Biomedical subjects

Koichi Ito

Publications and source records attributed to Koichi Ito.

At least 55 records · Page 3Linked to original sources

PubMed2005

[Method of component assay of alpha-glucosyltransferase-treated stevia (enzymatically modified stevia) products using enzymatic hydrolysis].

We have developed an analytical method for components of alpha-glucosyltransferase-treated stevia, a food additive product. Suitable conditions to separate additional sugar from alpha-glucosyltransferase-treated stevia by using glucoamylase were found (55 degrees C for 3 hr with 250 U of glucoamylase in 10 mL of reaction solution). By solid-phase extraction using a C18 cartridge column, polysaccharides were excluded from the sample, and the glycosides and sugar obtained after hydrolysis with glucoamylase were separated on another C18 cartridge column. The glycosides and sugar contents were determined by HPLC. By this method, additional sugar was detected in all of three product samples tested and the sugar was glucose. The contents of glucose and total glycosides (minus unreacted glycoside) were 25-42% and 35.7-52.5%, respectively. In alpha-glucosyltransferase-treated stevia, the sum of total glycosides and glucose amounted to 77.5-80.4% of the total and their recoveries from samples from which polysaccharide had been excluded by C18 cartridge column processing were over 85%. The contents of alpha-glucosyltransferase-treated stevia obtained by multiplying the sugar content by the coefficient (0.9) for hydrolysis and converting on dry weight basis were all over 80.0% and met the standard set by the Japan Food Additives Association.

Carbohydrates↗

PubMed2005

[Structural analysis of unknown dyes detected in hajikami (ginger pickled in vinegar)].

Hajikami (ginger pickled in vinegar) is often used as a relish for grilled fish, and it often contains coloring agents. We detected Rose Bengal (R105) and two unknown dyes in some Hajikami by thin layer chromatography. In this study, we tried to characterize these unknown dyes by HPLC with photodiode array detection (PDA-HPLC), liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). PDA-HPLC analysis showed that the spectra of the unknown dyes resembled that of R105, suggesting the molecular structures of these two dyes are similar to that of R105. Furthermore, LC/MS analysis suggested that the these dyes are R105 in which one or two iodines are replaced by hydrogen. Finally, the two dyes were determined by 1H-NMR and 13C-NMR to be 4,5,6,7-tetrachloro-3',6'-dihydroxy-2',7'-diiodo spiro[isobenzofuran- 1 (3H),9'-[9H]xanthen]-3-one disodium salt and 4,5,6,7-tetrachloro-3',6'-dihydroxy-2',7'-diiodo spiro[isobenzofuran-1(3H),9'[9H]xanthen]-3-one disodium salt.

Chromatography, High Pressure Liquid↗

PubMed2004

Clinical evaluation of color Doppler imaging for the differential diagnosis of thyroid follicular lesions.

Ultrasonography (US) of the thyroid gland has recently proved to be a useful clinical diagnostic method, and the newly developed high-resolution US with a color Doppler flow mapping function can reveal fine details of the thyroid gland and the hemodynamic features of a thyroid neoplasm. US can yield a diagnostic accuracy of more than 90% for thyroid carcinoma, especially papillary carcinoma. However, neither conventional B-mode US imaging nor aspiration biopsy cytology has delivered satisfactory results for follicular carcinoma. The aim of this study was to evaluate the clinical usefulness of color Doppler imaging for the differential diagnosis of thyroid follicular lesions. A color Doppler scan was performed in 310 patients with a solitary cold nodule, and a combination of B-mode US and color Doppler findings, including tumor vascularity and results of a fast Fourier transform (FFT) analysis were used to create the following diagnostic grading system for differential diagnosis of follicular lesions: grade 1, benign follicular lesion [no color flow mapping (CFM) inside the nodule]; grade 2, benign peripheral type [CFM only in peripheral area, pulsatility index (PI) < 1.0]; grade 3, suspected follicular carcinoma (penetrating CFM, vascularity moderate); grade 4, follicular carcinoma (high-velocity penetrating CFM, PI = 1.0). All patients were subjected to surgical resection, and histologic examination was used to confirm the diagnosis. The grades of the 177 adenomatous nodules were as follows: grade 1, 46.9%; grade 2, 48.0%; grade 3, 5.1%; grade 4, 0%. The corresponding percentages for the 89 follicular adenomas were 16.9%, 49.4%, 30.3%, and 3.4%; and for the 44 follicular carcinomas they were 0%, 13.6%, 45.5%, and 40.9%. On the assumption that grade 1 and 2 lesions are benign and grade 3 and 4 lesions are malignant, 38 of the 44 follicular carcinomas and 227 of the 266 benign tumors had been accurately diagnosed, yielding a sensitivity of 88.9%, a specificity of 74.2%, and an accuracy of 81.0% for the grading system. Color Doppler imaging of 310 follicular tumors has revealed that high-velocity pulsatile blood flow penetrating the tumor is a characteristic finding of follicular carcinoma and is a new diagnostic criterion for performing color Doppler imaging. The differential diagnostic grading scores for color Doppler examinations and the results of FFT analysis demonstrate that US with the color Doppler function can play an important role in the differential diagnosis of thyroid tumors.

