Protein tyrosine phosphatase activity in human endometrium.

Protein tyrosine phosphatase (PTP) activity was demonstrated in human endometrium by a histochemical method using phosphotyrosine as substrate. For comparative purposes, non-specific acid phosphatase (AcP) activity was also examined. Protein tyrosine phosphatase activity was very low in proliferative and atrophic endometrium, but its activity was increased 9-fold in glandular epithelium during the secretory phase, and 48-fold in predecidual endometrium, induced by a progestagen-releasing intrauterine device, compared with the proliferative endometrium. Thus, PTP activity appeared to be progesterone-induced. Endometrial PTP appeared to be cellular rather than secretory in origin; its activity was inhibited by vanadate, and its histochemical properties were different from those of lysosomal AcP, but similar to those of prostatic-type AcP. Endometrial PTP may functionally counteract the effects of protein tyrosine kinases (PTKs) associated with growth factor receptors and cellular oncoproteins. Cyclic endometrial proliferation and differentiation are thought to be regulated by the autocrine and paracrine pathways by growth factors such as epidermal growth factor, insulin-like growth factor I and platelet-derived growth factors, and their receptors. However, cessation of proliferation could not be explained by the amounts of these growth factors present or their receptors, in that no constant changes at the interface of the late proliferative and early secretory phases were found. Down-regulation of stimulatory-signalling pathways of PTKs by endometrial PTP induced by progesterone may explain the decrease observed in proliferative activity of glandular cells in cyclic endometrium.

Structural determinants for ligand binding and catalysis of triosephosphate isomerase.

The crystal structure of leishmania triosephosphate isomerase (TIM) complexed with 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate (IPP) highlights the importance of Asn11 for binding and catalysis. IPP is an analogue of the substrate D-glyceraldehyde-3-phosphate, and it is observed to bind with its aldehyde oxygen in an oxyanion hole formed by ND2 of Asn11 and NE2 of His95. Comparison of the mode of binding of IPP and the transition state analogue phosphoglycolohydroxamate (PGH) suggests that the Glu167 side chain, as well as the triose part of the substrate, adopt different conformations as the catalysed reaction proceeds. Comparison of the TIM-IPP and the TIM-PGH structures with other liganded and unliganded structures also highlights the conformational flexibility of the ligand and the active site, as well as the conserved mode of ligand binding.

Alkaline fixation-resistant acid phosphatases in human tissues: histochemical evidence for a new type of acid phosphatase in endothelial, endometrial and neuronal sites.

The effect of pH during formalin fixation on acid phosphatases in human tissues was studied. Lysosomal-type acid phosphatase was sensitive to alkaline fixation, being completely inactive after fixation at pH 9.0. Prostatic and tartrate-resistant osteoclastic/macrophagic types were alkaline fixation-resistant, as was an acid phosphatase localized in endothelium, endometrial stromal cells and intestinal nerves. The latter activity was further separable into fluoride- and tartrate-sensitive beta-glycerophosphatase and fluoride-sensitive, tartrate-resistant alpha-naphthyl phosphatase. The activities appeared to represent either different, tightly associated enzymes or separate activity centres of a single enzyme. Alkaline fixation-resistant alpha-naphthyl phosphatase at endothelial, endometrial and neuronal sites was also well demonstrated in unfixed or neutral formalin-fixed sections as tartrate-resistant activity similar to classical tartrate-resistant acid phosphatase, but these phosphatases appear to be antigenically different. Alkaline fixation-resistant acid phosphatase showed a restricted tissue distribution both in endothelium (mainly in vessels of abdominal organs) and at neuronal sites (only in intestinal nerves). Alkaline fixation-resistant acid phosphatase appears to represent a previously unknown or uncharacterized enzyme activity whose chemical properties could not be classified as any previously known type of acid or other phosphatases.

Developmental changes in the expression of neuronal ceroid lipofuscinoses-linked proteins.

Neuronal ceroid lipofuscinoses (NCL) form a distinct group of storage diseases where the normal development of the central nervous system is interrupted and neurons of the neocortex begin to degenerate. Mutations in genes encoding three lysosomal enzymes are the causes for three early-onset forms of NCLs: palmitoyl-protein thioesterase 1 (PPT1) is deficient in human infantile NCL, tripeptidyl peptidase 1 (TTP1) in late-infantile NCL, and cathepsin D in congenital ovine NCL. We wanted to compare the developmental expression profiles of these enzymes in rat brain. In conclusion, the PPT1 expression pattern differed from the two other lysosomal enzymes implicated in NCL diseases, thus suggesting a distinctive role for PPT1 in brain development.

