Pharmacology of the biological response modifier bropirimine (PNU-54461) on experimental autoimmune encephalomyelitis (EAE) in mice.

In murine severe experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis (MS), we tested the efficacy of a 5-halo-6-phenyl pyrimidinone compound, bropirimine (PNU-54461). We observed that the compound is active in suppressing EAE when administered orally, a significant pharmacological advantage compared to some current therapies for the treatment of MS. Furthermore, bropirimine was most efficacious when dosing was begun 5-10 days after injection of myelin basic protein, the protein isolated from the central nervous system and used for inducing EAE in our model. This is a period of time following the initial immunological events leading to the disease, when large-scale leukocyte infiltration into the central nervous system begins. Following oral dosing, bropirimine peaked in the blood within 3 h and was cleared to undetectable concentrations within 16-18 h. Despite the pharmacokinetics in the blood, bropirimine was fully efficacious when dosed orally every two or three days. Surprisingly, bropirimine treatment did not result in a statistically significant decrease in leukocyte infiltration into the lower spinal cord, unless the compound was dosed daily at a high concentration. We also observed the concentration and time course of alpha-interferon in blood following oral dosing of bropirimine. The kinetics of interferon in the blood are similar to, but clearly distinguishable from, the pharmacokinetics of bropirimine in the blood. It is not clear whether or not the induction of interferon plays a key role in the efficacy of bropirimine. Nevertheless, the results using bropirimine in EAE suggest that the compound may be useful for the treatment of multiple sclerosis.

Pharmacokinetic properties, induction of interferon, and efficacy of selected 5-halo-6-phenyl pyrimidinones, bropirimine analogues, in a model of severe experimental autoimmune encephalomyelitis.

We showed previously that a 5-halo-6-phenyl-pyrimidinone, bropirimine (PNU-54461), inhibited progression of severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. In the work presented here, we examined the activity of a group of chemically-related bropirimine analogues. First, the pharmacokinetic properties of the bropirimine analogues were examined in normal mice following oral dosing. After equal oral doses, both PNU-56169 and PNU-63693 were found in the blood of normal mice at equal or higher concentrations than bropirimine, but PNU-54462 and PNU-56359 were present in blood only at very low concentrations. Next, we examined the bropirimine analogues for activity in our model of severe EAE. At a dose of 400 mg/kg administered orally every second day PNU-56169 nearly completely blocked EAE progression, but was ineffective at 100 mg/kg. PNU-63693 was effective in EAE at concentrations of 200 mg/kg, 100 mg/kg, 50 mg/kg, and as low as 25 mg/kg. Histopathology was examined by observing leukocyte infiltration into the lower spinal cords of the mice. Treatment with 400 mg/kg of PNU-56169 and doses of 25, 50, 100, and 200 mg/kg of PNU-63693 significantly inhibited leukocyte infiltration into the lower spinal cord of treated mice in a dose-dependent manner. Orally administered PNU-56169 and PNU-63693 also stimulated significant concentrations of IFNalpha in the serum of treated mice, which may be related to the efficacy of the compounds in EAE. However, the correlation between IFNalpha in the blood and efficacy in treating EAE was not exact. Thus, PNU-56169 and PNU-63693 were delivered to the blood following oral dosing, induced significant concentrations of IFNalpha in the blood, and were equally or more potent than PNU-54461 in inhibiting clinical signs of EAE. The results suggest that 5-halo-6-phenyl-pyrimidinones are an interesting class of compounds to investigate for development in the treatment of multiple sclerosis.

Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6.

Somites, segmented mesodermal units of the vertebrate embryo, are the precursors of adult skeletal muscle, bone and cartilage. During embryogenesis, somite progenitor cells ingress through the primitive streak, move laterally to a paraxial position (alongside the body axis) and segment into epithelial somites. Little is known about how this paraxial mesoderm tissue is specified. We have previously described a mouse T-box gene, Tbx6, which codes for a putative DNA-binding protein. The embryonic pattern of expression of Tbx6 in somite precursor cells suggests that this gene may be involved in the specification of paraxial mesoderm. We now report the creation of a mutation in Tbx6 that profoundly affects the differentiation of paraxial mesoderm. Irregular somites form in the neck region of mutant embryos, whereas more posterior paraxial tissue does not form somites but instead differentiates along a neural pathway, forming neural-tube-like structures that flank the axial neural tube. These paraxial tubes show dorsal/ventral patterning that is characteristic of the neural tube, and have differentiated motor neurons. These results indicate that Tbx6 is needed for cells to choose between a mesodermal and a neuronal differentiation pathway during gastrulation; Tbx6 is essential for the specification of posterior paraxial mesoderm, and in its absence cells destined to form posterior somites differentiate along a neuronal pathway.

Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos.

Mutations of the human BRCA1 and BRCA2 genes encoding tumor suppressors have been implicated in inherited predisposition to breast and other cancers. Disruption of the homologous mouse genes Brca1 and Brca2 by targeting showed that they both have indispensable roles during embryogenesis, because nullizygous embryos become developmentally retarded and disorganized, and die early in development. In Brca1 mutants, the onset of abnormalities is earlier by one day and their phenotypic features and time of death are highly variable, whereas the phenotype of Brca2 null embryos is more uniform, and they all survive for at least 8.5 embryonic days. Observations with Brca1/Brca2 double nullizygotes raise the possibility that the two developmental pathways could be linked. Interestingly, the impact of the Brca1 or Brca2 null mutation is less severe in a p53 null background.

Tbx6, a mouse T-Box gene implicated in paraxial mesoderm formation at gastrulation.

The T-box genes constitute an evolutionarily conserved family of putative transcription factors which are expressed in discrete domains during embryogenesis, suggesting that they may play roles in inductive interactions. Members have been identified by virtue of their homology to the prototypical T-box gene, T or Brachyury, which is required for mesoderm formation and axial elongation during embryogenesis. We have previously reported the discovery of six new mouse T-box genes, Tbx1-Tbx6, and described the expression patterns of Tbx1-Tbx5 (Bollag et al., 1994; Agulnik et al., 1996; Chapman et al., 1996; Gibson-Brown et al., 1996). We have obtained cDNA clones encoding the full-length Tbx6 protein from screens of gastrulation-stage mouse cDNA libraries and determined the spatial and temporal distribution of Tbx6 transcripts during embryogenesis. The gene codes for a 1.9-kb transcript with an open reading frame coding for a 540-amino acid protein, with a predicted molecular weight of 59 kDa. Tbx6 maps to chromosome 7 and does not appear to be linked to any known mutation. Unlike other members of the mouse T-box gene family which are expressed in a wide variety of tissues derived from all germ layers, Tbx6 expression is quite restricted. Tbx6 transcripts are first detected in the gastrulation stage embryo in the primitive streak and newly recruited paraxial mesoderm. Later in development, Tbx6 expression is restricted to presomitic, paraxial mesoderm and to the tail bud, which replaces the streak as the source of mesoderm. Expression in the tail bud persists until 12. 5 days postcoitus. Tbx6 expression thus overlaps that of Brachyury in the primitive streak and tail bud, although Brachyury is expressed earlier in the primitive streak. Brachyury is also expressed in a second domain, the node and notochord, that is not shared with Tbx6. The onset of Tbx6 expression is not affected in homozygous null Brachyury mutant embryos at 7.5 days postcoitus. However, Tbx6 expression is extinguished in mutant embryos as soon as the Brachyury phenotype becomes evident at 8.5 days postcoitus, indicating that the continued expression of Tbx6 is directly or indirectly dependent upon Brachyury expression.

Eliminating sleep-associated hypoxemia improves growth in infants with bronchopulmonary dysplasia.

OBJECTIVE: Infants with bronchopulmonary dysplasia (BPD) have been previously reported to have a decrease in growth velocity after stopping supplemental oxygen (SO). SO was stopped after a short-term recording (20-30 minutes) of pulse oxygen saturation (Sao2) of 92% or greater in room air. Other studies have documented that Sao2 decreases further during feedings and sleep in infants with BPD. Two questions were asked: (1) whether short-term, awake Sao2 studies would reliably predict prolonged sleep Sao2; and (2) how Sao2 sustained at 88% to 91% vs 92% or greater in room air would impact growth velocity in infants with BPD. METHODOLOGY: Short-term Sao2 studies were prospectively compared with prolonged sleep Sao2 (n = 63) and the growth velocity of infants who had SO discontinued after a prolonged sleep Sao2 recording of 88% to 91% (group 1; n = 14) versus 92% or greater (group 2; n = 34) in room air. RESULTS: Failure to maintain Sao2 at predetermined levels occurred in 18 (29%) of 63 infants during their first prolonged sleep study. There was no correlation between short-term awake Sao2 and prolonged sleep Sao2 recordings (r = .02). Body weight, height, weight for height, and rate of weight gain were similar for all study infants before SO was stopped and remained constant for group 2 infants after SO was stopped. However, group 1 infants had a significant decrease in the rate of weight gain (17.3 +/- 13.1 vs 3.7 +/- 6.1 g/kg per day), and the mean z scores for weight gain and weight for height also decreased significantly for group 1 infants. Energy intake, incidence of acute infection, hematocrit values, and medication use did not differ before or after stopping SO in either group. CONCLUSIONS: This study indicated that short-term, awake Sao2 measurements do not predict prolonged sleep Sao2, and overall, infants with BPD continued a positive growth trend when Sao2, remained greater than 92% during prolonged sleep.