Adenoma↗

PubMed2004

IL-1alpha affects mineralized nodule formation by rat osteoblasts.

The present study was conducted to determine the effect of the inflammatory mediator interleukin 1alpha (IL-1alpha) on osteogenesis using rat osteoblasts. We examined the effect of IL-1alpha on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in rat osteosarcoma cell lines. The cells were cultured with alpha-minimum essential medium containing 10% fetal bovine serum with and without 0, 1, 10, and 100 units/ml of IL-1alpha for up to 14 days. The mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a Calcium C-Test kit. The expression of extracellular matrix proteins was estimated by determining levels of mRNAs using the semiquantitative reverse transcription-polymerase chain reaction. The mineralized nodule formation and the calcium content in mineralized nodules were remarkably suppressed by IL-1alpha after 5 days of culture. The ALPase activity decreased in a dose-dependent manner in the presence of IL-1alpha after 7 days of culture. The expression of type I collagen was decreased after 3 days of culture with IL-1alpha. The expression of bone sialoprotein was slightly decreased at days 3 and 5, and the expression of osteopontin was increased at days 3, 5, and 7 of culture with IL-1alpha. These results suggest that IL-1alpha suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.

Alkaline Phosphatase↗

PubMed2004

SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease.

Autoimmune thyroid disease (AITD) is caused by an immune response to self-thyroid antigens and has a significant genetic component. Antisense RNA transcripts have been implicated in gene regulation. Here we have identified a novel zinc-finger gene, designated ZFAT (zinc-finger gene in AITD susceptibility region), as one of the susceptibility genes in 8q23-q24 through an initial association analysis using the probands in the previous linkage analysis and a subsequent association analysis of the samples from a total of 515 affected individuals and 526 controls. The T allele of the single-nucleotide polymorphism (SNP), Ex9b-SNP10 located in the intron 9 of ZFAT, is associated with increased risk for AITD (dominant model: odds ratio = 1.7, P = 0.000091). The Ex9b-SNP10 falls into the 3'-UTR of truncated-ZFAT (TR-ZFAT) and the promoter region of the small antisense transcript of ZFAT (SAS-ZFAT). In peripheral blood lymphocytes, SAS-ZFAT is exclusively expressed in CD19+ B cells and expression levels of SAS-ZFAT and TR-ZFAT seemed to correlate with the Ex9b-SNP10-T-associated ZFAT-allele, inversely and positively, respectively. The Ex9b-SNP10 is critically involved in the regulation of SAS-ZFAT expression in vitro and this expression results in a decreased expression of TR-ZFAT. These results suggested that the SNP-associated ZFAT-allele plays a critical role in B cell function by affecting the expression level of TR-ZFAT through regulating SAS-ZFAT expression and that this novel regulatory mechanism of SNPs might be involved in controlling susceptibility or resistance to human disease.

B-Lymphocytes↗

PubMed2004

Attenuated plasticity of postsynaptic kainate receptors in hippocampal CA3 pyramidal neurons.

