MicroRNAs in pathophysiology of acute myocardial infarction and cardiogenic shock.

AIM: Levels of circulating miRNA are considered to be potential biomarkers of acute myocardial infarction and disease progression. METHODS: In this study, the expression levels of circulating miRNA-1, miRNA-133 and miRNA-124a were investigated in a group of patients with acute myocardial infarction (STEMI) and cardiogenic shock (CS) compared to controls. RESULTS: During the hospitalization period, miRNA-133 showed a significant up-regulation in the serum of STEMI and CS patients compared to controls, while the expression of miRNA-1 was significantly different only in CS. The expression of miRNA-124 was significantly higher in STEMI and CS. Furthermore, miRNA-1 expression was related to the level of circulating glucose in patients with STEMI. We also found a negative correlation between miRNA-133 and MMP-9 levels. MiRNA-124 expression was significantly related to the level of soluble ST2; the marker correlated to cardiac damage. CONCLUSION: All selected miRNAs are potential markers of cardiac injury in cardiogenic shock, whereas miRNA-124a and -133 are markers of injury in STEMI. MiRNA-1 expression is related to circulating glucose in STEMI. None of miRNAs could be correlated to the extent of injury, progress of the disease, or prognosis of patient outcome. Therefore, the levels of circulating miRNA have no potential for becoming a biomarker of myocardial damage and as such would bring no further benefit compared to current markers (Tab. 4, Fig. 1, Ref. 47).

The human L-myc gene encodes multiple nuclear phosphoproteins from alternatively processed mRNAs.

The human proto-oncogene L-myc generates at least four different mRNAs by alternative RNA processing. We have identified two phosphorylated L-myc proteins with molecular masses of 60,000 and 66,000 daltons [p60L-myc(human) and p66L-myc(human)] in a small-cell carcinoma line expressing high levels of L-myc mRNA. These proteins have a short half-life and are localized to the nuclear matrix fraction, as previously reported for the c-myc and N-myc proteins. In vitro translation experiments demonstrated that both the p60 and p66 species are encoded by a 3.9-kilobase (kb) mRNA which retains intron 1, while only the p60 protein is translated from a 3.6-kb L-myc mRNA which has had intron 1 removed. While L-myc proteins [p32L-myc(human) and p37L-myc(human)] could be synthesized in vitro from 2.2-kb mRNA templates, no such proteins were detected by immunoprecipitation in vivo. These observations suggest that alternative RNA processing of the L-myc transcript could play a role in determining the steady-state levels of the p60L-myc and p66L-myc proteins.

Analysis of human small cell lung cancer differentiation antigens using a panel of rat monoclonal antibodies.

The antigen expression of human small cell lung cancer (SCLC) was studied using a panel of 21 independent rat monoclonal antibodies. The panel was selected by isolating hybridomas producing antibodies reactive with two SCLC lines but not with autologous B-lymphoblastoid lines. The antibodies were then tested in radiobinding assays against a panel of 17 SCLC lines, 13 non-small cell lung cancer lines, 6 SCLC necropsy specimens, 13 neuroectodermal lines (melanomas, neuroblastomas, glioblastomas), 15 other human lines, the glycolipid extracts of SCLC, human meconium, and human red blood cells. Using immunohistochemical assays, 14 of the antibodies were tested against normal lung, liver, and kidney, and lung cancer biopsies and xenografts. These analyses revealed the following: (a) SCLC elicited predominantly immunoglobulin M antibodies despite hyperimmunization; (b) the 21 antibodies displayed distinct binding and immunohistochemical phenotypes, indicating that they recognized many different epitopes; (c) 14 of the 21 antibodies reacted with glycolipid determinants; (d) the 21 determinants were expressed on over 80% of SCLC cell lines, necropsy samples, and xenografts; (e) the determinants were also expressed on normal adult bronchial epithelium, proximal tubules of adult kidney, and in a few instances on other normal cell types; (f) the antigens were expressed less frequently on nonsmall cell lung cancer samples but did not clearly distinguish SCLC from non-small cell lung cancer; (g) biochemical and morphological variants of SCLC exhibiting more malignant and undifferentiated behavior and containing greatly amplified c-myconcogenes failed to express several determinants or expressed them at lower levels; (h) and finally, while many human cell lines failed to express the antigens including human melanoma and glioblastoma lines, human neuroblastoma lines frequently did express the SCLC antigens. These detailed studies utilizing a panel of distinct monoclonal antibodies define a series of antigens on the surface of the majority of SCLC undescribed previously.

Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation.

We investigated the immunocytochemical staining and immunoblotting characteristics of 33 different p53 mutant proteins identified in lung cancer cell lines (18 small-cell lung cancer and 15 non-small-cell lung cancer) using monoclonal antibodies pAbs 240, 421 and 1801. The p53 mutants studied were representative of those found in lung cancer and included three deletions, four nonsense, seven splicing and 19 missense lesions. Control cell lines included six B-lymphoblastoid cell lines and two lung cancer cell lines without p53 mutations. Immunocytochemistry demonstrated 16 cell lines (48%) with definite overexpression of p53 protein (the high-expresser group of mutants), while in the remainder of cases either no p53 expression or low levels of p53 protein expression were found (the low-expresser group of mutants). The type of p53 mutation correlated with the expresser group. High expressers all had p53 missense mutations in exons 5-8, and immunocytochemistry identified 16/17 (94%) of these mutants. Several classes of p53 mutations occur in the low-expresser groups: deletions, splicing mutants, nonsense mutants and missense mutations outside of exons 5-8 all resulted in very low or undetectable levels of p53 protein. We conclude that there are low- and high-expression groups of p53 mutants in lung cancer and that the detection of protein expression in tumor cells by immunocytochemistry and immunoblotting is dependent upon the type of mutation of the p53 tumor-suppressor gene.

RB protein status and clinical correlation from 171 cell lines representing lung cancer, extrapulmonary small cell carcinoma, and mesothelioma.

We have studied RB protein expression in 171 cell lines derived from patients with small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), pulmonary carcinoid, mesothelioma, and extrapulmonary small cell cancer (EPSC) and have correlated this data with clinical outcome. We detected absent or aberrant RB protein expression in 66/75 SCLC, 12/80 NSCLC, 1/6 carcinoid, 0/5 mesothelioma, and 4/5 EPSC samples. In addition, we observed integration of human papilloma virus (HPV) DNA in the single EPSC cell line that retained wildtype RB protein. We did not detect integration of HPV, SV40 or adenoviral DNA in other tumor samples with wildtype RB status. We also noted a stable, hypophosphorylated mutant RB in 12 SCLC and 3 NSCLC samples which might have been falsely interpreted as wildtype by current immunohistochemical techniques. Analysis of the matched clinical data showed no associations between RB status and age, sex, extent of disease, performance status, smoking history, and previous treatment. In addition, retrospective analyses showed no consistent correlation of RB protein expression with either best clinical response, overall survival, or in vitro chemotherapeutic drug sensitivity. The stable expression of RB after gene transfection into RB(-) SCLC cells, however, resulted in a trend toward increased in vitro resistance to etoposide, cisplatin and doxorubicin.

High frequency of somatically acquired p53 mutations in small-cell lung cancer cell lines and tumors.

We analysed the p53 open reading frame (ORF) in 16 small-cell lung cancer (SCLC) cell lines by direct sequencing of cDNA/PCR products and in 20 SCLC tumors by chemical cleavage and single-strand conformation polymorphism analyses of genomic DNA/PCR products. Abnormalities of p53 were found in 16/16 cell lines (100%) and in 16/20 tumors (80%). In the SCLC cell lines, mutations (59% missense, 18% nonsense and 23% splicing) changing the coding sequence were dispersed between amino acids 68 and 342. In the tumor samples, while the mutations occurred predominantly in exons 5-8, other mutations were located outside these regions. G to T transversions were common, occurring in 32% of the cases. We found no p53 mutations in the corresponding normal tissue from 19 patients whose tumors had p53 lesions, indicating that the mutations were all somatically acquired. In analysing the clinical data of the patients we found no correlation between tumor response to therapy or survival and the location or type of mutations. We conclude from these data that: (1) p53 mutations are found in SCLC with high frequency; (2) p53 mutations in a significant fraction of cases generate cDNAs with nonsense or splicing mutations; and (3) to date, these mutations have all been somatically acquired events.

In vitro and in vivo characterization of recombinant human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger.

