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Women with stage III ovarian cancer and with < or = 2 cm residual tumour were randomly assigned to receive either conventionally dosed chemotherapy (group A) or HDCT (group B). Patients allocated to group A received 6 cycles of paclitaxel (T) 135 mg/m2 and cisplatin (P) 75 mg/m2 every 3 weeks, and those allocated to HDCT received 3 TP cycles followed by peripheral blood stem cell mobilisation with cyclophosphamide (C) 3000 mg/m2 and T 175 mg/m2, and subsequently HDCT with carboplatin 1500 mg/m2, C 120 mg/kg, and mitoxantrone 75 mg/m2. The trial was closed early after 42 patients were entered due to slow accrual. The median follow-up time of patients who were alive was 81 months. The median progression-free survival time was 15.9 and 16.6 months (hazard ratio, HR 0.83; 95% CI 0.41-1.69, P = 0.61) and the median overall survival time was 43.7 and 64.3 months (HR, 0.74; 95% CI 0.34-1.61, P = 0.44) in groups A and B, respectively. Although one patient died of HDCT-related toxicity, the regimen was otherwise relatively well tolerated. We conclude that the HDCT regimen used was feasible, but did not result in significantly improved survival in this prematurely closed trial. A clinically important survival benefit cannot be excluded due to the small sample size.

Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS, fluorescent subunits with diffusion times of 0.2 ms were observed. Additional treatment with DTT caused a drop in the diffusion time to 0.1 ms. Changes in the amplitude of the autocorrelation function suggested a 3-fold increase in fluorescent particle number when viral particles were treated with SDS, and a further 1.5-fold increase with additional treatment with DTT. Thus, the data showed that an average of 4.5 molecules of gp64-GFP was incorporated in the membrane of the modified baculovirus. Further, this suggests that each fluorescent gp64 trimer carries on average 1.5 fluorescent units. Similar experiments were carried out with two non-enveloped fluorescent virus-like particles (fVLPs) that displayed enhanced green fluorescent protein (EGFP). These, fVLPs of canine and human B19 parvoviruses were treated with 6 M urea and 5 mM SDS, respectively. Correspondingly, the original hydrodynamic radii of 17 and 14 nm were reduced to 9 and 5 nm after treatment. Here, the change in the amplitude of the autocorrelation curve suggested a 10-fold increase in particle number when viral particles of CPV were treated with 6 M urea at 50 degrees C for 10 min. For EGFP-B19, there was a decrease in the amplitude, accompanied by a 9-fold increase in the number of fluorescent units with SDS treatment. The results showed that approximately 10 and 9 fluorescent units were associated with the corresponding CPV and B19 VLPs. In summary, we were able to estimate the number of fluorescent subunits in a baculovirus containing a GFP-fusion with its gp64 envelope protein and in two different parvo-VLPs containing EGFP-fused with their VP2 capsid proteins.

Although sharing a T=1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.

A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.

Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A(2) like domain in N-terminus of VP1. In this study we characterized the role of PLA(2) activity on CPV entry process. PLA(2) activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA(2) inhibitors inhibited the viral proliferation suggesting that PLA(2) activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA(2) activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A(1), brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A(1), brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA(2) activity of the virus. These results suggest that parvoviral PLA(2) activity is essential for productive infection and presumably utilized in membrane penetration process of the virus, but CPV also needs other pH-dependent changes or factors to be released to the cytoplasm from endocytic vesicles.

Canine parvovirus (CPV), a model virus for the study of parvoviral entry, enters host cells by receptor-mediated endocytosis, escapes from endosomal vesicles to the cytosol, and then replicates in the nucleus. We examined the role of the microtubule (MT)-mediated cytoplasmic trafficking of viral particles toward the nucleus. Immunofluorescence and immunoelectron microscopy showed that capsids were transported through the cytoplasm into the nucleus after cytoplasmic microinjection but that in the presence of MT-depolymerizing agents, viral capsids were unable to reach the nucleus. The nuclear accumulation of capsids was also reduced by microinjection of an anti-dynein antibody. Moreover, electron microscopy and light microscopy experiments demonstrated that viral capsids associate with tubulin and dynein in vitro. Coprecipitation studies indicated that viral capsids interact with dynein. When the cytoplasmic transport process was studied in living cells by microinjecting fluorescently labeled capsids into the cytoplasm of cells containing fluorescent tubulin, capsids were found in close contact with MTs. These results suggest that intact MTs and the motor protein dynein are required for the cytoplasmic transport of CPV capsids and contribute to the accumulation of the capsid in the nucleus.

