Birth Control in Clinical Trials: Industry Survey of Current Use Practices, Governance, and Monitoring.

The Health and Environmental Sciences Institute (HESI) Developmental and Reproductive Toxicology Technical Committee sponsored a pharmaceutical industry survey on current industry practices for contraception use during clinical trials. The objectives of the survey were to improve our understanding of the current industry practices for contraception requirements in clinical trials, the governance processes set up to promote consistency and/or compliance with contraception requirements, and the effectiveness of current contraception practices in preventing pregnancies during clinical trials. Opportunities for improvements in current practices were also considered. The survey results from 12 pharmaceutical companies identified significant variability among companies with regard to contraception practices and governance during clinical trials. This variability was due primarily to differences in definitions, areas of scientific uncertainty or misunderstanding, and differences in company approaches to enrollment in clinical trials. The survey also revealed that few companies collected data in a manner that would allow a retrospective understanding of the reasons for failure of birth control during clinical trials. In this article, suggestions are made for topics where regulatory guidance or scientific publications could facilitate best practice. These include provisions for a pragmatic definition of women of childbearing potential, guidance on how animal data can influence the requirements for male and female birth control, evidence-based guidance on birth control and pregnancy testing regimes suitable for low- and high-risk situations, plus practical methods to ascertain the risk of drug-drug interactions with hormonal contraceptives.

Neural changes in the auditory cortex of awake guinea pigs after two tinnitus inducers: salicylate and acoustic trauma.

Tinnitus, also called phantom auditory perception, is a major health problem in western countries. As such, a significant amount of effort has been devoted to understanding its mechanisms, including studies in animals wherein a supposed "tinnitus state" can be induced. Here, we studied on the same awake animals the effects of a high-dose of salicylate and of an acoustic trauma both at levels known to induce tinnitus. Recordings of cortical activity (local field potentials) from chronically implanted electrodes in the same animals under each condition allowed direct comparison of the effects of salicylate and trauma (noise trauma was carried out several days after full recovery from salicylate administration). Salicylate induced a systematic and reversible increase in amplitude of cortical responses evoked by tone bursts over a wide range of frequencies and intensities. The effects of noise trauma, though much more variable than those of salicylate, resulted in both increases and decreases in the amplitude of cortical responses. These alterations of cortical response amplitudes likely reflect associated hypoacusis and hyperacusis. The effects of salicylate administration and noise trauma on spontaneous activity were also studied. Fourier analysis did not reveal any increase in power within any given frequency band; rather, both treatments induced a decrease of power spectrum over a relatively broad frequency band (approximately 10-30 Hz). Entropy rate of spontaneous activity, a measure of complexity (temporal correlations), was found to decrease after salicylate but not after acoustic trauma. The present data on evoked potentials confirm salicylate effects at the cortical level and partially extend such effects to acoustic trauma. While the present study showed that both salicylate and noise trauma induced some changes of spontaneous activity in auditory cortex, none of these changes are interpretable in terms of potential neural correlate of tinnitus.

Effects of hearing aid fitting on the perceptual characteristics of tinnitus.

Restoration of auditory input through the use of hearing aids has been proposed as a potentially important means of altering tinnitus among those tinnitus sufferers who experience significant sensorineural hearing loss. In animal models of neural plasticity induced by noise trauma, high-frequency stimulation in deafferented regions of the auditory spectrum has been shown to modulate cortical reorganization after hearing loss, a result which suggests that the neural basis of tinnitus is subject to interference by acoustic stimulation. This study drew on deafferentation models to investigate the effect of hearing aids on the psychoacoustic properties of the tinnitus sensation, using both conventional amplification and high-bandwidth amplification regimes. The tinnitus percept was affected only weakly in the conventional amplification group, and was not at all affected in the high-bandwidth group. The changes observed under conventional, low-to-medium frequency amplification may indicate that the perceptual characteristics of tinnitus depend on the pattern of sensory inputs - notably a contrast in activity between adjacent central auditory regions of more and less afferent activity - while the absence of modifications in the high-bandwidth amplification group suggests limit on the tractability of the tinnitus percept. This limit to the malleability of the tinnitus percept may arise from either the extent of hearing deficits or the duration and robustness of the neuroplastic changes that originally give rise to tinnitus.

