Bendamustine, etoposide and dexamethasone to mobilize peripheral blood hematopoietic stem cells for autologous transplantation in patients with multiple myeloma.

Chemotherapeutic agents without cross-resistance to prior therapies may enhance PBSC collection and improve patient outcomes by exacting a more potent direct antitumor effect before autologous stem cell transplant. Bendamustine has broad clinical activity in transplantable lymphoid malignancies, but concern remains over the potential adverse impact of this combined alkylator-nucleoside analog on stem cell mobilization. We performed a prospective, nonrandomized phase II study including 34 patients with multiple myeloma (MM) (n=34; International Staging System (ISS) stages I (35%), II (29%) and III (24%); not scored (13%)) to evaluate bendamustine's efficacy and safety as a stem cell mobilizing agent. Patients received bendamustine (120 mg/m2 IV days 1, 2), etoposide (200 mg/m2 IV days 1-3) and dexamethasone (40 mg PO days 1- 4) (bendamustine, etoposide and dexamethasone (BED)) followed by filgrastim (10 µg/kg/day SC; through collection). All patients (100%) successfully yielded stem cells (median of 21.60 × 106/kg of body weight; range 9.24-55.5 × 106/kg), and 88% required a single apheresis. Six nonhematologic serious adverse events were observed in 6 patients including: neutropenic fever (1, grade 3), bone pain (1, grade 3) and renal insufficiency (1, grade 1). In conclusion, BED safely and effectively mobilizes hematopoietic stem cells.

Wnt-signalling pathway in ovarian epithelial tumours: increased expression of beta-catenin and GSK3beta.

Beta-catenin is involved in both cell-cell adhesion and in transcriptional regulation by the Wingless/Wnt signalling pathway. Alterations of components of this pathway have been suggested to play a central role in tumorigenesis. The present study investigated, by immunohistochemistry and immunoblotting, the protein expression and localisation of beta-catenin, adenomatous polyposis coli (APC), glycogen synthase kinase 3beta (GSK3beta) and lymphocyte enhancer factor-1 (Lef-1) in normal human ovaries and in epithelial ovarian tumours in vivo and in vitro. Immortalised human ovarian surface epithelium and ovarian cancer cell cells (OVCAR-3) expressed beta-catenin, APC, GSK3beta and Lef-1. Nuclear staining of beta-catenin and Lef-1 were demonstrated only in OVCAR-3 cells. There were significant increases of beta-catenin and GSK3beta, while APC was reduced in ovarian cancer compared to the normal ovary. Beta-catenin and Lef-1 were coimmunoprecipitated in ovarian tumours, but not in the normal ovary. Nuclear localisation of beta-catenin or Lef-1 could not be demonstrated in the normal ovary or in the ovarian tumours. The absence of nuclear localisation of beta-catenin could be due to an increased binding to the cadherin-alpha-catenin cell adhesion complex. In fact, we have earlier reported an increased expression of E-cadherin in ovarian adenocarcinomas. In summary, this study demonstrates an increase in the expression of components of the Wingless/Wnt pathway in malignant ovarian tumours. The increase suggests a role for this signalling pathway in cell transformation and in tumour progression. However, it remains to be demonstrated whether it is an increased participation of beta-catenin in transcriptional regulation, or in the stabilisation of cellular integrity, or both, that is the crucial event in ovarian tumorigenesis.

Acetazolamide inhibits stimulated feline liver and gallbladder bicarbonate secretion.

