Tailored UPRE2 variants for dynamic gene regulation in yeast.

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

Advances in recombinant protease production: current state and perspectives.

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in proteins, are important in the food industry, biotechnology, and medical fields. With increasing demand for proteases, there is a growing emphasis on enhancing their expression and production through microbial systems. However, proteases' native hosts often fall short in high-level expression and compatibility with downstream applications. As a result, the recombinant production of proteases has become a significant focus, offering a solution to these challenges. This review presents an overview of the current state of protease production in prokaryotic and eukaryotic expression systems, highlighting key findings and trends. In prokaryotic systems, the Bacillus spp. is the predominant host for proteinase expression. Yeasts are commonly used in eukaryotic systems. Recent advancements in protease engineering over the past five years, including rational design and directed evolution, are also highlighted. By exploring the progress in both expression systems and engineering techniques, this review provides a detailed understanding of the current landscape of recombinant protease research and its prospects for future advancements.

Microglia sense and suppress epileptic neuronal hyperexcitability.

Microglia are the resident immune cells of the central nervous system, undertaking surveillance role and reacting to brain homeostasis and neurological diseases. Recent studies indicate that microglia modulate epilepsy-induced neuronal activities, however, the mechanisms underlying microglia-neuron communication in epilepsy are still unclear. Here we report that epileptic neuronal hyperexcitability activates microglia and drives microglial ATP/ADP hydrolyzing ectoenzyme CD39 (encoded by Entpd1) expression via recruiting the cAMP responsive element binding protein (CREB)-regulated transcription coactivator-1 (CRTC1) from cytoplasm to the nucleus and binding to CREB. Activated microglia in turn suppress epileptic neuronal hyperexcitability in a CD39 dependent manner. Disrupting microglial CREB/CRTC1 signaling, however, decreases CD39 expression and diminishes the inhibitory effect of microglia on epileptic neuronal hyperexcitability. Overall, our findings reveal CD39-dependent control of epileptic neuronal hyperexcitability by microglia is through an excitation-transcription coupling mechanism.

Overexpression of genes by stress-responsive promoters increases protein secretion in Saccharomyces cerevisiae.

Recombinant proteins produced by cell factories are now widely used in various fields. Many efforts have been made to improve the secretion capacity of cell factories to meet the increasing demand for recombinant proteins. Recombinant protein production usually causes cell stress in the endoplasmic reticulum (ER). The overexpression of key genes possibly removes limitations in protein secretion. However, inappropriate gene expression may have negative effects. There is a need for dynamic control of genes adapted to cellular status. In this study, we constructed and characterized synthetic promoters that were inducible under ER stress conditions in Saccharomyces cerevisiae. The unfolded protein response element UPRE2, responding to stress with a wide dynamic range, was assembled with various promoter core regions, resulting in UPR-responsive promoters. Synthetic responsive promoters regulated gene expression by responding to stress level, which reflected the cellular status. The engineered strain using synthetic responsive promoters P4UPRE2 - TDH3 and P4UPRE2 - TEF1 for co-expression of ERO1 and SLY1 had 95% higher α-amylase production compared with the strain using the native promoters PTDH3 and PTEF1. This work showed that UPR-responsive promoters were useful in the metabolic engineering of yeast strains for tuning genes to support efficient protein production.

A Conway-Maxwell-Poisson-Binomial AR(1) Model for Bounded Time Series Data.

Binomial autoregressive models are frequently used for modeling bounded time series counts. However, they are not well developed for more complex bounded time series counts of the occurrence of n exchangeable and dependent units, which are becoming increasingly common in practice. To fill this gap, this paper first constructs an exchangeable Conway-Maxwell-Poisson-binomial (CMPB) thinning operator and then establishes the Conway-Maxwell-Poisson-binomial AR (CMPBAR) model. We establish its stationarity and ergodicity, discuss the conditional maximum likelihood (CML) estimate of the model's parameters, and establish the asymptotic normality of the CML estimator. In a simulation study, the boxplots illustrate that the CML estimator is consistent and the qqplots show the asymptotic normality of the CML estimator. In the real data example, our model takes a smaller AIC and BIC than its main competitors.