Background: Ovarian cysts are found in some premenopausal women with autosomal dominant polycystic kidney disease (ADPKD). Liver cysts are the major extrarenal manifestations, frequently found in postmenopausal women. Female steroid hormones may regulate hepatic cystogenesis, but ovarian manifestations in postmenopausal women are unknown. The aim of the present study is to investigate possible ovarian manifestations associated with ADPKD in premenopausal and postmenopausal women., Methods: The study group included 19 premenopausal or postmenopausal women with ADPKD and 19 age- and parity-matched control women with no history of renal disease. Ovarian structures and volumes were measured by means of transvaginal ultrasonography., Results: Three of 8 premenopausal and 1 of 11 postmenopausal women with ADPKD had a small unilateral single ovarian cyst; there were none in controls. Eight women with ADPKD and 8 controls of premenopausal age had similar ovarian volumes (4.92 +/- 2.97 versus 3.92 +/- 2.60 cm3). Eleven postmenopausal women with ADPKD had an increased mean ovarian volume compared with 11 postmenopausal controls (4.11 +/- 2.60 versus 1.66 +/- 0.96 cm3; P < 0.01). Ovarian volume was not associated with the use of hormonal replacement therapy or impaired renal function in postmenopausal women with ADPKD., Conclusion: ADPKD is not associated with cystic ovaries, although single ovarian cysts are occasionally found. Equal ovarian volumes between premenopausal and postmenopausal women with ADPKD suggest a possible indirect effect of polycystic kidneys on gonads in aging women., (Copyright 2002 by the National Kidney Foundation, Inc.)

Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes resembling recycling endosomes. Later, CPV was found to enter, via late endosomes, a perinuclear vesicular compartment, where it colocalized with lysosomal markers. There was no indication of CPV entry into the trans-Golgi or the endoplasmic reticulum. Similar results were obtained both with full and with empty capsids. The data thus suggest that CPV or its DNA was released from the lysosomal compartment to the cytoplasm to be then transported to the nucleus. Electron microscopy analysis revealed endosomal vesicles containing CPV to be associated with microtubules. In the presence of nocodazole, a microtubule-disrupting drug, CPV entry was blocked and the virus was found in peripheral vesicles. Thus, some step(s) of the entry process were dependent on microtubules. Microinjection of antibodies to dynein caused CPV to remain in pericellular vesicles. This suggests an important role for the motor protein dynein in transporting vesicles containing CPV along the microtubule network.

Objective: To evaluate endometrial thickness and uterine arterial flow measurement as predictors of endometrial cancer., Design: Prospective study among a cohort of women invited to age-adjusted, population-based breast cancer screening by mammography., Setting: City of Turku, Finland., Population: 1074 postmenopausal women aged 57-61 years (mean 59 years)., Methods: Conventional and colour Doppler sonography. Endometrial biopsy was taken when the endometrial thickness (double layer) was > or = 4.0 mm, if the uterine artery pulsatility index was < or = 1.0 or if there was a fluid accumulation in the endometrial cavity., Main Outcome Measures: Detection of endometrial cancer in endometrial biopsy. Record linkage with the files of the Finnish Cancer Registry three and a half years after the first ultrasound examination. Major statistical results are based on the analysis of variance and logistic regression models., Results: An endometrial biopsy was taken from 291 women (27%). One woman had endometrial tuberculosis, three an endometrial polyp, 16 endometrial hyperplasia, three endometrial carcinoma (Stage Ib), and one had cervical carcinoma (Stage Ib). One woman was diagnosed as having endometrial cancer Stage Ib two and a half years after screening; she had refused further examination after a positive screen. A second endometrial cancer (Stage Ib) was diagnosed three years after a negative screening result., Conclusion: Transvaginal sonography is confirmed to have a very high sensitivity for the detection of early endometrial carcinoma, but the specificity remains low. If endometrial cancer is to be detected at an early stage, further examinations should be carried out when the endometrial thickness is > or = 4.0 mm, especially when the woman has risk factors such as obesity, late menopause or current use of hormonal replacement therapy. Doppler sonography does not improve the detection of premalignant and malignant endometrial lesions compared with normal ultrasound.

