Human Beta Subunit Research Articles

Personalized diagnostic approach and indirect quantification of extravasation in human anaphylaxis.

Anaphylaxis is the most acute and life-threatening manifestation of allergic disorders. Currently, there is a need to improve its medical management and increase the understanding of its molecular mechanisms. This study aimed to quantify the extravasation underlying human anaphylactic reactions and propose new theragnostic approaches. Molecular determinations were performed in paired serum samples obtained during the acute phase and at baseline from patients presenting with hypersensitivity reactions. These were classified according to their severity as Grades 1, 2 and 3, the two latter being considered anaphylaxis. Tryptase levels were measured by ImmunoCAP, and serum protein concentration was quantified by Bradford assay. Human serum albumin (HSA) and haemoglobin beta subunit (HBB) levels were determined by Western blot and polyacrylamide gel electrophoresis, respectively. A total of 150 patients were included in the study. Of them, 112 had experienced anaphylaxis (83 and 29 with Grade 2 and 3 reactions, respectively). Tryptase diagnostic efficiency substantially improved when considering patients' baseline values (33%-54%) instead of the acute value threshold (21%). Serum protein concentration and HSA significantly decreased in anaphylaxis (p < .0001). HSA levels dropped with the severity of the reaction (6% and 15% for Grade 2 and 3 reactions, respectively). Furthermore, HBB levels increased during the acute phase of all hypersensitivity reactions (p < .0001). For the first time, the extravasation underlying human anaphylaxis has been evaluated based on the severity of the reaction using HSA and protein concentration measurements. Additionally, our findings propose new diagnostic and potential therapeutic approaches for this pathological event.

Characterization of the promoter of the human farnesyltransferase beta subunit and the impact of the transcription factor OCT-1 on its expression.

Farnesyltransferase (FTase) enables about 100 proteins to interact with cellular membranes by catalyzing the posttranslational addition of a farnesyl group. Farnesylated proteins provide important functions and inhibitors against the β-subunit of the heterodimer of FTase are intensively studied in clinical and preclinical trials. However, very little is known about the transcriptional regulation of the β-subunit. The examined promoter region of the human FTase β-subunit gene (FNTB) showed significant basal promoter activity in HEK-293 and in HeLa cells. We were able to locate the core promoter at -165 to -74. Ten potential binding sites of the transcription factor OCT-1 were detected. Three could be confirmed using EMSA super shift experiments. OCT-1 overexpression and knockdown confirmed it as an important regulator of FNTB expression. Our results provide a basis for further research on FNTB/OCT-1 regulation, its inhibitors and diseases influenced by both such as colon carcinoma or diabetes mellitus.

Blocking the human common beta subunit of the GM-CSF, IL-5 and IL-3 receptors markedly reduces hyperinflammation in ARDS models

Acute respiratory distress syndrome (ARDS) is triggered by various aetiological factors such as trauma, sepsis and respiratory viruses including SARS-CoV-2 and influenza A virus. Immune profiling of severe COVID-19 patients has identified a complex pattern of cytokines including granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-5, which are significant mediators of viral-induced hyperinflammation. This strong response has prompted the development of therapies that block GM-CSF and other cytokines individually to limit inflammation related pathology. The common cytokine binding site of the human common beta (βc) receptor signals for three inflammatory cytokines: GM-CSF, IL-5 and IL-3. In this study, βc was targeted with the monoclonal antibody (mAb) CSL311 in engineered mice devoid of mouse βc and βIL-3 and expressing human βc (hβcTg mice). Direct pulmonary administration of lipopolysaccharide (LPS) caused ARDS-like lung injury, and CSL311 markedly reduced lung inflammation and oedema, resulting in improved oxygen saturation levels in hβcTg mice. In a separate model, influenza (HKx31) lung infection caused viral pneumonia associated with a large influx of myeloid cells into the lungs of hβcTg mice. The therapeutic application of CSL311 potently decreased accumulation of monocytes/macrophages, neutrophils, and eosinophils without altering lung viral loads. Furthermore, CSL311 treatment did not limit the viral-induced expansion of NK and NKT cells, or the tissue expression of type I/II/III interferons needed for efficient viral clearance. Simultaneously blocking GM-CSF, IL-5 and IL-3 signalling with CSL311 may represent an improved and clinically applicable strategy to reducing hyperinflammation in the ARDS setting.

