Adiponectin multimers in maternal plasma.

OBJECTIVE: Adiponectin is an anti-diabetic, anti-atherogenic, anti-inflammatory, and angiogenic adipokine that circulates in oligomeric complexes including: low molecular weight (LMW) trimers, medium molecular weight (MMW) hexamers, and high molecular weight (HMW) isoforms. The aim of this study was to determine whether there are changes in adiponectin multimers in pregnancy and as a function of maternal weight. STUDY DESIGN: In this cross-sectional study, plasma concentrations of total, HMW, MMW, and LMW adiponectin were determined in women included in three groups: (1) normal pregnant women of normal body mass index (BMI) (n = 466), (2) overweight pregnant women (BMI >or=25; n = 257), and (3) non-pregnant women of normal weight (n = 40). Blood samples were collected once from each woman between 11 and 42 weeks of gestation. Plasma adiponectin multimer concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Non-parametric statistics were used for analysis. RESULTS: (1) The median HMW adiponectin concentration and the median HMW/total adiponectin ratio were significantly higher, and the median LMW adiponectin concentration was significantly lower in pregnant women than in non-pregnant women. (2) Among pregnant women, the median plasma concentration of total, HMW, and MMW adiponectin was significantly higher in normal weight women than in overweight patients. (3) Maternal HMW was the most prevalent adiponectin multimer regardless of gestational age or BMI status. (4) There were no significant differences in the median concentration of total, MMW, and LMW adiponectin and their relative distribution with advancing gestation. CONCLUSION: Human pregnancy is characterized by quantitative and qualitative changes in adiponectin multimers, especially the most active isoform, HMW adiponectin.

Tumor markers (pregnancy protein 1, placental-specific tissue protein 10, placental-specific tissue protein 12, alpha1-fetoprotein) in patients with bronchial carcinoma.

The concentrations of pregnancy protein 1 (SP1), placental-specific tissue protein 10 (PP10), placental-specific tissue protein 12 (PP12) and alpha1-Fetoprotein (AFP) were analyzed in serum samples of 83 patients with bronchial carcinoma at stages II-IV Protein levels were determined by means of single radial immunodiffusion, rocket immuno-electrophoresis, radioimmunoassay and enzyme- immunoassay. PP12 and AFP serum concentrations were significantly increased in the cancer group compared with the control group. PP12 (control group: x=54.08 microg/l; s=61. 70; tumor group: x = 122.52 microg/l; s = 131.16); AFP (control group: x=3.05 microg/l; s=3. 76; tumor group: x = 8.29 microg/l; s = 17.75). SPI was found in only 22 cases of the tumor group. PP10 (control group: x = 2.25 microg/l; s = 0.866; tumor group: x = 2.503 microg/l; s=1.508). Given that at least two of the tested parameters were determined to be in the pathological range, the sensitivity amounted to 0.64 and the specificity to 0.92.

Cloning, sequencing, structural and molecular biological characterization of placental protein 20 (PP20)/human thiamin pyrophosphokinase (hTPK).

Full-length cDNAs of placental protein 20 (PP20) were cloned by screening a human placental cDNA library, which encode a 243 amino acid protein, identical to human thiamin pyrophosphokinase (hTPK) as confirmed by protein sequence analysis. Genomic alignment showed that the PP20/hTPK gene contains 9 exons. It is abundantly expressed in placenta, as numerous EST clones were identified. As thiamine metabolism deficiencies have been seen in placental infarcts previously, these indicate that PP20/hTPK may have a role in placental diseases. Analysis of the 1kb promoter region showed numerous putative transcription factor binding sites, which might be responsible for the ubiquitous PP20/hTPK expression. This may also be in accordance with the presence of the protein in tissues responsible for the regulation of the exquisite balance between cell division, differentiation and survival. TPK activity of the purified and recombinant protein was proved by mass spectrometry with electrospray ionization. By Western blot, PP20/hTPK was found in all human normal and tumorous adult and fetal tissues in nearly equal amounts, but not in sera. By immunohistochemical and immunofluorescent confocal imaging methods, diffuse labelling in the cytoplasm of the syncytiotrophoblasts and weak staining of the trophoblasts were observed, and the amount of PP20/hTPK decreased from the first trimester to the end of gestation. A 3D model of PP20/hTPK was computed (PDB No.: 1OLY) by homology modelling. A high degree of structural homology showed that the thiamin binding site was highly similar to that of the mouse enzyme, but highly different from the bacterial ones. Comparison of the catalytic centre sequences revealed differences, raising the possibility of designing new drugs which specifically inhibit bacterial and fungal enzymes without affecting PP20/hTPK and offering the possibility for safe antimicrobial therapy during pregnancy.

