Pregnancy outcomes in women with rheumatic diseases: a real-world observational study in Japan.

OBJECTIVES: We aimed to evaluate the obstetric complications and the risk factors for these events in pregnant women with rheumatic diseases (RDs). METHODS: A single-center retrospective study of women with RDs at Hokkaido University Hospital between 2007 and 2016 was conducted. Clinical features and maternal and fetal outcomes were retrospectively collected. The rate of pregnancy complications was compared with the general obstetric population (GOP) in Japan. RESULTS: Overall, 132 pregnancies in 95 women with RDs were recorded. Underlying RDs were systemic erythematosus (SLE) (n = 57), antiphospholipid syndrome (APS) (n = 35), rheumatoid arthritis (n = 9), and other RDs (n = 31). Antiphospholipid antibodies (aPL) were detected in 44 pregnancies (32%). Glucocorticoid was used in 82 pregnancies (62%), and tacrolimus in 20 pregnancies (15%). There were 24 disease flares (18%), but no RD-related death was documented. We recorded 112 live births, 6 abortions, 8 miscarriages, and 6 stillbirths. Pregnancies with RDs appeared to have frequent, emergency cesarean sections and preterm deliveries compared with GOP (30% vs 15% and 21% vs 14%, respectively). The median [interquartile range] birthweight in SLE and APS was lower than GOP (2591 [2231-2958] g and 2600 [2276-2920] g vs 2950 [2650-3250] g, respectively). In pregnancies with SLE, low complement levels presented the risk of maternal complications (odds ratio [95% CI]; 3.9 [1.0-14.9], p = 0.046) and anti-DNA antibody positivity was significantly correlated with the risk of fetal complications (3.5 [1.1-11.2], p = 0.036). In pregnancies with APS, maternal age over 35 years and duration of disease longer than 9 years (7.4 [1.3-40.8], p = 0.021, and 11.16 [1.1-118.8], p = 0.046, respectively) were significantly correlated with the risk of fetal complications. CONCLUSION: Pregnancies with RDs were at increased risk of having both maternal complications and adverse neonatal outcomes, indicating these pregnancies should be closely monitored.

Downregulation of miRNA-31 induces taxane resistance in ovarian cancer cells through increase of receptor tyrosine kinase MET.

Ovarian cancer is one of the most aggressive female reproductive tract tumors. Paclitaxel (PTX) is widely used for the treatment of ovarian cancer. However, ovarian cancers often acquire chemotherapeutic resistance to this agent. We investigated the mechanism of chemoresistance by analysis of microRNAs using the ovarian cancer cell line KFr13 and its PTX-resistant derivative (KFr13Tx). We found that miR-31 was downregulated in KFr13Tx cells, and that re-introduction of miR31 re-sensitized them to PTX both in vitro and in vivo. miR-31 was found to bind to the 3'-UTR of mRNA of MET, and the decrease in MET correlated to higher sensitivity to PTX. Furthermore, co-treatment of KFr13Tx cells with MET inhibitors sensitized the tumor cells to PTX both in vitro and in vivo. In addition, lower levels of miR31 and higher expression of MET in human ovarian cancer specimens were significantly correlated with PTX chemoresistance and poor prognosis. This study demonstrated miR31-dependent regulation of MET for chemoresistance of ovarian cancer, raising the possibility that combination therapy with a MET inhibitor and PTX will increase PTX efficacy.

Mutant p53 gain-of-function induces epithelial-mesenchymal transition through modulation of the miR-130b-ZEB1 axis.

The tumor suppressor gene p53 has been implicated in the regulation of epithelial-mesenchymal transition (EMT) and tumor metastasis by regulating microRNA (miRNA) expression. Here, we report that mutant p53 exerts oncogenic functions and promotes EMT in endometrial cancer (EC) by directly binding to the promoter of miR-130b (a negative regulator of ZEB1) and inhibiting its transcription. We transduced p53 mutants into p53-null EC cells, profiled the miRNA expression by miRNA microarray and identified miR-130b as a potential target of mutant p53. Ectopic expression of p53 mutants repressed the expression of miR-130b and triggered ZEB1-dependent EMT and cancer cell invasion. Loss of an endogenous p53 mutation increased the expression of miR-130b, which resulted in reduced ZEB1 expression and attenuation of the EMT phenotype. Furthermore, re-expression of miR-130b suppressed mutant p53-induced EMT and ZEB1 expression. Importantly, the expression of miR-130 was significantly reduced in EC tissues, and patients with higher expression levels of miR-130b survived longer. These data provide a novel understanding of the roles of p53 gain-of-function mutations in accelerating tumor progression and metastasis through modulation of the miR-130b-ZEB1 axis.