Kainate receptor-mediated components of postsynaptic currents at hippocampal mossy fiber synapses have markedly slower kinetics than currents arising from AMPA receptors. Here, we demonstrate that other aspects of kainate and AMPA receptor function at this synapse are distinct; in particular, kainate receptor currents are less sensitive to short- and long-term increases in presynaptic strength. EPSCs arising predominantly from AMPA receptors exhibited well characterized paired-pulse facilitation, frequency facilitation, and NMDA receptor-independent long-term potentiation, whereas isolated kainate receptor synaptic currents (KA-EPSCs) exhibited attenuated facilitation and long-term potentiation. In addition, KA-EPSCs varied in their sensitivity to a low-affinity competitive antagonist, suggestive of a synaptic heterogeneity greater than that of EPSCs comprised predominantly of AMPA receptors. These data suggest that the proportional contribution of AMPA and kainate receptors to ensemble synaptic currents will vary depending on the firing frequency of mossy fiber afferents. These synaptic features may be a mechanism for limiting activation of kainate receptors at mossy fiber synapses, which has been shown to be involved in seizurogenic firing of the CA3 network.

Animals↗

PubMed2004

Crystal structure and functional analysis of the eukaryotic class II release factor eRF3 from S. pombe.

Translation termination in eukaryotes is governed by two interacting release factors, eRF1 and eRF3. The crystal structure of the eEF1alpha-like region of eRF3 from S. pombe determined in three states (free protein, GDP-, and GTP-bound forms) reveals an overall structure that is similar to EF-Tu, although with quite different domain arrangements. In contrast to EF-Tu, GDP/GTP binding to eRF3c does not induce dramatic conformational changes, and Mg(2+) is not required for GDP binding to eRF3c. Mg(2+) at higher concentration accelerates GDP release, suggesting a novel mechanism for nucleotide exchange on eRF3 from that of other GTPases. Mapping sequence conservation onto the molecular surface, combined with mutagenesis analysis, identified the eRF1 binding region, and revealed an essential function for the C terminus of eRF3. The N-terminal extension, rich in acidic amino acids, blocks the proposed eRF1 binding site, potentially regulating eRF1 binding to eRF3 in a competitive manner.

Amino Acid Motifs↗

PubMed2004

Effect of beta-alanyl-L-histidinato zinc on the differentiation pathway of human periodontal ligament cells.

We examined the effect of beta-alanyl-L-histidinato zinc (AHZ) on the differentiation of human periodontal ligament (HPDL) cells. HPDL cells were cultured with alpha-minimum essential medium containing 10% fetal bovine serum with or without 10(-4) or 10(-5) M AHZ for up to 10 days. The expression of runt-related transcription factor-2/core binding factor alpha-1 (RUNX2/Cbfa1), Sox9, bone morphogenetic proteins (BMPs), and BMP receptors was measured using semiquantitative reverse transcription-polymerase chain reactions. Phosphorylation of Smad1 was determined using a Western blot analysis. RUNX2/Cbfa1 expression increased markedly in cells cultured with AHZ, while Sox9 expression increased slightly. BMP-7 expression was much higher than that of controls in cultures with AHZ, whereas BMP-2 expression was only slightly higher. The expression of BMP receptors increased markedly in cells cultured with AHZ. The phosphorylation of Smad1, a signal-transducing molecule for BMP-2 and BMP-7, was increased markedly in cultures with AHZ. The results suggest that AHZ diverts the differentiation pathway of HPDL cells to the osteoblast lineage via BMP-2 or BMP-7.

Animals↗

PubMed2004

Association of the T-cell regulatory gene CTLA4 with Graves' disease and autoimmune thyroid disease in the Japanese.

Autoimmune thyroid disease (AITD) is caused by an immune response to self-thyroid antigen. The cytotoxic T-lymphocyte antigen-4 ( CTLA4) gene, encoding a negative regulator of the T-lymphocyte immune response, had been reported to be associated and/or linked to AITD. Recently, AITD susceptibility in the Caucasians was mapped to the 6.1-kb 3'UTR of the CTLA4 gene, in which the three single-nucleotide polymorphisms (SNPs) CT60, JO31, and JO30 were strongly associated with AITD. In order to determine the association of the CTLA4 gene with AITD in the Japanese, case-control association analysis for the four SNPs of the CTLA4 gene using 380 AITD patients and 266 healthy controls was done. Among the SNPs examined, the SNP JO31 was most significantly associated with AITD in the Japanese, whereas the association of the JO30 with AITD was not observed. The frequency of the disease-susceptible G allele of the JO31 of the Japanese control was higher than that of the Caucasians (67.1% vs 50.2%); however, the G allele of the JO31 was associated with Graves' disease (GD) (67.1% vs 76.3%, P=0.0013) and AITD in the Japanese (67.1% vs 74.2%, P=0.0055). Furthermore, the G allele of the JO31 was associated with the increased risk for GD [ P=0.0051, odds ratio (OR)=1.7] and AITD ( P=0.016, OR=1.5) in a dominant model. These results suggested that the CTLA4 gene is involved in the susceptibility for GD and AITD in the Japanese.

