Effects of polypharmacy on the prevalence of adverse drug events resulting in outpatient visits and hospitalization.

A high proportion of hospitalizations is attributable to the prevalence of adverse drug events. This retrospective study included outpatients and inpatients to determine the prevalence of adverse drug events and if polypharmacy increases it. The prevalence, classification, and causality of adverse drug events were assessed based on medical records, laboratory values, and other data. Multivariate analysis (multiple logistic regression analysis) was performed with the presence or absence of adverse drug events at the time of the visit as the dependent variable and items for which the P-value was <0.25 in the univariate analysis as independent variables. The prevalence of adverse drug events was 13.0%, 10.9%, and 16.0% among all patients, the outpatient group, and the inpatient group, respectively. Multivariate analysis showed that polypharmacy (≥5 drugs) significantly increased the risk of adverse drug events in all patients. The prevalence of adverse drug events significantly increased with each additional drug used. We expect that minimizing the number of medications through moderation of the number of prescription drugs and elimination of polypharmacy will reduce the number of outpatient visits and hospitalizations due to adverse drug events.

Temperature dependence of the radiative recombination time in ZnO nanorods under an external magnetic field of 6 T.

The Temperature dependence of the exciton radiative decay time in ZnO nanorods has been investigated, which is associated with the density of states for the intra-relaxation of thermally excited excitons. The photoluminescence decay time was calibrated by using the photoluminescence intensity in order to obtain the radiative decay time. In the absence of an external magnetic field, we have confirmed that the radiative decay time increased with temperature in a similar manner to that seen in bulk material (∼ T1.5). Under an external magnetic field of 6 T parallel to the c-axis, we found that the power coefficient of the radiative decay time with temperature decreased (∼ T1.3) when compared to that in the absence of a magnetic field. This result can be attributed to an enhancement of the effective mass perpendicular to the magnetic field and a redshift of the center-of-mass exciton as a consequence of perturbation effects in the weak-field regime.

Underestimation of COVID-19 cases in Japan: an analysis of RT-PCR testing for COVID-19 among 47 prefectures in Japan.

BACKGROUND: Under the unique Japanese policy to restrict reverse transcriptase-polymerase chain reaction (RT-PCR) testing against severe acute respiratory syndrome coronavirus 2, a nationwide number of its confirmed cases and mortality remains to be low. Yet the information is lacking on geographical differences of these measures and their associated factors. AIM: Evaluation of prefecture-based geographical differences and associated predictors for the incidence and number of RT-PCR tests for coronavirus disease 2019 (COVID-19). DESIGN: Cross-sectional study using regression and correlation analysis. METHODS: We retrieved domestic laboratory-confirmed cases, deaths and the number of RT-PCR testing for COVID-19 from 15 January to 6 April 2020 in 47 prefectures in Japan, using publicly available data by the Ministry of Health, Labour and Welfare. We did descriptive analyses of these three measures and identified significant predictors for the incidence and RT-PCR testing through multiple regression analyses and correlates with the number of deaths through correlation analysis. RESULTS: The median prefectural-level incidence and number of RT-PCR testing per 100 000 population were 1.14 and 38.6, respectively. Multiple regression analyses revealed that significant predictors for the incidence were prefectural-level population (P < 0.001) and the number of RT-PCR testing (P = 0.03); and those for RT-PCR testing were the incidence (P = 0.025), available beds (P = 0.045) and cluster infections (P = 0.034). CONCLUSION: Considering bidirectional association between the incidence and RT-PCR testing, there may have been an underdiagnosed population for the infection. The restraint policy for RT-PCR testing should be revisited to meet the increasing demand under the COVID-19 epidemic.

Regulation of the yeast Yap1p nuclear export signal is mediated by redox signal-induced reversible disulfide bond formation.

Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H(2)O(2) or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H(2)O(2) and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H(2)O(2) or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H(2)O(2) and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H(2)O(2)-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys(598)) and second (Cys(620)) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.

Selective interaction of vitamin D receptor with transcriptional coactivators by a vitamin D analog.

