Solution formulation and lyophilisation of a recombinant fibronectin fragment.

The 9th-10th type III fibronectin domain pair shows promise in tissue engineering and tumour vasculature targeting. Calorimetry and structure-function analysis were used to investigate the effects of solution formulation and lyophilisation of a mutant ((9-10)FNIII-P). A single endothermic transition for (9-10)FNIII-P in solution was observed at pH<8, irrespective of addition of sucrose or PEG. The temperature at the maximum heat capacity (T(m)) and enthalpy (deltaH) of the transition increased for increasing sucrose concentrations but decreased for increasing PEG concentrations. The transition was fitted to a single two-state unfolding mechanism (in contrast to unfolding in guanidine. x HCl) and was partially reversible only at pH 4, with increasing concentrations of sucrose causing a marked fall in deltaH between scans. Circular dichroism spectra for the thermal unfolding of (9-10)FNIII-P at pH 4 showed loss of native beta-sheet structure and loss of aromatic contributions to the peak centred around 226 nm yielding an intermediate conformation, which in the presence of sucrose was more disordered. Despite a glass transition (T(g)') for (9-10)FNIII-P(aq) of -70 degrees C, primary drying at -30 degrees C did not perturb its conformation upon reconstitution or its biological activity following lyophilisation; the addition of sucrose or PEG had no influence on structure or activity. The main consideration in the formulation of (9-10)FNIII-P was therefore pH.

Placental arylamine N-acetyltransferase type 1: potential contributory source of urinary folate catabolite p-acetamidobenzoylglutamate during pregnancy.

Human arylamine N-acetyltransferase type 1 (NAT1), better known as a drug-metabolising enzyme, has been proposed to acetylate the folate catabolite p-aminobenzoylglutamate (p-abaglu) to N-acetamidobenzoylglutamate (ap-abaglu) which is a major urinary folate catabolite. Using mass spectroscopic analysis, we demonstrate the formation of ap-abaglu by recombinant human NAT1 and human placental homogenates. Using density gradient centrifugation the placental enzymic activity which acetylates p-aba and the placental enzymic activity acetylating p-abaglu both have an S(20,w) value of 3.25 S. This is the expected value for a monomer of human NAT1 (33 kDa). The specific NAT1 inhibitor 5-iodosalicylate inhibits acetylation of both p-aba and p-abaglu catalysed by either recombinant human NAT1 or placental samples as the source of enzyme. These data demonstrate that NAT1 is the major placental enzyme involved in acetylating p-abaglu.

Module-module interactions in the cell binding region of fibronectin: stability, flexibility and specificity.

The structure of mosaic proteins depends on the nature and strength of interactions between individual modules. Here we investigated the structural significance of module-module interactions in the RGD-dependent cell binding region of human fibronectin, comprising the ninth and tenth fibronectin type III. A combination of protein engineering, thermodynamics and nuclear magnetic resonance methods was employed to establish a relationship between intermodular protein-protein interactions and the structural properties of the module pair. A poly(glycine) peptide link connecting the C terminus of the ninth and the N terminus of the tenth module was introduced to probe the range of the interaction. NMR studies (Chemical shifts and 15N relaxation) together with equilibrium and kinetic unfolding experiments were carried out on five different single and double module constructs. The results show that non-specific protein-protein interactions provide the bulk of the thermodynamic stabilization and the motional constraint of the two modules. Specific interactions between the two modules are restricted to the wild-type module pair and decline very rapidly with the insertion of additional linker residues. This low level of specificity is nonetheless sufficient to fine-tune the precise module-module orientation and to provide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree of motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfaces.

A tissue-specific protein in rat osteogenic tissues.

A tissue-specific protein fraction has been detected in rat osteogenic tissue. Dissociative extraction of adult rat bone matrix with 4 M guanidinium chloride solution was followed sequentially by gel chromatography and polyacrylamide gel electrophoresis. By the latter procedure a prominent protein component of molecular weight 19,000 was isolated from the low molecular weight fraction, and antibodies directed against this protein were raised in rabbits. The antibodies were mainly against antigenic sites on this protein, as shown by protein blotting techniques. By embedding rat tissues in hydrophilic plastic and by using immunohistochemical procedures the presence of this protein was demonstrated specifically in bone matrix in vivo, in osteogenic tissue developing in diffusion chamber culture, and in a malignant osteoblast cell line (UMR 106). Soft tissues (liver, kidney, spleen, gut, skin, thymus, eye) showed no reactivity with the antiserum and in vitro a further malignant osteoblast cell line (ROS 17/2.8) did not synthesize the 19,000 molecular weight protein. This protein appears to be expressed solely by osteogenic tissue and may be used as a biochemical criterion of osteogenic differentiation.

Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor.

In previous studies using specific G alpha s antibodies we have identified several human myometrial G alpha s protein isoforms with molecular masses of 45, 46, 47, 54, and 58 kDa, respectively. During pregnancy, levels of the 46- and 54-kDa proteins are significantly increased compared to those in nonpregnant myometrium and then decreased at the onset of labor. In this study we investigated the expression of G alpha s messenger ribonucleic acid (mRNA) splice variants, which are generated as a result of alternative splicing of a single mRNA precursor, in term pregnancy and parturition to determine whether there was any correlation with the observed changes in G alpha s protein isoforms. A myometrial G alpha s complementary DNA was synthesized using RT-PCR and cloned into pCRtmII suitable for preparation of riboprobes for use in ribonuclease protection assays. Using this technique, we identified at least three myometrial G alpha s mRNAs, including two forms of G alpha s-Large (with or without the serine at amino acid 87) and one form of G alpha s-Small (with the serine at amino acid 72). G alpha s Small (with the serine) and G alpha s-Large (with the serine) mRNAs encode for the 46- and 54-kDa G alpha s protein isoforms, respectively, and were increased in term pregnancy and then subsequently decreased after the onset of labor. Our data suggest that posttranscriptional regulation of G alpha s mRNAs may be important in the differential expression of G alpha s protein isoforms during pregnancy and labor.

The role of the ninth and tenth type III domains of human fibronectin in cell adhesion.

Fibronectins (FN) contain sites, in addition to the cell recognition site RGD in the tenth type III domain (FIII10), that are required for adhesive activity. The role of FIII10 and the adjacent FIII9 was analysed in functional cell adhesion assays recombinant FIII domains in which the domain boundaries were strictly conserved. FIII9 had no adhesive activity. FIII10, and FIII9 plus FIII10 had less activity than FN, whereas the activity of FIII9-10 was similar to FN. We conclude that FIII9 acts synergistically with FIII10 in cell adhesion, and that this synergy is dependent upon the structural integrity of the FIII9-10 pair of domains.

Expression of human myometrial G alpha s messenger ribonucleic acid transcript during pregnancy and labour: involvement of alternative splicing pathways.

We have shown previously that expression of 46 and 54 kDa human myometrial G alpha s protein isoforms is increased during gestation and then subsequently decreased during labour. These proteins appear to be coded for by G alpha s-Small (with a serine residue at position 72) and G alpha s-Large (with a serine residue at position 87) mRNA splice variants respectively. In the study presented here we have used a G alpha s cDNA template to generate [32P]cytidine cRNA riboprobes for use in RNase protection assays, so as to measure total myometrial G alpha s mRNA levels in relation to the pattern of expression of G alpha s mRNA splice variants during pregnancy and labour. We report that total levels of human myometrial G alpha s mRNA remain similar in non-pregnant and pregnant women but are substantially reduced during parturition. Our data also provide strong evidence that alternative splicing of G alpha s precursor mRNA has a primary role in regulating expression of G alpha s protein isoforms during pregnancy and labour. The inclusion of an additional serine codon in G alpha s mRNAs during pregnancy involves a switch in alternative splicing pathways. We speculate that this switch may be due to a change in specificity of splicing factors that are modulated during pregnancy and labour.

Identification of G alpha s messenger ribonucleic acid splice variants in human granulosa cells.

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by G alpha s-linked receptors. In this paper we have investigated the expression of G alpha s mRNA splice variants in relation to expression of G alpha s protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all G alpha s mRNA isoforms as well as quantifying total amounts of G alpha s mRNA. Granulosa cells express the message for G alpha s-Large and G alpha s-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of G alpha s-Large and G alpha s-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in G alpha s variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between G alpha s variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

Expression of granulocyte-colony stimulating factor and its receptor is regulated during the development of the human placenta.