The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18 degrees C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membrane fusion steps in the entry process. Microinjection experiments showed that CPV particles which were injected directly into the cytoplasm, thus avoiding the endocytic pathway, were unable to initiate progeny virus production. CPV treated at pH 5.0 prior to microinjection was unable to initiate virus production, showing that factors of the endocytic route other than low pH are necessary for the initiation of infection by CPV.

We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4-13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor-mediated process). Transport into the nucleus was an energy-dependent and temperature-dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.

We evaluated the utility of a single CA 125 measurement in combination with transvaginal sonography for early detection of ovarian and endometrial cancer in asymptomatic postmenopausal women. A sample of peripheral blood was taken from 1291 apparently healthy postmenopausal women, who were examined by conventional and color Doppler ultrasound for early detection of ovarian and endometrial cancer. Serum CA 125 was determined in all samples 3 years later by the IMx CA 125 assay (Abbott Laboratories, Abbott Park, IL). The cutoff level based on the 99th percentile was 30 U/ml. Elevated values were controlled by repeat sonography and an additional determination of CA 125. Record linkage with the files of the Finnish Cancer Registry was performed 3 1/2 years after the primary sonographic screening. The mean CA 125 concentration was 8.1 U/ml (range 0-1410 U/ml). Fourteen of the 1291 women had a CA 125 level greater than 30 U/ml. None of these had signs of either endometrial or ovarian malignancy in the primary sonography screening. Among the other women three cases of endometrial carcinoma (all stage Ib) and one ovarian carcinoma (stage Ia with borderline malignancy) were detected by sonography. All these patients had a CA 125 value <30 U/ml, the mean value being 11.4 U/ml (range 7.5-16.7 U/ml). During follow-up of 3.5 years, one stage Ia ovarian carcinoma, one abdominal carcinomatosis, and two endometrial carcinomas (both stage Ib) were diagnosed. In these patients the mean value for CA 125 was 12.7 U/ml (range 2.5-30.9 U/ml) at the primary sonography screening. A single CA 125 measurement provides no advantage in the early detection of ovarian and endometrial cancer in asymptomatic postmenopausal women compared with transvaginal sonography. The vast majority of women with an elevated CA 125 value have some reason other than an ovarian or endometrial malignancy for this finding.

Certain methicillin-resistant Staphylococcus aureus strains contain a 230-kDa cell-wall protein which is not present on the surface of other staphylococci. The presence of this 230-kDa protein is associated with a negative test result in commercial assays designed to detect fibrinogen-binding proteins and/or protein A on the staphylococcal surface. We have purified and partially characterised the 230-kDa protein from a lysostaphin digest of a non-agglutinating methicillin-resistant S. aureus strain. Partial amino acid sequence data obtained from the purified protein did not reveal any significant similarities to known proteins which indicates that the protein is novel. The 230-kDa protein was very sensitive to proteolysis; soluble plasmin, or plasmin formed on the bacterial-cell surface, rapidly degraded the 230-kDa protein to a 175-kDa form. The finding that the 230-kDa protein bound to lectins allowed its purification by affinity chromatography on immobilised wheat germ agglutinin. Furthermore, the degradation of the 230-kDa protein was associated with an increased adherence of non-agglutinating methicillin-resistant S. aureus cells to solid-phase fibronectin, fibrinogen or IgG.

We describe a rapid immunochromatographic method for the quantitation of progesterone in bovine milk. The method is based on a 'competitive' assay format using the monoclonal antibody to progesterone and a progesterone-protein conjugate labelled with colloidal gold particles. The monoclonal antibody to progesterone is immobilized as a narrow detection zone on a porous membrane. The sample is mixed with colloidal gold particles coated with progesterone-protein conjugate, and the mixture is allowed to migrate past the detection zone. Migration is facilitated by capillary forces. The amount of labelled progesterone-protein conjugate bound to the detection zone, as detected by photometric scanning, is inversely proportional to the amount of progesterone present in the sample. Analysis is complete in less than 10 min. The method has a practical detection limit of 5 ng of progesterone per ml of bovine milk.