Bile acidification is a key factor in preventing calcium carbonate precipitation and gallstone formation. Carbonic anhydrase II (CA II), that is inhibited by acetazolamide, plays a role in regulation of the acid-base balance in many tissues. This study examines the effect of acetazolamide on secretin- and vasoactive intestinal peptide (VIP)-stimulated gallbladder mucosal bicarbonate and acid secretion. Gallbladders in anaesthetized cats were perfused with a bicarbonate buffer bubbled with CO2 in air. In 20 experiments VIP (10 microg kg(-1) h(-1)) and in 10 experiments secretin (4 microg kg(-1) h(-1)) were infused continuously intravenous (i.v.). Hepatic bile and samples from the buffer before and after perfusion of the gallbladder were collected for calculation of ion and fluid transport. During basal conditions a continuous secretion of H+ by the gallbladder mucosa was seen. Intravenous infusion of vasoactive intestinal peptide (VIP) and secretin caused a secretion of bicarbonate from the gallbladder mucosa (P < 0.01). This secretion was reduced by intraluminal (i.l.) acetazolamide (P < 0.01). Bile flow was enhanced by infusion of VIP and secretin (P < 0.01) but this stimulated outflow was not affected by i.v. acetazolamide. The presence of CA II in the gallbladder was demonstrated by immunoblotting. Biliary CA activity has an important function in the regulation of VIP- and secretin-stimulated bicarbonate secretion across the gallbladder mucosa.

Higher levels of soluble E-cadherin in cyst fluid from malignant ovarian tumours than in benign cysts.

A major diagnostic dilemma in the clinical gynaecological oncology setting is to preoperatively determine whether a complex ovarian mass is benign or malignant. The cell-cell adhesion molecule E-cadherin has previously been localised in biopsies from both benign and malignant epithelial ovarian tumours. In this study, soluble E-cadherin levels was measured with ELISA-technique in peripheral blood, ascites and cystic fluids from patients (n = 33) undergoing surgery for ovarian cystic masses. The levels of soluble E-cadherin were significantly higher in cystic fluid from cystadenocarcinomas (p < 0.001) and borderline tumours (p < 0.05) as compared to cystic fluid from cystadenomas. In ascites fluid and peripheral blood no significant differences were seen. However, ratios of cystic fluid/peripheral blood levels were significantly higher in cystadenocarcinoma (p < 0.001) and borderline tumours (p < 0.05) as compared to benign tumours. In conclusion, measurements of soluble E-cadherin in cystic fluid from patients presenting with complex ovarian masses may be beneficial in increasing the accuracy of preoperative diagnosis.

Domestic violence during pregnancy. The prevalence of physical injuries, substance use, abortions and miscarriages.

BACKGROUND: The aim of this study was to estimate the prevalence of physical injuries, alcohol and tobacco use, abortions and miscarriages due to domestic violence during pregnancy and to compare socio-economic background factors between abused and non abused women. METHOD: Personal interview combined with a standardized questionnaire involving 207 pregnant Swedish born women married to or cohabiting with Swedish born men. The women were consecutively chosen from three different antenatal clinics in Göteborg, Sweden. RESULTS: Overall 30 women were abused during the current pregnancy as defined from the category 'symbolic violence' in the Severity of Violence Against Women Scale (SVAW). The most frequent targets for physical abuse were: the upper arm, the forearm, and the face and neck region. Ninety-five percent of women abused during pregnancy had been abused prior to the pregnancy. Notable was the finding that 4.3% of the pregnant women had been exposed to serious violence. Abused women were significantly younger and single, had lower income and education compared to the non abused women. In the group of abused women a higher proportion of women had undergone one or more abortions than in the non-abused group. Smoking and alcohol use among partners were strongly correlated with physical and sexual abuse. CONCLUSIONS: The results suggest that in antenatal and obstetric clinics, emphasis should be focused on previous history of abuse and a complete physical examination of the women. Since bruises often were located at hidden areas of the body, it is of importance to scrutinize those sites as part of a routine examination. It is also important to look for common defensive marks on the forearms. The partner's cigarette and alcohol use is also an important piece of information regarding risk factors connected to domestic violence.

Increased expression of the transcription factors CCAAT-enhancer binding protein-beta (C/EBBeta) and C/EBzeta (CHOP) correlate with invasiveness of human colorectal cancer.