Glucuronidation of nonylphenol and octylphenol eliminates their ability to activate transcription via the estrogen receptor.

Both p-nonylphenol (NP) and p-octylphenol (OP) exhibit weak estrogen-like activity in in vitro and in some rodent assays. To help understand the biochemical and molecular basis for these effects, and thus to permit extrapolation of risk to human health, it is important to establish whether these activities are retained by their metabolites. These data are particularly important in light of the knowledge that both NP and OP are rapidly and extensively metabolized to their glucuronide conjugates in rats. The activity of these glucuronide metabolites, however, is unknown. These studies investigated the intrinsic ability of NP, OP, and their principal mammalian metabolites, nonylphenol glucuronide (NPG) and octylphenol glucuronide (OPG), to affect estrogen receptor (ER)- or androgen receptor (AR)-mediated transcription in a yeast transcriptional activation system. Specifically, the estrogen-, anti-estrogen-, androgen-, and anti-androgen-like activities of NP, OP, NPG, and OPG have been assessed using recombinant yeast strains that express either human ER or AR. The two parent compounds, NP (EC(50) 110 nM) and OP (EC(50) 700 nM), exhibited intrinsic estrogen-like activity in this system, and consistent with numerous studies with these chemicals, they were 3-4 orders of magnitude less potent than 17beta-estradiol (EC(50) 500 pM). However, in contrast to the parent molecules, neither NPG nor OPG exhibited any evidence of estrogen-, antiestrogen-, androgen-, or anti-androgen-like activity in these recombinant yeast strains. Therefore, the weak estrogen-like activity noted for NP and OP in vivo at high doses is likely to reflect saturation of parent molecule glucuronidation. At anticipated levels of human exposure to NP and OP such a saturation of detoxification is highly unlikely; therefore, these in vitro data support the conclusion that the potential endocrine hazard posed by NP and OP to humans is likely to be negligible.

The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin.

An overview is provided of the cancer chemoprevention actions of phenolic antioxidants and 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin). These agents principally appear to exert their beneficial effects through induction of phase II drug-metabolizing enzymes such as glutathione S-transferase (GST). The requirement for oxidative metabolism of the synthetic antioxidants to carbonyl-containing compounds, including quinones, in order that they can induce gene expression is discussed. Previous work has shown that the basic leucine zipper transcription factor Nrf2 is involved in induction of GST by the phenolic antioxidant butylated hydroxyanisole (BHA). Evidence is provided from a mouse possessing a targeted disruption of the Nrf2 gene that, in murine liver, the transcription factor regulates basal expression of several class Alpha and class Mu GST subunits, but not class Pi GST. In the Nrf2 knock-out mouse, hepatic induction of class Alpha and class Mu GST by BHA and the synthetic antioxidant ethoxyquin is similarly impaired, suggesting that these agents affect gene activation by a related mechanism. Significantly, residual induction of GST by antioxidants is apparent in the Nrf2 mutant mouse, indicating the existence of an alternative mechanism of gene activation.

Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver.

Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.

Pi-class glutathione S-transferase: regulation and function.

Our laboratory has been involved in the study of Glutathione S-transferase pi (GST pi) for many years, both in terms of regulation of gene expression and in trying to understand the endogenous function(s) of this enzyme and also what role it may play in the carcinogenic process [1]. Over-expression of GST pi has been associated with carcinogenesis and the development of many different human tumours, for example testis [2], ovarian [3] and colorectal [4] and is often inversely correlated with prognosis or patient survival [5,6]. In addition, GST Pi has been implicated in the acquisition of antineoplastic drug resistance [7-9]. In order to study the transcriptional regulation of this gene, we have utilised a multi-drug resistant derivative (VCREMS) of the human mammary carcinoma cell line, MCF7, in which GST P1 mRNA and protein are significantly elevated in the absence of gene amplification [10-13]. Interestingly, we have recently reported the discovery of polymorphisms at the GSTP1 locus, resulting in two alleles GSTP1a and GSTP1b. In the study, the GSTP1b allele was found with increased frequency in bladder and testicular cancer, while the GSTP1a allele was significantly decreased in cases of prostate cancer [14]. In an attempt to elucidate the endogenous role(s) of GST pi, we have used homologous recombination in embryonic stem (ES) cells to inactivate both murine GST Pi genes and create a mouse strain completely deficient in the expression of this enzyme. This provides us with a unique animal model with which to study the effects of the absence of GST pi expression on the metabolism and pharmacokinetics of xenobiotics.