Weekly paclitaxel/5-fluorouracil followed by platinum retreatment for patients with recurrent ovarian cancer: a single institution experience.

PURPOSE: Since the prognosis of recurrent ovarian cancer patients is still poor, we need to establish a useful treatment strategy to achieve their long-term survival. We treated recurrent ovarian cancer patients with weekly paclitaxel (PTX)/5-fluorouracil (5-FU) followed by platinum retreatment to investigate its clinical efficacy in a preliminary manner. METHODS: Sixteen patients with recurrent ovarian cancer, pretreated with taxane and platinum, were treated with weekly paclitaxel (PTX)/5-fluorouracil (FU). PTX (80 mg/m2) on day 1, 8, and 15 was combined with a bolus injection of 5-FU (500 mg/m2) on day 2, 9, and 16. Chemotherapy was given every four weeks. Patients with stable disease or progressive disease were subsequently retreated with a platinum-containing regimen. Response was evaluated by RECIST criteria or CA125 criteria. Toxicities were evaluated according to the National Cancer Institute-common toxicity criteria (NCI-CTC) version 3. RESULTS: Among five patients with sensitive disease, one of four patients with measurable tumor and one without measurable tumor responded to weekly PTX/5-FU. Among 11 patients with resistant disease, none of five patients with measurable tumor and three of six patients without measurable tumor responded to weekly PTX/5-FU. Overall objective response rate by weekly PTX/5-FU was 31.3% (5/16). Among 16 patients, 13 patients who showed no response or progressive disease (three with sensitive disease, ten with resistant disease) received platinum retreatment after weekly PTX/5FU. All three patients with sensitive disease and three of ten patients with resistant disease revealed response to platinum retreatment. Overall objective response rate by platinum retreatment after weekly PTX/5-FU was 46.2% (6/13). CONCLUSIONS: Weekly PTX/5FU followed by platinum retreatment could be a useful treatment strategy for recurrent ovarian cancer patients. We need to establish the standard treatment strategy for recurrent ovarian cancer patients with a poor prognosis.

Rapid detection of the cariogenic pathogens Streptococcus mutans and Streptococcus sobrinus using loop-mediated isothermal amplification.

INTRODUCTION: Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. In this study, we developed a rapid, sensitive method for detecting these major cariogenic pathogens using loop-mediated isothermal amplification (LAMP). The assay procedure is quite simple: the amplification is carried out in a single tube under isothermal conditions at 63 degrees C, and the result can be obtained in less than 1 h. METHODS: Initially, a set of six primers was designed by targeting S. mutans-specific and S. sobrinus-specific regions, identified using the genomic subtractive hybridization technique. We evaluated the specificities and sensitivities of these assays. Furthermore, we detected and quantified these bacteria in saliva and carious dentin from eight children. RESULTS: The sensitivities of the S. mutans-specific and S. sobrinus-specific LAMP methods, examined using agarose gel electrophoresis, were each one cell for a 30-min reaction. The detection limits using real-time turbidimetry analysis were 1 to 10(7) cells (3.28 x 10(1) to 3.28 x 10(8) fg S. mutans template DNA) per reaction tube and 1 to 10(5) cells (2.72 x 10(3) to 2.72 x 10(8) fg S. sobrinus template DNA) per reaction tube. Using these assays, we detected and quantified these cariogenic bacteria for evaluation of the LAMP assay for clinical diagnosis. CONCLUSIONS: Our results suggest that the LAMP-based assay in combination with subtractive hybridization is valuable for preparing species-specific primers for closely related species. Furthermore, the LAMP-based assay will be a useful tool for the rapid and sensitive prediction of dental caries.

Rapid detection of Actinobacillus actinomycetemcomitans using a loop-mediated isothermal amplification method.