Evolution of mouse T-box genes by tandem duplication and cluster dispersion.

The T-box genes comprise an ancient family of putative transcription factors conserved across species as divergent as Mus musculus and Caenorhabditis elegans. All T-box gene products are characterized by a novel 174-186-amino acid DNA binding domain called the T-box that was first discovered in the polypeptide products of the mouse T locus and the Drosophila melanogaster optomotor-blind gene. Earlier studies allowed the identification of five mouse T-box genes, T, Tbx1-3, and Tbr1, that all map to different chromosomal locations and are expressed in unique temporal and spatial patterns during embryogenesis. Here, we report the discovery of three new members of the mouse T-box gene family, named Tbx4, Tbx5, and Tbx6. Two of these newly discovered genes, Tbx4 and Tbx5, were found to be tightly linked to previously identified T-box genes. Combined results from phylogenetic, linkage, and physical mapping studies provide a picture for the evolution of a T-box subfamily by unequal crossing over to form a two-gene cluster that was duplicated and dispersed to two chromosomal locations. This analysis suggests that Tbx4 and Tbx5 are cognate genes that diverged apart from a common ancestral gene during early vertebrate evolution.

Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development.

A novel family of genes, characterized by the presence of a region of homology to the DNA-binding domain of the Brachyury (T) locus product, has recently been identified. The region of homology has been named the T-box, and the new mouse genes that contain the T-box domain have been named T-box 1-6 (Tbx1 through Tbx6). As the basis for further study of the function and evolution of these genes, we have examined the expression of 5 of these genes, Tbx1-Tbx5, across a wide range of embryonic stages from blastocyst through gastrulation and early organogenesis by in situ hybridization of wholemounts and tissue sections. Tbx3 is expressed earliest, in the inner cell mass of the blastocyst. Four of the genes are expressed in different components of the mesoderm or mesoderm/endoderm during gastrulation (Tbx1 and Tbx3-5). All of these genes have highly specific patterns of expression during later embryogenesis, notably in areas undergoing inductive tissue interactions. In several cases there is complementary expression of different genes in 2 interacting tissues, as in the lung epithelium (Tbx1) and lung mesenchyme (Tbx2-5), and in mammary buds (Tbx3) and mammary stroma (Tbx2). Tbx1 shows very little overlap in the sites of expression with the other 4 genes, in contrast to a striking similarity in expression between members of the 2 cognate gene sets, Tbx2/Tbx3 and Tbx4/Tbx5. This is a clear reflection of the evolutionary relationship between the 5 genes since the divergence of Tbx1 occurred long before the relatively recent divergence of Tbx2 and 3 and Tbx4 and 5 from common ancestral genes. These studies are a good indication that the T-box family of genes has important roles in inductive interactions in many stages of mammalian embryogenesis.

Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity.

Tetrapod fore-and hindlimbs have evolved from the pectoral and pelvic fins of an ancient vertebrate ancestor. In this ancestor, the pectoral fin appears to have arisen following the rostral homeotic recapitulation of an existing pelvic appendage (Tabin and Laufer (1993), Nature 361, 692-693). Thus the basic appendage outgrowth program is reiterated in both tetrapod fore- and hindlimbs and the pectoral and pelvic fins of extant teleost fishes (Sordino et al. (1995) Nature 375, 678-681). Recently a novel family of putative transcription factors, which includes the T (Brachyury) locus, has been identified and dubbed the "T-box' family. In mice, all of these genes have expression patterns indicative of involvement in embryonic induction (Chapman et al. (1996) Dev. Dyn., in press), and four (Tbx2-Tbx5) are represented as two cognate, linked gene pairs (Agulnik et al., (1996), Genetics, in press). We now report that, whereas Tbx2 and Tbx3 are expressed in similar spatiotemporal patterns in both limbs, Tbx5 and Tbx4 expression is primarily restricted to the developing fore- and hindlimb buds, respectively. These observations suggest that T-box genes have played a role in the evolution of fin and limb morphogenesis, and that Tbx5 and Tbx4 may have been divergently selected to play a role in the differential specification of fore- (pectoral) versus hind- (pelvic) limb (fin) identity.

