Humanα 2-macroglobulin gene is located on chromosome 12

A cDNA clone encoding amino acids 809–1451 of the protease inhibitor ₂-macroglobulin has been isolated from an adult human liver cDNA library. This cDNA was used to examine DNA samples prepared from a panel of human-mouse somatic cell hybrids with different numbers and combinations of human chromosomes for the presence of the human ₂-macroglobulin gene. The cosegregation of this gene and chromosome 12 in the cell hybrid panel indicated that the ₂-macroglobulin structural gene (designated A2M) is on human chromosome 12.     

Proteases and antiproteases related to the coagulation system in plasma and ascites — An approach to differentiate between malignant and cirrhotic ascites

Summary The concentrations of several proteases and antiproteases known to be present in ascites were tested in plasma and ascitic fluid with regard to their ability to separate ascites according to malignant or nonmalignant disease. Seventeen patients with proven malignant ascites and 37 with ascites due to liver cirrhosis were included. Activities of plasminogen, ₂-antiplasmin, antithrombin-III, and factor V, and the concentration of ₁-protease inhibitor were significantly higher in the plasma of patients with malignant ascites than in cirrhotic patients. Fibronectin, plasminogen, ₂-macroglobulin, ₁-protease inhibitor, antithrombin-III, and albumin revealed higher concentrations or activities in malignant ascites than in cirrhotic ascites. Due to a wide variation of most parameters, only fibronectin, antithrombin III, and ₁-protease inhibitor in ascites had a sensitivity and specificity higher than 90% for malignant ascites. When the specific protein/albumin ratio was used, only the accuracy of fibronectin was increased reaching a sensitivity and specificity of 100%. The plasma/ascites gradients of the proteins assessed differed significantly, that of fibronectin being much higher (22±7) than that of all other proteins. In malignant ascites fibronectin concentration was only correlated with ₁-protease inhibitor concentration but not with the concentration or activity of all other proteins, while in cirrhotic ascites most proteins revealed a positive correlation.The determination of the fibronectin concentration or the fibronectin/albumin ratio in ascites can differentiate malignant and nonmalignant ascites. All other proteases and antiproteases assessed are of lesser value for this purpose, although most are significantly increased in ascites and plasma of patients with malignant disorders.Abbreviations ₂AP ₂-Antiplasmin - ₁PI ₁-Protease inhibitor - AT III Antithrombin III - FDP Fibrin(ogen) degradation products - FM Fibrin monomers - ₂MG ₂-Macroglobulin - PTT Partial thromboplastin time - RT Reptilase time     

Hypomethylation of the human HLA-DRα gene in breast carcinomas and autologous metastases

The methylation pattern of the human HLA-DR gene was analyzed in normal breast tissues, breast primary tumors and lymphonodal metastases isolated from patients carrying breast carcinomas. In breast adenomas and also in normal tissues (including breast, muscle, brain, sperm and T- and B-lymphocytes), the HLA-DR gene is hypermethylated at the CCGG and GCGC sites. In all tissues studied, the only constantly unmethylated region is located in the 5 portion of the gene, near the promoter sequence. Further, the results indicate that the HLA-DR gene is hypomethylated in carcinomas and in the relative metastatic lymph nodes. It is suggested that hypomethylation of the human HLA-DR gene could be proposed as a molecular marker of malignant breast tumors.     

Effect of dehydroepiandrosterone on radioligand binding of [<Superscript>3</Superscript>H]-testosterone by androgen receptors in rat hypothalamus

Intramuscular injections of dehydroepiandrosterone in a dose of 0.7 mg/kg for 10 days significantly increased nuclear and cytoplasmic fractions of androgen receptors in the preoptic/ anterior hypothalamic area. Presumably, the effect of the neurosteroid is mediated by 5-reductase transformation of dehydroepiandrosterone into 5-dehydroepiandrosterone, which initiates the synthesis of androgen receptors.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 10, pp. 435–438, October, 2004     

