Adverse event profiles of hypomagnesemia caused by proton pump inhibitors using the Japanese Adverse Drug Event Report (JADER) Database.

Proton pump inhibitors (PPIs) are commonly used for the prevention or treatment of gastric ulcers, but they can induce hypomagnesemia. Little is known about the onset duration and risk factors related to patient characteristics of this adverse event in Japanese patients. Therefore, we analyzed the time-to-onset of PPI-induced hypomagnesemia and evaluated the association between hypomagnesemia and PPIs using the Japanese Adverse Drug Event Report (JADER) database. We analyzed hypomagnesemia cases between 2004 and 2021. The time-to-onset analysis was performed using the Weibull distribution, and the adjusted reporting odds ratio (aROR) or 95% confidence interval (95% CI) was calculated using a multiple logistic regression analysis. The analysis database comprised 236,525 cases, with 188 cases associated with hypomagnesemia. The median onset duration (interquartile range) of PPI-induced hypomagnesemia was 99.0 (51.8-285.5 ) days, which is considered the random failure type. The multiple logistic regression analysis revealed that hypomagnesemia is significantly associated with male sex (aROR, 95% CI: 1.66, 1.23-2.25) , age < 60 (1.59, 1.14-2.21) , estimated body-mass index (eBMI) (0.94, 0.91-0.98) , PPIs (1.66, 1.18-2.30) , and the interaction of age (<60)*PPIs (1.58, 1.13-2.19) . However, diuretics were not significantly associated with hypomagnesemia. Our results suggest that serum magnesium levels should be measured regularly regardless of the duration of PPI use, especially in patients with male sex, age < 60, or low BMI. These findings will assist health professionals in the adequate use of PPIs. These findings need to be evaluated by cohort studies and long-term clinical investigations.

Time-dependent changes in serum magnesium levels in patients receiving cetuximab with low baseline serum sodium levels.

Cetuximab causes electrolyte abnormalities, such as hypomagnesemia, hypokalemia, and hypocalcemia. However, little is known about the relationships between the onset of hypomagnesemia, patient background before administration, and time-dependent changes in serum magnesium levels. Therefore, we examined the patient backgrounds that influenced the onset of hypomagnesemia and the time-dependent changes in serum magnesium levels in patients receiving cetuximab. A retrospective study was performed to investigate patients with advanced or recurrent colorectal cancer or head and neck cancer, treated with a cetuximab regimen from 2012 to 2020 at Kindai University Nara Hospital. In total, 52 patients who met the inclusion criteria were enrolled in this study. The serum magnesium level was significantly lower in the hyponatremia before the administration group than in the non-hyponatremia group (p < 0.001). Univariate logistic regression analysis revealed that the baseline serum sodium levels (odds ratio [OR]: 0.741, 95% confidence interval [CI]: 0.588-0.934) and the combination of magnesium oxide tablet (OR: 0.997, 95% CI: 0.995-0.999) were one of the independent factors for hypomagnesemia. These results indicated that hyponatremia before administration may be an indicator of serum magnesium levels after administration of cetuximab. Cetuximab-induced hypomagnesemia may be predicted using baseline serum sodium levels, and hypomagnesemia may be prevented by administration of magnesium oxide tablets. Our findings provided new evidence for the management of serum magnesium levels in patients receiving cetuximab.

Phenotypic modulation of smooth muscle cells in lymphoedema.