Transcriptional and post-transcriptional mechanisms can regulate cell-specific expression of the human Pi-class glutathione S-transferase gene.

Previous studies from this laboratory have identified transcriptional mechanisms that are utilized to increase expression of the human glutathione S-transferase gene GSTP1 in a multidrug-resistant derivative (VCREMS) of the human mammary carcinoma cell line MCF7 [Moffat, McLaren and Wolf (1994) J. Biol. Chem. 269, 16397-16402]. The data presented here provide strong evidence that post-transcriptional mechanisms can also play an important role in determining cell-specific expression of the GSTP1 gene. GSTP1 mRNA levels were shown to be elevated 3.1-fold in the human bladder carcinoma cell line EJ compared with VCREMS cells. Despite this observation, transient transfection assays revealed a decreased rate of GSTP1 promoter activity in EJ cells. Indeed, GSTP1 transcriptional repressor activity, mediated by a region located between nucleotides -105 and -86 (as we have previously described in MCF7 cells), was observed in EJ cells. However, in contrast with our results in MCF7 cells, the EJ repressor activity did not displace the essential nuclear complex bound to the C1 promoter element (-73 to -54) in vitro. In addition, competition experiments indicated that an AP-1-like protein is an integral component of the C1-bound complex in EJ cells. Interestingly, experiments utilizing actinomycin D to inhibit transcription demonstrated significantly greater stability of GSTP1 mRNA in EJ cells than in VCREMS cells. These findings suggest that cell-specific differences in the rates of GSTP1 mRNA decay provide the predominant mechanism responsible for elevated expression of the GSTP1 gene in EJ cells.

Functional characterization of the transcription silencer element located within the human Pi class glutathione S-transferase promoter.

We have previously demonstrated enhanced transcriptional activity of the human Pi class glutathione S-transferase (GSTP1) promoter in a multidrug-resistant derivative (VCREMS) of the human mammary carcinoma cell line, MCF7 (Moffat, G. J., McLaren, A. W., and Wolf, C. R. (1994) J. Biol. Chem. 269, 16397-16402). Furthermore, we have identified an essential sequence (C1; -70 to -59) within the GSTP1 promoter that bound a Jun-Fos heterodimer in VCREMS but not in MCF7 cells. These present studies have examined the negative regulatory element (-105 to -86), which acted to suppress GSTP1 transcription in MCF7 cells. Mutational analysis of this silencer element further defined the repressor binding site to be located between nucleotides -97 and -90. In vitro DNA binding assays suggested that the repressor exerted its action by causing displacement of the essential non-AP-1-like MCF7 C1 complex. However, the addition of MCF7 nuclear extract did not disrupt binding of the VCREMS Jun-Fos C1 complex to the GSTP1 promoter. Furthermore, upstream insertion of the GSTP1 silencer element failed to inhibit activity of a heterologous promoter in MCF7 cells. These results highlighted the cell and promoter specificity of the GSTP1 transcriptional repressor and implicated a functional requirement for contact between the repressor and C1 complex. In this regard, the introduction of half-helical turns between the silencer and the C1 element abrogated repressor activity, thus leading to the hypothesis that a direct interaction between the repressor and C1 complex was required to suppress GSTP1 transcription. Moreover, these findings suggest that cell-specific differences in the composition of the C1 nuclear complex may dictate repressor activity.