Regulation of cell differentiation is most often impaired in malignant tumors and may represent a key mechanism for the progression of the disease. CCAAT-enhancer binding protein (C/EBP) is a family of transcription factors involved in the regulation of embryonic gut development in rodents, which has also been detected in various malignancies, e.g., liposarcomas and breast and ovarian epithelial tumors. We studied the relationship between C/EBP and tumor histology (Duke's invasive stage and pathological grade) in colorectal cancer. Immunoblotting techniques were used on microdissected fresh frozen tumor specimens, and expression of C/EBPalpha, C/EBPbeta and C/EBPzeta (CHOP) was analyzed in addition to that of the cell-cycle regulator p53 and the proliferation marker PCNA. Expression of C/EBPbeta (LAP isoforms) was markedly increased in all tumors compared with normal colon mucosa. Although the inter-patient variability was large, we found that LIP, the isoform of C/EBPbeta known to inhibit transcription, was expressed at higher levels in Duke's stage B tumors compared with Duke's stage A, whereas Duke's C tumors had the lowest LIP expression. A similar relationship was seen for CHOP. The cell-cycle regulator gene p53 was the only factor that clearly correlated with pathological grade: a decrease in p53 expression was demonstrated. Our data suggest that genetic and cellular events involving C/EBPbeta and CHOP are important for tumor invasion and that these events do not appear to be related to the pathological grade of the tumor.

The invisible wounds: the occurrence of psychological abuse and anxiety compared with previous experience of physical abuse during the childbearing year.

The aim of this study was to measure the prevalence, effects and character of psychological abuse in women visiting antenatal clinics. A standardized questionnaire based on four different established scales (PMWI, SVAW, TSC-33, and STAI) was used to estimate the frequency of psychological, physical and sexual abuse, anxiety and depression. In the study 207 pregnant Swedish born women married to or cohabiting with Swedish born men were consecutively chosen from three different antenatal clinics from the city of Göteborg, Sweden. Personal interviews were conducted in connection to their regular visit to the antenatal clinic, ranging from the first to the third trimester. Fifty-one (24.5%) women out of 207 reported threats and/or acts of violence during the last year according to the Severity of Violence Against Women Scale (SVAW). There was 89.4% who had experienced dominance/isolation according to the Psychological Maltreatment of Women Inventory (PMWI) and 44.4% of the women reported emotional/verbal abuse. Occupational status, but not age income or education, was found to be significantly correlated to physical violence, dominance/isolation and to emotional/verbal factor according to Psychological Maltreatment of Women Inventory (PMWI). Threats of moderate violence' and 'serious violence' were strongly correlated to physical violence (correlation coefficient 0.9433 and 0.9405, respectively). Sexual abuse demonstrated a high correlation to physical violence and emotional/verbal factor. The results indicate that sexual violence is highly represented in the abusive relationship and also that depression and anxiety in the childbearing year may be caused by domestic violence. This study emphasises the importance of incorporating screening for threats and actual acts of psychological, physical and sexual abuse into routine care for women, enabling health care providers to identify high-risk patients and improve quality of care.

The expression of CCAAT/enhancer binding protein (C/EBP) in the human ovary in vivo: specific increase in C/EBPbeta during epithelial tumour progression.

The CCAAT/enhancer binding protein (C/EBP) family of transcription factors is involved in metabolism and differentiation of cells, especially in rodent liver cells and adipocytes. Their roles in vivo and in particular during pathophysiological conditions in humans are largely unknown. We have investigated the presence of C/EBPalpha, -beta, -delta and -zeta in normal ovaries and in epithelial ovarian tumours of different stages. Immunohistochemical experiments demonstrated that C/EBPalpha and C/EBPbeta were preferentially expressed in epithelial/tumour cells irrespective of stage or grade of the tumour. C/EBPbeta was located in the nuclei of the cells, in contrast to C/EBPalpha, which was present only in the cytoplasm of these cells. The nuclear localization of C/EBPbeta indicates an active role of this transcription factor in tumour cells, whereas the cytoplasmic distribution suggests a more passive function of C/EBPalpha. C/EBPdelta and -zeta demonstrated a more diverse distribution with predominant localization to epithelial cells, but stromal distribution was also noted. The intracellular distribution was confined to both the nucleus and the cytoplasm for C/EBPdelta and -zeta. Western blotting demonstrated that C/EBPalpha, -beta, -delta and -zeta were present in a majority of the samples. The amount of C/EBPbeta increased markedly with malignancy, i.e. with degree of dedifferentiation, while the other members of the C/EBP family displayed a more constant expression level. These results demonstrate an association between the expression of members of the C/EBP family and the formation of epithelial ovarian tumours, with C/EBPbeta as a potential marker for these tumours. As C/EBPbeta is known to be expressed during proliferation of cells in vitro, it may participate in the proliferative process of ovarian epithelial tumour cells in vivo and play a central role in tumour progression.