Novel Mutations in Human Luteinizing Hormone Beta Subunit Related to Polycystic Ovary Syndrome among Sudanese Women

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry

The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGβ, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGβ glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGβ glycoforms, which allowed for the first time the detection of 33 hCGβ glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.

Analysis of External Quality Assessment of Maternal Serum Prenatal Screening for Down Syndrome in the First Trimester in China.

We analyzed the 2019 external quality assessment (EQA) results to evaluate the analytical performance of maternal serum prenatal screening for Down Syndrome (DS) in the first trimester in China. In each round, five lyophilized EQA samples with detailed clinical information were distributed to participants and used to test the concentration of human chorionic gonadotropin free beta subunits (free β-HCG) and pregnancy-associated plasma protein-A (PAPP-A) to calculate the risk value of DS. The robust coefficient of variability (CV) was calculated or test results for free β-HCG and PAPP-A using analytes and major measurement platforms. For DS risk values, the robust CV was calculated using a professional calculation platform. Failed EQA results were analyzed using serum marker testing results and DS risk values. EQA results were collected from 242 laboratories in round 1 and 239 laboratories in round 2. Total acceptable rates of testing results for free β-HCG and PAPP-A ranged from 95.04% to 97.91%. Overall acceptable rates of DS risk values ranged from 97.52% to 97.90%. The proportion of laboratories exhibiting satisfactory performance was higher in round 2 than in round 1 for serum marker testing results and DS risk values. The robust CV for risk values for each sample were significantly higher than those of serum markers. Three EQA result failure types were found, including result reporting errors, serum marker concentration testing errors, and DS risk calculation errors. The analytical performance of maternal serum prenatal screening for DS in the first trimester in China can be improved further.

Autologous Mesenchimal Stem Cell Therapy in Patients with Unexplainable Low Ovarian Response

Background: In Mexico there are 17% of women of reproductive age with infertility problems, equivalent to 1.4 million couples who need assisted reproductive techniques; 9 to 24% of these patients present low ovarian response, mainly in women older than 38 years, due to ovarian aging. Objective: To design a human method to administer autologous adipose tissue mesenchymal stem cells directly into the ovaries to elucidate the underlying mechanisms of ovarian rejuvenation and point the way for the development of future therapies. Method and Result: We present a case of a 39-year-old woman, classified as low or suboptimal ovarian response. included in group 2, subgroup 2a, of the Poseidon criteria; to which a human method was designed to administer autologous adipose tissue mesenchymal stem cells (AD-MSCs) directly into the ovaries prior to ovarian stimulation; under written informed consent to the patient; It was carried out in a procedure where 14 oocytes were obtained, which were fertilized with donor semen (normozoospermic) and a biopsy was performed of three blastocysts obtained, to carry out a preimplantation genetic study that reported an euploid embryo; This is placed in the mother’s uterus in order to achieve pregnancy and a healthy newborn. Once transferred to the mother, the euploid blastocyst, the human gonadotropin hormone beta subunit test was positive 6572mUI / mL and it was completed, obtaining a male newborn obtained by caesarean section at 39.2 weeks of healthy gestation with adequate evolution. Conclusions: Stem cell therapy obtained from bone marrow is an alternative for women with low ovarian response to achieve pregnancy, by improving the quality of oocytes; it is the first report in Mexico, in women with low idiopathic ovarian response.