The development of the placenta is dependent upon the regulated proliferation, invasion and differentiation of trophoblast. Expression of cytokines at the feto-maternal interface suggests that these molecules may participate in placentation. The expression of granulocyte-colony stimulating factor (G-CSF) and G-CSF receptor (G-CSFR) during the development of the human placenta was studied by immunohistochemistry using an anti-G-CSF monoclonal antibody (mAb) and two novel anti-G-CSFR mAbs. G-CSF was present in the stroma of fetal chorionic villi and maternal decidual tissues throughout pregnancy. G-CSFR was detected at high levels in fetal first and third, but not second trimester placental tissues. Staining for G-CSFR was undetectable in maternal decidual tissue from all gestational stages. In first trimester tissues, staining for placental G-CSFR was strongest in differentiated syncytiotrophoblast and invasive, extravillous cytotrophoblast, and weak staining was evident in undifferentiated cytotrophoblast. Immunohistochemical data suggesting temporal regulation of G-CSFR were corroborated by Western blotting and amplification by reverse transcription and PCR of G-CSFR mRNA. These data suggested that expression of G-CSFR in the human placenta is regulated both temporally and spatially, and that placental G-CSF is involved in paracrine regulation, and indicate a role for G-CSF and G-CSFR in trophoblast growth or function during placentation.

Endometriosis in monozygotic twins.

OBJECTIVE: To describe the occurrence of endometriosis in monozygotic twins. DESIGN: Postal questionnaire plus confirmation of disease status. SETTING: Twins were recruited via the American Endometriosis Association and the National Endometriosis Society of Great Britain and via British gynecologists. RESULT(S): Fourteen twin pairs were concordant for endometriosis, and two were discordant. Nine pairs of twins had moderate-severe endometriosis. CONCLUSION(S): These findings contribute to the growing body of literature that suggests endometriosis has a genetic basis.

Acetylation of arylamines by the placenta.

The N-acetylation of arylamines and hydrazines used as drugs may alter their pharmacological or toxicological activity. Arylamine N-acetyltransferase (NATs) are involved in drug metabolism, as they catalyse the N-acetylation of arylamine and mono-substituted hydrazine substrates. Placental metabolism regulates the nature of the chemicals which reach the developing fetus. The study of drug metabolism during pregnancy is important in determining the effect on the fetus of drugs administered to the mother and the maternal drug dose required, important if the treatment is to be effective. There are two forms of NAT in humans, NAT1 and NAT2, which are encoded at multi-allelic loci. There is inter-individual variation in both NAT1 and NAT2 activity, which has implications in drug dosage. Using a combination of enzyme activity measurements and Western blotting, this study has characterised the arylamine N-acetylation capabilities of placenta and cord blood. NAT1 activity in placenta and cord blood demonstrated inter-individual variation and the variation was in the range expected for adult NAT1 activity. The genotypes of both NAT1* and NAT2* were determined using DNA prepared using placental blood clots (maternal DNA) and cord blood (fetal DNA). The results indicate that placental NAT activity is an important factor when considering N-acetylation during pregnancy.

Mutations in fibrillin-1 cause congenital scleroderma: stiff skin syndrome.

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

Self-assembling multimeric integrin alpha5beta1 ligands for cell attachment and spreading.

Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin alpha5beta1 requires the synergy site on the 9th type III fibronectin domain (FIII) in addition to RGD on the 10th FIII domain. Here, we have designed and expressed soluble protein chimeras consisting of an N-terminal 9th-10th FIII domain pair, IgG-derived hinge and leucine zipper-derived helix; the latter mutated to yield di-, tri- and tetrameric coiled coils and thus self-assembling, multimeric integrin alpha5beta1 ligands. A unique C-terminal cysteine was appended to the helix to facilitate 'anchoring' of the chimeras with a defined orientation on a surface. Size-exclusion chromatography and circular dichroism demonstrated that the chimeras self-assembled as multimers in solution with defined secondary structures predicted from theoretical calculations. Biotinylation via a thioether bond was used to selectively bind the chimeras to streptavidin-coated surfaces, each of which was then shown to bind integrin alpha5beta1 by surface plasmon resonance. Spreading of fibroblasts to surfaces derivatised with the chimeras was found to proceed in the order: tetramer > trimer > dimer > monomer. Thus, we describe novel polyvalent integrin alpha5beta1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro.

Familial endometriosis.

PURPOSE: The study aimed to identify families with endometriosis and to document disease severity within the families and the clinical characteristics of the affected women. RESULTS: Two hundred and thirty women with surgically confirmed endometriosis in 100 families were identified. The families consisted of 19 mother-daughter pairs, 1 set of cousins and 56 sister pairs. There were 5 families with 3 affected sisters, 1 family with 5 affected sisters, and 18 families with > or = 3 affected members in more than one generation. The mean age at the onset of symptoms and the mean age at surgical diagnosis was 22.1 +/- 8.8 SD (range 10-46) and 31.8 +/- 7.9 SD (range 15-56) years respectively. Seventy-nine women (34.3%) had revised AFS Stage I-II disease, and 151 (65.7%) had revised AFS Stage III-IV disease. CONCLUSION: The study confirms a familial tendency for endometriosis and supports the hypothesis that endometriosis has a genetic basis.