Programmed cell death in the absence of c-Fos and c-Jun.

Programmed cell death, or apoptosis, is a normal process in the development of a variety of embryonic and adult tissues, and is also observed in several pathological conditions. Several recent studies, using both expression and functional assays, have implicated the transcription factor, AP-1, in the regulation of programmed cell death, and specifically implicate the genes c-fos and c-jun, as well as some other family members. If the products of the c-fos and/or c-jun genes are essential components in the cascade of events that leads to programmed cell death in mammalian cells, it follows that cell death would not occur in mice lacking functional copies of these genes. We have made use of null mutations in the c-fos and c-jun genes that were produced by gene targeting (Johnson, R.S., Spiegelman, B.M. and Papaioannou, V.E. (1992). Cell 71, 577-586; Johnson, R.S., Van Lingen, B., Papaioannou, V.E. and Spiegelman, B.M. (1993). Genes Dev. 7, 1309-1317) to investigate this possibility. Cell death was assayed using an in situ apoptosis assay in c-fos null embryos and adults, c-jun null embryos, and c-fos/c-jun double null embryos compared with control mice. The occurrence of cell death in c-fos null mice was also assessed in two experimental conditions that normally lead to neuronal cell death. The first was unilateral section of the sciatic nerve in neonates, which leads to the death of anterior horn cells of the spinal cord on the operated side. The second was a genetic cross combining the weaver mutation, which causes death of cerebellar granule cells, with the c-fos mutation. Our results show that programmed cell death occurs normally in developing embryonic tissues and adult thymus and ovary, regardless of the absence of a functional c-fos gene. Furthermore, absence of c-fos had no effect on neuronal cell death in the spinal cord following sciatic nerve section, or in heterozygous weavers' cerebellae. Finally, the results show that programmed cell death can take place in embryos lacking both Fos and Jun.

Effect on protein phosphatase activity of peptide backbone modification and truncation of the autoinhibitory domain peptide of calcineurin.

Solid-phase synthesis of the autoinhibitory domain of calcineurin, CaN A467-491, also produced [aspartimide477]CaN A467-491 and [iso-Asp477]CaN467-491 when Boc-based chemistry was employed. In addition, the truncated peptide CaN A467-488 was obtained when Fmoc-based chemistry was employed. All four peptides proved to be effective inhibitors of protein phosphatase activity of calcineurin. The full-length peptide and the C-terminally truncated peptide (CaN467-488) were indistinguishable, with Ki values of 28 +/- 3 and 31 +/- 5 mu M, respectively. The internally modified peptides, [iso-Asp477]CaN A467-491 and [aspartimide477]-CaN A467-491, possessed lower inhibitory potencies (Ki values of 87 +/- 10 and 55 +/- 3 mu M, respectively).

Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue.

The expansion of CAG triplet repeats in the translated region of the human HD gene, encoding a protein (huntingtin) of unknown function, is a dominant mutation leading to manifestation of Huntington's disease. Targeted disruption of the homologous mouse gene (Hdh), to examine the normal role of huntingtin, shows that this protein is functionally indispensable, since nullizygous embryos become developmentally retarded and disorganized, and die between days 8.5 and 10.5 of gestation. Based on the observation that the level of the regionalized apoptotic cell death in the embryonic ectoderm, a layer expressing the Hdh gene, is much higher than normal in the null mutants, we propose that huntingtin is involved in processes counterbalancing the operation of an apoptotic pathway.

Regulation of M-phase promoting factor activity during development of mouse male germ cells.

While the role of M-phase promoting factor (MPF) in controlling meiosis in oocytes has been well documented, very little is known about its function in male germ cells. Previous studies have localized transcripts for cyclin B1, the regulatory subunit of MPF, in male germ cells, with highest levels in postmeiotic, early round spermatids and much reduced levels in the meiotically dividing pachytene spermatocytes. The present study describes the localization of the regulatory and the catalytic subunits of MPF, CycB1 and Cdc2, respectively, to specific cell types within the testis. Immunoblotting revealed that both CycB1 and Cdc2 were present at highest levels in pachytene spermatocytes, with lower levels observed in the postmeiotic compartment. To assay for MPF activity, kinase complexes were isolated from lysates of testicular cells using p13suc1 agarose and antibodies directed against Cdc2 and CycB1. Activity of these kinase complexes was analyzed using histone H1 as an exogenous substrate. Cdc2 and CycB1-associated kinase activities were localized to the meiotically dividing pachytene spermatocytes, but not to postmeiotic spermatids.