Valproic acid suppresses cuprizone-induced hippocampal demyelination and anxiety-like behavior by promoting cholesterol biosynthesis.

Myelin consists of several layers of tightly compacted membranes that form an insulating sheath around axons. These membranes are highly enriched in cholesterol, which is essential for the myelination process. Proper myelination is crucial for various neurophysiological functions while demyelination may cause CNS disease, such as multiple sclerosis (MS). Recent studies demonstrated that demyelination occurs not only in the white matter but also in the grey matter, such as the hippocampus, which may cause cognitive deficits and mental disorders. Valproic acid (VPA) is an anticonvulsant agent prescribed for the treatment of epilepsy and seizure. Recently, VPA was reported to alter cholesterol metabolism in neural cells, suggesting that it may play an important role in myelin biogenesis. Here in this study, we found significant demyelination in the hippocampus of the mouse cuprizone model, which is accompanied by reduced cholesterol biosynthesis and increased anxiety-like behavior. VPA treatment, however, suppressed cuprizone-induced hippocampal demyelination and anxiety-like behavior by promoting cholesterol biosynthesis. These data identify an important role of VPA in the hippocampal demyelination process and the hippocampal demyelination-related behavior deficit via regulation of cholesterol biosynthesis, which provides new insights into the mechanisms of VPA as a protective agent against CNS demyelination.

Acoustofluidic dynamic interfacial tensiometry.

The interfacial tension (IFT) of fluids plays an essential role in industrial, biomedical, and synthetic chemistry applications; however, measuring IFT at ultralow volumes is challenging. Here, we report a novel method for sessile drop tensiometry using surface acoustic waves (SAWs). The IFT of the fluids was determined by acquiring the silhouette of an axisymmetric sessile drop and applying iterative fitting using Taylor's deformation equation. Owing to physiochemical differences, upon interacting with acoustic waves, each microfluid has a different streaming velocity. This streaming velocity dictates any subsequent changes in droplet shape (i.e., height and width). We demonstrate the effectiveness of the proposed SAW-based tensiometry technique using blood plasma to screen for high leptin levels. The proposed device can measure the IFT of microscale liquid volumes (up to 1 µL) with an error margin of only ±5% (at 25 °C), which deviates from previous reported results. As such, this method provides pathologists with a solution for the pre-diagnosis of various blood-related diseases.

Progesterone receptor membrane component 1 and 2 regulate granulosa cell mitosis and survival through a NFΚB-dependent mechanism†.

Progesterone receptor membrane component 1 (PGRMC1) interacts with PGRMC2, and disrupting this interaction in spontaneously immortalized granulosa cells (SIGCS) leads to an inappropriate entry into the cell cycle, mitotic arrest, and ultimately cell death. The present study revealed that PGRMC1 and PGRMC2 localize to the cytoplasm of murine granulosa cells of nonatretric follicles with their staining intensity being somewhat diminished in granulosa cells of atretic follicles. Compared to controls (Pgrmc1fl/fl), the rate at which granulosa cells entered the cell cycle increased in nonatretic and atretic follicles of mice in which Pgrmc1 was conditionally deleted (Pgrmc1d/d) from granulosa cells. This increased rate of entry into the cell cycle was associated with a ≥ 2-fold increase in follicular atresia and the nuclear localization of nuclear factor-kappa-B transcription factor P65; (NFΚB/p65, or RELA). GTPase activating protein binding protein 2 (G3BP2) binds NFΚB/p65 through an interaction with NFΚB inhibitor alpha (IκBα), thereby maintaining NFΚB/p65's cytoplasmic localization and restricting its transcriptional activity. Since PGRMC1 and PGRMC2 bind G3BP2, studies were designed to assess the functional relationship between PGRMC1, PGRMC2, and NFΚB/p65 in SIGCs. In these studies, disrupting the interaction between PGRMC1 and PGRMC2 increased the nuclear localization of NFΚB/p65, and depleting PGRMC1, PGRMC2, or G3BP2 increased NFΚB transcriptional activity and the progression into the cell cycle. Taken together, these studies suggest that PGRMC1 and 2 regulate granulosa cell cycle entry in follicles by precisely controlling the localization and thereby the transcriptional activity of NFΚB/p65.