Complete structure of the murine p36 (annexin II) gene. Identification of mRNAs for both the murine and the human gene with alternatively spliced 5' noncoding exons.

p36 (also termed annexin II) is a 39 kDa Ca2+/phospholipid-binding, membrane-associated protein that is a protein-tyrosine kinase substrate. We report here studies of the noncoding exons of p36, which combined with our earlier studies of the coding exons, allow us to conclude that the murine p36 gene is 34 kb in length with 14 exons. Comparison of the genes coding for mouse and human p36 (annexin II) and mouse, rat and human p35 (annexin I) and pigeon cp35 (an annexin I-related protein) shows strong genomic structural conservation supporting the hypothesis that these genes had a common ancestor. Both human and murine p36 mRNAs were found to be alternatively spliced in their 5' noncoding region. In both cases exon 2 is a cassette exon, which is present in a small fraction of p36 mRNAs. In type 1 mouse p36 mRNA the first noncoding 44 base exon 1 is joined to exon 3, the first of the 12 coding exons. In type 2 mRNA a 70 base noncoding exon (exon 2) is inserted between exon 1 and exon 3. Type 1 mRNA was present in all cell types studied as revealed by Northern analysis and primer extension, whereas type 2 mRNA could only be detected by RACE or PCR, indicating that it is of very low abundance. The major transcription start site of the mouse p36 gene was mapped by primer extension to be 61 bp upstream of the AUG initiation codon, which corresponds to type 1 mRNA, The murine p36 gene enhancer/promoter region contains a putative TATA box and several other potential regulatory sequences. The two alternatively-spliced human p36 mRNAs differ by the presence or absence of a noncoding 81 base exon (exon 2) inserted after exon 1, with exon 2-containing mRNAs representing approximately 10% of total p36 mRNA. The 300 bp spanning the promoter and exons 1-3 of the human and murine p36 genes show strong sequence homology immediately before and after the major transcription start site except in the region corresponding to exon 2, where homology is more limited.

Sp1-mediated transcriptional activation of the human Pi class glutathione S-transferase promoter.

Previous studies in this laboratory have identified an essential AP-1 recognition sequence (C1 region; -69 to -63) in th human Pi class glutathione s-transferase (GSTP1) promoter and a negatively acting regulatory element (-105 to -86) that acts to suppress GSTP1 transcription in the human mammary carcinoma cell line, MCF7 (1). The data presented here further delineate the functional characteristics of the GSTP1 promoter by examining the significance of two potential binding sites for the transcription factor, Sp1 (-57 to -49 and -47 to -39). The introduction of mutations within these Sp1-like elements and the use of Sp1 antisera in electrophoretic mobility shift assays demonstrated that Sp1 was bound to this region of the GSTP1 promoter in three different cell lines, MCF7, VCREMS, and EJ. Moreover, these in vitro studies indicated that only one of the two putative Sp1 response elements was utilized. Transient transfection assays using GSTP1 promoter constructs that incorporated mutations of the Sp1 elements clearly demonstrated that binding of Sp1 to the GSTP1 promoter was absolutely required for optimal levels of GSTP1 transcription. In particular, disruption of the distal Sp1 recognition motif (-57 to -49) markedly reduced GSTP1 promoter activity in each cell line, thus indicating preferential binding of Sp1 to the distal site. However, insertion of the repressor binding site (-105 to -86) into these constructs suggested that Sp1 was not involved in mediating the suppressive effects of the GSTP1 transcriptional repressor in MCF7 cells, because inhibition of Sp1 binding did not alleviate repressor activity. Therefore, these studies provide strong evidence that Sp1 plays a central role in regulating basal levels of GSTP1 transcription.

Involvement of Jun and Fos proteins in regulating transcriptional activation of the human pi class glutathione S-transferase gene in multidrug-resistant MCF7 breast cancer cells.