INTRODUCTION: Actinobacillus actinomycetemcomitans has been implicated in the etiology of aggressive periodontitis. In this study, we applied a novel nucleic acid amplification method, called loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions, allowing the rapid detection of A. actinomycetemcomitans. METHODS: We designed the primers for detecting A. actinomycetemcomitans and evaluated the specificity and sensitivity of the assay. RESULTS: The LAMP primers used in this study successfully amplified serotypes a-e of A. actinomycetemcomitans, while other oral bacteria were not amplified. By measuring the precipitation of magnesium pyrophosphate, we could quantify the chromosomal DNA of A. actinomycetemcomitans. The detection limits using the real-time turbidimetry analysis were 5.8 x 10(2)-5.8 x 10(7) copies of A. actinomycetemcomitans template DNA per reaction tube. In addition, the LAMP assay was used for the rapid detection of A. actinomycetemcomitans in clinical specimens from eight individuals. The results with the LAMP method were similar to those using conventional polymerase chain reaction. CONCLUSION: Our results suggest that the LAMP-based assay is very useful for the rapid detection of A. actinomycetemcomitans.

Diabetes mellitus is a multivariate independent prognostic factor in endometrial carcinoma: a clinicopathologic study on 313 patients.

OBJECTIVE: The aim of this study was to analyse the influence of diabetes mellitus as a prognostic factor for overall survival in endometrial cancer. MATERIALS AND METHODS: Charts were reviewed from patients with endometrial carcinoma from 1985 to 2003. Data on clinicopathologic variables, adjuvant treatment, site of recurrence and survival were collected. The chi-square test was used to examine associations between variables. The Kaplan-Meier method was used for survival analysis and Cox's proportional hazards model for multiple regression analysis. RESULTS: Multivariate analysis revealed that diabetes mellitus, FIGO stage and depth of myometrial invasion were significantly associated with overall survival.

Loop-mediated isothermal amplification method for the rapid detection of Enterococcus faecalis in infected root canals.

INTRODUCTION: Enterococcus faecalis is a major pathogen in the etiology of apical periodontitis after root canal treatment. A loop-mediated isothermal amplification method, which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand-displacement activity, was developed for the rapid detection of E. faecalis in clinical specimens from root canals. METHODS: Primers for detecting E. faecalis from the azoA gene were designed. The specificity of this assay was evaluated using various oral bacteria and the sensitivity was evaluated using serially diluted E. faecalis chromosomal DNA. In addition, loop-mediated isothermal amplification assays were applied to the rapid detection of E. faecalis from endodontic samples. RESULTS: The loop-mediated isothermal amplification products amplified with the primer set were specific for E. faecalis. To confirm the specificity of the amplicon, the amplified products were digested with the restriction endonuclease Sau3AI. The lower detection limit of the E. faecalis primer set without the loop primer set was 10 microg/tube for a 50-min loop-mediated isothermal amplification reaction. Using loop primers increased the detection sensitivity by several orders of magnitude. Furthermore, E. faecalis was detected with the loop-mediated isothermal amplification assay in four root canals from 18 individuals and the detection results were consistent with those of conventional polymerase chain reactions. CONCLUSION: These results indicate that the loop-mediated isothermal amplification assay is very useful for rapid detection of E. faecalis and diagnosis of endodontic infection.

Urodynamic study on postsurgical bladder function in cervical cancer treated with systematic nerve-sparing radical hysterectomy.

The objective of this study was to assess the postsurgical bladder function by urodynamic study in patients with cervical cancer treated with nerve-sparing radical hysterectomy. A total of 27 consecutive patients were included in the study. Of the 27 patients, autonomic nerves had been completely preserved at least on one side in 22 patients (group A), and autonomic nerves could not be successfully preserved in five patients (group B). In group A, there was no significant difference in compliance at the moment of strong desire to void, maximum flow rate, and residual urine volume between before the operation and at 12 months after the operation. However, abdominal pressure at maximum flow had significantly increased in patients of group B than of group A. Detrusor contraction pressure at maximum flow had significantly decreased in patients of group B than of group A. Bladder sensation was diminished in three cases (60%) of group B but preserved in all the patients of group A. Although it is still preliminary, our surgical technique described in this report is thought to be effective for preservation of bladder function. For further evaluation of the efficacy of nerve-sparing radical hysterectomy in terms of quality of life and survival of patients, a prospective randomized trial needs to be performed.

Relationship between oxygen consumption and sex of bovine in vitro fertilized embryos.