Homology modelling and molecular dynamics studies of human placental tissue protein 13 (galectin-13).

The primary structure of the newly sequence analysed placental tissue protein 13 (PP13) was highly homologous to several members of the beta-galactoside-binding S-type lectin (galectin) family. By homology modelling, the three-dimensional structure of PP13 was built based on high-resolution crystal structures of homologues and also their characteristic 'jellyroll' fold was found in the case of PP13. Our model has been deposited in the Brookhaven Protein Data Bank. By multiple sequence alignment and structure-based secondary structure prediction, we underlined the structural similarity of PP13 with its homologues. The secondary structure of PP13 was identical with 'proto-type' galectins consisting of a five- and a six-stranded beta-sheet, joined by two alpha-helices, and galectins' highly conserved carbohydrate-recognition domain (CRD) was also present in PP13. Of the eight consensus residues in the CRD, four identical and three conservatively substituted were shared by PP13. By docking simulations PP13 possessed sugar-binding activity with highest affinity to N-acetyllactosamine and lactose typical of most galectins. All ligands were docked into the putative CRD of PP13. Based on several lines of evidence discussed in this paper demonstrating that PP13 is a novel galectin, PP13 was also designated galectin-13. These computational results provide some new insights into the possible role and importance of PP13 in various processes of the human body and can be of help in the initial steps of further functional research.

Overexpression of placental tissue protein 17b/TIP47 in cervical dysplasias and cervical carcinoma.

In our previous Western- and Northern-blot investigations, high over-expression of placental protein 17b/TIP47 was detected in extracts of human cervical carcinoma tissues compared to normal conditions of the cervical tissue. PP17b serum levels were also elevated in untreated cervical carcinoma patients compared to healthy controls. In the present study, the expression pattern of PP17 proteins was investigated in various cervical dysplasias and in cervical carcinoma tissue specimens by the streptavidin-biotin immunoperoxidase technique using PP17-specific antiserum. In normal third-trimester human placentas, which served as positive controls, mainly cytoplasmic PP17 immunostaining of syncytiotrophoblasts and chorionic trophoblasts was observed. Normal human uterine cervical squamous and glandular epithelia were negative or weakly positive, while in low grade dysplasias (CIN I-II) only the cytoplasms of dysplastic cells were weakly positive or positive; in high grade dysplasias (CIN III/ISC) cytoplasms of the dysplastic cells were strongly positive. Normal and superficial cells in the differentiated zones were negative in all tissue specimens. In cases of invasive epithelial cervical carcinomas, small basal-type tumour cells were mostly negative whilst cells with squamous differentiation were strongly positive for PP17. Our hypotheses for this newly detected phenomenon are briefly discussed.

Molecular cloning and characterization of placental tissue protein 18 (PP18a)/human mitochondrial branched-chain aminotransferase (BCATm) and its novel alternatively spliced PP18b variant.

Five different insert-length cDNAs encoding for soluble placental tissue protein 18 (PP18) variants were isolated by screening a human placental cDNA library using monospecific anti-PP18 serum. Sequence analysis of the longest clone showed that the insert contains an open reading frame encoding for a 392 residue-long protein with a 27 amino acid mitochondrial targeting sequence. The mature protein-designated PP18a-is 41.264 kDa consisting of 365 residues and is identical to the previously isolated and characterized PP18 antigen described in 1985. We also found a new, alternatively spliced cDNA encoding for a 300 residue-long, 33.776 kDa protein, which was designated PP18b. Alignment search of the protein databank showed that PP18a is almost entirely identical to the human mitochondrial branched-chain aminotransferase, while PP18b is its newly discovered splicing variant. We detected the two PP18 variants in normal adult and fetal human tissues besides the mitochondrial (only PP18a) and cytosolic (only PP18b) fractions of term placenta with chemiluminescence Western blot analysis. The 41 kDa PP18a variant was expressed ubiquitously, while the 33 kDa PP18b variant was found in smaller amounts in nearly all tissues. Trace amounts of the variants were present in the sera of non-pregnant healthy controls, as well as in pregnant women, but there was no real change in serum levels during pregnancy. In conclusion, PP18 variants are not specific for the placenta. Aminotransferase activity of placental origin PP18 antigens was verified by structural analysis and by a coupled branched-chain aminotransferase/glutamate dehydrogenase assay.