Synthesis and secretion of α2-macroglobulin by human glioma established cell lines

Summary Human ₂-macroglobulin (₂M) is a high molecular weight plasma proteinase inhibitor exhibiting a broad specificity; in fact it is capable of binding endopeptidases from all known classes of proteases (Barret 1981). Two human glioma cell lines, namely an astrocytoma and a glioblastoma, were found to synthesize and secrete in the culture medium a protein which resembles the serum ₂M for immunological, biochemical and biological features. Using polyclonal antibodies to serum ₂M, an ₂M-like factor could be detected in the cytoplasm and in the culture medium of the tumor cells. Furthermore this factor accumulated in cytoplasmic granules if cells were incubated with monensin and its production was dramatically reduced following a treatment with cycloheximide. This protein behaved like the serum ₂M in immunoblotting analysis and exhibited the same antiproteolytic activity. Its role in human brain is unknown at present. Since interactions of proteinases and proteinase-inhibitors appear to influence the hosttumor immune response and to play a crucial role during the migration of metastasizing tumor cells, ₂M expression observed in these glioma cells could be involved in tumor cell proliferation and invasion.     

Erythromycin and cycloheximide sensitivities of protein and RNA Synthesis in sporulating cells of Saccharomyces cerevisiae: Environmentally induced modifications controlled by chromosomal and mitochondrial genes

Summary The purpose of the experiments reported below was to examine the response in sporulation medium of the three diploid cell types MAT MAT, MAT MAT (asporogenic diploids) and MAT MAT (sporogenic diploid) to erythromycin, a specific inhibitor of mitochondrial protein synthesis (MPS) in vegetative cultures, and cycloheximide, a specific inhibitor of cytosol protein synthesis (CPS) in vegetative cultures. When MAT MAT diploids are transferred to sporulation medium a significant fraction of total protein synthesis (CPS + MPS) becomes sensitive to erythromycin in contrast to the behavior of MATa MATa and MAT MAT diploids in which the resistance of CPS to erythromycin is maintained. The decompartmentalization of erythromycin sensitivity is thus cell type specific. Erythromycin stimulates total RNA synthesis of MAT MAT cells in sporulation medium but not of MAT MAT and MAT MAT cells. Cycloheximide inhibits protein synthesis and stimulates RNA synthesis in all three diploid cell types. An erythromycin resistant mutant, shown to be due to a mutation of the mitochondrial genome, exhibited only partial resistance of CPS to erythromycin in sporulation medium in the background of the MAT MAT mating type genotype. Total RNA synthesis in this mutant was not stimulated. The results reported indicate that mitochondrial functions during sporulation are not restricted to those involving respiratory metabolism.     

CD34<Superscript>+</Superscript> fibrocytes in tubular carcinomas and radial scars of the breast

The present study was undertaken to analyze whether and to what extent CD34⁺ fibrocytes and -smooth muscle actin (-SMA)-positive myofibroblasts occur in the stroma of radial scars and tubular carcinoma of the breast. We investigated a total of 24 radial scars obtained from 23 females and a total of 43 tubular carcinomas. All tubular carcinomas showed a virtually complete loss of CD34⁺ fibrocytes paralleled by a gain of -SMA-positive myofibroblasts. The peripheral parts of radial scars harbored CD34⁺ fibrocytes comparable to normal breast tissue. In 23 of 24 radial scars, the central core was characterized by a loss of CD34⁺ fibrocytes accompanied by the presence of -SMA-positive myofibroblasts in six cases, a pattern that up to now has only been described in malignant breast lesions. This finding underlines the close relationship between tubular carcinomas and radial scars. We conclude CD34- and -SMA immunohistochemistry to be valuable adjunctive tools in distinguishing radial scars from tubular carcinomas. In the present study, the presence of CD34⁺ fibrocytes excluded malignancy. The absence of CD34⁺ fibrocytes paralleled by the presence of -SMA myofibroblasts indicated malignancy in most cases, although it has to be carefully considered that in a minority of cases (6 of 24) the central core of radial scars may disclose this stromal composition.This revised version was published online September 2003 with corrections to size and format of Fig. 1     