Previous studies in rodents and nonhuman primates have demonstrated that pretreatment with cholinesterases can provide significant protection against behavioral and lethal effects of nerve agent intoxication. Human butyrylcholinesterase (HuBuChE) purified from plasma has been shown to protect against up to 5 x LD50s of nerve agents in guinea pigs and non-human primates, and is currently being explored as a bioscavenger pretreatment for human use. A recombinant form of HuBuChE has been expressed in the milk of transgenic goats as a product called Protexia. Protexia was supplied by Nexia Biotechnologies (Que., Canada) as a purified solution with a specific activity of 600 U/mg. Initial in vitro studies using radiolabeled 3H-soman or 3H-DFP (diisopropyl fluorophosphate) demonstrated that these inhibitors specifically bind to Protexia. When Protexia was mixed with soman, sarin, tabun or VX using varying molar ratios of enzyme to nerve agent (8:1, 4:1, 1:1 and 1:4, respectively), the data indicated that 50% inhibition of enzyme activity occurs around the 1:1 molar ratio for each of the nerve agents. Protexia was further characterized for its interaction with pyridostigmine bromide and six unique carbamate inhibitors of cholinesterase. IC50 and Ki values for Protexia were determined to be very similar to those of HuBuChE purified from human plasma. These data suggest that Protexia has biochemical properties very similar to those HuBuChE when compared in vitro. Together these data the continued development of the goat milk-derived recombinant HuBuChE Protexia as a potential bioscavenger of organophosphorus nerve agents.

Gastrin-releasing peptide gene-associated peptides are expressed in normal human fetal lung and small cell lung cancer: a novel peptide family found in man.

Mammalian gastrin-releasing peptide (GRP) is found in cells of neuroendocrine and neural origin, and GRP mediates a variety of physiological and trophic responses when it binds to high affinity cell surface receptors on effector cells. Analysis of cDNA clones derived from prepro-GRP mRNAs predict the concurrent expression of a unique series of peptide hormones, the GRP gene-associated peptides (GGAPs). Alternative RNA splicing of the primary GRP gene transcript results in mRNAs that could encode 3 distinct forms of GGAPs. Using specific antisera directed against synthetic peptides representing portions of the predicted GGAPs, we found multiple GGAP forms in the endocrine cells of human fetal lung and in human small cell lung cancer cells (SCLC). Within a single pulmonary endocrine cell, at least 2 of these 3 predicted forms could be demonstrated using immunohistochemical techniques. In addition, concordant expression of GRP and GGAPs was found in 10 SCLC cell lines and 3 human SCLC tumors. These findings establish GGAPs as a novel peptide family in man and warrant further investigation into their potential role in normal and malignant growth.

Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess.

Conjugates of monoclonal antibodies with radioactive isotopes, drugs or toxins have great potential for specific radiolocalization and inactivation of tumor cells. Because the conjugation procedure may adversely alter the antibody, quality control procedures must be applied to determine important characteristics of the conjugated antibody. One such property is how much of the conjugated antibody is able to bind to the relevant antigen. Based on theoretical considerations, we have developed a binding assay for radiolabeled monoclonal antibodies in which the fraction of immunoreactive antibody is determined by linear extrapolation to conditions representing infinite antigen excess. This ensures that the true value of the immunoreactive fraction is obtained, as opposed to the apparent immunoreactive fraction determined under conditions of limited antigen excess. The described assay is based on a double-inverse plot of the binding data which may be considered a modification of the Lineweaver-Burk plot. We established the method using 125I- and 111In-labeling of the 2 monoclonal antibodies T101 and 9.2.27 which currently are undergoing radioimaging trials at the National Cancer Institute. For properly performed conjugation procedures, immunoreactive fractions of about 0.9 were obtained, but a prolonged chloramine-T reaction for 125I-labeling resulted in an immunoreactive fraction of only 0.6. Due to its principle of determining binding at infinite antigen excess, the present method is quite insensitive to variation in the actual amounts of cells and antibody used, as well as the incubation time. We therefore recommend it as a quality control procedure for radiolabeled antibodies. Under certain conditions, this procedure is also applicable for quality control of drug- and toxin-conjugated monoclonal antibodies.

Counterimmunoelectrophoresis of blood cultures. Temporal Relationship of positive Gram stain to positive counterimmunoelectrophoresis.

Counterimmunoelectrophoresis (CIE), Gram staining, and quantitative measurements were performed on simulated blood cultures at hourly intervals after inoculation with Streptococcus pneumoniae, Klebsiella pneumoniae, or Haemophilus influenzae. The CIE became positive either at the same time as the Gram stain or within the ensuing five hours. In no case was CIE positive when the Gram stain was negative. The numbers of colony-forming units milliliter necessary for a positive CIE result varied with the types of microorganisms. The procedure of CIE appears useful as a rapid screening technic for identification of bacteria in blood cultures when microorganisms are seen in Gram-stained smears.