BACKGROUND: Lymphoedema is a debilitating progressive condition that is frequently observed following cancer surgery and severely restricts quality of life. Although it is known that lymphatic dysfunction and obstruction underlie lymphoedema, the pathogenic mechanism is poorly understood. Smooth muscle cells (SMCs) play pivotal roles in the pathogenesis of various vascular diseases, including atherosclerosis. OBJECTIVES: We analysed SMCs in lymphatic vessels from the lymphoedematous legs of 29 patients. METHODS: Expression of smooth muscle α-actin (SMαA) and smooth muscle myosin heavy chain (SM-MHC) isoforms SM1 and SM2 was investigated using immunohistochemistry. RESULTS: Compared with normal lymphatic vessels, all affected lymphatic vessels in chronic lymphoedema showed marked wall thickening. In addition to increases in the numbers of rows of SMαA(+) SM1(+) SMCs in the tunica media, SMCs were also observed in the subendothelial region (tunica intima). While most intimal and medial cells were positive for SMαA and SM1, staining for SM1 and particularly SM2, a marker of mature SMCs, progressively declined in lymphatic vessels in increasingly severe lymphoedema lesions. Consequently, the SM1(+) and SM2(+) cell fractions were significantly reduced in the tunica media and intima of lymphatic vessels. CONCLUSIONS: These observations indicate that the lymphatic tunica media and tunica intima consist mainly of phenotypically modulated SMCs, and that SMCs play a key role in the development of lymphoedema.

Responsiveness to interleukin 4 and interleukin 2 of peripheral blood mononuclear cells in atopic dermatitis.

Although the pathogenesis of atopic dermatitis (AD) is unknown, many immunologic abnormalities such as high levels of serum IgE and increase of IgE Fc receptor-positive lymphocytes have been demonstrated. Recently, interleukin 4 (IL-4) has been shown to induce enormously the production of IgE and to enhance the expression of IgE Fc receptor by B cells, suggesting the possible involvement of IL-4 in the pathogenesis of AD. We examined IL-4 responsiveness or interleukin 2 (IL-2) responsiveness of peripheral blood mononuclear cells of 31 patients with AD, 19 healthy individuals, and seven patients with other skin diseases. We found that IL-4 responsiveness of AD was higher than that of non-AD control, although IL-2 responsiveness of AD showed no significant change. However, the value of the IL-4 responsiveness did not significantly correlate with the clinical severity, personal history of respiratory allergy, serum IgE level, or clinical course of patients with AD. The hyperresponsiveness to IL-4 detected in AD was not likely to be due to the effects of steroids or anti-mast cell drugs because the value of IL-4 responsiveness was significantly low, compared to AD, in patients with other skin diseases who were treated similarly. Because the T-cell-enriched population, but not the B-cell-enriched population, showed significant proliferation in response to exogenous IL-4 or IL-2, T cells were the main population that reacted in our proliferation assay. These results indicate that IL-4-driven proliferative response may be efficiently operative in T cells in patients with AD.

Estradiol-stimulated proteolytic cleavage of the estrogen receptor in mouse uterus.

Increased proteolytic degradation of the estrogen receptor (ER) was detected in uterine cytosol of estradiol-treated ovariectomized mice compared to saline controls. Estradiol had no direct effect on the proteinase activity or susceptibility of the ER to the enzyme. The proteolytic activity gradually increased after a single injection of estradiol with early increases at 2 and 8 h followed by a progressive increase which reached a maximum at 36 h. The proteinase(s) activity resulted in cleavage of the native ER form of 65,000 (65 K ER) to a product of limited proteolysis having an apparent molecular weight of 54,000 (54 K ER). The pH optimum for this proteinase activity was 6.0. The proteinase was inhibited by 2.5 mM p-chloromercuribenzoic acid and 2.5 mM p-chloromercuriphenylsulfonate and partially inhibited by 2.5 mM iodoacetamide but not by 1 mg/ml leupeptin, 0.1 mg/ml antipain, 0.1 mg/ml chymostatin, 0.1 mg/ml pepstatin, 0.1 mg/ml E-64, 2.5 mg/ml soybean trypsin inhibitor, 2.5 mM phenylmethylsulfonylfluoride, 2.5 mM diisopropylfluorophosphate, and 10 mM EGTA. The results suggested that the proteinase(s) had a thiol group essential for its activity. Estrogen receptor in the mouse uterine cytosol fraction appears to be degraded sequentially in two steps in which 65 K ER is cleaved to a 54 K ER which upon longer incubation is further degraded to a 37 K form. The second step was inhibited by leupeptin, antipain, chymostatin, E-64, and p-chloromercuribenzoic acid. A possible function of the 54 K ER under physiological conditions is discussed since the 54 K ER was also found in nuclear samples. This form of the ER still retains the ability to bind estradiol and DNA.