Autologous Mesenchimal Stem Cell Therapy in Patients with Unexplainable Low Ovarian Response

Background: In Mexico there are 17% of women of reproductive age with infertility problems, equivalent to 1.4 million couples who need assisted reproductive techniques; 9 to 24% of these patients present low ovarian response, mainly in women older than 38 years, due to ovarian aging. Objective: To design a human method to administer autologous adipose tissue mesenchymal stem cells directly into the ovaries to elucidate the underlying mechanisms of ovarian rejuvenation and point the way for the development of future therapies. Method and Result: We present a case of a 39-year-old woman, classified as low or suboptimal ovarian response. included in group 2, subgroup 2a, of the Poseidon criteria; to which a human method was designed to administer autologous adipose tissue mesenchymal stem cells (AD-MSCs) directly into the ovaries prior to ovarian stimulation; under written informed consent to the patient; It was carried out in a procedure where 14 oocytes were obtained, which were fertilized with donor semen (normozoospermic) and a biopsy was performed of three blastocysts obtained, to carry out a preimplantation genetic study that reported an euploid embryo; This is placed in the mother’s uterus in order to achieve pregnancy and a healthy newborn. Once transferred to the mother, the euploid blastocyst, the human gonadotropin hormone beta subunit test was positive 6572mUI / mL and it was completed, obtaining a male newborn obtained by caesarean section at 39.2 weeks of healthy gestation with adequate evolution. Conclusions: Stem cell therapy obtained from bone marrow is an alternative for women with low ovarian response to achieve pregnancy, by improving the quality of oocytes; it is the first report in Mexico, in women with low idiopathic ovarian response.

Small Molecule Follicle-Stimulating Hormone Receptor Agonists and Antagonists.

The follicle-stimulating hormone receptor (FSHR) has been targeted therapeutically for decades, due to its pivotal role in reproduction. To date, only purified and recombinant/biosimilar FSH have been used to target FSHR in assisted reproduction, with the exception of corifollitropin alfa; a modified gonadotropin in which the FSH beta subunit is joined to the C-terminal peptide of the human choriogonadotropin beta subunit, to extend serum half-life. Assisted reproduction protocols usually entail the trauma of multiple injections of FSH to initiate and promote folliculogenesis, which has prompted the development of a number of orally-available low molecular weight (LMW) chemical scaffolds targeting the FSHR. Furthermore, the recently documented roles of the FSHR in diverse extragonadal tissues, including cancer, fat metabolism, and bone density regulation, has highlighted the potential utility of LMW modulators of FSHR activity. Despite these chemical scaffolds encompassing a spectrum of in vitro and in vivo activities and pharmacological profiles, none have yet reached the clinic. In this review we discuss the major chemical classes of LMW molecules targeting the FSHR, and document their activity profiles and current status of development, in addition to discussing potential clinical applications.

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

0265: A novel SCN5A mutation associated with exercise-induced polymorphic ventricular arrhythmias resembling CPVT

Cardiac sodium channel is a complex which includes the poreforming alpha subunit and regulatory proteins that allow sodium influx during the depolarization phase of the ventricular action potential. The alpha subunit, Nav1.5, is encoded by SCN5A . Mutations in this gene are responsible for a wide spectrum of hereditary arrhythmias such as cardiac conduction disease and atrial fibrillation. A mutation in SCN5A , leading to a p.I141V substitution, was identified in a large multigenerational family with an arrhythmia syndrome resembling catecholaminergic polymorphic ventricular tachycardia through whole-exome sequencing. The purpose of this study was to identify the molecular mechanisms linking the p.I141V mutation to this arrhythmia. To evaluate the incidence of this substitution on Nav1.5 function, whole-cell patch-clamp experiments were performed on HEK293 cells transfected with the human alpha and beta subunits. The presence of the p.I141V mutation shifted the voltage-dependence of steady state activation towards more negative potentials. Thus, we studied the effect of this mutation on the sodium window current. Compared to the WT, the p.I141V window current exhibited a larger peak and this peak was shifted towards more negative potentials. To investigate the functional consequences of the cardiac hyperexcitability due to the p.I141V mutation, we incorporated biophysical properties of the mutant into atrial, Purkinje and ventricular cell models. The computational analyses imply that the biophysical changes caused by the p.I141V mutation accelerates the spontaneous rate in the Purkinje cell model, and reduce the excitation threshold leading to an increase of the excitability of the cardiac cells. In conclusion, the present study demonstrates that mutations in SCN5A also may result in exercise-induced polymorphic ventricular premature complexes and tachycardia resembling CPVT.