Elevated levels of the human pi class glutathione S-transferase (GSTP1-1) have been implicated in the development of antineoplastic drug resistance. Using GSTP1 promoter deletion constructs we have shown that enhanced GSTP1 transcription (up to 18-fold) is the predominant mechanism responsible for increased GSTP1-1 levels in a multidrug resistant derivative (VCREMS) of the human mammary carcinoma cell line MCF7. Furthermore, disruption of a putative AP-1 response element within the GSTP1 promoter (nucleotides -69 to -63) abrogated GSTP1 transcription in both cell lines. In addition, band shift assays demonstrated binding of a VCREMS nuclear complex to the promoter region C1 (-73 to -54) which could be competed for by a DNA fragment containing a known AP-1 binding site from the human collagenase promoter. However, no such competition was observed for the major MCF7 C1 complex. The role of a Fos-Jun-like complex in regulating GSTP1 transcription in VCREMS cells was further emphasized by the introduction of point mutations within the C1 region which were known to inhibit AP-1 binding and the interaction of antisera raised against human c-Jun and c-Fos with the major C1 complex in VCREMS cells. These studies therefore highlight cell-specific differences in the binding pattern of Jun and Fos proteins to the GSTP1 promoter which are likely to play an important role in regulating transcriptional activation of the GSTP1 gene in drug-resistant breast cancer cells.

Regulation of C4b-binding protein gene expression by the acute-phase mediators tumor necrosis factor-alpha, interleukin-6, and interleukin-1.

C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. During an acute-phase response, we have shown that hepatic levels of murine C4BP mRNA are elevated 2.5-fold while rat liver C4BP gene expression exhibits a 4-fold induction. Furthermore, a survey of different mouse tissues showed that during acute inflammation C4BP gene expression was confined to the liver. To gain a better understanding of the acute-phase regulation of C4BP gene expression we utilized the rat hepatoma cell line FAO in which tumor necrosis factor-alpha (TNF-alpha) produced a 2.7-fold induction of C4BP mRNA levels. In the absence of TNF-alpha, interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6) had little effect on C4BP gene expression but when all three cytokines were used together a synergistic 4-fold induction of C4BP mRNA levels was observed. In contrast the synthetic glucocorticoid dexamethasone inhibited TNF-alpha-induced C4BP gene expression. Cycloheximide-mediated inhibition of inducible C4BP gene expression demonstrated the requirement for ongoing protein synthesis. Rapid induction of C4BP mRNA levels by TNF-alpha and IL-6 (within 1 h) and the observation that stimulation was inhibited by actinomycin D provided evidence that regulation of C4BP gene expression during the acute-phase response is regulated at the transcriptional level. Isolation of a genomic clone extending into the 5' regulatory region of the rat C4BP gene enabled us to identify the major transcriptional start site and putative response elements through which TNF-alpha, IL-6, IL-1 alpha, and dexamethasone may exert their effects on C4BP gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete structure of the murine C4b-binding protein gene and regulation of its expression by dexamethasone.

C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. A murine genomic library was screened, and five clones were selected that covered the remaining four exons in the 5'-region of the C4BP gene. Together with previous work (Barnum, S. R., Kristensen, T., Chaplin, D. D., Seldin, M. F., and Tack, B. F. (1989) Biochemistry 28, 8312-8317), the entire C4BP gene has now been shown to be 23 kilobases (kb) long and comprised of 10 exons ranging in size from 86 to 442 base pairs (bp). Primer extension analysis revealed the major transcription start site to be 46 bp upstream of the published cDNA start site. Northern blot analysis of RNA isolated from several mouse tissues demonstrated that the C4BP gene is expressed in a liver-specific manner. Several regions homologous to known response elements were identified upstream of the C4BP gene including a strong hepatocyte nuclear factor 1 binding site and four putative glucocorticoid response elements. Furthermore, dexamethasone increased C4BP mRNA and protein levels in the mouse liver cell line, NMuLi. The stimulation of C4BP gene expression was rapid and independent of protein synthesis. These results suggest dexamethasone induction of the C4BP gene is a primary response and therefore a transcriptional effect. Inhibition of the dexamethasone effect on C4BP by actinomycin D supports this theory. These studies also provide evidence that, for optimal induction of the C4BP gene, the glucocorticoid receptor complex may cooperatively interact with accessory transcription factors. It is likely that stimulation of C4BP gene expression by dexamethasone may allude to a mechanism by which glucocorticoids exert their anti-inflammatory effects.