Quantitative dot-blot enzyme immunoassay for serum amyloid A protein.

A dot-blot enzyme immunoassay is described which tests for serum amyloid A protein (SAA) using a commercially available antiserum. Heat treatment of the serum caused a marked increase in the apparent concentration of SAA. Pre-heated serum was directly bound to a nitrocellulose membrane using 'Bio-Dot' microfiltration apparatus to ensure uniformity. Then the SAA was stained using an 'Immun-Blot' assay kit (Bio-Rad, Laboratories, U.S.A.), and the optical density of the stained SAA spots was measured directly by a densitometer. The detection limit was 1.25 micrograms/ml of apoSAA1 (an isotype of SAA) dissolved in fetal bovine serum. The method had sufficient sensitivity to measure SAA in 76% of normal subjects. The method is ideally suited to the rapid processing of a large number of samples (up to 96 samples) using only a small amount of the antiserum.

Subunit compositions of trypsin inhibitors from tubers of taro, Colocasia antiquorum var. nymphaifolia?

Several trypsin inhibitors with different mobilities on polyacrylamide gel electrophoresis occur in the tubers of taro (Colocasia antiquorum), and they each have a dimeric molecular weight of 40,000. Of all the constituent subunits, molecular weight 20,000, of the taro trypsin inhibitor (TTI), three major subunit components were separated by chromatography on SP-Sephadex C-25 in 8 M urea, and they were named protomers alpha, beta, and gamma in the order of their elution from the SP-Sephadex column. After removal or dilution of the urea, the three protomers could be either reassociated individually or hybridized with each other to form dimeric inhibitors. All of the reassociated dimers were powerful inhibitors of trypsin. Among them, each dimer derived from protomers alpha and gamma was a weak inhibitor of chymotrypsin, whereas the dimer of protomer beta did not inhibit the enzyme. Therefore TTI is presumed to be a mixture of heterogeneous and homogenous dimers whose properties reflect those of their constituent protomers. It was also proved that the major three trypsin inhibitors (TTI-I, TTI-II, and TTI-III) previously isolated from taro tubers are composed of protomers alpha and gamma, i.e., TTI-II is a heterogeneous dimer of protomers alpha and gamma, and TTI-I and TTI-III are homogeneous dimers of protomers alpha and gamma, respectively. The molecular weight of a trypsin-TTI complex saturated with trypsin was found to be 79,000, suggesting the formation of a tetrameric complex.

Isolation and characterization of trypsin inhibitors from tubers of taro, Colocasia antiquorum var. nymphaifolia?

Three trypsin inhibitors were isolated from tubers of taro (Colocasia antiquorum var. nymphaifolia?) and named taro trypsin inhibitors (TTI)-I, -II, and -III. The final preparations were homogeneous by polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The three inhibitors showed strong and stoichiometric inhibition against bovine trypsin and the inhibitor constants (Ki) were estimated to be of the order of 10(-9) to 10(-10) M. In contrast, they had only a weak capacity to inhibit bovine alpha-chymotrypsin and did not inhibit subtilisins (BPN' and Carlsberg), porcine pepsin, or papain. Each inhibitor appeared to be a protein with a molecular weight of 40,000 which could be dissociated into two subunits, both of which had a molecular weight of 20,000. The inhibitors formed complexes with trypsin at molar ratios of 1:2, suggesting that each subunit of these inhibitors can react with the enzyme in a 1:1 molar ratio. The three inhibitors had similar amino acid compositions and none of them contained carbohydrate or free sulfhydryl group. The antitryptic activity of all three inhibitors was suppressed by treatment with 1,2-cyclohexanedione (CHD) but not with 2,4,6-trinitrobenzenesulfonate (TNBS), thus demonstrating each of the inhibitors to contain an arginyl residue at the reactive site.

Regions of the molecule responsible for substrate specificity of monoamine oxidase A and B: a chimeric enzyme analysis.

