Structure of pp32, an acidic nuclear protein which inhibits oncogene-induced formation of transformed foci.

pp32 is a nuclear protein found highly expressed in normal tissues in those cells capable of self-renewal and in neoplastic cells. We report the cloning of cDNAs encoding human and murine pp32. The clones encode a 28.6-kDa protein; approximately two-thirds of the N-terminal predicts an amphipathic alpha helix containing two possible nuclear localization signals and a potential leucine zipper motif. The C-terminal third is exceptionally acidic, comprised of approximately 70% aspartic and glutamic acid residues; the predicted pI of human pp32 is 3.81. Human and murine pp32 cDNAs are 88% identical; the predicted proteins are 89% identical and 95% similar. Although the structure of pp32 is suggestive of a transcription factor, pp32 did not significantly modulate transcription of a reporter construct when fused to the Gal4 DNA-binding domain. In contrast, in cotransfection experiments, pp32 inhibited the ability of a broad assortment of oncogene pairs to transform rat embryo fibroblasts, including ras + myc, ras + jun, ras + E1a, ras + mutant p53, and E6 + E7. In related experiments, pp32 inhibited the ability of Rat 1a-myc cells to grow in soft agar, whereas it failed to affect ras-induced focus formation in NIH3T3 cells. These results suggest that pp32 may play a key role in self-renewing cell populations where it may act in the nucleus to limit their sensitivity to transformation.

pp32 overexpression induces nuclear pleomorphism in rat prostatic carcinoma cells.

Nuclear pleomorphism is an important diagnostic factor in tumour pathology. Traditionally, nuclear pleomorphism is evaluated qualitatively or semiquantitatively, often as a component of tumour grade; the molecular basis of nuclear pleomorphism, however, remains unclear. In this study, we investigated the quantitative effects on nuclear morphology of overexpressing pp32, a recently described nuclear phosphoprotein highly expressed in self-renewing and neoplastic cell populations. Assessment of Feulgen-stained transfected and control lines of AT3.1, a rat prostatic carcinoma cell line, using a computerized Cellular Image Analysis System (BD CAS-200) showed that stable overexpression of human pp32 in AT3.1 cells is accompanied by marked increases in the coefficient of variation of nuclear shape, nuclear size and chromatin textures but not in DNA content. In contrast, stable transfection with control vector, with ras, or with bcl-2 failed to affect nuclear morphology. Cell cycle analysis further showed that pp32-related increases in variation of nuclear structure manifested principally in G1. These studies suggest that pp32 plays a role either directly or indirectly in the control of nuclear shape of G1 cells.

Inhibition of fatty acid synthesis delays disease progression in a xenograft model of ovarian cancer.

One of the key limiting factors in the treatment of advanced stage human epithelial malignancies is the lack of selective molecular targets for antineoplastic therapy. A substantial subset of human ovarian, endometrial, breast, colorectal, and prostatic cancers exhibit increased endogenous fatty acid biosynthesis and overexpress certain enzymes in the pathway. Cell lines derived from these tumors use endogenously synthesized fatty acids for cellular functions, whereas normal cells and tissues appear to utilize dietary lipids preferentially. We have previously shown that the difference in fatty acid biosynthesis between cancer and normal cells is an exploitable target for metabolic inhibitors in vitro. Here, we report observations in vivo using the i.p. model of the multiply drug-resistant OVCAR-3 human ovarian carcinoma in nude mice which demonstrate that: (a) fatty acid synthase overexpression in OVCAR-3 is comparable to levels in primary human tumors assessed by immunohistochemistry; (b) fatty acid synthetic activity of OVCAR-3 is comparably elevated in vitro and in vivo and is 4 to >20-fold higher than normal murine tissues; (c) treatment with the specific fatty acid synthase inhibitor, cerulenin, markedly reduces tumor cell fatty acid biosynthesis in vivo; (d) fatty acid synthase inhibition produces regression of established ascites tumor; and (e) treatment with cerulenin causes reduction in ascites incidence, delay in onset of ascites, and significantly increased survival (P<0.04).

Intermolecular homologies of human interferon-alpha.

Human interferon-alpha 2 (IFN) was analyzed by homology search computer program with the use of protein primary structures data bases. Results indicate that four domains with heightened ability to form homology pairs with different proteins exist in the IFN molecule. These domains occupy regions 35-56, 72-85, 97-110 and 124-136, mainly between the alpha-helical cylinders on the tertiary structure models. Additionally, results show in IFN structure the presence of amino-acid motifs that create the opportunity for this cytokine to influence directly the processes of DNA functioning in cell nuclei.