Isoform-specific regulation of the CCAAT/enhancer-binding protein family of transcription factors by 3',5'-cyclic adenosine monophosphate in Sertoli cells.

The C/EBP (CCAAT/enhancer-binding protein) family of transcription factors is important for differentiation, lipid biosynthesis, and metabolism. Here, we demonstrate for the first time the presence of C/EBP alpha, beta, delta, and zeta messenger RNA (mRNA) and protein in Sertoli cell primary cultures. Treatment with FSH or 8-CPTcAMP strongly induced C/EBP beta mRNA above basal levels with rapid and transient kinetics in Sertoli cell primary cultures as well as in whole testes from hypophysectomized rats. Whereas C/EBP beta mRNA was induced approximately 50-fold, C/EBP delta mRNA was induced 5- to 8-fold by cAMP in Sertoli cells. Messenger RNA for C/EBP beta and delta were induced by inhibition of protein synthesis with cycloheximide and cycloheximide acted synergistically with cAMP. Immunoblots with C/EBP antibodies demonstrated a strong induction of C/EBP beta, delta, and zeta by cAMP. Electrophoretic mobility shift analysis of nuclear proteins from cAMP-treated Sertoli cells using a C/EBP consensus oligonucleotide and antibodies revealed specific binding of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP beta antibody. Transfections of Sertoli cells with a C/EBP reporter construct showed approximately 3-fold induction of reporter gene activity by cAMP. In contrast, the reporter gene vector with a mutated form of the C/EBP binding site, was almost unresponsive to cAMP in transfections of Sertoli cells. Furthermore, C/EBP beta expression increased the activities of two promoters known to be cAMP-responsive in Sertoli cells. Thus, the early induction of C/EBP isoforms by cAMP may play a role in FSH-dependent regulation of late response genes in Sertoli cells.

Role of cyclooxygenase-2 for fluid secretion by the inflamed gallbladder mucosa.

Inflammatory fluid secretion by the gallbladder mucosa in experimental cholecystitis is induced by activation of cyclooxygenase, which leads to an increase in prostaglandin formation. Cyclooxygenase exists as a constitutive (cyclooxygenase-l) and an inducible (cyclooxygenase-2) isoform. The aim of this study was to demonstrate the role of cyclooxygenase-2 in inflammatory fluid secretion of the feline gallbladder. Experiments were performed 10 weeks after a surgical procedure in which chronic cholecystitis was induced in cats by ligation of the cystic duct and implantation of a gallstone in the gallbladder. Gallbladder fluid transport was continuously monitored via a perfusion system. In inflammed gallbladders the continuous fluid secretion was reversed to absorption by intravenous injection of the selective cyclooxygenase-2 blocker, NS 398 (P <0.001). Increased levels of the inducible cyclooxygenase-2 were shown by immunoblotting in inflamed gallbladders. Selective pharmacologic blockage of cyclooxygenase-2 reduced the prostaglandin E2 release to the inflamed gallbladder lumen (P <0.01). These data suggest that cyclooxygenase-2 is involved in the inflammatory response during chronic cholecystitis. Selective cyclooxygenase-2 blockers may offer an alternative to traditional nonsterodial anti-inflammatory drugs with fewer side effects in patients with cholecystitis who are awaiting operation.

The chemotactic cytokine interleukin-8--a cyst fluid marker for malignant epithelial ovarian cancer?