The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor. A family of cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and activates the transactivation function of nuclear receptors in a ligand-dependent way. We examined interaction of VDR with these coactivators that was induced by several vitamin D analogs, since they exert differential subsets of the biological action of vitamin D through unknown mechanisms. Unlike other vitamin D analogs tested, OCT (22-oxa-1alpha,25-dihydroxyvitamin D3) induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1. Consistent with these interactions, only TIF2 was able to potentiate the transactivation function of VDR bound to OCT. Thus, the present findings suggest that the structure of VDR is altered in a vitamin D analog-specific way, resulting in selective interactions of VDR with coactivators. Such selective interaction of coactivators with VDR may specify the array of biological actions of a vitamin D analog like OCT, possibly through activating a particular set of target gene promoters.

[Coronary artery bypass grafting to left anterior descending coronary artery diagnosed by multidetector-row computed tomography].

A 64-year-old male received coronary angiography because of chest pain. Although coronary angiography showed total occlusion of right coronary artery (RCA) # 2 and left anterior descending branch (LAD) #6, and a significant stenosis of left circumflex (LCx) #11, it could not visualize LAD distal to LAD # 6. Since coronary multidetector-row computed tomography (MD CT) could visualize the distal LAD, coronary artery bypass grafting (CABG) was indicated for this patient. Left internal thoracic artery (LITA) was anastomosed to LAD and saphenous vein graft (SVG) was used for distal anastomoses to obtuse marginal branch (OM) and 4-posterior descending branch (# 4 PD). Postoperative course was uneventful. LITA anastomosed to LAD and SVG to OM and # 4 PD were visualized by postoperative coronary angiography. MD CT in addition to coronary angiography was demonstrated useful to assess precise lesions of the coronary artery disease in this case.

[C-reactive protein, white blood cell and body temperature following cardiovascular surgery, as predicting factors of postoperative infection].

The aim of this study is to clarify the relationship between CRP and postoperative infection after cardiovascular surgery. We had 5 cases of surgical site infection, and 3 cases of infective endocarditis (IE) among 57 patients selected for this study out of 405 patients who had undergone cardiovascular surgery from May 1995 to March 2005. CRP, WBC and body temperature (BT) were evaluated during 1 week after the operation. Our results showed not only that the mean value of CRP level in the 49 non-infection patients attained the peak on the 2nd or 3rd day after the operation (18.2 +/- 4.7 and 17.7 +/- 5.7 mg/dl), but also that each patient in this group showed the same pattern of CRP sequence. CRP in the 5 cases of postoperative infection showed different patterns from that in the non-infection group. CRP in 3 cases of valve replacement for IE showed significantly higher level than that in 16 cases of valve replacement without IE through 1 week after the surgery. WBC level in the non-infection group reached the peak just after the operation (11.3 +/- 4.4 x 10(3)/microl) and then decreased gradually during 1 week after the operation. WBC in the 3 cases of valve replacement for IE, did not show different sequence pattern from that in the 16 cases of valve replacement without IE. WBC in a case of postoperative mediastinal infection showed a similar pattern of sequence to that in the non-infection group although it showed a remarkably high level of CRP sequence through 1 week after the surgery. BT in the non-infection group became the lowest just after the operation and reached the peak 8 hours after the operation. It then decreased gradually during 1 week after the operation. Our study demonstrates that CRP sequence after the surgery might be useful to detect postoperative infection after cardiovascular surgery.

Positive and negative regulations of the renal 25-hydroxyvitamin D3 1alpha-hydroxylase gene by parathyroid hormone, calcitonin, and 1alpha,25(OH)2D3 in intact animals.

Reflecting the prime role of 1alpha,25(OH)2D3 in calcium homeostasis, the activity of 25-hydroxyvitamin D3 1alpha-hydroxylase, a key enzyme for 1alpha,25(OH)2D3 biosynthesis, is tightly regulated by 1alpha,25(OH)2D3, PTH and calcitonin. Its significant activity is found in kidney, though the enzymatic activity is also reported in extra-renal tissues. In the present study, we found that the 1alpha-hydroxylase gene abundantly expresses in kidney, and at low levels in other tissues and in some cell lines. Positive and negative regulations of 1alpha-hydroxylase gene by PTH, calcitonin, or 1alpha,25(OH)2D3 were observed at transcriptional levels in kidneys of animals and in a mouse proximal tubule cell line. Moreover, the protein kinase A inhibitor abrogated the PTH-mediated positive regulation. In mice lacking the vitamin D receptor, the 1alpha-hydroxylase gene expression was overinduced, and the inducible effect of either PTH or calcitonin, but not the repression by 1alpha,25(OH)2D3, was evident. Thus, vitamin D receptor is essential for the negative regulation by 1alpha,25(OH)2D3. Moreover, we demonstrate that renal 1alpha-hydroxylase gene expression in chronic renal failure model rats was decreased and the positive effect by PTH and calcitonin was diminished. The present study demonstrates that PTH and calcitonin positively regulate renal 1alpha-hydroxylase gene expression via PKA-dependent and independent pathway, respectively, and that 1alpha,25(OH)2D3 negatively regulates it mediated by vitamin D receptor. Furthermore, in a moderate state of chronic renal failure, renal cells expressing the 1alpha-hydroxylase gene appear to have diminished potential in response to PTH and calcitonin.