CGB activates ERK and AKT kinases in cancer cells via LHCGR-independent mechanism

Expression of human chorionic gonadotropin free beta subunit (hCGβ) and its hyperglycosylated variant (hCGβ-H) is a phenomenon confirmed for tumors of different origin. Despite numerous studies, the mechanism of hCGβ action in cancer remains unknown especially that not all tumors secreting hCGβ express the receptor for human chorionic gonadotropin (LHCGR). In the presented study, we verified the hypothesis of hCGβ potential to activate signaling pathways involving extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) kinases with and without the contribution of LHCGR. To achieve this goal, human ovarian carcinoma cells OVCAR-3 expressing LHCGR and SKOV-3 not expressing LHCGR were either transfected with a vector coding for hCGβ or stimulated with recombinant hCGβ and the level of pERK and pAKT was measured. The results of the experiments showed that hCGβ action leads to the increase in ERK and AKT kinases phosphorylation in cancer cells and indicate that these biological effects can be achieved independently of LHCGR presence. The study also demonstrated that the presence of the receptor is a key factor influencing the magnitude of cells' response.

CDC50A plays a key role in the uptake of the anticancer drug perifosine in human carcinoma cells

Functional aminophospholipid translocases are composed of at least two proteins: an alpha subunit from the P4 subfamily of P-type ATPases and a beta subunit from the CDC50-Lem3p family. Over-expression and knockdown of the human beta subunit CDC50A in KB cells enhanced and decreased, respectively, the uptake of both fluorescent aminophospholipid analogues and the anticancer alkyl-phospholipid perifosine. Confocal microscopy showed that CDC50A-V5 was localized at the endoplasmic reticulum and the Golgi complex of both KB (perifosine-sensitive) and KB PER-R (perifosine-resistant, alkyl-phospholipid uptake deficient) cells, but was only widely distributed in the early and late endosomes in KB cells. Biotinylation of cell surface proteins allowed CDC50A-V5 to be detected in the plasma membrane of KB cells but not in KB PER-R cells, thereby suggesting a defect in CDC50A trafficking that could explain the inability of KB PER-R to uptake perifosine. Over-expression of CDC50A in HeLa and HEK293T cells did not increase uptake, since the protein was retained at the endoplasmic reticulum and Golgi. However, when CDC50A was co-expressed with the P4-ATPase Atp8b1, the two proteins co-localized at the plasma membrane and the uptake of aminophospholipids and perifosine increased strikingly in both cell lines. These findings suggest that CDC50A plays a key role in perifosine uptake in human cells, presumably by forming a functional plasma membrane translocator in combination with a P4-ATPase.

Divergent sodium channel defects in familial hemiplegic migraine.

Familial hemiplegic migraine type 3 (FHM3) is a severe autosomal dominant migraine disorder caused by mutations in the voltage-gated sodium channel Na(V)1.1 encoded by SCN1A. We determined the functional consequences of three mutations linked to FHM3 (L263V, Q1489K, and L1649Q) in an effort to identify molecular defects that underlie this inherited migraine disorder. Only L263V and Q1489K generated quantifiable sodium currents when coexpressed in tsA201 cells with the human beta(1) and beta(2) accessory subunits. The third mutant, L1649Q, failed to generate measurable whole-cell current because of markedly reduced cell surface expression. Compared to WT-Na(V)1.1, Q1489K exhibited increased persistent current but also enhanced entry into slow inactivation as well as delayed recovery from fast and slow inactivation, thus resulting in a predominantly loss-of-function phenotype further demonstrated by a greater loss of channel availability during repetitive stimulation. In contrast, L263V exhibited gain-of-function features, including delayed entry into, as well as accelerated recovery from, fast inactivation; depolarizing shifts in the steady-state voltage dependence of fast and slow inactivation; increased persistent current; and delayed entry into slow inactivation. Notably, the two mutations (Q1489K and L1649Q) that exhibited partial or complete loss of function are linked to typical FHM, whereas the gain-of-function mutation L263V occurred in a family having both FHM and a high incidence of generalized epilepsy. We infer from these data that a complex spectrum of Na(V)1.1 defects can cause FHM3. Our results also emphasize the complex relationship between migraine and epilepsy and provide further evidence that both disorders may share common molecular mechanisms.