Structural features of the human C3 gene: intron/exon organization, transcriptional start site, and promoter region sequence.

The third component of human complement (C3) is a key molecule in the activation of the complement cascade. C3 cDNA fragments were used to identify seven cosmid clones that covered all but 1 kilobase pair (kb) of the C3 gene. The remainder of the gene was cloned by using the polymerase chain reaction. These clones were used to identify the intron/exon boundaries and to map the gene. The C3 gene is 42 kb in length and comprises 41 exons ranging in size from 52 to 213 base pairs (bp). The transcription start site was identified by primer extension, and approximately 1 kb of DNA upstream of this site was sequenced. Putative TATA and CAAT boxes were identified along with a number of regions that shared homology with known regulatory sequences. These include responsive elements for interferon-gamma, interleukin-6, nuclear factor kappa B, estrogen, glucocorticoids and thyroid hormone. Several of these agents have been shown to affect C3 synthesis and mRNA levels. The sizes of the exons in C3 were compared to those of C4 and alpha 2-macroglobulin (alpha 2M). Thirty-nine of 41 exons in C4 were found to be of similar size to the analogous ones in C3, and two-thirds of those in alpha 2M were also similarly sized, supporting the hypothesis that these genes arose from a common ancestor.

Complement biosynthesis in human synovial tissue.

Molecular biological and immunochemical techniques have been used to study the synthesis of complement components by synovial tissue from patients with rheumatoid arthritis or osteoarthritis and by normal synovial tissue from a patient undergoing patellectomy. Using Northern and dot-blot analyses, mRNAs coding for C1-inhibitor, C2, C3, C4 and factor B have been detected, but not for C5. Quantitative analyses of the data have not shown any significant differences in the steady state levels of any of the mRNAs in synovium from rheumatoid arthritis and osteoarthritis patients. When synovial membrane fragments from rheumatoid arthritis, osteoarthritis patients or normal synovium were cultured in vitro, synthesis of C1-inhibitor, C2, C3, C4 and factor B detected by ELISA and C2, C3 and factor B were shown to be functionally active. This study thus provides conclusive evidence that synthesis of complement components occurs locally within normal and inflamed synovial tissue. The local synthesis of complement within normal synovial joints may be of importance in their defence against infection, whereas in inflamed joints it may contribute to the inflammatory response.

Uterine chromatin template activity during the early stages of pregnancy in the rat.

1. The template activity of chromatin prepared from rat uterine nuclei during dioestrus, oestrus and the first 7 days of pregnancy has been examined. 2. The DNA, RNA, histone and non-histone protein contents of uterine chromatin remained constant during early pregnancy. 3. The rate of RNA synthesis on Day 1 uterine chromatin was 8.61 +/- 0.59 (mean +/- S.E.) pmol of UMP incorporated/mg DNA per 10 min. When compared with DNA prepared from rat liver nuclei, 13.20 +/- 0.27% (mean +/- S.E.) of the Day 1 chromatin DNA was available for transcription by Escherichia coli RNA polymerase. 4. Uterine chromatin from rats in early dioestrus had significantly less template activity than during oestrus. 5. Chromatin prepared from whole uterus on Day 5 and from implantation sites on Days 6 and 7 of pregnancy had a significantly higher template activity than chromatin obtained from uteri on Day 1. Chromatin from interimplantation tissue on Day 6 had a lower template activity than that from uteri on Day 1. 6. RNA - DNA hybridisation of RNA transcribed from chromatin obtained on Days 2, 5 and 7 of pregnancy showed that RNA transcribed from Day 5 chromatin obtained species not present (or present in very small amounts) in RNA transcribed by chromatin from uteri on Day 2 and from implantation tissue on Day 7 of pregnancy. 7. The results are discussed in relation to the cellular changes occurring in the stroma immediately before implantation and it is postulated that the appearance of a new species of RNA on Day 5 is related to the preparation of the stromal cells for decidualisation.