Histochemically demonstrable acid phosphotyrosine phosphatase activity in human tissues.

A histochemical method for the demonstration of activity of acid phosphatase(s) hydrolyzing o-phospho-L-tyrosine at pH 6.2 is described. Due to the instability of ordinary acid phosphatase incubation media containing phosphotyrosine and lead salt, the latter was dissolved in citrate solution (possibly forming a chelate) to obtain a stable incubation mixture. Localization of enzyme activity at the cellular level was good. Phosphotyrosine is known to be hydrolyzed at slightly acid pH by prostatic, macrophagic, and low-molecular weight cytoplasmic acid phosphatases and phosphotyrosyl protein phosphatases. These enzymes have been suggested or are established to be involved in cellular regulation by dephosphorylation of tyrosyl residues of proteins formed by various protein tyrosine kinases. An unexpected finding from various tissues was that the widely distributed lysosomal acid phosphatase did not contribute to the histochemical reaction, possibly due to poor hydrolysis of phosphotyrosine by lysosomal-type acid phosphatase. Various human tissues were studied for their acid phosphotyrosine phosphatase activities, which were then quantitated photometrically. Highest activity was found in the prostate glands, seminal vesicle epithelium, endometrial secretory glands, alveolar macrophages, stratum granulosum of the skin, and lymph nodes. The contribution of various enzymes hydrolyzing phosphotyrosine to histochemically detected activity in different tissues is discussed here.

Histochemically demonstrable protein tyrosine phosphatase in human breast and colorectal cancer: large decrease in its activity in colorectal cancer suggests a tumor suppressor role in colorectal mucosal cells.

Many oncogene products and growth factor receptors are protein tyrosine kinases, and exert their cellular effects by the phosphorylation of tyrosyl residues of effector proteins. The balance and dynamic renewal of phosphotyrosine proteins are also regulated by protein tyrosine phosphatases (PTPs), whose inhibition under experimental conditions causes cellular proliferation and transformation, with a concomitant increase in phosphotyrosine protein content. Inverse effects are obtained by increasing PTP activity. On the basis of these effects, PTPs might also function as tumor suppressors in human tissues. This possibility was further investigated here by demonstrating PTP and phosphotyrosine protein content with histochemical techniques. In normal human breast tissue PTP activity was low and in the majority of breast cancers the activity was increased and exhibited great variation between different cases. When the relationship of phosphotyrosine protein content with PTP was evaluated, no inverse dependence was detected, suggesting that in human breast tissue and cancer PTP may not show tumor suppressor activity. In normal colorectal mucosae PTP activity was high, while in all colorectal cancers it was very low, constituting only 14% of the activity present in normal mucosal cells. The great drop in PTP activity together with reported alterations in a gene encoding a PTP and in a chromosome containing a PTP gene in colorectal cancer strongly suggest that PTP may function as a tumor suppressor in human colorectal mucosae. The decrease in PTP activity may be one factor stimulating or causing neoplastic proliferation in multistep colorectal carcinogenesis.

Immunohistochemically demonstrable pp60(c-SRC) in human breast-cancer.

Both elevated protein-tyrosine kinase activity of pp60(c-src) and the association of its high activity with a short disease-free period have been reported in patients with human breast cancer. In another human malignancy, colon cancer, tyrosine kinase activity and a protein level of pp60(c-src) have been observed to be correlated directly with the malignancy grade of tumors. In the present work, pp60(c-src) was demonstrated immunohistochemically in 48 human breast cancers, and the level demonstrated in this way was compared with prognostic histopathological factors. A weakly positive granular reaction product was detected in the plasma membrane of normal ductal epithelial cells and of cancer cells in 40% of cases of infiltrating breast cancer. In 60% of breast cancers, the pp60(c-src) level was increased, the reaction intensity was elevated, and a positive reaction was seen in cytoplasma and in the nuclear area. When the cancers with a low level of pp60(c-src) were compared with those showing an increased level with respect to prognostic factors (tumor size, axillary metastases and histopathological maligancy grade), no statistically significant differences were found. It seems that although pp60(c-src) is not primarily associated with the malignant growth of all human breast cancers, it may contribute to malignant cell proliferation in many of them.