Structural characterization of human general transcription factor TFIIF in solution

Human general transcription factor IIF (TFIIF), a component of the transcription pre-initiation complex (PIC) associated with RNA polymerase II (Pol II), was characterized by size-exclusion chromatography (SEC), electrospray ionization mass spectrometry (ESI-MS), and chemical cross-linking. Recombinant TFIIF, composed of an equimolar ratio of alpha and beta subunits, was bacterially expressed, purified to homogeneity, and found to have a transcription activity similar to a natural one in the human in vitro transcription system. SEC of purified TFIIF, as previously reported, suggested that this protein has a size >200 kDa. In contrast, ESI-MS of the purified sample gave a molecular size of 87 kDa, indicating that TFIIF is an alphabeta heterodimer, which was confirmed by matrix-assisted laser desorption/ionization (MALDI) MS of the cross-linked TFIIF components. Recent electron microscopy (EM) and photo-cross-linking studies showed that the yeast TFIIF homolog containing Tfg1 and Tfg2, corresponding to the human alpha and beta subunits, exists as a heterodimer in the PIC, so the human TFIIF is also likely to exist as a heterodimer even in the PIC. In the yeast PIC, EM and photo-cross-linking studies showed different results for the mutual location of TFIIE and TFIIF along DNA. We have examined the direct interaction between human TFIIF and TFIIE by ESI-MS, SEC, and chemical cross-linking; however, no direct interaction was observed, at least in solution. This is consistent with the previous photo-cross-linking observation that TFIIF and TFIIE flank DNA separately on both sides of the Pol II central cleft in the yeast PIC.

Attenuation of Oxidative Stress After Varicocelectomy in Subfertile Patients With Varicocele

We examined changes due to oxidative damage to spermatozoa and alterations in antioxidant capacity in subfertile patients with varicocele before and after varicocelectomy in a prospective study. A total of 30 young subfertile male patients with varicocele were recruited in this study. Varicocele was diagnosed by physical examination and Doppler ultrasound. Semen analysis was performed in the 30 patients before and 6 months after varicocelectomy using a computer assisted semen analyzer. The parameters for evaluating oxidative stress changes were 4977 bp deletion of mitochondrial DNA in sperm, as detected by polymerase chain reaction, the 8-OHdG (8-hydroxy-2'-deoxyguanosine) content in spermatozoa DNA, as measured by a high performance liquid chromatography electrochemical method, and seminal plasma protein thiols and ascorbic acid, as measured by spectrophotometric methods. Semen quality, including motility, morphology and sperm density, was improved in 22 patients (73.3%) after varicocelectomy. The incidence of 4977 bp deletion of mitochondrial DNA in sperm was 40% (12 of 30 patients) and 13.3% (4 of 30) before and after surgery, respectively. Mean +/- SD 8-OHdG content in sperm DNA, and seminal plasma protein thiols and ascorbic acid were 10.27 +/- 2.24/10(5) 2'-deoxyguanosine, 0.77 +/- 0.75 nmole/ml and 1.87 +/- 0.40 mg/dl before operation, and 5.95 +/- 1.46/10(5) 2'-deoxyguanosine, 3.00 +/- 1.17 nmole/ml and 3.12 +/- 0.94 mg/dl after surgery, respectively. The incidence of 4977 bp deletion of mitochondrial DNA in sperm and the level of 8-OHdG in sperm DNA were decreased, and seminal plasma protein thiols and ascorbic acid were increased significantly in all 30 patients after varicocelectomy. Also, in the 8 patients in whom semen quality did not improve after surgery a significant decrease in 8-OHdG in sperm DNA, and a significant increase in seminal plasma protein thiols and ascorbic acid were observed. Subfertile patients with varicocele had a significant decrease in oxidative damage in sperm DNA and an increase in antioxidant capacity in seminal plasma after varicocelectomy, indicating that surgery is effective treatment in such patients.