Upregulation of CBP by PLY can cause permeability of blood-brain barrier to increase meningitis.

BACKGROUND: Streptococcus pneumoniae causes many human diseases including bacterial meningitis. Previous study proposed that pneumolysin (PLY), a cytotoxin from pneumococcus, is related to the infection across blood-brain barrier (BBB). However, the mechanism of how PLY break through BBB remains elusive. The present study showed that PLY can increase the permeability of BBB both in vitro and in vivo in our experiments. RESULTS: Further we found out that PLY leads to the high expression of CERB-binding protein (CBP) which can lead to releasing of tumor necrosis factor α then enhance apoptosis of cells which is a significant factor leading to permeabilization of BBB. CONCLUSION: Our findings demonstrate that CBP plays an important role in the pneumococcus infection in the brain and could be a potential therapeutic target against pneumococcal meningitis.

Superhydrophilic Phytic-Acid-Doped Conductive Hydrogels as Metal-Free and Binder-Free Electrocatalysts for Efficient Water Oxidation.

Recently, metal-free, heteroatom-doped carbon nanomaterials have emerged as promising electrocatalysts for the oxygen evolution reaction (OER), but their synthesis is a tedious process involving energy-wasting calcination. Molecular electrocatalysts offer attractive catalysts for the OER. Here, phytic acid (PA) was selected to investigate the OER activity of carbons in organic molecules by DFT calculations and experiments. Positively charged carbons on PA were very active towards the OER. The PA molecules were fixed into a porous, conductive hydrogel with a superhydrophilic surface. This outperformed most metal-free electrocatalysts. Besides the active sites on PA, the high OER activity was also related to the porous and conductive networks on the hydrogel, which allowed fast charge and mass transport during the OER. Therefore, this work provides a metal-free, organic-molecule-based electrocatalyst to replace carbon nanomaterials for efficient OER.

Boosting Electrochemical Hydrogen Evolution of Porous Metal Phosphides Nanosheets by Coating Defective TiO2 Overlayers.

Nonprecious transition metal phosphides (TMPs) have emerged as robust electrocalysts for the hydrogen evolution reaction (HER). However, the TMPs suffer from low activity for water dissociation, which greatly limits the efficiency for alkaline HER. Here, a facile yet robust strategy is reported to boost the HER of metal phosphides by coating defective TiO2 overlayers. The oxygen vacancies (Ov ) on defective TiO2 overlayers are found to possess high activity for adsorption and dissociation of water, thereby significantly promoting the initial Volmer step of HER to generate the reactive hydrogen atoms. Moreover, the porous (Co, Ni)2 P (i.e., Co2 P and Ni2 P) nanosheets provide enough active sites for adsorption and recombination of reactive hydrogen atoms to produce hydrogen gas. The catalytic synergy of (Co, Ni)2 P and Ov coupled with the hierarchically porous structure renders the porous (Co, Ni)2 P@0.1TiO2 nanosheet arrays excellent electrocatalysts for HER, showing a small overpotential (92 mV) to yield a current density of 10 mA cm-2 , a small Tafel slope (49 mV dec-1 ), and an outstanding stability. This work demonstrates a surface decoration route for enhancing the activity of nonprecious metal-based electrocatalysts for HER.