Schistosoma japonicum in the black rat, Rattus rattus mindanensis, from Leyte, Philippines in relation to Oncomelania snail colonies with reference to other endoparasites.

This study examined the prevalence of Schistosoma japonicum infections in field rats, Rattus rattus mindanensis, according to different trapping locations. Between October 1995 and January 1996, traps were set in the municipality of Palo, Leyte, Philippines to determine the correlation of rats infected with schistosomiasis to the proximity of the intermediate snail host, Oncomelania hupensis quadrasi, colonies. Of the 22 rats that were caught within a snail colony, 21 (95.5%) were positive for schistosomiasis. Of the 23 rats that were caught 100 meters from a snail colony, 13 (56.5%) were positive for schistosomiasis. Of the 17 rats that were caught approximately 1 km from a snail colony, zero (0%) were positive for schistosomiasis. Infection rates were highest within the habitat of the intermediate host and lowest in rats captured far from snail colonies. Captured rats were also examined for the presence of other endoparasites. Infections of the following were found: Angiostrongylus cantonensis, Gonylonema neoplasticum, Hymenolepis diminuta, Nippostrongylus muris, Strongyloides ratti, Syphacia obvelata, Taenia crassicollis and Trichuris muris, but there was no correlation between trapping location and prevalence. None of the rats were infected with Moniliformis moniliformis, Trichinella spiralis, Trypanosoma lewisi or Vampirolepis nana.

In vitro activity of coumermycin A1.

The in vitro activity of coumermycin A(1) was compared with that of novobiocin, ampicillin, and minocycline. Coumermycin was found to be the most active antibiotic of the four against Staphylococcus aureus. It was about 50 times more active than novobiocin or minocycline against the strains tested. Coumermycin also showed good activity against group A streptococci and pneumococci, moderate activity against Escherichia coli, indole-positive Proteus species, and Pseudomonas aeruginosa, and poor activity against Klebsiella-Enterobacter and enterococci. Against P. mirabilis, however, coumermycin activity was almost equal to that of ampicillin. The new antibiotic was further found to be greatly reduced in activity in the presence of plasma, but its minimal inhibitory concentration was not greatly affected by inoculum size. Coumermycin was found to be bacteriostatic in its action, and resistance to it developed slowly. Also, cross-resistance was present with novobiocin but absent with ampicillin or minocycline.

Properties of monoclonal antibodies specific for determinants of a protein antigen, myoglobin.

Monoclonal hybridoma antibodies specific for the protein antigen sperm whale myoglobin were produced using hyperimmune spleen cells from mice with the genetic trait of high responsiveness to myoglobin. Antibodies from the several clones tested were found to produce linear Scatchard plots, as predicted for homogeneous antibodies, and to possess high affinities for the immunogen (KA congruent to 10(9) M-1). None of the monoclonal antibodies tested reacted with either fragment (1-55) or fragment (132-153) of sperm whale myoglobin. Competitive binding assays using human and horse myoglobins suggested that several of these monoclonal antibodies, which can readily distinguish these myoglobins, recognize different antigenic determinants on the myoglobin molecule. Studies using additional myoglobin sequence variants as competitors should be able to more closely define these antigenic determinants.

Counterimmunoelectrophoresis of staphylococcal antibody.

Modifications of the staphylococcal counterimmunoelectrophoresis technique were evaluated to determine how variations in the procedure affected results. Neither a buffer pH range of 7.8 to 9.0 nor buffer molarity of 0.015 or 0.025 when tested in combinations caused appreciable differences. However, use of different agar preparations or delay in addition of antigen to the test slide altered the location of the precipitin band. Agarose was found to be more sensitive in determining the serum precipitin titer and provided a better photographic record than either Ionagar or Noble agar.

jun-B inhibits and c-fos stimulates the transforming and trans-activating activities of c-jun.