The present study was conducted to examine the relationship between the oxygen consumption rate and sex ratio of bovine in vitro fertilized embryos on each day of blastocyst formation. The quality of blastocysts collected on day 7, 8, and 9 after in vitro fertilization (IVF) were categorized as ranks A and B (excellent and good, respectively) based on microscopic observation of the morphology. The oxygen consumption rate and sex of individual blastocysts were evaluated using two novel techniques: scanning electrochemical microscopy (SECM) and loop-mediated isothermal amplification (LAMP), respectively. The oxygen consumption rates of embryos of rank A were significantly higher (p < 0.05) than those of rank B, irrespective of the day of blastocyst appearance after IVF. Neither did the proportion of male embryos of ranks A and B differ significantly from each other at any of the days examined, nor from the average proportion (53%). The oxygen consumption rate of embryos of rank B collected on day 8 was significantly higher (p < 0.05) in female embryos than in male embryos collected on the same day. However, there were no apparent differences of oxygen consumption rates at each day of blastocyst appearance between male and female embryos of rank A. These results indicate that the oxygen consumption rate of individual embryos reflects their quality but does not correlate with the sex ratio of embryos of excellent quality.

Multivariate analysis for prognostic significance of histologic subtype, GST-pi, MDR-1, and p53 in stages II-IV ovarian cancer.

It has been suggested that histologic subtype of ovarian cancer is a factor that determines the chemoresponsiveness of tumor. In this study, we wanted to clarify the prognostic significance of histologic subtype and its correlation to expression of chemoresistance-related proteins (CRPs) in ovarian cancer. A total of 93 stage II-IV ovarian cancers, where the proportion of serous, endometrioid, mucinous, and clear cell subtype was 61.3%, 14.0%, 7.5%, and 17.2%, respectively, were investigated for glutathione S-transferase-pi (GST-pi), MDR (multidrug resistance)-1, and p53 expression using immunohistochemistry. GST-pi expression was detected in 62.4% of the tumors and was not related to histologic subtype of tumor. MDR-1 expression was observed in 12.9% of the tumors tested and was more frequently detected in clear cell adenocarcinomas than other histologic subtypes of tumor (10/ 16 vs. 2 / 77, P < 0.001). P53 expression was found in 49.1% of serous, 53.8% of endometrioid, and 50% of mucinous adenocarcinomas. In contrast, none of 16 clear cell adenocarcinomas showed positive p53 staining. In univariate analysis, no direct correlations were found between CRPs and overall survival. Histology of mucinous/clear cell tumors (P = 0.0063), as well as FIGO stage III/IV (P = 0.0091) and residual tumor >or= 2 cm (P = 0.0045), was found to have independent prognostic value in multivariate analysis. In conclusion, histologic subtype proved to be the significant independent prognostic factor in addition to FIGO stage and residual tumor in stage II-IV ovarian cancer. GST-pi, MDR-1, and p53 expression pattern is closely related to histologic subtype of ovarian cancer, although they are not significant predictors of survival.

Application of the transition state theory to water transport across cell membranes.

We have applied the transition state theory of Eyring et al. (The Theory of Rate Processes, McGraw-Hill, 1941) to water transport across cell membranes. We have then evaluated free energy (Delta F(not equal)), enthalpy (Delta H(not equal)) and entropy (Delta S(not equal)) of activation for water permeation across membranes, such as Arbacia eggs, Xenopus oocytes with or without aquaporin water channels, mammalian erythrocytes, aquaporin proteoliposomes, liposomes and collodion membrane. Delta H(not equal) was found to be correlated with Delta S(not equal). This is so-called Delta H(not equal) and Delta S(not equal) compensation over the ranges of Delta H(not equal) and Delta S(not equal) from 2 to 22 kcal/mol and from -26 to 45 e.u., respectively, indicating that low Delta H(not equal) values correspond to negative Delta S(not equal). Large positive Delta S(not equal) and high Delta H(not equal) values might be accompanied by reversible breakage of secondary bonds in the membrane, presumably in membrane lipid bilayer. Largely negative Delta S(not equal) and low Delta H(not equal) values for aquaporin water channels, aquaporin proteoliposomes and porous collodion membrane could be explained by the immobilization of permeating water molecules in the membrane, i.e., the partial loss of rotational and/or translational freedoms of water molecules in water channels.

Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes.

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.

Determinants of NPC1 expression and action: key promoter regions, posttranscriptional control, and the importance of a "cysteine-rich" loop.

