Study of molecular forms of ovine and human prolactin]

After labelling of human and ovine prolactine by the lactoperoxydase method, each prolactine shows at least three molecular forms : amorphous aggregates of large molecular weight, one dimere (M.W. no. 50 000) and one monomere (M.W. no. 25 000). The monomere and the dimere can spontaneously give rise to the aggregates. But the dimere never give monomere and monomere never give dimere. Urea 8 M alone have not dissociating action on the polymeres or on the dimere. But urea 8 M plus mercaptoethanol transforms polymeres into dimeres and monomeres.     

Heterogeneity of ovine prolactin after labeling with iodine 125: value of labeling with lactoperoxidase]

The authors have shown an heterogeneity of the ovine prolactine molecule after labelling with iodine 125. As well with chloramine T as with lactoperoxydase, it appears three molecular species which react with the immune serum antiprolactine (I.S.). The first species is of high molecular weight and is probably constituted of aggregates. Their combination with the I.S. is non specific and give blanks of high value. The second species is a dimere and the third one is the monomere. The two last species react with the I.S. and can give competition curves when they are choosen as tracer. However, if one uses as tracer a product obtained by labelling with chloramine T, the competition appears for high concentrations of native hormone. As if the I.S. recognizes much more the labelled protein than the native one. But if one uses the same species but labelled with lactoperoxydase, the competition appears for concentrations lower than five nanogrammes. In the same time one can see that the specificity toward different I.S. is modified. The authors think that the labelling with lactoperoxydase better preserves the tertiary structure of the native protein than do the labelling with chloramine T.     

Immunofluorescent identification of somatotropic and prolactin cells in the anterior lobe of the hypophysis (pars distalis) of the monkey,Macacus irus

Summary Using antibodies against human growth hormone and ovine prolactine, it is possible to identify somatotropic and prolactin cells in the anterior lobe of the hypophysis (pars distalis) of the monkey, Macacus irus. These cells are present in both adult and infant male and female monkeys. The density of the somatotropic cells is greater at the periphery of the anterior lobe. Prolactin cells are of two types: one type is dispersed throughout the pars distalis and consists of small and large cells, the other type forms clusters of large cells.We wish to express our thanks to Mrs. M. Raccurt (CNRS) for her highly skilled technical assistance     

Sex difference in the relationship between the hypothalamic-pituitary-adrenal axis and sex hormones in obesity

Objective: This study was carried out to investigate the role of sex in the regulation of the hypothalamic‐pituitary‐adrenal (HPA) axis and its relationship with testosterone levels in male and female obesity. Research Methods and Procedures: Twenty‐two obese men (OB‐M) and 29 obese women (OB‐W) participated in the study. Two groups of normal weight men (NW‐M) and women (NW‐W), respectively, served as controls. In basal conditions, blood concentrations of major androgens, sex hormone—binding protein, and gonadotropins were assessed, and the free androgen index (testosterone ×100/ sex hormone‐binding globulin) was calculated. All subjects underwent a combined corticotropin‐releasing hormone plus arginine‐vasopressin stimulation test. Results: OB‐M and NW‐M had higher basal adrenal cortical tropic hormone (ACTH) and cortisol levels than their female counterparts. In addition, ACTH, but not cortisol basal, levels were significantly higher in obese than in normal weight controls in both sexes. OB‐W had a higher response than OB‐M to the combined corticotropin‐releasing hormone plus arginine‐vasopressin test of both ACTH and cortisol [expressed as incremental percentage of area under the curve (AUC%)]. The same finding was present between NW‐W and NW‐M. Basal luteinizing hormone levels were negatively correlated to ACTH_AUC% in both OB‐W and OB‐M. In the OB‐W, however, a positive correlation was found between cortisol_AUC% and testosterone (r = 0.48; p = 0.002), whereas a tendency toward a negative correlation was present in OB‐M. Discussion: In conclusion, we have shown a significant positive relationship between the activity of the HPA axis and testosterone in obese women, which suggests a partial responsibility of increased HPA axis activity in determining testosterone levels. In addition, it clearly seems that, as reported in normal weight subjects, a sex difference in the HPA axis activity still persists even in the presence of obesity.     