No enzyme activity of 25-hydroxyvitamin D3 1alpha-hydroxylase gene product in pseudovitamin D deficiency rickets, including that with mild clinical manifestation.

Pseudovitamin D deficiency rickets (PDDR) is an autosomal recessive disorder caused by defect in the activation of vitamin D. We recently isolated 25-hydroxyvitamin D3 1alpha-hydroxylase gene and identified four homozygous inactivating missense mutations in this gene by analysis of four typical cases of PDDR. This disease shows some phenotypic variation, and it has been suspected that patients with mild phenotypes have mutations that do not totally abolish the enzyme activity. To investigate the molecular defects associated with the phenotypic variation, we analyzed six additional unrelated PDDR patients: one with mild and five with typical clinical manifestation. By sequence analysis, all six patients were proven to have mutations in both alleles. The mutations varied, and we identified four novel missense mutations, a nonsense mutation, and a splicing mutation for the first time. The patient with mild clinical symptoms was compound heterozygous for T321R and a splicing mutation. The splice site mutation caused intron retention. Enzyme activity of the T321R mutant was analyzed by overexpressing the mutant 1alpha-hydroxylase in Escherichia coli cells to detect the subtle residual enzyme activity. No residual enzyme activity was detected in T321R mutant or in the other mutants. These results indicate that all of the patients, including those of mild phenotype, are caused by 1alpha-hydroxylase gene mutations that totally abolish the enzyme activity.

The strong hydrophobic domain of the activated estrogen receptor of porcine uterus.

Basic estrogen receptor (ER) molecule (vero-ER) of porcine uterus, which was previously shown to be the activated ER necessary to translocate from the cytoplasm into the nucleus, possesses a strongly hydrophobic nature. The strong hydrophobicity of vero-ER was concealed through binding with ER-binding factors (ERBFs). Vero-ER lost its strong hydrophobicity and its capability to bind with ERBFs after limited proteolysis by endogenous protease. The strong hydrophobic domain of vero-ER, indispensable for the nuclear translocation, was assumed to be located near the binding site with ERBFs.

Expression of soybean glycinin subunit precursor cDNAs in Escherichia coli.

As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the NcoI/PstI-pKK233-2 expression vector in Escherichia coli MV1190, respectively. The resultant plasmids directed the expression of 57-kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl beta-D-thiogalactoside. These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits. This procedure makes glycinin subunits available as a model for studying structure-function relationships in seed proteins using site-directed mutagenesis. This is the first expression of glycinin-like storage protein in E. coli.

Correlation between 25-hydroxyvitamin D3 1 alpha-hydroxylase gene expression in alveolar macrophages and the activity of sarcoidosis.

PURPOSE: To demonstrate expression of the 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase) gene in human alveolar macrophages and measure the correlations among the 1 alpha-hydroxylase mRNA level, the activity of sarcoidosis, and calcium metabolism. SUBJECTS AND METHODS: We examined 7 patients with sarcoidosis and 6 control patients with other pulmonary disorders who underwent bronchoalveolar lavage. Levels of 1 alpha-hydroxylase mRNA were measured by semiquantitative polymerase chain reaction amplification. We measured serum levels of calcium, ionized calcium, parathyroid hormone, calcitriol (1,25-dihydroxyvitamin D3), and 25-hydroxyvitamin D3 to evaluate calcium metabolism. To estimate the activity of sarcoidosis, we measured the cell count, the CD4/CD8 ratio in bronchoalveolar lavage cells, and the serum angiotensin-converting enzyme (ACE) activity. RESULTS: Expression of 1 alpha-hydroxylase was demonstrated in purified human alveolar macrophages. The 1 alpha-hydroxylase mRNA levels in bronchoalveolar lavage cells were fivefold higher in sarcoidosis patients than in control patients (10.8 +/- 3.6 vs. 2.2 +/- 1.4, P <0.003). Among all patients studied, there were significant correlations between the 1 alpha-hydroxylase mRNA level in bronchoalveolar lavage samples and the percentage of alveolar lymphocytes (r = 0.83, P <0.005), the CD4/CD8 ratio (r = 0.77, P <0.02), serum ACE level (r = 0.58, P <0.05), serum ionized calcium level (r = 0.58, P <0.05), and the calcitriol/25-hydroxyvitamin D3 ratio (r = 0.57, P <0.05). In the sarcoidosis patients, a significant correlation was also observed between 1 alpha-hydroxylase mRNA and the percentage of alveolar lymphocytes (r = 0.82, P <0.05). CONCLUSION: There is a correlation between 1 alpha-hydroxylase gene expression in alveolar macrophages with the activity of sarcoidosis and its associated disturbances in calcium metabolism.

