Ingestion of Skippy, however, nearly abolished the increase in peanut-specific IgE and IgG and protected animals from developing anaphylaxis. Skippy-fed mice showed reduced numbers of T follicular helper (Tfh) cells and germinal center B cells in their draining lymph nodes, and single-cell RNA sequencing revealed a CD4+ T-cell population expressing cytotoxic T lymphocyte-associated protein 4 (CTLA-4) in these animals. Critically, blocking CTLA-4 with antibody increased levels of peanut-specific antibodies and reversed the protective effects of Skippy. CONCLUSION: Ingestion of a peanut product protects mice from peanut allergy induced by environmental exposure to peanuts, and the CTLA-4 pathway, which regulates Tfh cell responses, likely plays a pivotal role in this protection.

Diagnosis of non-immediate hypersensitivity to amoxicillin in children by skin test and drug provocation tests: A retrospective case-series study.

BACKGROUND: Skin rash often occurs upon oral administration of amoxicillin in children, due to non-immediate hypersensitivity. However, information on delayed hypersensitivity to amoxicillin is scarce. Moreover, the appropriate diagnostic method and actual diagnostic rate of delayed hypersensitivity to amoxicillin among Japanese children are unclear. We conducted intradermal tests (IDTs) and drug provocation tests (DPTs) and retrospectively investigated the proportion of children with a definitive diagnosis of non-immediate hypersensitivity to amoxicillin. We then evaluated the characteristics of patients with a positive allergic workup. METHODS: We enrolled children referred for suspected findings of mild or moderate non-immediate hypersensitivity to amoxicillin between August 2018 and March 2020. If the IDT in the delayed phase was negative, DPT with amoxicillin (60-90 mg/kg/day) was performed for 7 days. Non-immediate hypersensitivity to amoxicillin was defined when IDT or DPT was positive. We evaluated the potential of the drug-induced lymphocyte stimulation test (DLST) to reveal hypersensitivity to amoxicillin. RESULTS: This study enrolled 27 children. Fourteen children (52%) had hypersensitivity to amoxicillin, of whom 12 had positive IDTs and two had positive DPTs. No differences in age, sex, history of allergic disease, days from oral use to symptom onset, type of rash at symptom onset, generalized rash, and DLST results were observed between the hypersensitivity and non-hypersensitivity groups. CONCLUSIONS: Examination should be performed for children with mild or moderate reactions because positive cases have no significant features and half of the suspected cases are negative.

Mechanical Stimulation-Induced Calcium Signaling by Piezo1 Channel Activation in Human Odontoblast Reduces Dentin Mineralization.

Odontoblasts play critical roles in dentin formation and sensory transduction following stimuli on the dentin surface. Exogenous stimuli to the dentin surface elicit dentinal sensitivity through the movement of fluids in dentinal tubules, resulting in cellular deformation. Recently, Piezo1 channels have been implicated in mechanosensitive processes, as well as Ca2+ signals in odontoblasts. However, in human odontoblasts, the cellular responses induced by mechanical stimulation, Piezo1 channel expression, and its pharmacological properties remain unclear. In the present study, we examined functional expression of the Piezo1 channel by recording direct mechanical stimulation-induced Ca2+ signaling in dentin matrix protein 1 (DMP-1)-, nestin-, and dentin sialophosphoprotein (DSPP)-immunopositive human odontoblasts. Mechanical stimulation of human odontoblasts transiently increased intracellular free calcium concentration ([Ca2+]i). Application of repeated mechanical stimulation to human odontoblasts resulted in repeated transient [Ca2+]i increases, but did not show any desensitizing effects on [Ca2+]i increases. We also observed a transient [Ca2+]i increase in the neighboring odontoblasts to the stimulated cells during mechanical stimulation, showing a decrease in [Ca2+]i with an increasing distance from the mechanically stimulated cells. Application of Yoda1 transiently increased [Ca2+]i. This increase was inhibited by application of Gd3+ and Dooku1, respectively. Mechanical stimulation-induced [Ca2+]i increase was also inhibited by application of Gd3+ or Dooku1. When Piezo1 channels in human odontoblasts were knocked down by gene silencing with short hairpin RNA (shRNA), mechanical stimulation-induced [Ca2+]i responses were almost completely abolished. Piezo1 channel knockdown attenuated the number of Piezo1-immunopositive cells in the immunofluorescence analysis, while no effects were observed in Piezo2-immunopositive cells. Alizarin red staining distinctly showed that pharmacological activation of Piezo1 channels by Yoda1 significantly suppressed mineralization, and shRNA-mediated knockdown of Piezo1 also significantly enhanced mineralization. These results suggest that mechanical stimulation predominantly activates intracellular Ca2+ signaling via Piezo1 channel opening, rather than Piezo2 channels, and the Ca2+ signal establishes intercellular odontoblast-odontoblast communication. In addition, Piezo1 channel activation participates in the reduction of dentinogenesis. Thus, the intracellular Ca2+ signaling pathway mediated by Piezo1 channels could contribute to cellular function in human odontoblasts in two ways: (1) generating dentinal sensitivity and (2) suppressing physiological/reactional dentinogenesis, following cellular deformation induced by hydrodynamic forces inside dentinal tubules.