Macrophage inflammatory protein-1 alpha expression in non-neoplastic and neoplastic lung tissue

The chemokines are members of a bipartite superfamily of soluble proteins that have been implicated in a wide range of acute and chronic inflammatory processes, as well as other immunoregulatory functions. Macrophage inflammatory protein-1 alpha (MIP-1) belongs to the C-C subfamily of these chemokines and is primarily a potent chemoattractant and activator of monocytes. MIP-1 is also thought to play a role in host defence. We examined the expression of MIP-1 in normal lung, inflammatory lung tissue and lung cancer cells by the immunoperoxidase method using a MIP-1 monoclonal antibody. MIP-1 protein was found to be expressed not only by alveolar macrophages, but also by bronchial ciliated cells, hyperplastic alveolar type II cells and activated fibroblasts surrounding malignant tissue. Of 33 cases of lung cancer, 23 (70%) expressed MIP-1. These observations suggest that lung cancer cells, non-neoplastic alveolar type II cells and fibroblasts can participate in inflammatory cell recruitment via the production of MIP-1. Tumour derived MIP- may also affect the interaction between lung cancer and host inflammatory cells.     

Immunohistochemical studies on S-100 cells in the anterior pituitary gland of Sprague Dawley rats and spontaneous dwarf rats

The S-100 cells in the pituitary glands of adult male Sprague Dawley rats (SDs) and spontaneous dwarf rats (SDRs) were immunohistochemically examined using anti-S-100 and anti-S-100 monoclonal antibodies. The immunoreactive cells against S-100 protein were divided into three subtypes on the basis of their immunore-activity against subunits of S-100 protein: S-100 dominant type (the -type cell), S-100 dominant type (the \-type cell) and immunoreactive against both S-100 and S-100 (the -type cell). In the SD, -type cells represented 26% of the total S-100 immunoreactive cells (S-100 cells) and were localized in the peripheral area of the ventral region of the pituitary gland. This type of cell was observed forming clusters, with more abundant cytoplasm than the -type cell. The proportion of -type cells was 53%. They were diffusely distributed throughout the gland, and their processes were thicker than those of the -type cell. In the SDR, the proportion of -type cells was 55%, and they were observed throughout the gland. In contrast, -type cells totalled 12% and were localized in small areas of the central and peripheral region of the gland. The proportion of -type cells was 21% in the SD and 33% in the SDR and they were observed forming small clusters in both animal groups. The proportion of -type cells compared with the total of S-100-immunoreactive cells was significantly higher (P < 0.05) in the SDR than in the SD, while the proportion of -type cells was markedly lower (P < 0.05).     

Immunohistochemical studies on oncogene products (EGF-R,c-erbB-2) and growth factors (EGF,TGF-α) in human breast cancer: their relationship to oestrogen receptor status,histological grade,mitotic index and nodal status

Summary In this investigation, 83 human mammary carcinomas were examined for the expression of oestrogen receptor (ER), epidermal growth factor receptor (EGF-R), epidermal growth factor (EGF), transforming growth factor alpha (TGF-), c-erbB-2, histological grade, mitotic index and nodal status, all of which are reportedly prognostically significant factors (Bloom and Richardson 1957; Baak et al. 1985; Wright et al. 1989). ER expression was biochemically recognized in 43.4% of mammary carcinomas, and EGF-R, EGF, TGF- and c-erbB-2 were histochemically recognized in 25.3, 14.5, 27.7 and 18.0% of mammary carcinomas examined respectively, using conventional sections of buffered formalin-fixed, paraffin-embedded tissue and monoclonal or polyclonal antibodies. There were significant relationships between negative ER and positive EGF-R or TGF-; positive EGF-R and TGF-; positive EGF-R and c-erbB-2; and positive c-erbB-2 and TGF-. The single changes which were the negative ER and the positive c-erbB-2 correlated with histological grade and mitotic index. Co-expression of EGF-R and TGF- correlated with positive nodal status. Therefore, the present investigation indicates that the negative ER, single expression of c-erbB-2 and co-expression of EGF-R and TGF- are important markers which contribute indirectly to prognosis, which reconfirms previous findings on the former two while adding the new finding that immuno-histochemical demonstration of expression of EGF-R and TGF- may provide useful information for selecting the appropriate treatment.     