To examine regions of the monoamine oxidase (MAO, EC 1.4.3.4) molecule responsible for substrate recognition, a series of these enzymes, in which discrete regions in one molecule were substituted by corresponding sequences of the other, were constructed from the cDNAs of rat liver MAO A and MAO B and were expressed in yeast, Saccharomyces cerevisiae. Substrate specificities of the original and chimeric enzymes were examined in terms of the maximum activity (Vmax) and the affinity (Km) for serotonin, beta-phenylethylamine (PEA), and benzylamine. Chimeric enzymes with the amino-terminal portion (about 220 residues) and the amino-terminal and middle portions (about 400 residues) of MAO A and MAO B, respectively, exhibited substantially the same Km values as those of the parent enzymes. Extension of the substitution in the middle portion of a chimeric enzyme to the second half of the amino-terminal portion resulted in conversion of the Km values for serotonin to those of the counterpart. Data on relative Vmax values of the chimeric enzymes for the three substrates revealed that the relative catalytic activities were mainly determined by the presence of the middle portion. We conclude from these observations that the region between about residues 120-220 and about residues 50-400 is responsible for determination of the substrate specificity of MAO A and MAO B, respectively, while the middle portion, of about residues 220-400, may relate to the relative catalytic activity towards substrates.

Purification and characterization of an acidic amino acid-specific endopeptidase of Bacillus subtilis obtained from a commercial preparation (Protease Type XVI, Sigma).

An acidic amino acid-specific endopeptidase was purified from Protease Type XVI (Sigma), a commercial product from culture filtrate of Bacillus subtilis, by a series of column chromatographies on CM-Toyopearl (Fractogel) and Mono-S, guided by activity assay using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase HPLC. The molecular weight of the protease was estimated to be 18,000 by gel filtration on TSK gel G3000SWXL column using 6 M guanidine hydrochloride as an eluent, and 17,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the protease was 7.7. Studies on the substrate specificity with peptide p-nitroanilides and natural peptides revealed that the protease hydrolyzes the peptide bonds on the carboxyl-terminal side of acidic amino acids, especially of glutamic acid. The protease was completely inactivated by DFP, indicating the serine protease nature of the protease. The activity of the protease was also inhibited by EDTA and GEDTA, and reactivated by Ca2+. The protease contained 1.3 +/- 0.2 mol/mol protein of Ca2+. These results suggest that Ca2+ plays a vital role in the protease activity.

Purification and complex formation analysis of a cysteine proteinase inhibitor (cystatin) from seeds of Wisteria floribunda.

Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin). We purified and characterized one of these inhibitors, named WCPI-3. The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively. The isoelectric point was 5.7. WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM. Complex formation between WCPI-3 and Cys25-modified papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis. A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK. The VVAG sequence in this fragment corresponds to the conserved sequence QVVAG which is considered to be one of binding regions to cysteine proteinases. The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds.

Subsite mapping of an acidic amino acid-specific endopeptidase from Streptomyces griseus, GluSGP, and protease V8.

The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, GluSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitroanilide substrates which have a Glu residue at the P1 position. GluSGP and protease V8 favored Pro and Leu residues at S2, respectively, while the S3 subsite of GluSGP preferred Phe over either Ala or Leu. The S3 subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for GluSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a Km value of 0.41 mM (0.1 M Tris-HCl, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a Km value of 0.25 mM (0.1 M phosphate, pH 7.8), respectively. The kcat/Km values for these substrates obtained with GluSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.

Characterization of rat monoamine oxidase A with noncovalently-bound FAD expressed in yeast cells.