Effect of molybdate on the molecular organization of the estrogen receptor system of porcine uterus.

Molybdate was shown to have complex effects in modulating the molecular organization of the constituents of the estrogen receptor (ER) system of porcine uterus. We showed previously the presence of one basic ER molecule (vero-ER) (sedimentation coefficient, 4.5S; Stokes radius, 44 A) and ER-binding factors (ERBFs) ["8S" ER-forming factor ("8S" ER-FF), (component A) X (component B)6; "6S" ER-FF, (component B)6; "5S" ER-FF, component A] in the porcine uterus [Fukai, F. & Murayama, A. (1981) J. Biochem. 95, 1697-1704]. Molybdate regulates the specific interaction of vero-ER with ERBFs in a complex way. The apparent Kd value (6.7 X 10(-10) M) of vero-ER with "8S" ER-FF in the presence of molybdate (30 mM) was decreased remarkably as compared with that (2.7 X 10(-9) M) in the absence of molybdate. In contrast, the apparent Kd value (3.7 X 10(-9) M) of vero-ER with "5S" ER-FF observed in the presence of molybdate (30 mM) was increased over ten-fold as compared with that in the absence of molybdate. Meanwhile, the affinity (Kd, 5 X 10(-9) M) of vero-ER for "6S" ER-FF was scarcely influenced by molybdate. These results reveal the mechanism by which molybdate selectively stabilizes "8S" ER. Molybdate further affected the molecular constitution of ERBFs. The dissociation of "8S" ER-FF into component A and component B, which takes place under hypertonic (0.4 M KCl) conditions at higher temperature (25 degrees C), was suppressed almost completely by molybdate (30 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Association and dissociation of estrogen receptor with estrogen receptor-binding factors is regulated by Mg2+.

It was recently shown that uterine estrogen receptor (ER) is translocated from the cytoplasm into the nucleus in the form of vero-ER X E (basic ER molecule bound with estradiol), and the translocation is inhibited by the specific binding of vero-ER X E with the cytoplasmic protein factors designated as ER-binding factors (ERBFs) ["5S" ER-forming factor ("5S" ER-FF), "6S" ER-FF, "8S" ER-FF] [Murayama, A. & Fukai, F. (1983) J. Biochem. 94, 511]. It was found that the specific interaction of vero-ER X E with the ERBFs is regulated by Mg2+ at physiological concentrations. The apparent Kd values of vero-ER X E for the ERBFs ["5S" ER-FF, 2.7 X 10(-9) M; "6S" ER-FF, 6.0 X 10(-8) M; "8S" ER-FF, 3.5 X 10(-8) M] observed in the presence of 1 mM Mg2+ were all increased over 10 times as compared with in the absence of Mg2+. The inhibitory effects of the ERBFs on the nuclear translocation of vero-ER X E were reduced by Mg2+.

Cytoplasmic estrogen receptor system of gilt uterus. Partial purification of component B labeled with [14C]iodoacetic acid.