Effect of α1-Acidic Glycoprotein in the Ascitic Fluid of Cancer Patients on Human NK Cells: Selective Suppression of Interferon-Induced NK Activation

The in vitro effect of C-AGP (pure 1-acid glycoprotein from the ascitic fluid of cancer patients) on NK cell cytotoxicity was tested using normal healthy human PBMC. C-AGP had no inhibitory effect on basal NK cell activity. C-AGP selectively suppressed the augmentation of NK cell activity by rIFNA and rIFN, but C-AGP did not prevent the NK activation by rIL-2. NK cells in PBMC treated with C-AGP for 12 h and then washed just once, to remove the C-AGP, fully recovered the ability to respond to rIFNA. However, after the treatment of PBMC with C-AGP for 5 or 6 days, NK cells failed to respond to rIFNA, in spite of washing to remove C-AGP from the cultures. Monocytes were necessary for the suppressive effect of C-AGP on rIFNA activation of NK cells. Indomethacin restored the ability of NK cells to respond to rIFNA in C-AGP-treated PBMC. These results suggest that monocytes are able to selectively suppress the response of NK cells to IFNs in the presence of, or following treatment with C-AGP.     

Immunoreactivity of the JK-132 monoclonal antibody directed against basement membrane collagen in normal and diabetic glomeruli

The possible involvement of basement membrane-associated collagen (recognized by the monoclonal antibody JK-132) in the evolution of diabetic nephropathy was studied in kidney specimens from seven patients with noninsulin-dependent diabetes mellitus, and its distribution was compared with those of antibodies against 1 to 4 chains of type IV collagen. JK-132, a monoclonal antibody against basement membrane-associated collagen, reacted immunohistochemically exclusively with the mesangial matrix of the glomerular capillary. In contrast, antibodies to the 1 and 2 chains (IV) reacted strongly with mesangial matrix, and less strongly with the glomerular basement membrane (GBM). Antibodies to the 3 and 4 chains (IV) reacted mainly with GBM. In diabetes, JK-132 reacted most extensively with the expanded mesangial matrix, its staining intensity increasing with progression of the diabetic glomerulosclerosis. Antibodies to the 1 and 2 chains (IV) reacted prominently with the expanded mesangial matrix but less strongly with the GBM. Antibodies to the 3 and 4 chains reacted intensely with the thickened GBM. These results suggest that basement membrane-associated collagen differs from 1 to 4 chains of type IV collagen and that basement membrane-associated collagen is a good marker of mesangial expansion in diabetic nephropathy.     

Enhancement by TNF-α of reactivation and replication of latent herpes simplex virus from trigeminal ganglia of mice

Summary The influence of tumor-necrosis-factor- (TNF-), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukine-1 (IL-1) and IL-3 on the in vitro reactivation frequency and replication rate of trigeminal ganglia of mice latently infected with herpes simplex virus (HSV) strain KOS was studied. It could be demonstrated that TNF- and possibility GM-CSF, but not IL-1 and IL-3, enhanced the reactivation frequency and replication of HSV. Interferon / (IFN/) prevented reactivation and replication.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Acute Phase Responses and Cytokine Secretion in Chronic Fatigue Syndrome