Due to the difficulties in separating malignant and benign ovarian cysts by transvaginal ultrasound and other techniques, there is a need for biochemical markers in serum or cyst fluids. In the present study we have evaluated the levels of the chemokine interleukin-8 (IL-8) in ovarian cysts. IL-8 is known to be expressed in the normal ovary and to influence proliferation and angiogenesis of several nonovarian types of tumors. Cyst fluids from benign (n = 15) and malignant (n = 13) ovarian tumors were analyzed. The levels of IL-8 were found to be significantly (13-fold) higher in cyst fluids from malignant tumors (18.1 +/- 7.5 ng/ml; mean +/- SE) compared to benign cysts (1.3 +/- 0.7 ng/ml). The plasma levels of IL-8 were considerably lower (2.9 and 0.3% of levels in benign and malignant cyst fluids, respectively) than in cyst fluids. No difference in the plasma levels of patients with benign or malignant tumor could be detected. In contrast, the levels of CA 125 were significantly higher in plasma of patients with malignant disease with the inverse relation in cyst fluids. In conclusion, the levels of IL-8 are markedly elevated in cyst fluid from malignant tumors compared to benign. This specific increase indicates a role for this cytokine in ovarian tumor biology.

The transcription factor C/EBP-beta and its role in ovarian function; evidence for direct involvement in the ovulatory process.

Gonadotropins are responsible for maturation of the ovarian follicle and the oocyte. Ovulation is the ultimate step in this process and involves disintegration of the follicular wall and subsequent release of an oocyte into the oviduct. These events are triggered by a surge of luteinizing hormone (LH). Genes expressed in the ovary, that respond to LH, are likely to be involved in the biochemical pathways that regulate ovulation. The transcription factor C/EBP-beta is induced promptly in the ovary, as a response to an ovulatory dose of gonadotropins. We used an ex vivo perfusion system to demonstrate that a specific reduction in ovarian C/EBP-beta expression inhibits ovulation. In such ovaries the oocytes appeared to be entrapped within the follicle. We have found a correlation between the expression level of the activating isoform of C/EBP-beta and the number of oocytes ovulated in response to gonadotropins. Since a reduction in C/EBP-beta expression does not affect the level of the ovulatory mediator prostaglandin endoperoxide synthase-2 (PGS-2), these findings support the view of C/EBP-beta as an important factor in the ovulatory process and highlight a C/EBP-beta-dependent and PGS-2-independent pathway that takes part in regulation of ovulation.

E-cadherin expression in human epithelial ovarian cancer and normal ovary.

The ovarian surface epithelium (OSE) is the origin of the majority of human ovarian cancers. These adenocarcinomas are characterized by initial local growth followed by spreading into the peritoneal cavity at later stages of tumor progression. The cell-adhesion molecule E-cadherin (E-cad) plays an important role in maintaining tissue integrity. Disappearance or impaired function of E-cad have often been associated with tumor formation and invasion in vivo and in vitro. The cell-specific expression of E-cad was investigated in normal human ovaries (n = 12), in benign (n = 5) and borderline (n = 4) ovarian epithelial tumors and in adenocarcinomas of different stages and histological grades (n = 18), by immunohistochemistry and immunoblotting. An ovarian cancer cell line (NIH-OVCAR3) was used as a reference. The epithelial origin of the cells was confirmed with cytokeratin (AE1/AE3) staining. In normal ovaries, the expression of E-cad was limited to inclusion cysts or deep clefts lined with OSE, whereas no staining of the OSE could be demonstrated at the surface of the ovary. In contrast, benign and borderline tumors uniformly expressed E-cad. This was observed in malignant tumors of all stages despite their degree of differentiation. E-cad was also present in metastasis from such tumors. The cell-specific expression of E-cad in inclusion cysts of normal ovaries and in epithelial layers of borderline tumors indicates a role for E-cad in the early events of the progression to a malignant phenotype. E-cad was not downregulated in later stages of ovarian cancer progression.