Isolation and sequence analysis of a cDNA encoding human placental tissue protein 13 (PP13), a new lysophospholipase, homologue of human eosinophil Charcot-Leyden Crystal protein.

Expression of placental tissue protein 13 (PP13) in different human tissues was investigated by chemiluminescence Western blot analysis using monospecific anti-PP13 serum. In term placentae we detected a 16 kDa single protein band immunochemically identical to the purified PP13 antigen. After investigation of 26 types of human fetal and adult tissue, PP13 was also found in certain other normal and tumorous tissue extracts. It is not secreted into circulation as we could not find PP13 in sera of pregnant women. A full length cDNA with 578 bp insert was isolated by screening a human placental cDNA library with anti-PP13 serum. The open reading frame of the cDNA encodes for a 139-residue-long protein with a predicted molecular mass of 16.118 kDa, identical to the previously isolated and characterized PP13 antigen described in 1983. By alignment search of the protein databank PP13 is highly homologous (69 per cent) to the 16.5 kDa human eosinophil Charcot-Leyden Crystal protein, a unique dual-function lysophospholipase, a member of the beta-galactoside binding S-type animal lectin superfamily. Northern blot analysis revealed a 600 bp PP13 mRNA, detected only in placental tissue from 16 types of human healthy adult tissue. Lysophospholipase activity of PP13 was confirmed by(1)H and(31)P nuclear magnetic resonance (NMR) measurements.

Cloning and sequencing of human oncodevelopmental soluble placental tissue protein 17 (PP17): homology with adipophilin and the mouse adipose differentiation-related protein.

Using a monospecific anti-PP17 antiserum, we detected 4 different molecular-weight PP17 immunoreactive proteins (31,500 kD PP17a, 48, 000 kD PP17b, 60,900 kD PP17c and 74,000 kD PP17d) in different normal adult and fetal human tissues, and in term placenta, by chemiluminescence Western blot analysis. These proteins are overexpressed in cervix carcinoma tissue. Furthermore, increased amounts of PP17b are secreted into the circulation in cervix carcinoma patients; after radical surgery, PP17b serum levels are decreased, and the protein probably has an oncodevelopmental significance. cDNAs were isolated from a human placental cDNA library with the monospecific anti-PP17 antiserum. Sequence analysis of the clones showed that they encode for the 251 residue long PP17a variant, which is identical to the previously isolated and characterized PP17 antigen described in 1983. An alignment search of the protein databank showed that PP17a is homologous to human adipophilin and mouse adipose differentiation-related protein. PP17c turned out to be a dimer of PP17a, while PP17b and PP17d immunoreactive proteins recently detected on Western blots require further investigations.

Is placental tissue protein 17b/TIP47 a new factor in cervical cancer genesis?

We identified novel members of the placental tissue protein 17 (PP17) protein family which consists of different-size variants of PP17; cDNAs of PP17a and PP17b variants have also been cloned and sequence analyzed. By Western-blot analysis in cervical carcinoma tissues we found overexpression of PP17b. Compared to healthy controls a mean five-fold increase in the amount of PP17b was also detected in the sera of untreated cervical carcinoma patients, which declined after radical operations. In our recent findings, in sera of inoperable cervical carcinoma patients, we also found elevated PP17b levels which did not change after irradiation. By Northern-blot analyses we confirmed PP17b overexpression in cervical carcinoma tissues and also the alternative splicing of PP17 mRNAs in various normal human tissues. Presently, the amino acid sequence of TIP47--a mannose-6-phosphate receptor cargo selection device--turned out to be identical to that of PP17b. Due to its oncodevelopmental function, PP17b/TIP47 is more than likely to be connected to HSV-2 infection, which is probably one of the main etiopathogenic agents of cervical carcinoma along with the HPV virus, and may open a new trend in the research of pathological processes in human uterine cervical cancer.