Regulation of alternative splicing in the IIICS region of human fibronectin pre-mRNA encoding cell binding sites CS1 and CS5.

The cell binding sites CS1 and CS5 in the IIICS region of human fibronectin (FN) mediate the adhesion of specific cell types by interacting with the integrin alpha 4 beta 1. IIICS pre-mRNA is alternatively spliced via the use of three alternative splice acceptor sites and one alternative splice donor site. These alternative splicing pathways can potentially give rise to variant FN molecules which are CS1+,CS5+; CS1+,CS5-; CS1-,CS5+ or CS1-,CS5-. Here we show that selection of the acceptor site which incorporates mRNA encoding CS1 and CS5 is more frequent in foetal tissues compared to adult liver, whereas an alternative acceptor site and the alternative donor site, which exclude CS1 and CS5, are used at a higher level in adult liver compared to foetal tissue. All possible splice junctions were accurately processed, and selected at different levels in mRNA expressed from a IIICS minigene transiently transfected into a HeLa cell line which does not express FN, suggesting that all the cellular factors required for alternative processing of IIICS are present in this system. Furthermore, pre-mRNA expressed from a mutant construct lacking IIICS-1 intron sequence, was correctly processed in HeLa cells via selection of all possible splice sites. On the basis of our results we propose that regulation of splice site selection in IIICS and thus expression of CS1 and CS5 is achieved by subtle tuning of splicing systems involving the interaction of local cis elements and cellular factors which are not necessarily restricted developmentally or tissue-specifically, and that expression of CS1 and CS5 is independently regulated.

A role for exon sequences in alternative splicing of the human fibronectin gene.

Exon EDIIIA of the fibronectin (Fn) gene is alternatively spliced via pathways which either skip or include the whole exon in the messenger RNA (mRNA). We have investigated the role of EDIIIA exon sequences in the human Fn gene in determining alternative splicing of this exon during transient expression of alpha globin/Fn minigene hybrids in HeLa cells. We demonstrate that a DNA sequence of 81bp within the central region of exon EDIIIA is required for alternative splicing during processing of the primary transcript to generate both EDIIIA+ and EDIIIA- mRNA's. Furthermore, alternative splicing of EDIIIA only occurs when this sequence is present in the correct orientation since when it is in antisense orientation splicing always occurs via exon-skipping generating EDIIIA- mRNA.

Temporal and spatial regulation of expression of heparin-binding epidermal growth factor-like growth factor in the human endometrium: a possible role in blastocyst implantation.

The function of the endometrium in the implantation of the blastocyst depends on the regulated, cyclical regeneration of endometrial tissue and the expression of a receptive phenotype in response to steroid hormones. Experiments using animal and models suggest that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is important for endometrial receptivity, and that it may directly mediate blastocyst implantation We have investigated the expression of HB-EGF mRNA and protein in pregnant and nonpregnant human endometrium and placenta. Our data demonstrate that HB-EGF mRNA expression is low in the endometrium during the proliferative stage of the menstrual cycle and increases in the secretory stage, with highest expression immediately prior to the implantation window (day 19-21), after which levels decrease. Immunohistochemical detection of HB-EGF shows that it is present in the stroma of proliferative stage endometrium and that it is localized to the apical surface of the luminal epithelium of midsecretory stage endometrium. Levels of HB-EGF mRNA are low in pregnant endometrium and high in placental tissues at an early stage of development. Our data suggest that expression of human endometrial HB-EGF coincides with the expression of a receptive phenotype, and that H-EGF may have an important function in the implantation of the human blastocyst and early placental development.

Delay in the diagnosis of endometriosis: a survey of women from the USA and the UK.

We investigated the length of time between the onset of pain symptoms and the surgical diagnosis of endometriosis in women from the UK and the USA. A total of 218 women with surgically confirmed disease, recruited through endometriosis self-help groups, completed a postal questionnaire. The mean +/- SD delay in diagnosis for women from the USA was 11.73 +/- 9.05 years, significantly higher than the equivalent delay of 7.96 +/- 7.92 years for women from the UK (P < 0.01). The stage of disease did not effect the length of time between the onset of symptoms and diagnosis. Therefore there is considerable delay in the diagnosis of endometriosis for women from both the UK and the USA. Efforts to reduce this delay are required to minimize the suffering of women with this disease.