Surrogate markers of tumoral angiogenesis

BACKGROUND: Angiogenesis is a prerequisite for tumor growth and metastasis. Vascular cell adhesion molecule1 (VCAM-1) is expressed on endothelial cells as a result of vascular endothelial growth factor (VEGF) stimulation. PURPOSE: To determine if measurement in serum of VEGF or VCAM-1 provides an accurate measure of tumor angiogenesis. METHODS: VCAM-1 and VEGF were measured in the serum of women with early and advanced breast cancer by ELISA. Levels were compared to levels of VCAM-1 and VEGF in women with normal breasts and levels of the endothelial glycoprotein von Willebrand factor. Levels of VEGF and VCAM-1 in women with early breast cancer were correlated with established clinicopathological prognostic markers and intratumoral microvessel density (IMD). RESULTS: In early breast cancer serum VCAM-1 correlated closely with the microvessel density in tumors (r=0.61, p<0.001). Women with lymph node-positive and high-grade tumors had higher levels of serum VCAM-1 than women with lymph node-negative and low-grade tumors. Serum VEGF demonstrated no correlation with established prognostic features or IMD. Levels of VCAM-1 and VEGF were raised in women with advanced breast cancer. CONCLUSION: Serum VCAM-1 is a surrogate marker of angiogenesis in breast cancer and its measurement may help in the assessment of antiangiogenic drugs currently in phase II trials.     

Identification of a breast tumor-associated orosomucoid by concanavalin A affinity chromatography.

Con A-Sepharose affinity chromatography was utilized to examine the glycoproteins in phosphosaline extracts of normal and breast tumor tissues and breast patient sera. In extracts of normal breast tissue, normal sera and patient sera, all glycoproteins were eluted from the Con A-Sepharose with a linear gradient of 0.0-0.5 M alpha-methylmannose. Using breast tumor extracts, a glycoprotein peak which could not be eluted as with normal tissue extracts was observed. This tightly-binding peak could be eluted from the Con A-Sepharose with acetate buffer containing 1.0 M KCl. Polyacrylamide electrophoresis of this tightly-binding glycoprotein peak revealed one major glycoprotein and four minor glycoproteins. The major glycoprotein obtained from electrophoresis represented about 60% of the Con A-Sepharose tightly-binding protein and reacted with antiserum to human orosomucoid (alpha 1-acid glycoprotein). All glycoproteins isolated from tumor tissue extracts appeared to represent normal serum constituents as they were retained on an immunoadsorbent containing antibodies to normal serum proteins. The possible significance of the isolated tumor-associated orosomucoid is discussed.     

Cancer-associated serum galactosyltransferase activity. Demonstration in an animal model system.

Two different lines of solid tumors were produced in outbred hamsters by subcutaneous injection of polyoma transformed BHK cells. Growth of the tumors correlated with the appearance in serum of an electrophoretically distinct peak of galactosyltransferase: NeuAc-, Gal-free fetuin acceptor activity on polyacrylamide gels. This slow moving peak of enzyme activity (GT-HH) was detected before solid tumors could be grossly observed and the amount of activity in this peak was also found to be linearly related with growth of the tumor. GT-IIH was not detectable in control animals and separated from a faster migrating major area of serum galactosyltransferase activity (GT-IH) found in sera of both control and tumor-bearing hamsters. These two activities were shown to maintain their respective mobilities on re-electrophoresis. Solubilized enzyme derived from excised tumors demonstrated an electrophoretic mobility on polyacrylamide gels identical to that for GT-IIH present in serum from tumor-bearing animals. In contrast, enzyme activity solubilized from livers of both control or tumor-bearing hamsters showed a mobility similar to that of the faster moving serum galactosyltransferase enzyme activity, i.e. GT-IH. In addition, medium derived from nonconfluent BHKpy cells in tissue culture contained galactosyltransferase activity which co-electrophoresed with the slower migrating characteristics of galactosyltransferase activities derived from serum (control and tumor-bearing), solid tumors, liver and BHKpy cells in tissue culture were compared. All kinetic properties were similar with the exception that the Km UDP-galactose of GT-IIH (1.0 X 10(-5) M) was half that of GT-IH (2.0 X 10(-5) M).     