Phosphotyrosyl-proteins in human breast cancer.

The content of phosphotyrosyl-proteins, substrates of protein tyrosine kinases, was evaluated by immunohistochemical staining in human breast cancers, and its relation to the prognostic factors of cancers was studied. Both protein tyrosine kinase activity and the content of various growth factor receptors and oncoproteins with this activity have been shown to be elevated in breast cancers. The magnitude of the increase has been reported to have prognostic significance; greater increases were associated with poorer prognostic factors of cancers and with shorter survival of patients. Immunohistochemical staining demonstrated a low level of phosphotyrosyl-proteins in normal human breast tissue and in 44% of breast cancers. In 56% of breast cancers the level of phosphotyrosyl-proteins was increased; however, these cancers did not differ from cancers containing a low level with respect to prognostic factors such tumor size, histological differentiation grade or axillary lymph node metastases. The increased content of phosphotyrosyl-proteins in the majority of human breast cancers further supports the observations that growth factor receptors and oncoproteins with tyrosine kinase activities are activated in these cancers. The content of phosphotyrosyl-proteins, however, has no relation to the prognostic factors of cancers.

Sequential appearance of relaxin, prolactin and IGFBP-1 during growth and differentiation of the human endometrium.

Relaxin (RLX) is a product of the human corpus luteum, pregnancy decidua and placenta, prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) are products of the cyclic endometrium and of the pregnancy decidua. All three proteins are thought to function interdependently in endometrium/decidua as local factors within the uterus without reaching the systemic circulation. In this study, the avidin-biotin immunoperoxidase method for immunolocalization with monoclonal or polyclonal antibodies has been applied to serial sections of endometria obtained from patients at different stages of the menstrual cycle and in early and late gestation. This allowed the cellular localization of the three proteins to be followed simultaneously through the reproductive stages from cyclic endometrium to term gestational decidua. The production, as opposed to sequestration of RLX from an ovarian source was demonstrated by the application in parallel of an antibody to the processed hormone and its connecting peptide. RLX was shown localized to the glandular and luminal epithelia in the proliferative and secretory phases. The decidualized stromal cells also immunostained for RLX in the late secretory phase and in early and late pregnancy. PRL was localized first to the glandular epithelium and then stroma, appearing after RLX, IGFBP-1 appeared later in the secretory phase and predominantly in the decidualized stromal cells confirming previous studies. In contrast, all three proteins were immunostained in early pregnancy and increased to term gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of enoxaparin, a low-molecular-weight heparin, and unfractionated heparin, with or without dihydroergotamine, in abdominal hysterectomy.

The effects of administration of low-molecular-weight heparin (enoxaparin 20 mg) once a day, of unfractionated heparin (5000 IU twice a day, and of unfractionated heparin (2500 IU) plus dihydroergotamine (0.5 mg) twice a day were assessed in 100 patients undergoing abdominal hysterectomy. The test medications were given subcutaneously 2 hours before operation and for 3 days thereafter. There were no thromboembolic complications. Intraoperative blood loss, wound haematomas and blood loss via drains during four days after operation were similar in the three groups. None of the 37 patients receiving enoxaparin experienced major postoperative bleeding. Six out of 31 patients receiving unfractionated heparin without dihydroergotamine and two out of 32 patients receiving dihydroergotamine in addition experienced major bleeding necessitating re-operation and/or blood transfusion, (P < 0.05). Enoxaparin caused less major bleeding than unfractionated heparin with or without dihydroergotamine in patients undergoing hysterectomy.

Insulin-like growth factor binding proteins in human breast cancer tissue.