Breastfeeding support and barriers to women with gestational diabetes mellitus: a nationwide cross-sectional survey of hospitals in Japan.

BACKGROUND: Despite the benefits of breastfeeding for women with Gestational Diabetes Mellitus (GDM) and their infants, breastfeeding is less likely to be performed by this group. This study aimed to examine the current levels of implementation of breastfeeding support to women with GDM in Japan and to clarify barriers to promoting breastfeeding among this population. METHODS: A 25-item questionnaire was developed by the authors to investigate the current levels of implementation of breastfeeding support for women with GDM provided in hospitals, and to explore barriers for promoting breastfeeding among these women. The questionnaire was sent to all 1046 hospitals facilitating childbirth in Japan. Descriptive statistics were used to analyze the quantitative data, and content analysis was used to analyze qualitative data from the open-ended questions. RESULTS: All 296 respondents were included in this study. Regarding breastfeeding support, 95.2% of the respondents provided general information on breastfeeding to GDM women during antenatal midwife consultations. However, the benefits of breastfeeding for preventing type 2 diabetes were addressed by only 48.0%. Likewise, although follow-up services (e.g., telephone support or breastfeeding consultations) were conducted in 88.9% of hospitals, only 50.7% of hospitals informed women that breastfeeding decreases the risk of developing type 2 diabetes after GDM. Regarding barriers, seven categories and 20 subcategories about promoting breastfeeding for women with GDM were extracted and abstracted into the following three themes: Barriers associated with mother and infant, Barriers associated with health professionals, and Organizational barriers. CONCLUSIONS: In Japan, most hospitals that responded provided general breastfeeding support from the antenatal to postpartum periods. However, the benefits of breastfeeding in terms of preventing the incidence of type 2 diabetes following GDM were insufficiently communicated to women with GDM. Furthermore, there were numerous barriers to promoting breastfeeding among women with GDM.

Continuous interprofessional collaboration for women with gestational diabetes mellitus: A cross-sectional survey in Japan.

AIM: This study aimed to identify the current situation of interprofessional collaboration for gestational diabetes mellitus (GDM) management in Japan including the professionals involved, the collaboration methods employed, and the barriers perceived by nurses and midwives. METHODS: This nationwide cross-sectional survey of 1,046 total hospitals facilitating childbirth in Japan used an original 60-item questionnaire to investigate GDM management practice through interprofessional collaboration. The questionnaire required one responder to be a midwife or nurse who was familiar with the management practices for GDM women in their respective hospitals. Quantitative data were analyzed using descriptive statistics, and framework analysis was conducted for qualitative data collected by open-ended questions. RESULTS: All 308 respondents (response rate 29.4%) were included. The professionals included in interprofessional collaboration were limited, and the only strategy used for interprofessional collaboration by a majority (91.5%) of hospitals was medical charts. There were 50.8% of hospitals that provided postpartum GDM follow-ups and 54.5% of hospitals that did not engage in external collaboration for GDM management. The barriers to interprofessional collaboration extracted were seven categories and 23 subcategories, which were aligned within the following units of analysis: individual, team, organization, and community. CONCLUSIONS: This survey shows that interprofessional collaboration, including support for GDM postpartum follow-up in Japan remains insufficient. Furthermore, nurses and midwives perceive numerous barriers to interprofessional collaboration for continuous GDM management at the individual, team, organizational and community levels.

Factors associated with outcome of egg allergy 1 year after oral food challenge: A good baseline quality of life may be beneficial.