FADD is required for DR4- and DR5-mediated apoptosis: lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor family that can kill a wide variety of tumor cells but not normal cells. TRAIL-induced apoptosis in humans is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). What constitutes the signaling molecules downstream of these receptors, however, remains highly controversial. Using the FADD dominant negative molecule, several groups have reached different conclusions with respect to the role of FADD in TRAIL-induced apoptosis. More recently, using FADD-deficient (-/-) mouse embryonic fibroblasts, Yeh et al. (Yeh, W.-C., Pompa, J. L., McCurrach, M. E., Shu, H.-B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998) Science 279, 1954-1958) concluded that DR4 utilizes a FADD-independent apoptotic pathway. The latter experiment, however, involved transient overexpression, which often leads to nonspecific aggregation of death domain-containing receptors. To address this issue in a more physiological setting, we stably transfected mouse DR4/5, human DR4, or human DR5 into FADD(-/-) mouse embryonic fibroblast cells. We showed that FADD(-/-) MEF cells stably transfected with TRAIL receptors are resistant to TRAIL-mediated cell death. In contrast, TRAIL receptors stably transfected into heterozygous FADD(+/-) cells or FADD(-/-) cells reconstituted with a FADD retroviral construct are sensitive to the TRAIL cytotoxic effect. We conclude that FADD is required for DR4- and DR5-mediated apoptosis.     

Influence of gender and oral contraceptives intake on innate and inflammatory response. Role of neuroendocrine factors

The purpose of the present work was to determine differences between young men (M), and women in the follicular phase (W) and women taking oral contraceptives containing ethinylestradiol (CW) in the phagocytic process of neutrophils (chemotaxis, phagocytosis and microbicide capacity), in the serum concentrations of cytokines both pro-inflammatory (IFNgamma, TNFalpha, IL-12, IL-6, IL-8) and anti-inflammatory ones (IL-10 and IL-13), and in neuroendocrine factors with immunomodulatory capacity (estradiol, prolactin, cortisol, catecolamines and 72 kDa heat shock proteins, Hsp72). Men showed a lower phagocytosis and microbicide capacity than women, and less serum concentrations of the pro-inflammatory cytokines IL-6 and IL-8. CW neutrophils also showed a lower phagocytic capacity than W neutrophils, together with less serum IL-8 concentration. CW showed the highest serum concentration of IL-13. However, no statistical changes were observed in the pro-inflammatory cytokines: INF-gamma, TNF-alpha, IL-12 and in the anti-inflammatory cytokine IL-10. The greater anti-inflammatory status in CW than in W was parallel with lower concentrations of oestrogens. Cortisol, prolactin, and the extracellular Hsp72 seem to be involved in the gender- and contraceptives-induced differences in the inflammatory response. While cortisol (in general an immunosuppressive hormone) showed the highest values in CW, prolactin and Hsp72 (an immunopermissive factors) showed the lowest values in CW and M. Less clear is the participation of catecholamines in the gender-inflammatory differences.     

DNA and protein sequence conservation at the replication terminus in Bacillus subtilis 168 and W23.

Cloned DNA from the replication terminus region of Bacillus subtilis 168 was used to identify and construct a restriction map of the homologous region in B. subtilis W23. With this information, DNA from the terminus region of W23 was cloned and the sequence was determined for a 1,499-base-pair segment spanning the expected terC site. The position of the site was then located more precisely. Use of the cloned DNA from strain W23 as a probe for digests of DNA from exponentially growing cells of the same strain established the presence of the slowly migrating replication termination intermediate (forked DNA). The orientation and dimensions of the forked molecule were consistent with arrest of the clockwise fork at the terC site in W23, as has been shown to occur in strain 168. Thus, despite significant differences between the two strains, the same termination mechanism appears to be used. The DNA sequences spanning the terC site in strains 168 and W23 showed a high level of homology (90.2%) close to the site but very little at a distance of approximately 250 base pairs from the site in one particular direction. The overall sequence comparison emphasised the importance of the open reading frame for a 122-amino-acid protein adjacent to terC. Although there were 22 base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was completely conserved. It is suggested that the amino acid sequence conservation reflects a role for the protein in the clockwise fork arrest mechanism as proposed earlier (M.T. Smith and R.G. Wake, J. Bacteriol. 170:4083-4090, 1988).     