Breast tumor cell lines have been shown to secrete at least five distinct insulin-like growth factor (IGF) binding proteins (IGFBP), the secretion being related to the estrogen receptor (ER) content. In this study we investigated IGFBP mRNA expression and IGFBP content in relation to ER content in human breast tumors. Tissue specimens from 47 breast cancers were studied. In five cases the adjacent histologically normal tissue was also analyzed. IGFBP content in tissue homogenates was studied by Western ligand blot analysis, using [125I] IGF-I as a label, and IGFBP mRNA expression by reverse transcriptase polymerase chain reaction. The results show that human breast tumors express mRNAs encoding IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-5. The pattern of IGFBPs in different tumors varies. No correlation exists between ER content and IGFBPs with molecular weights 24,000 Mr, 28,000 Mr, 34,000 Mr or 43,000 Mr, whereas the 49,000 Mr IGFBP was more abundant in ER negative tumors (P less than 0.05). The IGFBP content was significantly (P less than 0.05) higher in five tumors than in their adjacent normal tissues suggesting that increased content of IGFBPs is a feature typically associated with the malignant transformation of breast tissue.

Decidual transformation of human extrauterine mesenchymal cells is associated with the appearance of insulin-like growth factor-binding protein-1.

Human extrauterine decidual cells were studied for the presence of insulin-like growth factor-binding protein-1 (IGFBP-1) by using an avidin-biotin complex immunoperoxidase method with a purified monoclonal antibody 6303 against IGFBP-1. It is well established that human decidua expresses mRNA for IGFBP-1 and synthesizes and secretes this protein. We now describe, for the first time, that cells morphologically similar to uterine decidual cells found beneath the peritoneal mesothelium in inguinal hernia and in the cervical mucosa during normal pregnancy and in the mesenchymal tissue of the Fallopian tubes and ovaries at term react with the monoclonal antibody for IGFBP-1 equally to endometrial decidual cells. These results imply that the extrauterine mesenchymal cells, which have undergone decidual transformation, are not only morphologically but also functionally, similar to their counterparts in the uterus. Secondly, the data suggest that IGFBP-1 expression is associated with a special type(s) of cells and a certain stage of differentiation rather than special organs in the body.

Effect of haem arginate on colony formation by haematopoietic progenitors in vitro.

The effect of haem arginate, a new stable haem compound, was studied on colony formation by erythroid (CFU-E, BFU-E) and granulocyte-macrophage (CFU-GM) progenitors from the bone marrow of 12 healthy persons. At the concentration of 10 mumol/l haem arginate significantly (p less than 0.05) increased and at the concentration of 200 mumol/l significantly decreased (p less than 0.005) the colony formation of CFU-E. A similar, though not significant, trend was seen in the BFU-E colony growth, while the CFU-GM colony formation was not influenced at any concentration between 1 and 200 mumol/l. We conclude that the stimulation of erythropoiesis in vitro is seen at lower concentrations with haem arginate than with former haemin preparations.

Binding of epidermal growth factor and insulin-like growth-factor I in renal carcinoma and adjacent normal kidney tissue.

The binding of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) to cell membranes was determined in 14 renal cancers and in 13 normal kidney tissues adjacent to the tumors. The soluble 34K IGF binding protein (34K IGF-BP) content and the phosphotyrosyl-protein phosphatase activity in renal cancer tissue and adjacent normal tissue were also determined. The specific EGF receptor binding in renal cancers was 12.7 +/- 2.5% (mean +/- SEM) as compared to 2.6 +/- 0.2% (mean +/- SEM) in normal tissues (p less than 0.01). Phosphotyrosyl-protein phosphatase activity in renal cancer tissue was less than half of that observed in normal renal tissue (p less than 0.01). The highest IGF-I binding was observed in 5 renal cancers although no consistent differences between IGF-I binding to tumor and normal tissues were observed. Both EGF and IGF binding to kidney tissue were higher than binding to gastro-intestinal tissue irrespective of whether normal or malignant tissues were compared. All normal kidney tissues and 7 of 8 kidney tumors contained measurable amounts of 34K IGF-BP as determined by RIA and the cross-linking technique. In 2 tumor tissue samples the 34K IGF-BP content was increased 8- and 15-fold over that seen in adjacent normal kidney tissue, whereas in the 6 other renal cancers the 34K IGF-BP was similar to that observed in normal kidney tissue.

Histochemically demonstrable phosphotyrosyl-protein phosphatase in normal human breast, in benign breast diseases and in breast cancer.