We have cloned the human jun-B gene and determined its sequence and transforming and trans-activating activities. jun-B is less potent that c-jun in transforming and immortalizing primary rat embryo cells in cooperation with activated ras (effects enhanced by c-fos and TPA); unlike c-jun, jun-B does not transform Rat-1A cells alone. However, cotransfection of c-jun and jun-B into primary rat embryo cells with c-Ha-ras results in a significant decrease in transformation compared with c-jun alone, an event reversed by TPA. Cotransfection of c-jun and jun-B with or without c-fos into F9 teratocarcinoma cells results in decreased trans-activation of AP-1 compared with either gene alone. Introduction of jun-B into primary rat c-jun/ras transformants or c-jun into jun-B/ras transformants also results in a decrease in trans-activation. These findings demonstrate that, whereas jun-B and c-jun each participate in AP-1 trans-activation and malignant transformation, interactions between them involve negative regulation.

Identification of a higher molecular weight DNA polymerase alpha catalytic polypeptide in monkey cells by monoclonal antibody.

A monoclonal antibody against purified calf DNA polymerase alpha (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) was used to immunoprecipitate proteins from a crude soluble extract of growing monkey BSC-1 cells. Immunoprecipitates contained familiar DNA polymerase alpha catalytic polypeptides of Mrs approximately equal to 115,000 and 70,000 and also a Mr 40,000 catalytic polypeptide; the major component in the immunoprecipitates, however, was a polypeptide of Mr approximately equal to 190,000 not previously identified as a DNA polymerase. This protein was capable of DNA polymerase activity after electroelution from NaDodSO4/polyacrylamide gels and renaturation. The highly purified enzyme so obtained was active with poly(dT).oligo(rA) as template.primer, resistant to dideoxy TTP (ddTTP), and inhibited by aphidicolin and butylphenyldeoxyguanosine 5'-triphosphate, thus identifying it as a DNA polymerase alpha. The results indicate that a polypeptide of Mr approximately equal to 190,000 is an abundant component among DNA polymerase alpha catalytic polypeptides in growing monkey cells.

Properties and applications of new monoclonal antibodies raised against calf DNA polymerase alpha.

A panel of 12 hybridoma cell lines secreting monoclonal antibodies against alpha-polymerase were prepared by fusion of mouse myeloma cells and spleen cells of a rat immunized with homogeneous calf thymus alpha-polymerase. Hybridomas were selected and cloned on the basis of immunobinding to pure alpha-polymerase in solid phase radioimmunoassay. Antibodies secreted by these cells eventually were purified in milligram quantities from ascites fluids. These antibodies, all of the rat immunoglobulin M class, cross-reacted with alpha-polymerases from calf and monkey cells as revealed by immunobinding in radioimmunoassay and by immunoprecipitation of DNA polymerase activity. The antibodies were not capable of neutralizing the enzyme activity. With the methods described these antibodies may be used to immunoprecipitate alpha-polymerase from crude extracts of mammalian cells and to measure levels of the enzyme protein.

Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer.

The autocrine hypothesis proposes that a cell produces and secretes a hormone-like substance that can interact with specific membrane receptors on its surface to induce effects such as proliferation. Thus, a cancer cell could act to stimulate its own growth. Bombesin and bombesin-like peptides (BLPs) such as gastrin-releasing peptide (GRP) cause various physiological responses in mammals, including stimulation of proliferation of 3T3 mouse fibroblasts and normal human bronchial epithelial cells in vitro and induction of gastrin cell hyperplasia and increased pancreatic DNA content in vivo in rats. Human small-cell lung cancer (SCLC) cell lines produce and secrete BLPs and can express a single class of high-affinity receptors for BLPs. Exogenously added BLPs can also stimulate the clonal growth and DNA synthesis of SCLC in vitro. These findings suggest that BLPs function as autocrine growth factors for this tumour. One way to test this hypothesis is to interrupt the function of the endogenously produced BLPs. Here, we demonstrate that a monoclonal antibody to bombesin binds to the C-terminal region of BLPs, blocks the binding of the hormone to cellular receptors and inhibits the clonal growth of SCLC in vitro and the growth of SCLC xenografts in vivo. These results demonstrate that BLPs can function as autocrine growth factors for human SCLC.

Novel and potent tacrine-related hetero- and homobivalent ligands for acetylcholinesterase and butyrylcholinesterase.

Based upon synthetic and biochemical results, a novel and potent tacrine analogue and heterobivalent analogues of tacrine, were designed. The role played by the amino groups of homo- and heterobivalent ligands in the interaction with the peripheral and catalytic sites of AChE and BuChE were investigated. The syntheses of these materials together with the results of AChE/BuChE inhibition assays are detailed.