The FAD-binding cysteine of rat liver monoamine oxidase A (MAO A), Cys406, was converted to an alanine by site-directed mutagenesis of the cDNA. The wild-type and mutated enzymes were expressed in yeast cells and catalytic activities were assayed, using as substrates serotonin, tyramine, and kynuramine. Specific activities of the Ala-mutant for these substrates, calculated as the activities per pargyline-sensitive molecule, were about half of those of the wild-type enzyme. The Km values of the mutant enzyme for the substrates were similar to those of the wild-type enzyme. An adduct between FAD and pargyline, a mechanism-based inhibitor, was attached to the apoprotein in the wild-type enzyme, while in the Ala-mutant it was detached from the apoprotein, thereby indicating the presence of noncovalently bound FAD in the mutant enzyme. The Ala-mutant rapidly lost activity during incubation, whereas the wild-type enzyme retained the initial activity. Partial protection from inactivation occurred in the presence of FAD, but not of FMN. Recovery of the enzyme activity was nil when FAD was added after the inactivation. Thus, while the covalent attachment of FAD in MAO A is not required for the catalytic activity, it may function as a structural core for the active conformation in the membrane.

Hyperresponsibility to exogeneous interleukin 4 in atopic dermatitis.

Many humoral and cellular immunological abnormalities have been reported in atopic dermatitis (AD). Since interleukin 4 (IL-4) enhances IgE production and IgE-Fc receptor expression by B cells as well as [3H]-TdR incorporation by T cells, we hypothesized that IL-4 may play an important role in the regulation of the immune response in AD. We examined [3H]-TdR incorporation by peripheral blood lymphocytes from AD patients or from non-AD controls in the presence or absence of IL-4 or interleukin 2 (IL-2). We found that IL-4/IL-2 responsiveness was significantly higher in AD than controls, although the IgE levels did not seem to be correlated with IL-4/IL-2 responsiveness. It is possible that most humoral and cellular immunological abnormalities of AD may be due to this hyperresponsibility to IL-4.

CD56-Positive cutaneous lymphoma with multicentric Castleman's disease-like systemic manifestations.

We report a 55-year-old Japanese male with CD56+ cutaneous lymphoma. The patient had multiple cervical lymphadenopathy, a red nodule on his neck, and parotid gland nodularity. Histologic features of the biopsied cervical lymph node showed follicular hyperplasia with numerous plasma cells. A biopsied skin specimen of the nodule on his neck demonstrated dense infiltration of atypical large lymphocytes into the dermis. Immunohistochemical study of this specimen revealed CD3+, CD4+, and CD56+ expression in the majority of neoplastic cells. Polymerase chain reaction assays for the detection of Epstein-Barr virus sequences were positive for lymph node and skin DNA. Laboratory examinations showed polyclonal gammopathy, pancytopenia, and high serum interleukin-6 levels. These clinical and histological findings resembled those of multicentric Castleman's disease.

[Cellular immunoregulation in atopic dermatitis--hyperresponsiveness to interleukin-4 and hyporesponsiveness to interleukin-2].

We examined the responsiveness to Interleukin-4 (IL-4) and Interleukin-2 (IL-2) of peripheral blood lymphocytes from patients with atopic dermatitis (AD) and non-AD controls. The IL-4 responsiveness of the AD patients was found to be higher than that of the non-AD controls. In contrast, the IL-2 responsiveness of the AD patients was relatively lower than that of the non-AD controls. When IL-4/IL-2 responsiveness ratio was compared, the IL-4/IL-2 responsiveness ratio of the AD patients (54.1 +/- 18.5) was significantly higher than that of the non-AD controls (24.8 +/- 9.9, p less than 0.001). The high IL-4/IL-2 responsiveness ratio found in the AD patients may possibly be related to the pathogenesis of AD.

Rat monoamine oxidase B expressed in Escherichia coli has a covalently-bound FAD.

Rat liver monoamine oxidase B (MAO B) was expressed in E. coli as catalytically active form, though inclusion bodies of the enzyme were also formed as a major protein in the cell. The active form of the recombinant MAO B exhibited similar properties as rat liver enzyme and localized in membrane of the bacteria. Covalent attachment of FAD to polypeptide chain of the recombinant enzyme was revealed by a labeling experiment with [3H]-pargyline, an irreversible mechanism-based inhibitor, indicating that the covalent linkage of FAD to the apoprotein was formed even in the prokaryotic cell. This observation suggests autocatalytic formation of the linkage in MAO B.