The activity of phosphotyrosyl-protein phosphatase enzyme was investigated by a histochemical method in the normal human breast and in breast diseases in order to evaluate its possible significance in the genesis and in the growth of benign and malignant epithelial proliferative. In normal human breast tissue only a weak enzyme activity was present. The activity was elevated in benign disease in actively proliferative lesions and in 71% of the cases of breast cancers. When enzyme activity of breast cancers was compared with the content of receptors for epidermal growth factor and insulin-like growth factor-I, no association was found. It is concluded that phosphotyrosyl-protein phosphatase is increased in actively proliferating human breast diseases. Thus the putative increase in phosphotyrosyl-proteins mediating benign and malignant epithelial proliferations is rather caused by an increase in protein-tyrosine kinase activity than by a decrease in phosphotyrosyl-protein phosphatase activity.

Spontaneous erythroid colony formation in the differential diagnosis of erythrocytosis.

Erythroid colony formation in vitro was studied in 80 patients with erythrocytosis. 43 of the patients had polycythaemia vera (PV), 18 patients had secondary erythrocytosis, 6 had normal red cell mass, and 13 patients were regarded as unclassified. Spontaneous erythroid colony formation, in the absence of exogenous erythropoietin in the cultures, was discovered in all patients with PV, whereas no patient with secondary erythrocytosis or with normal red cell mass showed this phenomenon. 8 of the 13 patients with unclassified erythrocytosis spontaneously formed erythroid colonies. 7 patient with unclassified erythrocytosis have been followed for 5 yr. 3 of the 4 patients with spontaneous colony growth but none of the 3 without it can now be classified as PV. Thus, spontaneous erythroid colony formation indicates PV even in early and atypical cases. Therefore, the culture of erythroid progenitors is very useful in the differential diagnosis of problematic cases with erythrocytosis.

Colony formation by megakaryocyte progenitors in myelodysplatic syndromes.

In vitro megakaryocytic colony formation by progenitors from the bone marrow was studied in 40 myelodysplastic syndrome patients. Megakaryocytic colonies were decreased in number or absent in 30 patients; only 10 showed normal colony growth. The growth correlated to some extent with the FAB-class. Patients with normal colony formation had either RA or RARS, but also in these two FAB-types half of the patients showed reduced megakaryocytic colony formation. Only 1 out of 15 patients with RAEB or RAEBt had normal megakaryocyte growth. The patients with CMML did not show any megakaryocytic colonies. The growth of erythroid colonies was normal in 3 patients and reduced or absent in the others. All 3 with normal erythroid colony formation also showed normal megakaryocyte growth, and all patients with normal megakaryocyte colony formation also had normal granulocyte-macrophage colony growth. Granulocyte-macrophage colony and cluster formation was normal in 17 patients. Defective formation of megakaryocytic, erythroid, and granulocyte-macrophage colonies was seen in 22 patients, compatible with a defect in a pluripotent stem cell. Megakaryocytic colony formation had no obvious correlation with any specific chromosome abnormality, and the distribution of the growth patterns was almost similar in patients with a normal and those with an abnormal karyotype.

Mechanism of anaemia in rheumatoid arthritis: demonstration of raised interleukin 1 beta concentrations in anaemic patients and of interleukin 1 mediated suppression of normal erythropoiesis and proliferation of human erythroleukaemia (HEL) cells in vitro.

The pathogenesis of the anaemia associated with rheumatoid disease is unclear. It has previously been shown that the degree of the anaemia correlates with the severity of the inflammatory disease and that serum from patients with arthritis inhibits erythropoiesis. This study was designed to examine whether interleukin 1 could be a mediator of the anaemia in rheumatoid arthritis. Radioimmunoassay of interleukin 1 beta in serum showed that patients with rheumatoid arthritis and associated anaemia had significantly higher interleukin 1 beta concentrations than patients with rheumatoid arthritis without anaemia. Pure recombinant human interleukin 1 alpha and interleukin 1 beta, in concentration ranges similar to those found in the arthritic patients, markedly suppressed the colony formation of the erythroid, but not the granulocyte-macrophage progenitor cells in cultures of normal bone marrow. Natural human interleukin 1 and recombinant interleukin 1 beta, but not interleukin 1 alpha, suppressed in a dose dependent manner the proliferation of the human erythroleukaemia cell line (HEL) in cultures, suggesting that the interleukin 1 effect is a direct one. The results show that interleukin 1 is a humoral inhibitor of erythropoiesis and suggests that interleukin 1 is involved in the development of anaemia in association with rheumatoid arthritis.