The risk of endometrial cancer (EC) in diabetics and hypertensives was studied as part of the planning of a trial of the efficacy of screening for EC. Two nationwide registers, the Finnish Cancer Registry and the Social Insurance Institution's (SII) population register, were linked to obtain 1715 EC patients diagnosed during 1970-1974 with individually matched controls. Data on the right for state-reimbursed medication for diabetes or hypertension prior to the case's cancer diagnosis was obtained from the SII's population register and was used as indicator of the diseases in question. Diabetes was a strong and hypertension a relatively weak risk factor for EC. The relative risk for the association of EC with diabetes was 4.1 (95% confidence limits 2.7 and 6.9) and with hypertension 1.6 (95% confidence limits 1.3 and 2.1). Diabetics and hypertensives are identifiable risk groups for EC in Finland. Diabetics form a suitable target population for a trial of the efficacy of screening for EC.

Immunochemical analysis of overlapping synthetic hexapeptides covering the entire length of the coat protein of potato virus Y (PVY) revealed immunodominant regions both at the N-terminal and at the C-terminal end of the coat protein. Immunization of rabbits with synthetic peptides representing N- and C-terminal regions of the coat protein resulted in production of antibodies that reacted with PVY. Antigenicity of PVY peptides was found to correlate with predicted beta turns, with hydrophilicity and with predicted chain flexibility. Characterization of the immunochemical properties of PVY will facilitate the development of detection methods for potyviruses.

Objective: Our purpose was to study the effects of postmenopausal hormone replacement therapy on the uterus and uterine circulation., Study Design: The study population consisted of 432 women, 58 to 59 years of age. Color Doppler ultrasonography with a transvaginal probe was used to measure the size of the uterus and the uterine artery pulsatility index., Results: The mean endometrial thickness in group 1 (controls without hormone replacement therapy) was significantly thinner compared with group 2 hormone replacement therapy and with group 3 after discontinuance of hormone replacement therapy. The mean uterine artery pulsatility index was lower both in group 2 and 3 compared with group 1. When hormone replacement therapy was initiated 2 to 10 years after menopause, the endometrial thickness did not differ from that among those who had started hormone replacement therapy earlier, but the pulsatility index was significantly higher. There was positive correlation between the size of the uterus and the pulsatility index in group 1, but the correlation was negative in group 2. In general, the duration of hormone replacement therapy had no effect on the pulsatility index. Estrogen users had a significantly thicker endometrium compared with estrogen-progestogen users. The pulsatility index was highest in the estrogen users with progestogen added every month., Conclusion: The duration, onset of treatment in relation to menopause, discontinuance of hormone replacement therapy, and mode of treatment modify both the normal postmenopausal endometrial thickness and the uterine vascular resistance.

We have recently demonstrated that many cancer cell lines produce a novel trypsinogen isoenzyme called tumor-associated trypsinogen 2 (TAT-2). It was found during a search of the target protease for tumor-associated trypsin inhibitor (TATI). We now show that degradation of subendothelial cell extracellular matrix (ECM) by four different cell lines (COLO 205 colon carcinoma, K-562 erythroleukemia, CAPAN-1 pancreatic carcinoma, and HT 1080 fibrosarcoma) can be partially inhibited by TATI or neutralizing trypsin antibodies. When cells were cultured in serum-free medium on ECM, TATI and trypsin antibodies inhibited the release of immunoreactive fibronectin fragments from ECM by 47-54 and 40%, respectively. Degradation of isotopically labeled ([3H]serine, [3H]proline, and [35S]sulfate) ECM was also significantly prevented by TATI. At its maximum, it exerted a 57% inhibition on the degradation of [3H]serine-labeled ECM. Plasminogen added exogenously to the culture medium further potentiated the proteolysis of ECM. Interestingly, addition of enteropeptidase, an activator of TAT-2, also enhanced cell-mediated proteolysis as assessed by degradation of purified fibronectin coated onto the surface of wells. Immunoblot analysis showed that enteropeptidase-mediated proteolysis generated a pattern of fibronectin fragments similar to that obtained by digestion of purified fibronectin by TAT-2. These results demonstrate the existence of a proteolytic system in tumor cells which is dependent on the activation of TAT-2. We suggest that TAT-2 is involved in a protease cascade-stimulating tumor cell invasion and degradation of extracellular matrix.