Cloning and sequence analysis of cDNAs encoding human placental tissue protein 17 (PP17) variants.

Using monospecific anti-PP17 serum with chemiluminescence Western-blot analysis, we detected different molecular-mass variants of human soluble placental tissue protein 17 (PP17) in different normal adult and fetal human tissues besides term placenta. 13 cDNAs with three different insert lengths encoding PP17 variants were isolated by screening a human placental cDNA library. Sequence analysis of the shortest clones showed that the inserts contain the same open reading frame encoding PP17a variant (28,129 kDa) consisting of 251 residues, which is identical to the previously isolated and characterised PP17 antigen described in 1983. The ubiquitous PP17b variant is encoded by longer clones and contains 434 residues with a predicted molecular mass of 47,208 kDa. Compared to normal conditions, these newly discovered PP17 variants are overexpressed in cervix carcinoma tissue, as are their three different-size messenger RNAs in HeLa cell line. Increased amounts of PP17b are secreted into the circulation in cervix carcinoma patients. We also observed a typical elevation in serum levels of PP17 variants during healthy pregnancy. An alignment search of the protein databank showed that PP17a and PP17b are homologous to adipose tissue differentiation and lipid-droplet-associated proteins: human adipophilin, mouse adipose differentiation-related protein and rat perilipin A and B.

Closure or non-closure of visceral peritoneums after abdominal hysterectomies and Wertheim-Meigs radical abdominal hysterectomies.

The effect of visceral peritoneal closure after conventional abdominal hysterectomies and Wertheim-Meigs radical operations was studied clinically. No considerable differences were found in the postoperative staying period; however, the incidence of complications were less in the peritoneal non-closure group (n = 91) than in the control, peritonealized group (n = 149). Significantly lower was the number of postoperative irregular pyelogram in cases without peritoneal closure (n = 25) of radical abdominal operations than in the control group (n = 49) with peritoneal suturing. We therefore suggest that the lack of suturing of visceral peritoneums has some advantages after abdominal hysterectomies and especially has benefits for Wertheim-Meigs operations.

Initial experience with placental protein 4 (PP-4) as tumor marker in cervical and endometrial cancer.

PP-4, a recently characterized glycoprotein from human placenta was studied using a specific double-antibody radioimmunoassay in sera of 130 volunteers, 74 cervical cancer patients and 43 endometrial cancer patients. Elevated levels (greater than 3 micrograms/l) were found in 35 (47.3%) cervical cancer patients and in 18 (41.9%) endometrial cancer patients. Degree of elevation were not correlated with clinical stage, histology, and histological degree of differentiation. 36 patients with cervical cancer and 20 patients with endometrial cancer were monitored on two to seven occasions during four to 50 weeks. Rising, remaining unchanged of falling levels of PP-4 correlated with progression, stabilization or regression of disease 55.5% in patients with cervical and 65.0% in patients with endometrial cancer. During and some months after external telecobalt irradiation therapy wide range of PP-4 levels were observed in some patients. The study suggest that PP-4 can be regarded as a tumor associated protein which most likely can serve as tumor marker in cervical and endometrial cancer.

Comparative study of enzyme-linked immunosorbent assay and radioimmunoassay techniques in determining serum placental protein 14 levels in gynecologic patients.

A comparative analysis of radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques in determining placental protein 14 (PP14) levels in gynecologic patients was performed. Serum PP14 levels were assessed in the patients in the last period of gestation, during menopause and in healthy nonpregnant controls. The possible discrepancy in PP14 levels was also examined in gynecologic patients with benign and malignant tumors. Both RIA and ELISA techniques proved to be sufficiently sensitive to measure PP14 concentration.

Placental protein 4 as a possible tumor marker in ovarian tumors.