The interaction of liver fatty-acid-binding protein (FABP) with anionic phospholipid vesicles: is there extended phospholipid anchorage under these conditions?

Liver FABP (fatty-acid-binding protein) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, lysophospholipids and bile acids. Liver FABP is also able to bind to anionic phospholipid vesicles under conditions of low ionic strength, and membrane binding results in the release of bound ligand. However, the molecular interactions involved in binding to the phospholipid interface and the mechanism of ligand release are not known. Ligand release could be due to a significant conformational change in the protein at the interface or interaction of a phospholipid molecule with the ligand-binding cavity of the protein resulting in ligand displacement. Two portal mutant proteins of liver FABP, L28W and M74W, have now been used to investigate the binding of liver FABP to anionic phospholipid vesicles, monitoring changes in fluorescence and also fluorescence quenching in the presence of brominated lipids. There is a large increase in fluorescence intensity when the L28W mutant protein binds to vesicles prepared from DOPG (dioleoyl-sn-phosphatidylglycerol), but a large decrease in fluorescence intensity when the M74W mutant binds to these vesicles. The Br(4)-phospholipid prepared by bromination of DOPG dramatically quenches both L28W and M74W, consistent with the close proximity of a fatty acyl chain to the tryptophan residues. The binding of liver FABP to DOPG vesicles is accompanied by only a minimal change in the CD spectrum. Overall, the results are consistent with a molecule of anionic phospholipid interacting with the central cavity of the liver FABP, possibly involving the phospholipid molecule in an extended conformation.     

Association of C/T polymorphism in the LRP5 gene with circulating follicle stimulating hormone in Caucasian postmenopausal women

The LRP5 gene is believed to be primarily associated with bone metabolism via Wnt signaling. The latter pathway, however, appears to control various other systems outside the skeleton. To find the relationships of the LRP5 gene to serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) in the cohort of normal postmenopausal women, we identified the C/T (c.4037:A1330V) polymorphism in the LRP5 gene using a restriction analysis of the PCR product in a cohort of 165 untreated pre- and post-menopausal women. In a subset of 111 post-menopausal women we analyzed the association between the LRP5 genotype and serum levels of sex-hormones including FSH and LH. The distribution of CC, TC and TT genotypes of the C/T polymorphism in the whole group was 73.9 %, 23.6 % and 2.4 %, respectively, which is comparable with other Caucasian populations. As no TT homozygote was found in the group of post-menopausal women, serum sex-hormones were compared between CC and TC genotypes. Women with the CT allele combination had markedly higher serum FSH levels as compared to carriers of the CC genotype (p<0.004). No differences between these genotypes were found in serum LH levels as well as the circulating sex-steroids such as estradiol, testosterone, dehydroepiandrosterone and/or its sulphate, androstenedione and SHBG. To conclude, the LRP5 gene is associated with circulating FSH in normal post-menopausal women in the present study. The mediating role of subtle undetectable variations in estrogen levels is discussed. We did not find any relationship between the LRP-5 genotype and serum LH levels.     

Identification of PSME3 as a novel serum tumor marker for colorectal cancer by combining two-dimensional polyacrylamide gel electrophoresis with a strictly mass spectrometry-based approach for data analysis

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550-6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.     

Thyroid Function And 3, 3'-Diiodothyronine Sulfate Cross-Reactive Substance (Compound W) in Maternal Hyperthyroidism With Antithyroid Treatment

ObjectiveTo test whether the serial measurement of maternal levels of compound W, a 3, 3′-diiodothyronine sulfate cross-reactive substance, can serve as a potential indicator of fetal thyroid function in pregnant women receiving antithyroid medication.MethodsCompound W was measured repeatedly in serum of pregnant women with hyperthyroidism treated with antithyroid medication. Free thyroxine levels of mothers and serum thyroid-stimulating hormone levels of 1-day-old neonates were analyzed by local clinical or state laboratories.ResultsUse of minimal antithyroid medication impaired the progressive increase of compound W seen in euthyroid mothers during pregnancy. At term, depressed compound W levels in maternal serum were found in 7 of 22 pregnancies; in 1 case, maternal compound W was suppressed and newborn thyroid-stimulating hormone was elevated. Seven mothers with treated hyperthyroidism failed to show an increase in serum levels of compound W after midterm.ConclusionNormal progression of maternal serum compound W may be an index of normal fetal thyroid development in mothers with hyperthyroidism treated with necessary antithyroid medication. (Endocr Pract. 2011;17:170-176)     

Characterization of different tumor antigens present in cells transformed by simian virus 40.