Alleles↗

PubMed2004

Adenolipoma of the thyroid gland: report of a case.

We report a case of adenolipoma of the thyroid gland, which is very unusual because thyroid adenoma rarely contains mature adipose tissue. A 55-year-old Japanese woman presented with a 1-month history of a neck mass. A blinded fine-needle aspiration biopsy showed benign follicular cells with no adipose cells, and we performed a left hemithyroidectomy. The specimen measured 7.0 x 4.4 cm and weighed 59.9 g. The cut surface of the tumor revealed a solid yellowish mass, and hematoxylin-eosin staining showed that the solid tumor was well separated from the adjacent thyroid tissue by a thin fibrous capsule. The tumor was found to be follicular adenoma composed of epithelial cells arranged in monotonous microfollicular patterns, intermingled with mature adipose tissue. No adipose tissue was found outside the tumor, and there were no signs of amyloid deposits in the tumor or adjacent thyroid tissue. The pathological diagnosis was adenolipoma of the thyroid gland. We discuss the pathogenesis of this entity and review the previously documented cases.

Adipose Tissue↗

PubMed2004

Gene expression in the developing rat mandible: a gene array study.

To analyse the molecular events that occur in the developing mandible, gene arrays containing probes for 1176 known genes were used. Total RNA was extracted from the mandibles of 1- and 6-day-old rats. Radiolabeled probes were then synthesised and used to probe the DNA arrays. Of the 1176 genes examined, 306 were detectable, and the latter were analyzed by bioinformatics algorithms. K-means clustering grouped 72 genes into Up, 56 genes into Down and 178 genes into NO change. A large number of genes related to cell receptors (by ligands) were grouped into the Down cluster. Gene array technology appears to be a useful tool for studying the complex process of mandibular development.

Animals↗

PubMed2004

Transient idling of posttermination ribosomes ready to reinitiate protein synthesis.

The fate of ribosomes between termination and initiation during protein synthesis is very basic, yet poorly understood. Here we found that translational reinitiation of the alkaline phosphatase gene occurs in Escherichia coli from an internal methionine codon when the authentic translation is prematurely terminated at a nonsense codon that is within seven codons upstream of the reinitiation codon (which we refer to as "reinitiation window"). Changing the reading frame downstream of the stop codon did not abolish the reinitiation, while inactivating the upstream initiation codon abolished the reinitiation. Moreover, depletion of the ribosome recycling factor (RRF), which disassembles posttermination ribosomes in conjunction with elongation factor G, did not influence the observed reinitiation. These findings suggest that posttermination ribosomes can undergo a transient idling state ready to reinitiate protein synthesis even in the absence of the Shine-Dalgarno (SD) sequence within the reinitiation window by evading disengagement from the mRNA.

Alkaline Phosphatase↗

PubMed2004

Surgical treatment of Graves' disease in children.

Thyroidectomy may be indicated in children with Graves' disease who have adverse reactions to antithyroid drugs or who relapse after antithyroid drug therapy. We investigated the characteristics of childhood Graves' disease from the standpoint of surgical outcome. Between 1989 and 1998, 1897 patients with Graves' disease underwent thyroidectomy and their thyroid function could be evaluated 2 to 3 years after thyroidectomy. The patients were divided into three groups according to age at thyroidectomy: 74 patients were 15 years old or less (children), 345 patients 16 to 20 years of age (adolescents), and 1478 patients 21 years of age or more (adults). The children included higher proportions of patients who had a large goiter (> 100 g), high thyrotropin-binding inhibitory immunoglobulin (TBII) level (> 50%), and small remnant thyroid (< 4 g). At 2 to 3 years after thyroidectomy, the overt recurrence rate of the children, adolescents, and adults was 9.5%, 4.9%, and 5%, respectively. The cumulative recurrence-free rate of the children, adolescents, and adults at 5 years after thyroidectomy was 82%, 90%, and 92%, respectively. Surgical complications were more frequently observed in children. Considering the aggressiveness of childhood Graves' disease, subtotal thyroidectomy with thyroid remnant less than 3 g is the procedure of choice for preventing recurrent hyperthyroidism.