The expression of c-myc during follicular growth and luteal formation in the rat ovary in vivo.

The processes of folliculogenesis and formation of corpora lutea involve proliferation and differentiation of the follicular cells. The expression of several oncogenes is associated with the proliferative phase in many cell types. The present study examined the expression and hormonal regulation of the c-myc proto-oncogene during follicular development and the luteal phase of pseudopregnancy. Follicular development was initiated by pregnant mare's serum gonadotropin (PMSG) in immature rats followed two days later by the injection of human chorionic gonadotropin (hCG) to induce ovulation and luteal formation. Ovaries were collected at different time points and the content and distribution of c-myc mRNA/protein were examined. C-myc increased rapidly after the administration of both PMSG and hCG, but the effect of PMSG was less pronounced. The increase after PMSG was transient and localized primarily to the granulosa cells of developing follicles. The ovulatory dose of hCG resulted in a rapid and substantial increase of c-myc mRNA and protein with maximal levels at 1 h and 2-4 respectively. At this stage, the c-myc protein was localized to the follicular cells, the surface epithelium and, to some extent, to the interstitial tissue. There was a subsequent decrease prior to ovulation. The luteal phase was characterized by decreasing levels of c-myc with increasing luteal age. In order to examine the involvement of specific hormones in the regulation of c-myc, hypophysectomized, immature rats were injected sequentially with estradiol (E2) and follicle-stimulating hormone (FSH). Hypophysectomy resulted in a decrease of c-myc compared with intact animals. The administration of E2 resulted in an increase of c-myc mRNA and protein. The subsequent treatment with FSH did not result in a further increase and the levels remained at the same level as with E2 only. However, an ovulatory dose of hCG to E2 and FSH primed animals resulted in an additional increase of c-myc mRNA and protein. The levels after E2 and FSH were considerably lower compared with those of untreated ovaries of intact, immature animals, suggesting the involvement of other endocrine and paracrine factors. The presence of proliferating cell nuclear antigen in cell extracts indicated that the expression of c-myc was associated with phases of increased proliferation of follicular cells after hormonal stimulation. The results demonstrate that c-myc is regulated by hormones (E2, gonadotropins) in the rat ovary during follicular development to the preovulatory stage. The pronounced increase prior to ovulation also suggests a role for c-myc in the regulation of proliferative events involved in luteal formation.

Prostaglandin synthesis is suppressed by progesterone in rat preovulatory follicles in vitro.

The inducible form of prostaglandin endoperoxide-2 (PGS-2) is transiently induced by activators of the protein kinase A and protein kinase C systems in rat preovulatory (PO) granulosa cells. This induction is suggested to play an important role in the ovulatory process, which shares many of the characteristics of an inflammatory reaction. The purpose of the present study was to explore the role of progesterone (P4) as an "anti-inflammatory" steroid for the regulation of PGS-2 and the synthesis of prostaglandins in the PO follicle. Isolated rat PO follicles were preincubated with different amounts of exogenous P4 before the addition of luteinizing hormone (LH) and 3-isobutyl-1-methylxanthine (IBMX) (LH + 1). Medium levels of prostaglandin E2 (PGE2) were measured by RIA and the protein contents of PGS-1 and PGS-2 were determined by immunoblotting. LH + I. Both the content of PGS-2 and the synthesis of PGE2 were decreased. The content of PGS-1 demonstrated only minor changes in response to P4. These results showed a dual regulation of PGS-2 in the rat PO follicle with both stimulatory and inhibitory pathways. One of the "anti-inflammatory" actions exerted by P4 in the present study was to reduce the expression of PGS-2 and the follicular production of prostaglandins. This action might be of importance for restriction and control of the inflammatory response in the ovulatory process in vivo.

Expression of CCAAT enhancer binding protein-alpha (C/EBP alpha) in the rat ovary: implications for follicular development and ovulation.