Mutations in the NPC1 gene cause Niemann-Pick type C disease, which is characterized by the accumulation of free cholesterol and other lipids in lysosomes. The NPC1 glycoprotein is located in a late endosomal compartment that transiently interacts with lysosomes. To identify factors regulating NPC1 expression and action, we analyzed the function of the human NPC1 promoter in human-derived ovarian, hepatic, and neuronal cells. A fragment containing the first 208 base pairs upstream from the major transcription initiation site was sufficient to drive near maximal NPC1 promoter activity. Deletion analysis revealed that sequences between base pairs -111 and -37 play an important role in controlling NPC1 transcription. Treatment of proliferating granulosa cells with 30 microM progesterone, which induces a reversible phenocopy of the cholesterol trafficking defect of Niemann-Pick type C disease, increased NPC1 mRNA levels threefold. The protein synthesis inhibitor, cycloheximide, also increased NPC1 mRNA levels, augmenting the progesterone-induced increase in NPC1 mRNA abundance. Progesterone treatment was shown to increase the mRNA half-life, but did not affect NPC1 promoter activity. Cysteine residues in a "cysteine-rich" loop predicted to reside in the intralumenal compartment of vesicles containing NPC1 were mutated, resulting in proteins that were incapable of correcting the cholesterol trafficking defect in CT60 cells, a Chinese hamster cell line in which the endogenous NPC1 gene is inactivated. Converting isoleucine 1061, also predicted to lie within the cysteine-rich loop, to a threonine residue inactivated the protein as well. The I1061T mutation is one of the most common mutations in Niemann-Pick type C disease. All of the cysteine-rich loop mutants were localized to cholesterol-engorged lysosomes in a pattern mimicking the distribution of NPC1 in progesterone-treated cells. A recombinant protein representing the cysteine-rich loop was shown to bind to a zinc-NTA agarose column. We conclude: (1) that cis elements residing in the first 111 base pairs upstream from the transcription start site are critical for transcription of the NPC1 gene; (2) that NPC1 expression is subject to posttranscriptional regulation in response to treatments that disrupt NPC1 function; and (3) that an intralumenal cysteine-rich loop with zinc-binding activity is critical to NPC1's ability to unload lysosomal cargo.

Lipopolysaccharide induces expression of genes encoding pro-inflammatory cytokines and the elastin-degrading enzyme, cathepsin S, in human cervical smooth-muscle cells.

OBJECTIVE: Vaginal and amniotic infection with gram-negative bacteria is associated with preterm birth. We previously reported that human cervical smooth-muscle cells (CSMC) respond to pro-inflammatory cytokines by expressing enzymes that degrade the extracellular matrix. Our objective was to characterize the effects of lipopolysaccharide (LPS) from Escherichia coli (E coli), Bacteroides fragilis, (B frag)and Fusobacterium nucleatum (F nuc)on the expression of pro-inflammatory cytokines and the elastin-degrading enzyme, cathepsin S, in human CSMC. METHODS: Human CSMC were exposed to LPS and the expression of mRNAs encoding pro-inflammatory cytokines and cathepsin S, and selected matrix metalloproteinases (MMPs) was analyzed by Northern blotting. The effect of cytokine-neutralizing antibodies on LPS-induced cathepsin S mRNA expression also was determined. RESULTS: E coli LPS increased expression of cathepsin S 12.5-fold after 12 hours; MMP-1 and MMP-3 mRNAs also were increased 2.9- and 3.5-fold, respectively. Tumor necrosis factor (TNF)-alpha, interleukin (IL-1)alpha, and IL-1beta mRNAs were markedly up-regulated after 3 hours of LPS treatment. B frag and F nuc LPS also induced TNF-alpha and cathepsin S mRNAs. E coli LPS caused a sevenfold increase in TNF-alpha secretion after 5 to 8 hours. Antihuman TNF-alpha monoclonal antibody, but not a monoclonal antibody to the low-density lipoprotein receptor, reduced the LPS-induced increase in cathepsin S mRNA by 27%, whereas neutralizing antibodies against IL-1alpha and IL-1beta did not suppress the response. Human CSMC were shown to express the toll-like receptor (TLR-2) and TLR-4 genes, which mediate the action of LPS. TLR-2 mRNA was up-regulated by TNF-alpha. CONCLUSION: CSMC respond to LPS with increased expression of pro-inflammatory cytokines and cathepsin S. Increases in cathepsin S mRNA result only in part from the rapid induction of TNF-alpha gene expression. TNF-alpha may also augment the CSMC response to LPS by increasing expression of the LPS signaling receptor, TLR-2, which probably directly mediates LPS action. These observations provide a mechanism by which gram-negative bacteria can precipitate cervical changes associated with preterm birth.