Nimesulide is a non-steroidal anti-inflammatory agent which has proved to be effective in reducing menstrual discomfort in dysmenorrhoeaic women. To determine the concentrations of this drug in the uterus (fundus, cervix), oviduct, and ovaries and to correlate these findings with plasma concentrations, a single oral dose of 100 mg nimesulide was administered 1 to 6 h before surgery to 12 women undergoing hysterectomy and salpingo-oophorectomy, mainly for fibroids. Tissue samples were taken, concentration of nimesulide measured by HPLC, and findings compared with plasma concentrations. One patient not undergoing treatment served as control. Nimesulide concentration in the tissues studied was highest 3 h after administration, as expected from the drug's pharmacokinetic profile. The highest tissue/plasma ratio (0.5) was also found at that time. Average tissue concentrations at 1, 2, 3, and 6 h after drug intake ranged from 0.3 to 1.8 micrograms g-1, and plasma concentrations from 2.6 to 4.1 micrograms ml-1. Nimesulide was evenly distributed in the tissues studied.

A competitive enzyme immunoassay for the determination of fibronectin in plasma is described. An enzyme conjugate prepared by coupling alkaline phosphatase to rabbit anti-human fibronectin antibodies by glutaraldehyde was used as principal reagent. The assay was performed by coating polystyrene tubes with purified fibronectin and reacting these coated tubes with a mixture of sample and enzyme-labeled antibodies. After overnight incubation, the amount of enzyme activity associated with the tube was determined. An assay range of 0.5-20 microgram/ml of fibronectin was obtained. The mean concentration of plasma fibronectin in female patients was found to be 270 microgram/ml (standard deviation 50 microgram/ml, n = 22). Denatured fibronectin had low activity in the assay. The presence of cross-reacting antigens in rat and guinea pig plasma was demonstrated by the enzyme immunoassay technique.

Fibronectin is a high-molecular-weight glycoprotein present in a soluble form in plasma and in other body fluids and as insoluble protein in connective tissue matrix. This study reports that soluble fibronectin is polymerized into filamentous structures and that polyamines stimulate this process and precipitate fibronectin. Fibronectin purified from human plasma under non-denaturing conditions appeared after negative staining as non-globular extended structures in the electron microscope. During storage of purified fibronectin at +4 degrees C, in particular a low ionic strength, increasing amounts of the protein appeared as protein filaments. These filaments had a diameter of 2--3 nm and a length of up to several micrometers. The filaments also formed bundles of variable thickness, apparently through lateral association. These structures could also be visualized by phase-contrast microscopy. Polyamines, at a concentration of 1--5 mM and at a low ionic strength, induced a rapid, extensive polymerization of fibronectin into filamentous structures. The effect increased in the order putrescine less than spermidine less than spermine. Polyamine-induced precipitation of fibronectin was reversible upon removal of the polyamine. Fibronectin secreted by normal and by malignant cells could be fairly selectively precipitated from the culture medium with polyamines. The observed filamentous polymers of soluble fibronectin resemble the filamentous fibronectin-containing pericellular structures in fibroblast cultures and may provide a model for studies on the deposition of fibronectin in matrix form.

Treatment of human immunoglobulin G, albumin and fibronectin with water-soluble carbodi-imide at pH4.75 in the presence of glycine ethyl ester resulted in an avid binding of (125)I-labelled native fibrinectin to the modified proteins. Succinoylation, reduction and alkylation or heat-denaturation had no such effect. In affinity chromatography under physiological conditions, serum was depleted of fibronectin when run through columns of the carbodi-imide-treated proteins coupled to agarose. Fractions eluted from such columns with urea were enriched in fibronectin. The binding of radiolabelled fibronectin to the carbodi-imide-treated proteins was inhibited by unlabelled fibronectin in relatively low concentrations, but also by albumin in higher concentrations. Heat-denatured albumin inhibited at concentrations approx. 10-30 times lower than native albumin. The binding reaction had a pH optimum of 6-8. It was inhibited at high ionic strength and in the presence of urea. Anionic detergents inhibited at millimolar concentrations, but non-ionic detergents did not inhibit the binding reaction. The results were interpreted as showing that: (1) fibronectin is capable of binding to itself, to immunoglobulin G and to albumin after a reduction of the negative surface charge of these proteins, and may have a general ability to bind such modified proteins; (2) this binding can take place under physiological conditions; (3) carboxy-group-modified proteins selectively bind fibronectin from serum. This novel binding phenomenon could be important in terms of the opsonin function of circulatory fibronectin. We propose that fibronectin may recognize modified (denatured) proteins and mediate their uptake by the reticuloendothelial system.