Expression of proliferating cell nuclear antigen in the mouse germ line and surrounding somatic cells suggests both proliferation-dependent and -independent modes of function.

The distribution of proliferating cell nuclear antigen (PCNA) in specific somatic and germ cells of the adult mouse ovary and testis was assessed using immunocytochemical staining and immunoblot analysis and was correlated with cellular proliferation and differentiation. In the adult ovary, immunocytochemical staining for PCNA within follicular cells varied depending on the stage of follicular growth. Since PCNA staining has proven to be a useful indicator of cells involved in DNA synthesis and repair, the pattern of PCNA staining in the ovary was compared to previous studies which used tritiated thymidine labeling as a marker for DNA synthesis. In the testis, PCNA was detected in the mitotically proliferating spermatogonia, but not in spermatocytes which had just entered meiosis. PCNA staining was again observed in spermatogenic cells in later stages of meiotic prophase, in particular zygotene and pachytene spermatocytes. As these cells are undergoing meiotic recombination, the presence of PCNA in these meiotic prophase cells could reflect a second function of PCNA, that of DNA excision repair.

Changes in lung lipid during spontaneous labor in fetal sheep.

The goals of this study were 1) to examine changes in lung liquid formation and composition during spontaneous labor in fetal lambs and 2) to determine the importance of beta-adrenergic stimulation and transepithelial Na+ flux in removing liquid from the lung lumen near birth. We measured net production of lung liquid (Jv), lung liquid composition, and transpulmonary electrical potential difference (PD) before and during labor in fetal sheep with chronically implanted tracheal and vascular catheters. We determined Jv by measuring rate of change in lung liquid concentration of 125I-albumin, an impermeant tracer that was mixed in lung liquid at the start of each study. In 17 paired experiments, Jv decreased from 11 +/- 2 ml/h (Jv > 0 = secretion) before labor to -1 +/- 2 ml/h (Jv < 0 = absorption) during labor; in 5 paired experiments, PD changed from -7 +/- 1 mV (lumen negative) before labor to -12 +/- 1 mV during labor. To determine whether absorption of lung liquid during labor is the result of beta-adrenergic stimulation, we studied the effect of propranolol on Jv during labor. When propranolol (40 microM) was added to lung liquid during active labor, Jv decreased from -2 +/- 2 to -8 +/- 3 ml/h (n = 9). Thus, propranolol did not inhibit lung liquid absorption during labor. To determine whether transepithelial Na+ movement provides the driving force for lung liquid clearance during labor, we tested the effects of amiloride, an Na+ transport inhibitor, on Jv and PD.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of an interleukin-1 receptor antagonist protein on type II collagen-induced arthritis and antigen-induced arthritis in mice.

OBJECTIVE: To investigate the anti-arthritic effect of recombinant human interleukin-1 receptor antagonist protein (IRAP) in two experimental models of arthritis. METHODS: Recombinant IRAP was administered daily to mice with type II collagen-induced arthritis (CIA) or with antigen-induced arthritis (AIA) provoked by methylated bovine serum albumin (mBSA). Disease incidence and severity were assessed by a clinical index and histologic features. Serum antibody to type II collagen, spleen cell proliferation to mBSA, and anti-IRAP antibodies were measured as indices of immune function. RESULTS: IRAP reduced the incidence and delayed the onset of CIA and suppressed the antibody response to type II collagen. In contrast, IRAP did not affect the pathogenesis of AIA and had no effect on either humoral or cellular immune responses to mBSA in arthritic mice. CONCLUSION: These observations suggest that interleukin-1 may play a prominent role in the development of some, but not all, forms of arthritis.

Regulation of expression of IL-1 receptor antagonist protein in human synovial and dermal fibroblasts.

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA > LPS > IL-1 alpha > TNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.

Isolation of the murine cyclin B2 cDNA and characterization of the lineage and temporal specificity of expression of the B1 and B2 cyclins during oogenesis, spermatogenesis and early embryogenesis.