NPC1-containing compartment of human granulosa-lutein cells: a role in the intracellular trafficking of cholesterol supporting steroidogenesis.

Steroidogenic cells represent unique systems for the exploration of intracellular cholesterol trafficking. We employed cytochemical and biochemical methods to explore the expression, regulation, and function of the Niemann-Pick C1 protein (NPC1) in human granulosa-lutein cells. NPC1 was localized in a subset of lysosome-associated membrane glycoprotein 2 (LAMP-2)-positive vesicles. By analyzing the sensitivity of NPC1 N-linked oligosaccharide chains to glycosidases and neuraminidase, evidence was obtained for movement of nascent NPC1 from the endoplasmic reticulum through the medial and trans compartments of the Golgi apparatus prior to its appearance in cytoplasmic vesicles. NPC1 protein content and the morphology and cellular distribution of NPC1-containing vesicles were not affected by treatment of the granulosa-lutein cells with 8-Br-cAMP, which stimulates cholesterol metabolism into progesterone. In contrast, steroidogenic acute regulatory (StAR) protein levels were increased by 8-Br-cAMP. Incubation of granulosa-lutein cells with low-density lipoprotein (LDL) in the presence of the hydrophobic amine, U18666A, caused accumulation of free cholesterol in granules, identified by filipin staining, that contained LAMP-2 and NPC1. These granules also stained for neutral lipid with Nile red, reflecting accumulation of LDL-derived cholesterol esters. LDL-stimulated progesterone synthesis was completely blocked by U18666A, leaving steroid output at levels similar to those of cells incubated in the absence of LDL. The hydrophobic amine also blocked the LDL augmentation of 8-Br-cAMP-stimulated progesterone synthesis, reducing steroid production to levels seen in cells stimulated with 8-Br-cAMP in the absence of LDL. Steroidogenesis recovered after U18666A was removed from the culture medium. U18666A treatment caused a 2-fold or more increase in NPC1 protein and mRNA levels, suggesting that disruption of NPC1's function activates a compensatory mechanism resulting in increased NPC1 synthesis. We conclude that the NPC1 compartment plays an important role in the trafficking of LDL-derived substrate in steroidogenic cells; that NPC1 expression is up-regulated when NPC1 action is blocked; and that the NPC1 compartment can be functionally separated from other intracellular pathways contributing substrate for steroidogenesis.

The steroidogenic acute regulatory protein (StAR): a window into the complexities of intracellular cholesterol trafficking.

Stimulation of steroid-producing cells of the gonads and adrenals with tropic hormone results in a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system. This process of cholesterol translocation is blocked by inhibitors of protein synthesis, suggesting that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory (StAR) protein appears to be the most likely candidate for the "labile" protein, based on the following observations: 1) Expression of StAR in COS-1 cells engineered to contain the cholesterol side-chain cleavage system substantially augments pregnenolone formation; 2) StAR protein is expressed almost exclusively in steroid-producing cells, except the trophoblast of the human placenta, and its presence is correlated with steroid hormone production; 3) StAR mRNA increases in response to cAMP; 4) StAR is a target for serine phosphorylation mediated by protein kinase A, a process that is essential for maximizing StAR activity; and 5) lack of functional StAR causes the autosomal recessive disease, congenital lipoid adrenal hyperplasia, characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. Studies on the mechanism of action of StAR revealed that import into mitochondria is not essential to its steroidogenesis-enhancing activity and more likely represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations established that the C-terminus of the StAR protein contains the functionally important domains. The demonstration of steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence confirmed that StAR import is not essential for its steroidogenic activity and suggested that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. Evidence that StAR functions as a cholesterol transfer protein raises the possibility that StAR acts directly on lipids of the outer mitochondrial membrane, probably stimulating cholesterol desorption from the sterol-rich outer membrane and its movement to the relatively sterol-poor inner membrane.

Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein.