Nonfunctional SCN1A Is Common in Severe Myoclonic Epilepsy of Infancy

Mutations in SCN1A, encoding the human Na(V)1.1 neuronal voltage-gated sodium channel, cause the syndrome of severe myoclonic epilepsy of infancy (SMEI). Most SMEI-associated mutations are predicted to truncate the SCN1A protein, likely causing a loss of sodium channel function. However, many missense or in-frame deletion SCN1A mutations have also been reported in this disorder, but their functional impact is largely unknown. Here we report the functional characterization of eight SCN1A mutations (G177E, I227S, R393H, Y426N, H939Q, C959R, delF1289, and T1909I) previously identified in SMEI probands. SCN1A mutants were constructed in a recombinant human SCN1A and then heterologously expressed in human tsA201 cells along with the human beta(1) and beta(2) sodium channel accessory subunits. Whole-cell patch-clamp recording was used to define biophysical properties of each mutant and for comparison with the wild-type (WT) channel. Six of the mutants were nonfunctional, but Y426N and T1909I generated measurable sodium channel activity. Cells expressing Y426N and T1909I had significantly lower current densities compared with WT-SCN1A. In addition, other biophysical abnormalities were observed for the two functional mutants including decreased channel availability (Y426N) and increased persistent sodium current (T1909I). We conclude that SMEI is caused either by complete loss of SCN1A function, or by dysfunctional sodium channels exhibiting mixed biophysical properties. This wide spectrum of functional defects observed among SCN1A mutations suggests that SMEI may result from more than a single molecular or cellular mechanism, or require other factors for pathogenesis.

Identification of a Prostaglandin-responsive Element in the Na,K-ATPase β1 Promoter That Is Regulated by cAMP and Ca2+: EVIDENCE FOR AN INTERACTIVE ROLE OF cAMP REGULATORY ELEMENT-BINDING PROTEIN AND Sp1

The Na,K-ATPase is a transmembrane protein responsible for maintaining electrochemical gradients across the plasma membrane in all mammalian cells, a process that is subject to regulation at the transcriptional as well as post-transcriptional level. Included among physiologic regulators in the kidney are prostaglandins. Previously, we demonstrated that prostaglandin E(1) (PGE(1)) increases the activity and expression of the Na,K-ATPase in Madin-Darby canine kidney cells (Taub, M., Borsick, M., Geisel, J., Matlhagela, K., Rajkhowa, T., and Allen, C. (2004) Exp. Cell Res. 299, 1-14; Taub, M. L., Wang, Y., Yang, I. S., Fiorella, P., and Lee, S. M. (1992) J. Cell. Physiol. 151, 337-346). In this work, we present evidence that transcription of the Na,K-ATPase beta(1) subunit is stimulated by PGE(1), an effect that may be mediated through the cAMP and Ca(2+) pathways. Transient transfection studies using 5'-deletion mutants of the human beta(1) subunit promoter indicated that region -100 to -92 containing the sequence AGTCCCTGC (a prostaglandin-responsive element (PGRE)) is required to elicit the stimulatory effects of PGE(1), 8-bromo-cAMP, phorbol 12-myristate 13-acetate, and okadaic acid. Electrophoretic mobility shift assays indicated that both the cAMP regulatory element-binding protein (CREB) and Sp1 bind to the PGRE present within this region of the beta(1) subunit promoter. The involvement of the PGRE and Sp1 sites in regulation by PGE(1) was further confirmed by the increased PGE(1) stimulation that was observed following insertion of the PGRE into a promoter/luciferase construct containing a portion of a heterologous promoter and the fibronectin promoter with four GC boxes. Further evidence suggesting an interaction between Sp1 and CREB was obtained from experiments conducted with pLuc-MCS-beta 72-167, which contains region -167 to -72 in the human beta(1) subunit promoter. The PGE(1) stimulation observed in Madin-Darby canine kidney cells transiently transfected with pLuc-MCS-beta 72-167 was reduced when the two GC boxes immediately upstream from the PGRE were translocated farther upstream. Also consistent with an interaction between CREB and Sp1 are the results of our immunoprecipitation studies indicating that CREB co-immunoprecipitated with Sp1 when an antibody against CREB, Sp1, or the CREB-binding protein was used.