[Determination of the amino acid composition of cartilage and creatine phosphokinase isoenzymes with the use of phenylthiocarbamyl amino acid derivatives]. / Opredelenie aminokislotnogo sostava khriashchevoi tkani i izofermentov kreatinfosfokinazy s ispol'zovaniem feniltiokarbamil'nykh proizvodnykh aminokislot.

A method for estimation of amino acid composition and collagen-specific amino acids was developed. The assay was based on the precolumn reaction of amino acids with phenylisothiocyanate. The phenylthiocarbamyl derivatives were analyzed by means of reverse-phase chromatography on octadecyl sorbents, which involved gradient elution with ethanol and sodium or ammonium acetate. The method was used for effective separation of collagen-specific and main amino acids in cartilage hydrolysate from healthy persons and of patients with funnel chest and Ehlers-Danlos syndrome and also for determination of amino acid composition in creatine phosphokinase B- and M-subunits.

[Isolation of the heterodimer of porcine MB creatine phosphokinase and the study of its binding with polyclonal antibodies]. / Poluchenie geterodimera MB-kreatinfosfokinazy svin'y i issledovanie ego sviazyvaniia s poliklonal'nymi antitelami.

Heterodimers of porcine creatine phosphokinase were obtained from dissociated MM and BB isoenzymes and investigated in the enzyme-linked immunosorbent assay. It has been shown that peroxidase conjugates of rabbit polyclonal antibodies against porcine enzymes have low affinity in the sandwich reaction.

[determination of the N-terminal sequence and amino acid composition of MM- and BB-isoenzymes of the swine creatine phosphokinase]. / Opredelenie N-kontsevoi posledovatel'nosti i aminokislotnogo sostava MM i BB-izofermentov kreatinfosfokinazy svin'i.

Amino-acid composition of porcine M and B creatine phosphokinase subunits and a sequence of 22 N-terminal amino acids have been determined. A comparative analysis with the structure of creatine phosphokinase from other sources made it possible to discover a sequence with 8 to 15 amino-acid residues, which is the most suitable for the preparation of polyclonal antibodies.

[Proteolytic resistance and thermostability of catalase and histidine decarboxylase from Micrococcus sp. n]. / Proteoliticheskaia ustoichivost' i termostabil'nost' katalazy i gistidindekarboksilazy iz Micrococcus sp. n.

Catalase from Micrococcus sp. n. is not hydrolyzed by trypsin at the protein/protease (pn/pe) exceeding 10/1. Histidine decarboxylase (HDC) loses 50% of activity after the first nine minutes of hydrolysis at the pn/pe ratio equal to 6/1, followed by a slow linear inactivation. Investigation of thermal stability at varying pH and temperatures demonstrated that catalase and HDC preserve 70-80% of activity after 5 minutes of incubation at 72 degrees C only at pH approaching the optimal pH of enzymatic activity (pH 7.45 for catalase and pH 5.55 for HDC).

[Physico-chemical characteristics of catalases from Micrococcus sp. n]. / Fiziko-khimicheskie kharakteristiki katalaz iz Micrococcus sp. n.

The physico-chemical properties of heme-containing and non-heme catalases isolated from the cell culture of Micrococcus sp. n. grown under intensive aeration were studied. The enzyme preparations were homogenous during polyacrylamide disc electrophoresis. The spectral and functional properties of the enzymes (e. g. specific activity, subunit molecular weight, quaternary structure, amino acid composition, immunoprecipitability, N-terminal amino acid sequences) were investigated. Monocrystals of non-heme catalase applicable for an X-ray analysis were grown and examined by X-ray spectroscopy. Both enzymes were stable upon storage in 40% ammonium sulfate for 2 months and resistant to lyophylization without any significant loss of their activity. Non-heme catalase is apparently an independent enzyme which is not derived from heme-containing catalase via dissociation, limited proteolysis or heme cleavage.

[Isolation from Micrococcus sp. n. of a homogeneous heme-containing catalase and a crystalline protein with catalase activity]. / Vydelenie iz Micrococcus sp. n gomogennykh gemsoderzhashchei katalazy i kristallicheskogo belka, obladaiushchego katalaznoi aktivnost'iu.

A method for isolation and purification of catalases from the culture of Micrococcus sp. n. grown under aeration conditions is described. Heme-containing catalase (I) and the protein possessing a catalase activity (II) were separated by fractionation with ammonium sulfate. The specific activity of the highly purified protein causing degradation of H2O2 is 200 times less than that of the heme-containing enzyme. The molecular weights of catalases I and II as determined by electrophoresis in polyacrylamide gel gradient 4/30% are 240000 and 130000, respectively. The method described is designed at rapid isolation of preparative amounts of catalases from Micrococcus sp. n.