Three Macaca irus monkeys were immunized with purified human CEA. One received unmodified CEA and two were immunized with haptenated (4-hydroxy-5-iodo-3-nitrophenyl acetyl) CEA. All three monkeys formed precipitating antibodies to CEA. The antisera did not precipitate the CEA-related normal glycoprotein antigen (NCA). In contrast, CEA-immunized rabbits, sheep and goats invariably form antibodies against NCA. Radio-immunoassays showed the monkey antisera to contain trace amounts of antibodies to NCA but the titers were about 10-1,000 times lower than those in rabbit and sheep sera with comparable anti-CEA titers. These results show that monkeys produce antibodies against CEA, but respond weakly to a normal, CEA-related antigen. The weak response to NCA suggests that monkeys may possess an NCA-related antigen. Antisera lacking reactivity to normal CEA-related antigens may be helpful in establishing a more specific diagnostic test for CEA.

Purified human plasma fibronectin at concentrations of about 30 microgram/ml was found to agglutinate trypsin-treated erythrocytes from certain species. The hemagglutination reaction was inhibited by specific antibodies to fibronectin, by relatively low concentrations of polyamines and by higher concentrations of basic amino acids and nonacetylated amino sugars. The divalent cations Ca2+ and Mg2+ and the chelating agent ethylenediaminetetraacetate did not affect the reaction. None of the neutral amino acids, neutral sugars or polyanions tested was inhibitory. The results imply that plasma fibronectin is capable of interacting with cell surfaces and support the idea of a similarity between cellular and plasma fibronectins.

We have earlier identified and characterized a tumor-associated trypsin inhibitor (TATI) in the urine of patients with gynecological cancer. Elevated levels of TATI occur in urine and serum of cancer patients (Stenman, U. H., Huhtala, M. L., Koistinen, R. & Seppälä, M. (1982) Int. J. Cancer 30, 53-57). To explain the elevation of TATI in cancer, we have postulated the existence of a tumor-associated protease reacting with TATI. Such a protease, tentatively called protease T, was found in cyst fluid from mucinous ovarian tumors. The protease occurs in complex with TATI, and its protease activity can be measured only after dissociation of the complex. This is achieved by reversed phase chromatography at low pH and elution with an isopropyl alcohol gradient. Protease T is inhibited by phenylmethanesulphonyl fluoride indicating that it is a serine protease. Its optimum activity at pH 9.1 and molecular mass of 24 kDa in gel chromatography are similar to those of trypsin but the substrate specificity is not identical and its isoelectric point (pI) is about 4.0, which is lower than the corresponding values of both cationic (pI 9) and anionic trypsin (pI 5). Protease T could be associated with the elevation of TATI seen in certain tumor patients.

The effect of chemical modification of arginine and lysine residues of fibronectin on its antigenic and gelatin-binding activity was studied by enzyme immunoassay techniques. Both modifications strongly reduced the gelatin-binding activity. Using conformation-specific antibodies it was shown that modification of lysines caused extensive conformational changes in the molecule. No such changes could be detected in arginine-modified fibronectin. The results suggest that arginine residues are directly involved in the binding of fibronectin to gelatin. Lysine residues seem to be important for maintaining a native conformation necessary for gelatin-binding.

Human plasma fibronectin has been investigated at physiological pH and ionic strength, by using small-angle X-ray and neutron scattering techniques. The results indicate that the molecule is disc shaped with an axial ratio of about 1:10. In fact, an ellipsoid of revolution with semiaxes a = 1.44 nm and b = c = 13.8 nm is in agreement with the experimental scattering data, and can also fully explain the rather extreme hydrodynamic parameters reported for fibronectin. The X-ray data gave a radius of gyration of 8.9 nm and a molecular weight of 510,000, whereas the neutron data gave slightly larger values, 9.5 nm and 530,000, respectively. From the volume of the best fitting ellipsoid we obtain a degree of hydration of 0.61 g H2O/g protein (dry weight). Neutron data, recorded at different D2O concentrations in the solvent, gave a match point of 43% D2O, which indicates that approximately 80% of the hydrogens bound to oxygen and nitrogen are exchangeable.

A carcinoembryonic antigen-like antigen(s) (CELIA) was isolated from normal human gastric jucie by immunoadsorbents and gel chromatography. The isolated CELIA reacted identically to CEA in immunodiffusion testa against anti-CEA and anti-CELIA. Other experiments suggested subtle differences between CELIA and CEA. Two out of six CELIA preparations tested in immunodiffusion did not react in a completely identical manner with CEA.In gel filtration, CELIA eluted slightly after CEA. Absorption of anti-CEA with CELIA resulted in antisera with greatly reduced reactivity to CELIA, but retained anti-CEA activity. Such antisera may be helpful in attempts to increase the cancer specificity of the CEA test.