Relationship between imbalanced synthesis of human chorionic gonadotropin alpha and beta subunits and pregnancy-induced hypertension

To study the relationship between imbalanced synthesis of human chorionic gonadotropin (hCG) alpha and beta subunits and the pathology of pregnancy-induced hypertension (PIH). Total hCG, free alphahCG and betahCG were measured in serum samples collected from 60 cases of PIH and 30 normal gravid women by radioimmunoassay. The ratio of total hCG/betahCG, betahCG/FalphahCG, (FalphahCG+betahCG)/betahCG were calculated. Immunohistochemical staining was performed in 13 placenta from each group. In PIH group the levels of FalphahCG, total hCG and betahCG were significantly higher than those of normal group (FalphahCG: 528 +/-421 IU/L compared with 222 +/-129 IU/L; betahCG: 39396 +/-6412 IU/L compared with 24265 +/-5575 IU/L; total hCG: 66597 +/-9294 IU/L compared with 36078 +/-4767 IU/L, all P<0.001). The betahCG/FalphahCG ratio in PIH was lower than that of normal group (91.23 +/-53.38 Compared with 119.4 +/-80.1, P<0.05); (FalphahCG+betahCG)/betahCG ratio and total hCG/betahCG ratio in two groups were (1.022 +/-0.026 compared with 1.015 +/-0.011; 1.802 +/-0.339 compared with 1.807 +/-0.258, respectively P>0.05). The immunohistochemical intensity of betahCG and FalphahCG in syncytiotrophoblast was significantly increased in 13 PIH compared with the control. These data suggested that the imbalanced synthesis of hCG alpha and beta subunits may cause hypertension.

A Functional Domain of the α1 Subunit of Soluble Guanylyl Cyclase Is Necessary for Activation of the Enzyme by Nitric Oxide and YC-1 but Is Not Involved in Heme Binding

Soluble guanylyl cyclase is a heterodimeric enzyme consisting of an alpha(1) and a beta(1) subunit and is an important target for endogenous nitric oxide and the guanylyl cyclase modulator YC-1. The activation of the enzyme by both substances is dependent on the presence of a prosthetic heme group. It has been unclear whether this prosthetic heme group is sandwiched between the alpha(1) and beta(1) subunits or whether it exclusively binds to the beta(1) subunit. Here we analyze progressive amino-terminal deletion mutants of the human alpha(1) subunit after co-expression with the human beta(1) subunit in the baculovirus/Sf9 system. Spectral, biochemical, and pharmacological analysis shows that the first 259 amino acids of the alpha(1) subunit can be deleted without loss of sensitivity to nitric oxide (NO) or YC-1 or loss of heme binding of the respective enzyme complex with the beta(1) subunit. This is in contrast to previous data indicating that NO sensitivity and a functional heme binding site requires full-length amino termini of bovine alpha(1) and beta(1) subunits. Further deletion of the first 364 amino acids of the alpha(1) subunit leads to an enzyme complex with preserved heme binding but loss of sensitivity to NO or YC-1 despite induction of the typical spectral shift by NO binding to the prosthetic heme group. We conclude that 1) the amino-terminal part of the alpha(1) subunit is not involved in heme binding and 2) amino acids 259-364 of the alpha(1) subunit represent an important functional domain for the transduction of the NO activation signal and likely represent the target for NO-sensitizing substances like YC-1.