Analysis of the cytoplasmic estrogen receptor (ER) system of gilt uterus by protecting ER from proteolysis showed that the ER system of gilt uterus is similar to that of cow uterus described previously. There was only one native unit molecule [native "4S" ER (sedimentation coefficient, 4.5S; Stokes radius, 45 A; molecular weight, 82,000)] with specific affinity towards estradiol. This molecule was further designated as vero-ER. "8S" ER-forming factor [("8S"ER)-FF] (Stokes radius, 51 A) was separated from the cytosol under hypotonic (low salt) conditions, and this was further dissociated into component A ["5S" ER-forming factor, ("5S"ER)-FF] (Stokes radius, 37 A) and component B (Stokes radius, 18.5 A) in the presence of sodium thiocyanate. Vero-ER was proteolyzed in the absence of Ca2+ ion by a cytoplasmic protease into modified "4S" ER (sedimentation coefficient, 4.5S; Stokes radius, 35 A; molecular weight, 65,000) which was further designated as secto-ER. The constituents of the cytoplasmic ER system of cow and gilt uteri cross-reacted with each other to undergo similar molecular assembly as in their own systems. When analyzed for various uterine specimens, fluctuation of ("8S"ER)-FF-level (1-50 unit/g tissue) was more remarkable than that of ER-level (3-6 x (10(-12) mol/g tissue). Component B labeled with [14C]iodoacetic acid was purified over 1,500-fold. Mixture of vero-ER and component B labeled with [14C]iodoacetic acid formed "6S' ER with 14C-activities under hypotonic conditions. This clearly excluded the possibility that component B is a catalyzer of self-association (dimerization) of vero-ER.

Studies on the molecular mechanism of the translocation of estrogen receptor from the cytoplasm into the nucleus.

A detailed study of the molecular mechanism of the translocation of estrogen receptor (ER) from the cytoplasm into the nucleus was undertaken in an in vitro system of porcine uterus. The capabilities of vero-ER . E (basic ER molecular bound with estradiol) (sedimentation coefficient 4.5S; Stokes radius 44 A) and the complexes ["5S" ER . E, (vero-ER . E) . (component A); "6S" ER . E, (vero-ER . E) . (component B)6; "8S" ER . E, (vero-ER . E) . (component B)6 . (component A)] with ER-binding factors (ERBFs) to translocate into the isolated nuclei were estimated by subtracting the amounts of ER adsorbed by the nuclear envelopes from those of ER bound to the whole nuclei. The results strongly supported our previous assumption that vero-ER . E translocates into the nuclei, and the complexes with ERBFs do not. The results suggested also that the binding site of vero-ER to ERBFs is required to be unoccupied in the process of the translocation of ER from the cytoplasm into the nucleus. The presence of a cytoplasmic factor (component C) which binds specifically with "5S" ER . E under low salt conditions was indicated. The complex, ("5S" ER . E) . (component C), was shown to possess relatively high affinity towards nuclear envelopes, but not to translocate into the nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the translocation of estrogen receptor from the cytoplasm into the nucleus by estrogen receptor-binding factors.

The molecular mechanism of the translocation of estrogen receptor (ER) from the cytoplasm into the nucleus was studied in an in vitro system taking into account the specific interactions of the basic constituents of the uterine ER-system which had been reported previously (Murayama, A. et al. (1980) J. Biochem. 88, 1457). It was found that ER is translocated from the cytoplasm into the nucleus in the form of vero-ER . estradiol (basic ER-unit bound with estradiol; sedimentation coefficient, 4.5S; Stokes radius, 44 A; Kd for estradiol, approximately 10(-10) M) dissociated from coexisting estrogen receptor-binding factors (ERBFs). ERBFs inhibited the translocation of vero-ER into nuclei through binding with it. The apparent equilibrium dissociation constants (Kd) of ERBFs for vero-ER were estimated by Scatchard analysis to be 2.6 X 10(-10) M ["5S" ER-forming factor ("5S" ER-FF)], 3.0 X 10(-9) M ("6S" ER-FF), and 2.5 X 10(-9) M ("8S" ER-FF), respectively. The inhibitory effects of ERBFs on the binding of vero-ER to the nucleus paralleled their association constants.

Estrogen receptor of cow uterus. III. Molecular constitution of estrogen receptor-binding factors.