The development of follicles from the antral to the preovulatory stage in the mammalian ovary involves both proliferation and differentiation of cells. These processes are coordinated by endocrine, paracrine, and autocrine factors in a time- and cell-specific pattern. Each stage of development is characterized by expression of specific genes the products of which are involved in different cellular processes, e.g., signal transduction (cAMP-dependent protein kinases), steroidogenesis (cytochrome P450 enzymes). At the nuclear level, the signaling pathway from external stimuli converges to modify the mechanisms of transcription factors. One such factor, CCAAT enhancer binding protein-alpha (C/EBPalpha), participates in differentiation processes of several organs, e.g., liver, adipose tissue, and gut. We have previously demonstrated that C/EBPalpha is expressed in rat ovarian follicles in a cell-, time-, and hormone-specific manner. This increase in granulosa cells was concomitant with the more differentiated phenotype. The aim of the present study was to explore the function of C/EBPalpha in the rat ovary. To achieve this, the expression of C/EBPalpha in follicular cells was attenuated in vivo by the local administration of antisense oligonucleotides (AS) into the ovarian bursa, i.e., the sac-like structure surrounding the ovary of immature rats. This administration resulted in an impaired response to subsequent injections of exogenous gonadotropins (PMSG, hCG) with an attenuated expression of C/EBPalpha protein and finally a decreased ovulation rate. Furthermore, the morphology of the AS-treated ovary was altered with large, oocyte-containing follicles at a time when ovaries exposed to sense (S) oligonucleotides demonstrated newly formed corpora lutea. AS also affected the expression of the proto-oncogene c-myc, which was elevated by this treatment. The administration of S was without effects. Thus, C/EBPalpha seems to be a necessary factor for follicular development in the rat ovary.

Regulation of the inducible form of prostaglandin endoperoxide synthase in the perfused rat ovary.

The regulation of the two isoforms of prostaglandin endoperoxide synthase (PGS-1 and PGS-2) and prostaglandin synthesis by luteinizing hormone (LH)/3-isobutyl-1-methylxanthine (IBMX) and progesterone was examined in granulosa cells and residual ovarian tissue of rat ovaries perfused in vitro. The endogenous progesterone synthesis was blocked by an inhibitor of 3 beta-dehydroxysteroid-dehydrogenase, compound A (CA), previously shown to reversibly inhibit ovulation in the in vitro perfused rat ovary. Preovulatory ovaries were perfused for 7 h, and soluble extracts from granulosa cells and residual ovarian tissue were obtained for immunoblotting and determination of the tissue contents of PGS-1/PGS-2. The tissue concentrations of prostaglandins (PGE2, PGF2 alpha and 6-keto-PGF1 alpha) were measured. The ovaries were perfused with medium alone (control) or medium containing LH (0.1 microgram/ml) and IBMX (0.2 mM), LH+IBMX+CA (10 micrograms/ml) or LH+IBMX+CA+progesterone (10 micrograms/ml). PGE2, PGF2 alpha and 6-keto-PGF1 alpha tissue concentrations were increased by LH+IBMX, with highest values detected for PGE2. The addition of CA alone or CA in combination with exogenous progesterone, did not change the values of prostaglandins increased by LH+IBMX. The content of PGS-1 was only marginally changed in both granulosa cells and residual ovarian tissue in the different treatment groups, compared to the control group. In contrast, PGS-2 was markedly increased by LH+IBMX, especially in the granulosa cells. The addition of CA, in combination with LH+IBMX, resulted in a small decrease of PGS-2, and progesterone further decreased its content. In the residual ovarian tissue, only minor changes of PGS-2 were detected. These results demonstrate that LH and progesterone selectively regulate the expression of PGS-2 in rat granulosa cells, whereas the hormonal regulation of PGS-1 is less pronounced. Progesterone inhibits PGS-2 in granulosa cells but has negligible effects on the total ovarian synthesis of prostaglandins during the ovulatory period.

Regulation of prostaglandin biosynthesis by luteinizing hormone and bradykinin in rat preovulatory follicles in vitro.