Adolescent↗

PubMed2004

Stimulation of thyroid-stimulating hormone (TSH) receptor antibody production following painless thyroiditis.

OBJECTIVE: Development or recurrence of Graves' disease (GD) following painless thyroiditis (PT) has been documented. Therefore, we measured titres of TSH receptor antibodies (TSHR Ab) using a novel sensitive TSHR Ab assay in patients with PT to determine whether PT enhances TSHR Ab production, possibly triggering the development or recurrence of GD. DESIGN AND MEASUREMENTS: Ninety-two patients who developed PT were studied. Group G consisted of 40 patients with a history of GD (19 patients in remission, 21 who had stopped taking antithyroid drugs during pregnancy). Group P consisted of 52 patients with no history of GD. Serum thyroid hormone levels, thyroid autoantibodies including TSHR Ab, and 123I uptake at 24 h (RAIU) were measured in these patients at the time of PT onset. TSHR Abs were measured by radioreceptor assay using porcine TSH receptors (pTBII) or human TSH receptors (hTBII). RESULTS: There were no significant differences in serum thyroid hormone levels or pTBII values between groups G and P. Nor was there any significant difference between p- and h-TBII values in group P. There was also no significant difference in pTBII levels before, compared to at the time of PT onset in group G patients. However, hTBII values at the PT onset were significantly higher in the group G than in the group P (7.7 +/- 9.8%vs. 1.4 +/- 5.4%, P = 0.0014). The rate of hTBII positivity was also significantly higher in group G than in group P (12/40 vs. 3/52, P = 0.002). Furthermore, the RAIU in group G patients was significantly higher than that in group P patients (2.8 +/- 2.4%vs. 1.3 +/- 0.9%, P = 0.0002). GD recurrence was observed in seven patients in group G, whose hTBII levels were significantly higher than those of other patients in this group (17.0 +/- 11.8%vs. 5.7 +/- 8.2%, P = 0.02). Of these seven with relapses, five had hTBII values exceeding 15%. CONCLUSIONS: TBII elevation at the onset of PT in patients with a history of GD was detected by a sensitive hTBII assay. Destruction of the thyroid by PT may trigger GD recurrence in patients with a history of GD.

Adult↗

PubMed2004

Ribosome recycling factor disassembles the post-termination ribosomal complex independent of the ribosomal translocase activity of elongation factor G.

Ribosome recycling factor (RRF) disassembles post-termination ribosomal complexes in concert with elongation factor EF-G freeing the ribosome for a new round of polypeptide synthesis. How RRF interacts with EF-G and disassembles post-termination ribosomes is unknown. RRF is structurally similar to tRNA and is therefore thought to bind to the ribosomal A site and be translocated by EF-G during ribosome disassembly as a mimic of tRNA. However, EF-G variants that remain active in GTP hydrolysis but are defective in tRNA translocation fully activate RRF function in vivo and in vitro. Furthermore, RRF and the GTP form of EF-G do not co-occupy the terminating ribosome in vitro; RRF is ejected by EF-G from the preformed complex. These findings suggest that RRF is not a functional mimic of tRNA and disassembles the post-termination ribosomal complex independently of the translocation activity of EF-G.

Escherichia coli↗

PubMed2004

Histological evidence of osseointegration in human retrieved fractured hydroxyapatite-coated screw-type implants: a case report.