PGRMC1/2 promotes luteal vascularization and maintains the primordial follicles of mice

To determine whether conditional depletion of progesterone receptor membrane component (PGRMC) 1 and PGRMC2 affected ovarian follicle development, follicle distribution was assessed in ovaries of young (≈3-month-old) and middle-aged (≈6-month-old) control (Pgrmc1/2fl/fl) and double conditional PGRMC1/2-knockout (Pgrmc1/2d/d) mice. This study revealed that the distribution of primary, preantral and antral follicles was not altered in Pgrmc1/2d/d mice, regardless of the age. Although the number of primordial follicles was similar at ≈3 months of age, their numbers were reduced by ≈80% in 6-month-old Pgrmc1/2d/d mice compared to age-matched Pgrmc1/2fl/fl mice. The Pgrmc1/2d/d mice were generated using Pgr-cre mice, so ablation of Pgrmc1 and Pgrmc2 in the ovary was restricted to peri-ovulatory follicles and subsequent corpora lutea (CL). In addition, the vascularization of CL was attenuated in Pgrmc1/2d/d mice, although mRNA levels of vascular endothelial growth factor A (Vegfa) were elevated. Moreover, depletion of Pgrmc1 and Pgrmc2 altered the gene expression profile in the non-luteal component of the ovary such that Vegfa expression, a stimulator of primordial follicle growth, was elevated; Kit Ligand expression, another stimulator of primordial follicle growth, was suppressed and anti-Mullerian hormone, an inhibitor of primordial follicle growth, was enhanced compared to Pgrmc1/2fl/fl mice. These data reveal that luteal cell depletion of Pgrmc1 and 2 alters the expression of growth factors within the non-luteal component of the ovary, which could account for the premature demise of the adult population of primordial follicles. In summary, the survival of adult primordial follicles is dependent in part on progesterone receptor membrane component 1 and 2.

Influences of cold atmospheric plasma on microbial safety, physicochemical and sensorial qualities of meat products.

Meat and meat products can be contaminated with pathogenic microorganisms, which cause serious health problems and economic loss. Recently, numerous novel non-thermal technologies have been developed to respond to growing consumer demand for high quality and safe meat products. Cold atmospheric plasma (CAP) is a novel and emerging non-thermal technology, showing great potential for applications in the food industry. This review presents recent advances on the developments and applications of CAP in meat products, including generation and microbial inactivation effects of CAP as well as its influences on physicochemical qualities and sensory attributes of meat products. Furthermore, the safety assessment of CAP-treated meat products and challenges in industrial application of CAP are also discussed.

Pharmacoeconomic evaluation of glimepiride combined with other drugs in the treatment of diabetes.

The goal of diabetic drug treatment is to stabilize the blood sugar for a long time to close to the normal level, to correct the metabolic disorder and eliminate the symptoms. At present, glimepiride has become commonly used drugs for the treatment of diabetes with obesity. Compared with metformin, acarbose and rosiglitazone, glimepiride has different mechanisms of drug action, clinical combination showed synergistic hypoglycemic effect, good clinical curative effect. So, we use three treatments to study as group A (glimepiride and metformin); group B (glimepiride and acarbose); Group C (glimepiride and rosiglitazone). From the analysis of drug economics, glimepiride and metformin scheme is better, has the lowest cost per unit cost effect. From the comparison of scheme is efficient, the best curative effect is rosiglitazone plus glimepiride, effective rate as 96.7%. At the same time, the drug can be rationally used to reduce the occurrence of some drug-induced diseases and adverse drug reactions.

AG 205, a progesterone receptor membrane component 1 antagonist, ablates progesterone's ability to block oxidative stress-induced apoptosis of human granulosa/luteal cells†.