"8S" estrogen receptor-forming factor [("8S"ER)-FF], a cytoplasmic factor of cow uterus, which binds specifically with native "4S" estrogen receptor (ER) to give "8S" ER under hypotonic (low salt) conditions was dissociated in the presence of 0.4 M NaSCN into two kinds of subunits designated as component A and component B. Characterization of components A and B was performed. The Stokes radius and molecular weight of component A were estimated to be around 37 A and 58,000, respectively. Those of component B were estimated to be around 18.5 A and 13,700, respectively. These components did not bind with estradiol, and underwent molecular assembly depending on the salt concentration of the medium to give complexes, which, in turn, bound with native "4S" ER to give ERs with higher sedimentation coefficients. Component A [designated as "5S" ER-forming factor, ("5S"ER)-FF] bound with native "4S" ER to give "5S" ER, which was stable under hypertonic (0.4 M KCl) conditions. Under hypotonic conditions, component B formed a hexamer [designated as "6S" ER-forming factor ("6S"ER)-FF], which bound with native "4S" ER to give "6S" ER. "4S" ER was dissociated from "6S" ER under hypertonic conditions. ("8S"ER)-FF was shown to be a 1 : 1 complex of component A and the hexamer of component B. These factors [("5S"ER)-FF, ("6S"ER)-FF and ("8S"ER)-FF] which bind specifically with native "4S" ER to give ERs with higher sedimentation coefficients were designated as estrogen receptor-binding factors (ERBFs).

Disorganization and in vitro assembly of the constituents of the cytoplasmic estrogen receptor system of cow uterus.

Analysis of estrogen receptor-binding factors (ERBFs) under isotonic (0.15 M KCl) conditions showed the presence of a factor designated as "7S" estrogen receptor-forming factor [("7S" ER)-FF], which binds with native "4S" estrogen receptor (ER) to form "7S" ER, besides the previously described component B and its hexamer ["6S" ER-forming factor, ("6S" ER)-FF]. Under hypertonic (0.4 M KCl) conditions at lower temperature (4 degrees C), ("7S" ER)-FF and the monomer of component B were present. Under hypertonic conditions at higher temperature (25 degrees C), ERBF existed as component A ["5S" ER-forming factor, ("5S" ER)-FF] and component B. ("7S" ER)-FF was estimated to be a 1 : 2 complex of component A and component B. Under isotonic conditions, approximately 30% of the cytoplasmic ER was "4S" form, and the rest was "6S" and "7S" forms. "5S" ER, "6S" ER, "7S" ER, and "8S" ER were reconstituted from partially purified native "4S" ER and the corresponding ERBFs. "5S" ER was estimated to be a 1 : 1 complex of native "4S" ER and ("5S" ER)-FF. "6S" ER was estimated to be a 1 : 1 complex of native "4S" ER and ("6S" ER)-FF. "7S" ER was estimated to be a 1 : 1 complex of native "4S" ER and ("7S" ER)-FF. "8S" ER was estimated to be a 1 : 1 complex of native "4S" ER and the "8S" ER-forming factor [("8S" ER)-FF] previously described, in which component A is bound to "4S" ER not directly, but via component B. A schematic molecular model of the in vitro dissociation and assembly of the constituents of the cytoplasmic ER system of cow uterus is presented.

Action of calpain on the basic estrogen receptor molecule of porcine uterus.

Basic estrogen receptor (ER) molecule (vero-ER) of the cytosol of porcine uterus was purified 1,200-fold after successive chromatographies on phenyl-Sepharose, hydroxylapatite, and DEAE-cellulose, followed by Sephadex G-150 gel filtration. The purified vero-ER was completely free from endogenous protease and ER-binding factor. The action of Ca2+-dependent cysteine proteinase (calpain) on vero-ER was studied by utilizing the purified receptor and calpains from porcine uterus (endogenous calpain), porcine kidney, and human erythrocytes. Proteolysis of vero-ER was followed by monitoring the disappearance of the binding capability of vero-ER with "8S" ER-forming factor. Vero-ER was proteolyzed by both the endogenous and the exogenous calpains in the presence of Ca2+. The calpains did not attack vero-ER in the absence of Ca2+. The results indicated the absolute requirement by calpain for Ca2+ for the limited hydrolysis of vero-ER. Uterine cytosol was shown to contain, in parallel with calpain, a protease which does not require Ca2+ for the limited proteolysis of vero-ER. The strongly hydrophobic domain of vero-ER, recently shown to be indispensable for the nuclear translocation of vero-ER (Murayama, A. & Fukai, F. (1983) FEBS Lett. 158, 255), was preferentially destroyed by both the Ca2+-requiring and -nonrequiring enzymes. It was assumed that calpain might intervene in the estrogen action by diminishing irreversibly the amount of the cytoplasmic ER capable of translocating into the nucleus.