Placental protein 4 (PP-4), a recently characterized glycoprotein from human placenta, was studied using a specific double-antibody radioimmunoassay in sera of 130 volunteers, 76 ovarian tumor patients and in ovarian tumor cyst fluid and ascites of 21 patients. Elevated levels (greater than 3 micrograms/l) were found in 45 of 52 ovarian cancer patients (86.5%). PP-4 levels correlated significantly with staging. 31 patients with malignant ovarian tumor were monitored on 2-9 occasions during 5-82 weeks. Rising or falling levels of PP-4 correlated with progression or regression of disease in 25 of 31 instances (80.6%). Elevated levels were found in 10 of 24 benign and borderline ovarian tumors. Elevated PP-4 level does not indicate malignancy in each case. PP-4 can be regarded as tumor-associated antigen and an tumor marker in oncological practice.

Circulating levels of placental protein 12 (PP 12) in diabetic pregnancy complicated by retinopathy.

Placental protein 12 (PP 12) is a soluble tissue antigen. Immunohistochemical studies have localized PP 12 in the placental syncytiotrophoblast, chorion and amnion, and also in the decidua. During normal pregnancy serum-PP 12 is already raised in the first trimester, there is then a peak at 18 weeks, a gradual fall until 32 weeks, and a moderate increase thereafter. The mean of the healthy control group at 18 weeks of pregnancy was 122.9 +/- 47.5 micrograms/l. The mean of the diabetic group with retinopathy at the same time was 192.2 +/- 78.8 micrograms/l. There was no significant difference between background retinopathy and the proliferative form of diabetic retinopathy. At all times during pregnancy the median values of PP 12 in diabetic pregnancies were significantly (p less than 0.01) above the control values. Increased PP 12 levels in diabetic pregnancy complicated by retinopathy are probably caused by decidual, placental and amniotic leakages.

Elevated sera levels of SP-1 induced by chemotherapy in patients with metastatic gestational trophoblastic diseases.

6 patients with invasive mole and 5 patients with choriocarcinoma were treated from 1983 till 1986. Serum samples were analyzed by simultaneous determining of pregnancy-specific beta-1-glycoprotein (SP-1) using enzyme-linked immunosorbent assay (ELISA) and beta subunit of human chorionic gonadotropin (beta-hCG) using Serono radioimmunoassay kit. In 2 patients with metastatic gestational trophoblastic disease (MGTD) SP-1 peaks were found during chemotherapy. In patients with MGTD with normalized beta-hCG levels a repeated, temporary elevation of isolated SP-1 levels was observed within some months following chemotherapy. After the last isolated peak of SP-1 the pulmonary metastases disappeared. This phenomenon was interpreted as a consequence of the oncolytic process in the affected tissue. In 1 patient with nonmetastatic choriocarcinoma SP-1 ELISA pseudoreaction was found. To recognize these pseudoreactions, a control plate with nonimmunized rabbit IgG was used, simultaneously with SP-1 determinations.

[Placental protein 12 (PP 12)/insulin-like growth factor binding protein (IGF-bp) in pregnancy in metabolically healthy patients and diabetic patients with retinopathy]. / Plazentaprotein 12 (PP 12)/Insulin like growth-factor binding protein (IGF-bp) in der Schwangerschaft von Stoffwechselgesunden und Diabetikerinnen mit Retinopathie.

The placental protein 12 (PP 12) was found to be identical with the insulin like growth-factor binding protein (IGF-bp). In present investigations serum samples of 420 healthy pregnant women between 7th and 40th weeks of gestation were proved regarding to their IGF-bp concentrations by means of a radioimmunoassay. For each pregnancy week means and standard deviations were estimated. IGF-bp serum concentrations continuously increased up to the 18th week (140 +/- 40 micrograms/l); followed by decreasing of the values up to the 30th week (102 +/- 44 micrograms/l) and then slightly increased up to term (121 +/- 46 micrograms/l). In addition the IGF-bp estimations were performed in maternal serum samples of 45 diabetic pregnant women complicated by retinopathia diabetica benigna as well as proliferativa; sum total 101 serum samples. In patients with benign retinopathy 82% (12/68) and in patients of proliferative retinopathy 85% (5/33) of the IGF-bp values were increased above the two times standard deviation.