BACKGROUND: Safely liberalizing the diet to include an allergenic food may accelerate resolution of food allergy. The outcome of liberalization, however, varies among patients. METHODS: We conducted a prospective observational study to identify factors associated with outcome for egg allergy 1 year after oral food challenge (OFC). We enrolled children <72 months old who had egg allergy and underwent OFC for determination of the safe intake quantity of egg allergen. Each child's baseline clinical background was recorded. Caregivers used the Food Allergy Quality of Life Questionnaire-Parent Form (FAQLQ-PF) to assess their children's QoL. Dietary advice based on the OFC result was provided to support safe egg consumption. At 1 year after OFC, the quantity of egg each child safely consumed in daily life was surveyed. We classified the outcome as Successful (Group S) if the quantity increased during the 1 year, or as Unsuccessful (Group U) if it did not. Factors associated with the outcome were investigated by multivariate logistic regression analysis. RESULTS: A total of 93 children were enrolled, and after 1 year, 57 finished in Group S and 36 in Group U. The mean FAQLQ-PF score at baseline was significantly lower (ie, a better QoL) in Group S than in Group U. Multivariate logistic regression analysis identified a good QoL and absence of comorbid asthma or atopic dermatitis as factors predicting a favorable outcome. CONCLUSION: QoL may affect food allergy outcome. Intervention focusing on the QoL may promote outgrowing of food allergies.

Three cases of severe adolescent asthma treated with mepolizumab: lung function trajectories.

Real-world experience with mepolizumab for pediatric asthma is still limited. We report 3 patients who were treated with mepolizumab for severe adolescent asthma. Two patients, a 12-year-old boy and a 14-year-old girl, responded well to mepolizumab and showed apparent improvement in lung function from a downward trend over time before treatment. The third patient, a 16-year-old boy, whose treatment was switched from omalizumab to mepolizumab, did not have satisfactory response. The 2 successful cases had eosinophil counts of 440 and 371/µL and multiple comorbid allergic diseases including food allergies. The clinical benefit to them included elimination of both exacerbation and exercise-induced asthma. Interestingly, the boy's food-induced gastrointestinal symptoms disappeared following start of mepolizumab treatment.

Washing with water alone versus soap in maintaining remission of eczema.

BACKGROUND: There is no consensus on the use of soap in skin care for atopic dermatitis in Japan. Thus, this study aimed to evaluate the efficacy of soap to maintain eczema remission in atopic dermatitis patients during the fall-winter period in Japan. METHODS: This assessor-blinded, pragmatic randomized, non-inferiority study enrolled atopic dermatitis patients whose eczema was controlled by regular steroid ointment application less than or equal to 2 days / week (tacrolimus ointment was permitted). For 8 ± 3 weeks, participants washed their upper and lower limbs on one side with soap (soap side) and on the other side with water alone (water side). The primary outcome was an Eczema Area and Severity Index score at week 8 ± 3. RESULTS: Twenty-nine participants were analyzed. The Eczema Area and Severity Index scores at week 8 ± 3 of the water and soap sides were 0.0 (0.0-0.4) and 0.0 (0.0-0.4), respectively (P = 0.18). The difference between both sides was -0.02 (-0.11-0.08), and the limits of the 95% confidence interval did not reach the prespecified non-inferiority margin. The average Patient-Oriented Eczema Measure score was 1.27 ± 1.7 and 1.32 ± 1.8 for the water and soap sides, respectively (P = 0.92). The total number of additional steroid ointment applications was four (0-20) times and six (0-23) times, respectively (P = 0.98). Participants were categorized according to self-assessments of the usefulness of soap, with 2, 24, and 3 participants in the water-effective, invariant, and soap-effective groups, respectively. CONCLUSIONS: For children with controlled atopic dermatitis, washing with water alone was not inferior to washing with soap for maintaining remission of eczema during the fall-winter period in Japan.

Six New Triterpenoids from the Aerial Parts of Maytenus diversifolia.

Five new ursane-type triterpenoids 1-5, and one new oleanane-type triterpenoids 7 were isolated from the MeOH extract of the aerial parts of Maytenus diversifolia. Their structures were elucidated by analyzing spectroscopic data and chemical transformation. Compounds 3 and 5 exhibited significant lethal activity in the brine shrimp lethality test (BST).

Altered strategy in short-term memory for pictures in children with attention-deficit/hyperactivity disorder: a near-infrared spectroscopy study.

Strategy in short-term memory for serially presented pictures shifts gradually from a non-phonological to a phonological method as memory ability increases during typical childhood development. However, little is known about the development of this strategic change in children with attention-deficit/hyperactivity disorder (ADHD). To understand the neural basis of ADHD, we investigated short-term memory strategies using near-infrared spectroscopy. ADHD children aged from 6 to 12 years and age- and sex-matched control children were assessed in this study. Regional activity was monitored in the left ventrolateral prefrontal cortex to assess strategies used during short-term memory for visual or phonological objects. We examined the hypothesis that the strategic methods used would be correlated with memory ability. Higher memory ability and the phonological strategy were significantly correlated in the control group but not in the ADHD group. Intriguingly, ADHD children receiving methylphenidate treatment exhibited increased use of phonological strategy compared with those without. In conclusion, we found evidence of an altered strategy in short-term memory in ADHD children. The modulatory effect of methylphenidate indicates its therapeutic efficacy.

Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria.

Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.

Hypertonic saline alters electrical barrier of the airway epithelium.

OBJECTIVE: The effect of tonicity changes in nebulizer solutions and irrigations on nasal mucosa is not well known. The present study aims to determine the basic mechanism of hypertonic solution on airway epithelial barrier. STUDY DESIGN AND SETTING: We investigated the electrical potential difference (PD) that is influenced by both active transport and the transepithelial electrical resistance of the epithelial mucosa in the human nose in vivo. The short circuit current (SCC) revealed net ion transport across the epithelium in the guinea pig trachea in vitro. Finally, the size dependency of macromolecules across the tracheal mucosa was determined in vitro using FITC-labeled dextrans of different sizes. RESULTS: PD was significantly decreased after topical application of hypertonic solution both in human and in guinea pig nose. SCC was significantly decreased after application of hypertonic solution. The transport of these dextrans from the basolateral to the apical side was not increased significantly after apical application of hypertonic saline. CONCLUSIONS: Hypertonic saline enhances the electrical permeability of the airway epithelial mucosa but not transport of macromolecule in the short term.

Measurement of water loss in human nasal mucosa.

BACKGROUND: Regulation of the barrier function in the nasal epithelium seems to be affected by the pathogenesis of allergic rhinitis. The measurement of transepidermal water loss has proven to be an important noninvasive method for assessing the efficiency of the skin as a protective barrier. Although the nasal mucosal epithelium also has such a protective function, the precise mechanism still is unknown. METHODS: We studied the alteration of nasal mucosal water loss in the basal state and after the nontraumatic applications of physiological saline, hypertonic saline (10% NaCl), nasal barrier cream, and 10% glycerol on the mucosal surface of the inferior turbinate. RESULTS: We observed that nasal mucosal water loss was increased by hypertonic saline and decreased by nasal cream and glycerol. CONCLUSION: For the first time, we showed the human nasal mucosal water loss both in the basal state and after topical application of various substances.

[Age distribution of transepithelial water loss in human nasal mucosa].

BACKGROUND: Dry nose is a common complaint in the elderly. Age distribution of transepithelial water loss of human nasal mucosa (TEWL) value was evaluated in this study. METHODS: Eighty-eight volunteers (50 men and 30 women) ranging from 10 to 75 years old were recruited for this study. Measurement of TEWL was performed on the inferior nasal turbinate. TEWL was measured with an evaporation meter applying Fick's law (Tewameter TM 300; Courage and Khazaka, Cologne, Germany). RESULTS: TEWL value tends to increase in order of age, indicating that the barrier function of epithelium may decline with age. CONCLUSION: The measurement of transepithelial water loss should be contributed to assess the efficiency of nasal mucosal barrier disorders in the elderly.

A comparison of raw starch-digesting glucoamylase production in liquid and solid cultures of Rhizopus strains.

Maximum growth for Rhizopus sp. A-11 was obtained at a zinc ion concentration of 0.7 ppm in a liquid medium. Glucoamylase (GA, EC 3.2.1.3) production in Rhizopus sp. A-11 was maximized at 710 U/ml, at the presence of 75 ppm for calcium and 0.7 ppm of zinc ions in liquid medium. Zinc ion is known as an essential biometal for Rhizopus growth; however, growth was inhibited by the zinc ion concentration, not maximized. Although calcium ion was not necessary to Rhizopus growth, GA production using Rhizopus sp. A-11 was markedly stimulated by calcium ion concentration over 75 ppm in the liquid medium. The GA productivity of the present liquid culture was about 4.4 times higher than that of the solid state culture, based on the unit starch amount in the liquid and solid media carbon source. The characteristics of the GA produced by the Rhizopus sp. A-11 liquid culture were interesting; that is, almost all the GA produced was classified as raw starch-digesting GA (GA-I). Secreted protein in the culture liquid after 30 h was nearly GA, and had a limited amount of impure protein. As a result, it was found that using a Rhizopus culture in a specified metal-ion regulated medium was an effective method for producing GA. Thus the present culture method was renamed the "metal-ion-regulated liquid culture method".