Purified human plasma fibronectin has been shown to agglutinate protease-treated red cells [Vuento, Hoppe-Seyler's Z. Physiol. Chem. 360, 1327-1333, (1979)]. The present report shows that the activity is inhibited by low concentrations of lectins and by macromolecular serum factors. Chemical modification of carboxyl groups of fibronectin strongly inhibited the activity, but modification of amino groups of guanidinium groups had little effect on the activity. The results suggest that fibronectin receptors on erythrocyte surface are carbohydrate-containing molecules. Humoral macromolecular factors may control the interaction of fibronectin with cell surfaces. Chemical modification studies indicate that the parts of the fibronectin molecule responsible for the hemagglutinin activity are different from those mediating the binding of fibronectin to collagen.

Degradation of fibronectin (FN) by subgingival and supragingival plaque and Bacteroides gingivalis (Bg) was studied in vitro. The degradation of FN by both types of plaque was relatively rapid, continuous but incomplete. Some differences were found between supra- and subgingival samples. Supragingival plaque extracts produced several FN fragments of 110-180 kd during short incubations of 15-60 min. The predominant fragment after overnight incubation was a 110 kd polypeptide. With subgingival plaque extract a more extensive degradation of FN was noted. The main degradation product was a 120 kd fragment after overnight incubation. Several peptide fragments were released from fibronectin by Bg extracts. Their molecular size was different from those produced by trypsin, elastase or dental plaque. When cell extracts of Bg were fractionated by high performance liquid chromatography, three separate peaks of fibronectin degrading activity were obtained. Two of those peaks also contained trypsin-like enzyme activity. The degradation of fibronectin and the subsequent formation of biologically active peptides may have many effects in periodontal pockets. These may include modifying effects on plaque growth and wound healing.

The polyamines spermine and spermidine were found to aggregate proteins from human plasma and serum, apparently due to electrostatic interactions between these cations and anionic proteins. Fibrinogen and fibronectin (cold insoluble globulin) were identified as the major molecular components of aggregates, and fibronectin could be quantitatively removed from plasma by aggregation with spermine. The results indicate that fibrinogen and fibronectin have anionic groups which react with polyamines and which are essential for the solubility of these proteins.

Ejaculated human spermatozoa were shown to have fibronectin polypeptides on their surface. Immunofluorescence studies revealed fibronectin as a belt-like fluorescent band on the post-acrosomal area of sperm heads, whereas none was found in sperm tails. The location of the fluorescent band corresponded to the equatorial segment of the spermatozoon. Fibronectin polypeptides were heterogeneous with Mr ranging from 35000 to 210000, as revealed by immunoblotting and by immunoprecipitation of detergent extracts from surface-radioiodinated spermatozoa.

CEA activity was studied using a three-layer bridge immunoperoxidase method in paraffin-embedded histological sections of gastric mucosa from patients with gastric cancer and from normal asymptomatic subjects. In all cases gastric cancer cells showed CEA or CEA-like activity irrespective of the histological type of cancer. The activity was located mainly in cellular membranes, on the brush border or in apical parts of the cell cytoplasm. CEA activity could not be demonstrated in mucosa specimens from normal controls. These results indicate that the appearance of CEA or CEA-like substances is regularly associated with the malignant transformation of epithelial cells of the gastric mucosa.

The quantitative analysis of circular dichroic spectra of native human plasma fibronectin according to the method of Provencher and Glöckner [Provencher, S. W., & Glöckner, J. (1981) Biochemistry 20, 33-37] indicated the presence of beta-sheet (79%), beta-turn (21%), but no alpha-helix or random coil in the secondary structure. The calf alveolar heparan sulfates induced a change in the conformation of fibronectin: the magnitude of the change depended on the molecular properties of the particular heparan sulfate preparations.

48 patients in the first trimester of pregnancy received a single oral dose of placebo or enisoprost (prostaglandin E1 analogue): 10, 25, 50, 100, 200 or 400 micrograms. Uterine activity was recorded for 1 h before and for 4 h after administration of the study drug with a Millar microtransducer. There was a dose-related increase in mean resting and active pressure, but not in frequency of pressure cycles. At the highest dose used, namely 400 micrograms, all 6 subjects bled. Enisoprost dilated the cervix and any further dilatation required was easy to accomplish.