Niemann-Pick type C (NPC) disease, characterized by accumulation of low density lipoprotein-derived free cholesterol in lysosomes, is caused by mutations in the NPC1 gene. We examined the ability of wild-type NPC1 and NPC1 mutants to correct the NPC sterol trafficking defect and their subcellular localization in CT60 cells. Cells transfected with wild-type NPC1 expressed 170- and 190-kDa proteins. Tunicamycin treatment resulted in a 140-kDa protein, the deduced size of NPC1, suggesting that NPC1 is N-glycosylated. Mutation of all four asparagines in potential N-terminal N-glycosylation sites to glutamines resulted in a 20-kDa reduction of the expressed protein. Proteins with a single N-glycosylation site mutation localized to late endosome/lysosomal compartments, as did wild-type NPC1, and each corrected the cholesterol trafficking defect. However, mutation of all four potential N-glycosylation sites reduced ability to correct the NPC phenotype commensurate with reduced expression of the protein. Mutations in the putative sterol-sensing domain resulted in inactive proteins targeted to lysosomal membranes encircling cholesterol-laden cores. N-terminal leucine zipper motif mutants could not correct the NPC defect, although they accumulated in lysosomal membranes. We conclude that NPC1 is a glycoprotein that must have an intact sterol-sensing domain and leucine zipper motif for cholesterol-mobilizing activity.

Pro-inflammatory cytokines induce expression of matrix-metabolizing enzymes in human cervical smooth muscle cells.

The process of cervical ripening has been likened to an inflammatory reaction associated with the catabolism of cervical extracellular matrix by enzymes released from infiltrating leukocytes. We hypothesized that smooth muscle cells in the cervix also participate in this process and that pro-inflammatory cytokines act on cervical smooth muscle cells (CSMC) to provoke the expression of matrix-degrading enzymes. We treated primary cultures of human CSMC with tumor necrosis factor-alpha (TNF-alpha) and examined expression of the elastinolytic enzyme, cathepsin S, the collagen metabolizing matrix metalloproteinases (MMP)-1, -3, -9, and the tissue inhibitor of metalloproteinase (TIMP)-1 and -2. A time course analysis revealed that 10 ng/ml of TNF-alpha induced cathepsin S, MMP-1, -3, and -9 mRNA expression with the maximal response observed after 24-48 hours. TNF-alpha induced cathepsin S, MMP-1, -3, and -9 mRNA expression in a dose-dependent manner: the maximal effect was observed at a concentration of 10 ng/ml, with appreciable increases observed at concentrations of 0.1 to 1.0 ng/ml. In contrast, TIMP-1 and -2 mRNAs were not significantly increased by TNF-alpha treatment. Interleukin-1beta produced a pattern of gene expression in the CSMC similar to that observed following TNF-alpha treatment. Western blot analysis and zymography confirmed the induction of proMMP-1, -3, and -9 in response to TNF-alpha, but MMP-2 immunoreactivity and zymographic activity were unaffected. TNF-alpha increased secretion of procathepsin S, but did not affect TIMP-1 and reduced TIMP-2 production. We conclude that CSMC are targets of pro-inflammatory cytokines, which induce a repertoire of enzymes capable of degrading the cervical extracellular matrix. The induction of these enzymes may facilitate the normal ripening of the cervix at term and participate in the premature cervical changes associated with preterm labor.

Niemann-Pick C1 protein: obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization.

Niemann-Pick type C (NPC) disease is an inherited lipid storage disorder that affects the viscera and central nervous system. A characteristic feature of NPC cells is the lysosomal accumulation of low density lipoprotein-derived cholesterol. To elucidate important structural features of the recently identified NPC1 gene product defective in NPC disease, we examined the ability of wild-type NPC1 and NPC1 mutants to correct the excessive lysosomal storage of low density lipoprotein-derived cholesterol in a model cell line displaying the NPC cholesterol-trafficking defect (CT60 Chinese hamster ovary cells). CT60 cells transfected with human wild-type NPC1 contained immunoreactive proteins of 170 and 190 kDa localized to the lysosomal/endosomal compartment. Wild-type NPC1 protein corrected the NPC cholesterol-trafficking defect in the CT60 cells. Mutation of conserved cysteine residues in the NPC1 N terminus to serine residues resulted in proteins targeted to lysosomal membranes encircling cholesterol-laden cores, whereas deletion of the C-terminal 4-aa residues containing the LLNF lysosome-targeting motif resulted in the expression of protein localized to the endoplasmic reticulum. None of these mutant NPC1 proteins corrected the NPC cholesterol-trafficking defect in CT60 cells. We conclude that transport of the NPC1 protein to the cholesterol-laden lysosomal compartment is essential for expression of its biological activity and that domains in the N terminus of the NPC1 protein are critical for mobilization of cholesterol from lysosomes.