A cDNA encoding the murine cyclin B2 (cycB2) was isolated from an adult mouse testis cDNA library as part of studies designed to identify cyclins involved in murine germ cell development. This cycB2 cDNA was then used to examine the pattern of cycB2 expression during male and female germ cell development and in early embryogenesis, and to compare this expression with the previously characterized expression of cycB1. A single 1.7 kb cycB2 transcript was detected by northern blot hybridization analysis of total RNA isolated from midgestation embryos and various adult tissues. Northern blot and in situ hybridization analyses revealed that cycB2 expression in the testis was most abundant in the germ cells, specifically in pachytene spermatocytes. This is in contrast to the highest levels of expression of cycB1 being present in early spermatids. In situ analysis of the ovary revealed cycB2 transcripts in both germ cells and somatic cells, specifically in the oocytes and granulosa cells of growing and mature follicles. The pattern of cycB1 and cycB2 expression in ovulated and fertilized eggs was also examined. While the steady state level of cycB1 and cycB2 signal remained constant in oocytes and ovulated eggs, signal of both appeared to decrease following fertilization. In addition, both cycB1 and cycB2 transcripts were detected in the cells of the inner cell mass and the trophectoderm of the blastocyst. These results demonstrate that there are lineage- and developmental-specific differences in the pattern of the B cyclins in mammalian germ cells, in contrast to the co-expression of B cyclins in the early conceptus.

Identification of a mouse B-type cyclin which exhibits developmentally regulated expression in the germ line.

To begin to examine the function of cyclins in mammalian germ cells, we have screened an adult mouse testis cDNA library for the presence of B-type cyclins. We have isolated cDNAs that encode a murine B-type cyclin, which has been designated cycB1. cycB1 was shown to be expressed in several adult tissues and in the midgestation mouse embryo. In the adult tissues, the highest levels of cycB1 transcripts were seen in the testis and ovary, which contain germ cells at various stages of differentiation. The major transcripts corresponding to cycB1 are 1.7 and 2.5 kb, with the 1.7 kb species being the predominant testicular transcript and the 2.5 kb species more abundant in the ovary. Examination of cDNAs corresponding to the 2.5 kb and 1.7 kb mRNAs revealed that these transcripts encode identical proteins, differing only in the polyadenylation signal used and therefore in the length of their 3' untranslated regions. Northern blot and in situ hybridization analyses revealed that the predominant sites of cycB1 expression in the testis and ovary were in the germinal compartment, particularly in early round spermatids in the testis and growing oocytes in the ovary. Thus cycB1 is expressed in both meiotic and postmeiotic cells. This pattern of cycB1 expression further suggests that cycB1 may have different functions in the two cell types, only one of which correlates with progression of the cell cycle.

IL-1 receptor antagonist protein production and gene expression in rheumatoid arthritis and osteoarthritis synovium.

IL-1 can participate in the perpetuation of arthritis through direct stimulation of synoviocytes and augmentation of matrix degradation. Hence, local production of the IL-1R antagonist protein (IRAP) might be an important negative feedback signal that regulates synovitis. We assessed synovial IRAP production in synovia from 30 individuals, by using a specific mAb and the immunoperoxidase staining method. IRAP was detected in 11 of 12 rheumatoid arthritis (RA) synovial tissues (ST) and was located primarily in the sublining, particularly in perivascular regions enriched for macrophages. Some staining was observed in the intimal lining of the synovium, although this was significantly less than in the sublining (p less than 0.05). Nine of 12 osteoarthritis (OA) tissues were positive for IRAP. In contrast to RA, the staining was observed primarily in the synovial lining in OA, with only minimal sublining IRAP being detected. Synovia from four patients without arthritis were negative (three autopsy specimens and one post-traumatic sample). Of the other two patients with miscellaneous diagnoses, one sample was negative (tenosynovitis) and one was positive (seronegative inflammatory arthritis) (sublining). Studies of serial sections and double-immunostaining experiments indicated that macrophages are the major cells containing immunoreactive IRAP. IRAP gene expression in vivo was determined by performing in situ hybridization on ST from 17 arthritis patients. RNA sense IRAP probes did not hybridize to any tissues. Anti-sense IRAP probes bound to two of nine RA tissues, two of six OA tissues, one of one seronegative inflammatory arthropathy tissue, and none of one flexor tenosynovitis tissue. As with immunoreactive protein, IRAP mRNA was primarily localized to cells in the synovial lining in OA but was more prominent in perivascular lymphoid aggregates in RA and seronegative inflammatory arthropathy. Northern blot analysis was performed on RNA isolated from nine ST. The appropriately sized IRAP band was identified in six of nine samples (five of six RA and one of three OA). Supernatants from cultured RA and OA ST cells contained immunoreactive and biologically active IRAP. Hence, IRAP gene expression and protein production occur in RA and OA synovium, albeit in different distributions.