This study addresses the hypothesis that clinical manifestations of chronic fatigue syndrome (CFS) are due in part to abnormal production of or sensitivity to cytokines such as interleukin-1 (IL-1) and IL-6 under basal conditions or in response to a particular physical stress: 15 min of exercise consisting of stepping up and down on a platform adjusted to the height of the patella. The study involved 10 CFS patients and 11 age-, sex-, and activity-matched controls: of these, 6 patients and 4 controls were tested in both the follicular and the luteal phases of the menstrual cycle, and the remainder were tested in only one phase, for a total of 31 experimental sessions. Prior to exercise, plasma concentrations of the acute phase reactant ₂-macroglobulin were 29% higher in CFS patients (P < 0.008) compared to controls. Secretion of IL-6 was generally higher for CFS patients (~38%), however, this difference was statistically significant only if all values over a 3-day period were analyzed by repeated-measures ANOVA (P = 0.035). IL-6 secretion correlated with plasma ₂-macroglobulin in control subjects at rest (R = 0.767, P = 0.001). Immediately after exercise, the CFS patients reported greater ratings of perceived exertion (P=0.027) compared to the healthy control subjects. Ratings of perceived exertion correlated with IL-1 secretion by cells from healthy control subjects (R = 0.603, P = 0.022), but not from CFS patients, and IL-1 secretion was not different between groups. Exercise induced a slight (<12%) but significant (P = 0.006) increase in IL-6 secretion, but the responses of the CFS patients were not different than controls. Furthermore, no significant exercise-induced changes in body temperature or plasma ₂-macroglobulin were observed. These data indicate that under basal conditions, CFS is associated with increased IL-6 secretion which is manifested by chronically elevated plasma ₂-macroglobulin concentrations. These modest differences suggest that cytokine dysregulation is not a singular or dominant factor in the pathogenesis of CFS.     

Combined effect of hydrogen peroxide induced oxidative stress and IL-1 alpha on IL-8 production in CaCo-2 cells (a human colon carcinoma cell line) and normal intestinal epithelial cells

Oxidative stress, IL-1, and IL-8 are known to contribute to mucosal inflammation of the gastrointestinal tract. We examined the IL-8 response after brief exposure to hydrogen peroxide induced oxidative stress in CaCo-2 cells (a human colon carcinoma cell line) and in human intestinal epithelial cells. In addition, we examined whether exposure to oxidative stress, followed by IL-1, could modulate IL-8 production. A transient up-regulation of IL-8 mRNA expression was observed after hydrogen peroxide treatment. Hydrogen peroxide induced oxidative stress was also observed to promote IL-8 secretion. Exposure to hydrogen peroxide, followed by IL-1, enhanced IL-8 production over that achieved with IL-1 alone. Thus, oxidative stress and IL-1 were observed to cooperatively enhance IL-8 production.     

Human T-cell leukemia/lymphoma virus I and/or Epstein-Barr virus-infected B-cell lines spontaneously produce acid-labile α-interferon

B-cell lines were established as spontaneous outgrowths of cell cultures from patients with adult T-cell leukemia (ATL). Three such lines were shown to have integrated human T-cell leukemia/lymphoma virus 1 (HTLV-I) proviral sequences as well as Epstein-Barr virus (EBV) infection. Supernatant fluids from these cultured cells were assayed for interferon (IFN) production. Acid-stable -IFN was found to be produced by one cell line (CF), and acid-labile -IFN by the other two (HS, MJB). In contrast, HTLV-I-infected T-cell lines did not produce IFN. Some EBV-infected B-cell lines produce acid-labile -IFN, while others do not. Since -IFN, both acid stable and acid labile, is found in sera from patients with the polyclonal activation of B cells as a constitutive part of the disease, the above observations suggest a possible role of polyclonal B-cell activation in -IFN production in these diseases.     