Luteinizing hormone (LH) stimulates prostaglandin biosynthesis and steroidogenesis in preovulatory (PO) follicles prior to ovulation. Since the ovulatory process shares many similarities with an inflammatory reaction, mediators of the inflammatory response, such as bradykinin (BK) have been suggested to modulate the effects of LH. In the present study the effect of BK (5 microM) on: 1) prostaglandin biosynthesis (PGE2, PGF2 alpha and 6-keto-PGF1 alpha), 2) the levels of two enzymes in the cyclo-oxygenase pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (PCS), and 3) cyclic adenosine 3'5'-monophosphate (cAMP) and progesterone response of PO follicles incubated in vitro were examined. LH (0.1 microgram/ml) stimulated the accumulation of cAMP and progesterone in the medium, while BK had no effect on these parameters. BK exerted a slight stimulatory effect on PGE2, and PGF2 alpha, (p less than or equal to 0.01) but not on 6-keto-PGF1 alpha synthesis, but no changes in PGS or PCS levels could be detected. The effect of LH on prostaglandin biosynthesis was much more pronounced, with an increase of PGE2, PGF2 alpha and 6-keto-PGF1 alpha. LH also induced PGS. The combination of LH and BK did not alter these responses compared to that of LH alone. This study demonstrates that BK stimulates prostaglandin biosynthesis in PO follicles. In contrast to LH, this effect of BK does not seem to involve the adenylate cyclase system, since BK did not stimulate cAMP production. BK did not affect the levels of PGS or PCS, and the stimulatory effect of BK is suggested to involve an increase in the availability of substrate for the cyclo-oxygenase pathway.

Regulation of prostaglandin endoperoxide synthase by cyclic adenosine 3',5'-monophosphate in the in vitro-perfused rat ovary.

The preovulatory regulation of two enzymes in the prostaglandin biosynthetic pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (ISN), was examined in granulosa cells and residual tissue of rat ovaries perfused in vitro. Ovaries from rats primed with pregnant mare's serum gonadotropin (20 IU) were perfused for up to 20 h starting the morning of induced proestrus. The amounts of PGS and ISN present were analyzed with immunoblotting techniques. Soluble extracts from granulosa cells and residual ovarian tissues were obtained at different times (0 h, 3 h, 7 h, 12 h) after treatment in vitro with luteinizing hormone (LH, 0.1 microgram/ml) and 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) and at 7 h in untreated control ovaries or after treatment with forskolin (30 microM) or LH (0.1 microgram/ml). The levels in the perfusion medium of cyclic adenosine 3',5'-monophosphate (cAMP), progesterone, testosterone, and estradiol were measured and the number of ovulations were examined. The levels of PGS after treatment with LH + IBMX increased up to 7 h and remained high at 12 h, a time that is close to the time of ovulation. The increase was more pronounced in the granulosa cells than in the residual tissue. Treatment with forskolin induced synthesis of PGS in granulosa cells, and the levels at 7 h were similar to those after stimulation with LH + IBMX. The levels of PGS were lower in granulosa cells of the group stimulated with LH alone than in granulosa cells from ovaries stimulated with LH + IBMX or forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of dihydrofolate reductase from trimethoprim-susceptible and trimethoprim-resistant Pseudomonas cepacia.

Trimethoprim resistance was investigated in cystic fibrosis isolates of Pseudomonas cepacia. Determination of the MIC of trimethoprim for 111 strains revealed at least two populations of resistant organisms, suggesting the presence of more than one mechanism of resistance. Investigation of the antibiotic target, dihydrofolate reductase, was undertaken in both a susceptible strain and a strain with high-level resistance (MIC, greater than 1,000 micrograms/ml). The enzyme was purified by using ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. Specific activities, molecular weights, isoelectric points, and substrate kinetics were similar for both enzymes. However, the dihydrofolate reductase from the trimethoprim-resistant strain demonstrated decreased susceptibility to inhibition by trimethoprim and increased susceptibility to inhibition by methotrexate, suggesting that these two enzymes are not identical. We conclude that the mechanism of trimethoprim resistance in this strain with high-level resistance is production of a trimethoprim-resistant dihydrofolate reductase.