The present studies were designed to determine whether progesterone (P4)-progesterone receptor membrane component 1 (PGRMC1) signaling is able to attenuate the apoptotic effects of oxidative stress induced by hydrogen peroxide (H2O2). To achieve this goal, freshly isolated human granulosa/luteal cells were maintained in culture. After several passages, the cells were treated with H2O2, which induced apoptosis within 2.5 h, while simultaneous treatment with P4 attenuated the apoptotic action of H2O2. AG 205, a PGRMC1 antagonist, eliminated P4's ability to prevent H2O2-induced apoptosis. AG 205 neither affected PGRMC1's cytoplasmic localization nor its interaction with PGRMC2, but appeared to reduce its presence within the nucleus. AG 205 also (1) increased the monomeric and decreased the higher molecular weight forms of PGRMC1 (i.e., dimers/oligomers) and (2) altered the expression of several genes involved in apoptosis. The most dramatic change was an approximate 8-fold increase in Harakiri (Hrk) mRNA. However, AG 205 did not induce apoptosis in the absence of H2O2. Taken together, these observations suggest that the higher molecular weight forms of PGRMC1 likely account in part for PGRMC1's ability to suppress the expression of Hrk. Harakiri is a BH-3 only member of the B-cell lymphoma 2 (BCL2) family that promotes apoptosis by binding to and antagonizing the antiapoptotic action of BCL2- and BCL2-like proteins. It is likely then that PGRMC1's ability to suppress Hrk is part of the mechanism through which P4-PGRMC1 signaling preserves the viability of human granulosa/luteal cells.

Progestin and AdipoQ Receptor 7, Progesterone Membrane Receptor Component 1 (PGRMC1), and PGRMC2 and Their Role in Regulating Progesterone's Ability to Suppress Human Granulosa/Luteal Cells from Entering into the Cell Cycle.

The present studies were designed to determine the role of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, progestin and adipoQ receptor 7 (PAQR7), and progesterone receptor (PGR) in mediating the antimitotic action of progesterone (P4) in human granulosa/luteal cells. For these studies granulosa/luteal cells of 10 women undergoing controlled ovarian hyperstimulation were isolated, maintained in culture, and depleted of PGRMC1, PGRMC2, PAQR7, or PGR by siRNA treatment. The rate of entry into the cell cycle was assessed using the FUCCI cell cycle sensor to determine the percentage of cells in the G1/S stage of the cell cycle. PGRMC1, PGRMC2, PAQR7, and PGR mRNA levels were assessed by real-time PCR and their interactions monitored by in situ proximity ligation assays (PLAs). These studies revealed that PGRMC1, PGRMC2, PAQR7, and PGR were expressed by granulosa/luteal cells from all patients, with PGRMC1 mRNA being most abundant, followed by PAQR7, PGRMC2, and PGR. However, their mRNA levels showed considerable patient variation. P4's ability to suppress entry into the cell cycle was dependent on PGRMC1, PGRMC2, and PAQR7 but not PGR. Moreover, PLAs indicated that PGRMC1, PGRMC2, and PAQR7 formed a complex within the cytoplasm. Based on these studies, it is proposed that these three P4 mediators form a complex within the cytoplasm that is required for P4's action. Moreover, P4's ability to regulate human follicle development may be dependent in part on the expression levels of each of these P4 mediators.

Progesterone receptor membrane component-1 (PGRMC1) and PGRMC-2 interact to suppress entry into the cell cycle in spontaneously immortalized rat granulosa cells.

Progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 are expressed in rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs) but their biological roles are not well defined. The present studies demonstrate that depleting either Pgrmc1 or Pgrmc2 in SIGCs increases entry into the cell cycle but does not increase cell proliferation. Rather, PGRMC1 and/or PGRMC2-deplete cells accumulate in metaphase and undergo apoptosis. Because both PGRMC1 and PGRMC2 localize to the mitotic spindle, their absence likely accounts for cells arresting in metaphase. Moreover, pull-down assays, colocalization studies and in situ proximity ligation assays (PLA) indicate that PGRMC1 binds PGRMC2. Disrupting the PGRMC1:PGRMC2 complex through the use of siRNA or the cytoplasmic delivery of a PGRMC2 antibody increases entry into the cell cycle. Conversely, overexpressing either PGRMC1-GFP or GFP-PGRMC2 fusion protein inhibits entry into the cell cycle. Subsequent studies reveal that depleting PGRMC1 and/or PGRMC2 reduces the percentage of cells in G0 and increases the percentage of cells in G1. These observations indicate that in addition to their role at metaphase, PGRMC1 and PGRMC2 are involved in regulating entry into the G1 stage of the cell cycle. Interestingly, both PGRMC1 and PGRMC2 bind GTPase-activating protein-binding protein 2 (G3BP2) as demonstrated by pull-down assays, colocalization assays, and PLAs. G3bp2 siRNA treatment also promotes entry into the G1 stage. This implies that dynamic changes in the interaction among PGRMC1, PGRMC2, and G3BP2 play an important protein regulating the rate at which SIGCs enter into the cell cycle.

Expression of progesterone receptor membrane component-2 within the immature rat ovary and its role in regulating mitosis and apoptosis of spontaneously immortalized granulosa cells.

Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular (3)H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development.

Acoustothermal transfection for cell therapy.

Transfected stem cells and T cells are promising in personalized cell therapy and immunotherapy against various diseases. However, existing transfection techniques face a fundamental trade-off between transfection efficiency and cell viability; achieving both simultaneously remains a substantial challenge. This study presents an acoustothermal transfection method that leverages acoustic and thermal effects on cells to enhance the permeability of both the cell membrane and nuclear envelope to achieve safe, efficient, and high-throughput transfection of primary T cells and stem cells. With this method, two types of plasmids were simultaneously delivered into the nuclei of mesenchymal stem cells (MSCs) with efficiencies of 89.6 ± 1.2%. CXCR4-transfected MSCs could efficiently target cerebral ischemia sites in vivo and reduce the infarct volume in mice. Our acoustothermal transfection method addresses a key bottleneck in balancing the transfection efficiency and cell viability, which can become a powerful tool in the future for cellular and gene therapies.

Plasminogen activator inhibitor 1 RNA-binding protein interacts with progesterone receptor membrane component 1 to regulate progesterone's ability to maintain the viability of spontaneously immortalized granulosa cells and rat granulosa cells.

Progesterone receptor membrane component 1 (PGRMC1) mediates the antiapoptotic action of progesterone (P4). PGRMC1 interacts with plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1), but the functional significance of this interaction is unknown. To examine the function of PGRMC1-PAIRBP1 interaction, PAIRBP1 was depleted from spontaneously immortalized granulosa cells (SIGCs) and the effects on the expression and localization of PGRMC1 as well as P4's ability to bind to SIGCs and prevent apoptosis was assessed. Depleting PAIRBP1 enhanced cellular (3)H-P4 binding and did not alter the expression or cellular localization of PGRMC1 but attenuated P4's antiapoptotic action. Transfection of a PGRMC1-green fluorescent protein (GFP) peptide mimic, which binds PAIRBP1 as demonstrated by in situ proximity assay, doubled the rate at which SIGCs undergo apoptosis compared to cells transfected with either the empty GFP expression vector or Pairbp1 small interfering RNA. Moreover, P4 did not prevent these cells from undergoing apoptosis. Similar studies conducted with granulosa cells isolated from immature rats also showed that PGRMC1 interacts with PAIRBP1 and that transfection of PGRMC1-GFP peptide mimic accelerates the rate of granulosa cell apoptosis by 4-fold even in the presence of serum and P4. These studies support the concept that the interaction between PAIRBP1-PGRMC1 is an essential component of the mechanism through which P4 inhibits apoptosis. Surprisingly, PGRMC1-PAIRBP1 interaction is not required for P4 binding or the cellular localization of PGRMC1 but rather appears to couple PGRMC1 to downstream components of the P4-PGRMC1 signal transduction pathway.