Analysis of adhesion molecules in Ki-1 anaplastic large-cell lymphoma

We analysed the expression of adhesion molecules on lymphoma cells in 13 patients with Ki-1 (CD30)-positive anaplastic large-cell lymphoma (Ki-1 ALCL; lymph nodes in 6, extranodal tumours in 6, and both lymph nodes and bone in 1). Very late activation antigen (VLA)-4 (CD49d) and Hermes lymph node homing receptor (CD44) were constantly expressed in all specimens, and intercellular adhesion molecule-1 (ICAM-1; CD54) was frequently expressed in 10 of the 14 specimens. The expressions of lymphocyte function-associated antigen-1 (LFA-1; CD11a) and VLA-5 (CD49e) occurred in 5 of 14 and 4 of 14 specimens, respectively. The expressions of VLA-2 (CD49b), endothelial leukocyte adhesion molecule-1, neural cell adhesion molecule (CD56) and E cadherin were always lacking. VLA-6 (CD49f) was absent in all but one specimen. The expression of VLA-5 on Ki-1 ALCL was high in subcutis-cutis but absent in lymph nodes. Furthermore, in one case, LFA-1 was detected in the primary lymph node, but was absent in a metastatic bone lesion. These results suggest that the expression of ICAM-1 is partially responsible for aleukemic behaviour in Ki-1 ALCL and, moreover, that the Ki-1 ALCL cells modify their expression of adhesion molecules at each of the involved organs.     

The effect of high altitude on the protein composition of human blood

Summary Twelve healthy young individuals had the protein composition of their blood determined in Stalinabad (850 m) and during their sojourn in the mountain of East Pamir between the months of May and October, 1958, at the altitude of 4200 m.During the first month of sojourn at high altitude, the total concentration of protein went up, while towards the end of the 4 month period it dropped somewhat, but still remained above the initial level. The relative and the absolute albumin content in the blood serum dropped immediately after the ascent and remained low for a month after descent from 4200 m to 850 m. The figures for ₁-, - and -globulins rose immediately after ascent. During the 4 month stay at high altitude, the ₁- -globulins went back to normal. During the first month after descent, figures for ₁-, - and -globulins were higher than the initial levels; ₂-globulin went up only after descent. Oncotic pressure of blood rose during the first month after ascent, then returned to normal at the expense of increased concentration of the globulin fraction, which compensates for the decreased albumin content in blood serum.(Presented by Active Member AMN SSSR S. R. Severin) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 50, No. 10, pp. 78–82, October, 1960.     

Comparative evaluation of integrin α- and β-chain expression in colorectal carcinoma cell lines and in their tumours of origin

The integrin family consists of broadly expressed cell surface adhesion receptors, each member of which is composed of a non-covalently linked / heterodimer. Integrin receptors are involved in the interaction with matrix proteins and may contribute to invasion and metastasis of carcinomas. To examine the biological role integrins play in colorectal carcinoma we compared the expression of integrin - and -subunits in situ and in vitro. Eight newly established cell lines derived from immunohistochemically characterized colorectal carcinomas together with two sublines obtained after nude mouse passage and the commonly used colon carcinoma lines HT-29, SW480, SW620, and COLO 205 were investigated by immunocytochemistry and flow cytometry. The carcinomas in situ expressed 1-, 2-, 3-, 6-, v-and 1-subunits in variable amounts while being devoid of 4, 5 and 3. The individual integrin profile of the tumour in tissue was essentially maintained in vitro. However, a neo-expression of the 5 chain was found, together with an induction or increase in 1, 2, 3, v and 1 levels. No decrease in integrin subunit expression was observed. Standard-serum and serum-free medium revealed no striking differences in - and -chain expression in the cell lines HT-29 and COLO 205. In serum-free medium, SW480 showed a slight increase of 1 and 5 and a decrease of 3 and v while SW620 expressed more 1. We conclude that the great variability of adhesion receptor expression of the integrin family in colorectal carcinomas in situ is essentially maintained in vitro, although culture conditions which are only marginally influenced by serum factors unpredictably lead to some increase in expression or even induction of several integrin subunits.This work is dedicated to Prof. Wilhelm Doerr on the occasion of this 80th birthday