Histological evidence of clinically successful dental implants is very rare. This case report presents histological evidence of osseointegration in human implants retrieved because of fractures at the connected portion between the abutments and fixtures due to a car accident. The duration of functional loading of the implants was 18 months. Two hydroxyapatite (HA)-coated screw-type implants were removed with part of the healthy bone from the mandibular left molar region. A block was prepared using cutting and grinding equipment to obtain a central section approximately 50 microm thick, which was stained with basic fuchsin and methylene blue. Histological examination revealed that the bone was dense and in close relation with the HA coating of the implants. The interspaces of each thread of the implant were filled with mineralized bone. Peri-implant soft tissue was not observed in the section. A high degree of osseointegration was noted, with a bone-to-implant contact of 87.5% (implant corresponding to tooth 36) and 97.4% (implant corresponding to tooth 37). The connection between the 30 and 50 microm HA coating and the metal was uniformly tight and constant. In conclusion, the histological evidence showed a high degree of osseointegration in two HA-coated screw-type dental implants retrieved after functional loading for 18 months.

Accidents, Traffic↗

PubMed2004

Effects of a new ultrasonic scaler on fibroblast attachment to root surfaces: a scanning electron microscopy analysis.

OBJECTIVE: A study was conducted to examine the effectiveness of scaling and root planing using a new ultrasonic scaler (Vector). METHODS: Eighty extracted teeth affected by periodontal disease were sorted into four groups of 20, each of which was subjected to one the following procedures: use of the Vector, Vector with polish, Enac and a Gracey scaler. The time spent on cleaning was measured. Half of the sample teeth were examined at random for surface roughness, and the surface texture was evaluated by means of the Remaining Calculus Index (RCI) and the Roughness and Loss of Tooth Substance Index (RLTSI). The remaining samples were incubated in dishes with a suspension of fibroblasts. After measuring the number of attached cells, the attachment of fibroblasts was observed by scanning electron microscopy. RESULTS: The RLTSI values in the Vector and Vector with polish groups were significantly lower than those in the Enac and Gracey groups, whereas the number of attached cells in the Vector with polish group was larger than in the Enac group. Cell attachment in the Vector and Vector with polish groups proved to be better than in the Enac and Gracey groups. CONCLUSION: Since use of the Vector with polish was able to provide scaling and root planing with minimal damage and tight attachment of fibroblasts, it is suggested that this may be a useful instrument for scaling and root planing.

Cell Adhesion↗

PubMed2004

Effect of carnosine on runt-related transcription factor-2/core binding factor alpha-1 and Sox9 expressions of human periodontal ligament cells.

BACKGROUND: Previous studies have reported that beta-alanyl-l-histidine (carnosine) enhanced the process of wound healing by stimulating effusion at the initial stage of inflammation, and also enhanced runt-related transcription factor-2/core binding factor alpha-1 (RUNX2/Cbfa1), Sox9, bone morphogenetic protein-2 (BMP-2) and BMP-7 expressions of human periodontal ligament cells. OBJECTIVES: In order to clarify the relationship between RUNX2/Cbfa1 or Sox9 expressions and BMP-2 or BMP-7 expressions enhanced by carnosine, we determined the effect of carnosine on the expression of BMP receptors and activation of their downstream signaling molecules in human periodontal ligament cells. MATERIAL AND METHODS: Human periodontal ligament cells were cultured with alpha-minimum essential medium containing 10% fetal bovine serum with or without 10(-4) or 10(-5) m carnosine for up to 10 days. The gene expression of BMP receptors, RUNX2/Cbfa1 and Sox9 was measured using semiquantitative reverse transcription-polymerase chain reaction. Phosphorylation of Smad1 was determined using a western blot analysis. RESULTS: Alkaline phosphatase activity increased in cultures with carnosine. Among the BMP receptors, expression of ActR-I and BMPR-II increased in cultures with carnosine, whereas expression of BMPR-IA, BMPR-IB, ActR-IIA and ActR-IIB was not affected. Culture with carnosine increased phosphorylation of Smad1, a signal-transducing molecule for BMP-2 and BMP-7. Noggin reduced carnosine-induced up-regulation of RUNX2/Cbfa1 and Sox9 mRNA, suggesting that BMPs were responsible for up-regulating RUNX2/Cbfa1 gene expression in human periodontal ligament cells. CONCLUSION: These results suggest that carnosine enhance RUNX2/Cbfa1 and Sox9 expression of human periodontal ligament cells via the autocrine action of BMP-2 or BMP-7 produced by the cells.

Alkaline Phosphatase↗