In addition to large T and small t antigens, cells transformed by simian virus 40 (SV40) commonly contain other proteins which specifically immunoprecipitate with SV40 anti-T serum and which are not detected in untransformed cells. The additional tumor antigens (T-Ags) fall into two groups: those having a close structural relationship with normal SV40 T-Ags, and those unrelated to large T and small t. The latter are probably nonviral T-Ags (NVT-Ags). The NVT-Ags comprise a family of proteins of molecular weight 50,000-55,000. Fingerprint analysis shows that NVT-Ags have few if any peptides in common with large T or small t, and that they lack the amino terminal tryptic peptide and the peptides unique to small t. NVT-Ags from different species have different fingerprints, but those isolated from different transformants of the same cell line are identical. The size of NVT is unaltered in cells transformed by mutants of SV40 with deletions in the region 0.60-0.55 map units. The mRNA for NVT does not hybridize to SV40 DNA. The other forms of T-Ag isolated from transformed cells fall into three classes: shortened forms of large T (truncated large T); multiple species of T-Ag with molecular weights very similar to, but distinct from, those of normal large T (large T doublets and triplets); and elongated forms of large T (super T). These proteins all contain the normal amino terminus of SV40 T-Ags, and the truncated forms of large T lack peptides from the carboxy terminal half of large T. One species of super T (molecular weight 130,000) contains only those methionine tryptic peptides present in normal large T, although it may contain some peptides in more than one copy.     

Intercellular invasion and the organizational stability of tissues: a role for fibronectin

Intracellular invasion is the movement of cells of one type into the fabric of other, contiguous tissues. Invasion is a signature behavior of the malignant tumor and also is found as part of the normal behavior of inflammatory blood cells and tissues engaged in the morphogenetic movements of normal embryogenesis and in a number of instances of normal and pathological tissue remodeling in the adult. Informed by the view that the underlying mechanisms of invasion will be similar for tumor cells and invasive blood and embryonic cells, this review adopts a comparative approach to the analysis of invasion. Invasion results in the development of a diffuse interface between contiguous tissues. Its alternative is the maintenance of stable, planar tissue boundaries. This is the more usual condition for contiguous tissues in the animal. This review will focus on the processes that, on the one hand, stabilize planar contact interfaces between tissues, and, on the other, promote the destabilization of tissue integrity by fostering intercellular invasion. Particular attention is devoted to a role for adhesive interactions mediated by the matrix adhesion molecule, fibronectin. In certain instances, fibronectin in the matrix promotes invasion whereas in others, the presence of fibronectin prevents invasion. The distinction appears to depend on whether the invasive tissue is migrating into an acellular extracellular matrix or whether invasion involves densely cellular tissues. In the first instance, fibronectin promotes invasion, whereas in the second, it stabilizes the interface of the contacting tissues and prevents invasion.     

Clinical use of metoclopramide test in the diagnosis of women with hyperprolactinemia

14 women with elevated prolactin (PRL) serum levels (greater than 25 ng/ml) were given 2.5 mg of metoclopramide, by bolus intravenous injection, to evaluate its diagnosic potential as a stimulus for PRL release. Following metoclopramide injection there was a prompt increase in serum PRL in normal subjects and in patients with moderate PRL elevations associated with galactorrhea-oligomenorrhea. The women with amenorrhea-galactorrhea regardless of the presence of absence of a pituitary tumor, showed a blunted response. Metoclopramide failed to induce TSH secretion in all cases. In conclusion: the use of the metoclopramide test provides no additional clinical information to that furnished by the basal serum PRL concentration for the hyperprolactinemic patient.