Constitutive production of phagocytosis inducing factor(s) in a monocyte/macrophage lineage cell line (THP-1) by retrovirus-transformed human T cell lines

Four human T cell lines, MT-2, TCL-Kan, TCL-As 2, and TCL-Haz, established from normal leukocytes by cocultivation with adult T-cell leukemia (ATL) virus (ATLV)-producing cells, produced constitutively phagocytosis inducing factor(s) (PIF) that induced phagocytosis in a human monocytic cell line, THP-1. These cell lines expressed ATLV-associated antigens (ATLA) as well as numerous virus particles, whereas the other twelve leukocyte cell lines tested, including T cell lines, B cell lines, and non-T and non-B cell lines, did not produce detectable amounts of the factor(s) in the culture supernatants. PIF was produced in the absence of serum and was not related to either ATLV-particles or viral structural proteins. Its activity was stable at 56 C for 30 min, but labile at 80 C for 30 min and at pH 2 for 20 hr. MT-2 and TCL-Kan produced large amounts of the factor(s) in the culture supernatants but little interferon-gamma (IFN-gamma) or colony stimulating factor (CSF) activity was detected; furthermore, the activity was not neutralized by rabbit anti-IFN-gamma sera. These observations suggest that some ATLV-transformed T cell lines produce PIF that is different from IFN-gamma and CSF.     

Identification and characterization of a human T cell line-derived lymphokine with MAF-like activity distinct from interferon-gamma

Culture supernatants of several human T cell leukemia cell lines were screened for macrophage-activating activity (MAF) as defined by induction of tumoricidal activity against human melanoma cells in a 72-hr assay. Two cell lines, MT-2 and C10/MJ2, were found to produce high levels of MAF activity constitutively, but the MT-2 cell line, unlike C10/MJ2, produced little IFN-gamma. This observation was confirmed by Northern blot analysis performed with specific IFN-gamma cDNA probe. The MT-2 cell line thus provides a useful system to evaluate the existence of lymphokines with MAF activity that are distinct from IFN-gamma. The MAF activity produced by MT-2 cells was distinguished from IFN-gamma by the following criteria. MAF activity was not removed by immunoaffinity chromatography with the use of immobilized specific polyclonal antibodies to IFN-gamma and was not neutralized by a monoclonal antibody to IFN-gamma. Heat or acid treatments of IFN-gamma resulted in loss of its antiviral activity, but these treatments had no effect on MAF activity. MAF activity was not abolished by polymyxin B sulfate, suggesting that this activity is not mediated by or dependent on LPS. Initial characterization studies performed by using membrane filtration, gel filtration chromatography, and isoelectric focusing indicate that the non-IFN-gamma MAF activity produced by MT-2 cells has an apparent m.w. of 55,000 and an isoelectric point of 5.5. Collectively, these data suggest that the MT-2 human T cell line constitutively produces high levels of MAF and low levels of IFN-gamma and offers a useful source for the further purification of a unique human lymphokine with macrophage-activating activity that is distinct from IFN-gamma.     

Oligo-2',5'-adenylate synthetase activity in cells persistently infected with human T-lymphotropic virus type I (HTLV-I).

Spontaneous production of interferon-gamma (IFN-gamma) was shown in several T-lymphoblastoid cell lines persistently infected with human T-lymphotropic virus (HTLV-1). However, the produced IFN-gamma was not always associated with the induction of the antivirus state. The induction of oligo-2',5'-adenylate synthetase (2-5AS) by IFN was studied in five human T-cell lines persistently infected with HTLV-I (MT-1, MT-2, SMT-1, HUT 102 and OKM-2). Four cell lines are able to produce IFN-gamma spontaneously, while the OKM-2 cell line is not. Poor induction of 2-5AS was recognized in three (MT-1, MT-2 and SMT-1) of the four cell lines producing IFN-gamma, though the poor induction was improved after long-term cultivation of cells with IFN-alpha. On the contrary, in the OKM-2 cell line, significant activity of the enzyme was induced by IFN-alpha. Induction of 2-5AS was not correlated with cell growth inhibition, but with the antivirus state. Furthermore, an inverse relationship between IFN-gamma production and 2-5AS induction was demonstrated in these cell lines with the exception of HUT 102 cells.     

Characterization of monoclonal B cell growth factor (BCGF) produced by a human T-T hybridoma

We previously demonstrated the development of a cloned human T cell hybridoma that secretes B cell growth factor (BCGF) in the absence of demonstrable interleukin 2 or B cell differentiation factor. Sephadex gel filtration chromatography demonstrated the m.w. of this factor to be 18 to 20K. The present studies were performed to further characterize the biochemical properties of the molecule and to determine its target cell specificity. Temperature stability studies showed the monoclonal BCGF to be stable at 37 degrees C for 12 hr and at 70 degrees C for 15 min; however, most (93%) of the activity was lost after incubation at 70 degrees C for 30 min. Aliquots of hybridoma supernatant were exposed to buffer solutions with variable pH with no diminution in activity over a pH range of 4.0 to 10.0 BCGF activity was not affected by 2-mercaptoethanol, neuraminidase, or nucleic acid denaturing enzymes. In contrast, all activity was destroyed by 10 M urea, trypsin, and chymotrypsin. Chromatofocusing demonstrated the isoelectric point of BCGF to be 6.3 to 6.6. Finally, absorption experiments demonstrated that BCGF activity was absorbed by large, activated B cells. Mitogen-stimulated T cell blasts, small resting B cells, and CESS cells failed to absorb BCGF activity from the hybridoma supernatant. These and future studies with purified monoclonal human BCGF should enhance our understanding of its immunochemical properties and of its role in the immunoregulation of human B cell responses.     

Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes

The interaction of 125I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4 degrees C to pH 3 resistance at 37 degrees C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37 degrees C led to nonlinear curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.     

A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens.

Several octopine strains of Agrobacterium tumefaciens were tested for Ti plasmid (pTi) transfer after induction by 400 micrograms of octopine per ml for 24 h. The strains could be divided into two groups, transfer efficient (Trae) and transfer inefficient (Traie); the respective rates of transfer were 0.77 x 10(-2) to 1.14 x 10(-2) and 0.33 x 10(-6) to 9.8 x 10(-6) plasmid transconjugant per donor cell. Transfer efficiencies of Traie strains were greatly increased when the time of induction was 72 h. A diffusible conjugation factor (CF) that can enhance conjugal transfer of pTi in A. tumefaciens was discovered when both Trae and Traie donor strains were induced in the same plate. The evidence indicates that CF is a key factor affecting transfer efficiency of pTi but is not sufficient by itself to induce transfer. Trac mutants can produce CF constitutively, and Trae strains can produce it after induction by low octopine concentrations. The transfer efficiency of Traie strains was greatly increased by adding CF to the induction medium. The thermosensitive strain B6S, which normally cannot conjugate at temperatures above 30 degrees C, could transfer pTi efficiently at 32 and 34 degrees C in the presence of CF. Production of CF is dependent on the presence of pTi but appears to be common for different opine strains; it was first detected in octopine strains, but nopaline strains also produced the same or a similar compound. CF is very biologically active, affecting donor but not recipient bacterial cells, but CF does not promote aggregation. Data suggest that CF might be an activator or derepressor in the conjugation system of A. tumefaciens. CF is a dialyzable small molecule and is resistant to DNase, RNase, protease, and heating to 100 degrees C for 10 min, but autoclaving (121 degrees C for 15 min) and alkaline treatment removed all activity.     

Suppressor factor secreted by T hybridoma established from peripheral blood lymphocytes of a bone marrow transplantation patient. I. Establishment of human T-cell hybridoma and partial characterization of suppressor factor

A stable human T-cell hybridoma was established by cell fusion between activated human peripheral blood lymphocytes from an allogeneic bone marrow transplantation patient and the JD1-17 cell line, a subclone of the human T leukemia Jurkat cell line. This hybrid clone 1-8, which bore the surface phenotype of suppressor cells (CD8+HNK1+), spontaneously secreted a factor which, at high dilutions, suppressed the responses of T and B cells induced by mitogens and alloantigens. This suppressor factor was found to be heat-resistant (56 degrees C, 30 min), stable at alkaline but not acid pH, unaffected by 2-mercaptoethanol, and sensitive to trypsin. Preparative isoelectric focusing revealed an isoelectric point of 5.35. The suppressor activity was selectively absorbed by blast T cells. By gel filtration on Sephacryl S-200 and HPLC, the suppressor activity was found in two peaks corresponding to 40-45 kDa (monomer) and 90-95 kDa (dimer).     

Regulation of thromboxane A2 biosynthesis in platelet-free human monocytes and the possible role of polypeptide growth factor(s) in the induction of cyclooxygenase system

It has previously been shown that platelet-free human monocytes, when properly incubated in the presence of animal and human sera, became capable of producing large amounts of thromboxane A2 and prostaglandin E2. The characteristics of these processes are reported here. Prostaglandin biosynthesis was time and cell concentration dependent; 24 h of incubation at 37 degrees C and 0.5 X 10(6) cells per ml medium were found to give the most reproducible results. Human monocytes produced thromboxane A2 and prostaglandin E2 in a typical ratio which ranged from 2.0 to 5.0 (28 experiments). Animal and human sera were similarly effective, while serum obtained from platelet-free blood was much less active. The activity of all sera tested was stable to heating (100 degrees C for 2-10 min) and extreme pH values (pH 2 and 11). It was unstable when the serum was heated at pH 11 and after 2-mercaptoethanol treatment. These observations prompted us to check the effect of polypeptide growth factors having properties similar to those reported above, such as platelet-derived growth factor, fibroblast growth factor, epidermal growth factor as well as insulin and transferrin. None of these, alone or in various combinations, was capable of eliciting a stimulation comparable with that of serum. Stimulation due to sera was, as expected, dose dependently inhibited by acetylsalicylic acid and more efficiently by indomethacin; unexpectedly it was also inhibited by protein synthesis inhibitors such as actinomycin D and cycloheximide in conditions under which no toxic effect of the drugs was evident. On the basis of these results we conclude that: (a) polypeptide growth factor(s) with a molecular weight at least 30 000 (as judged by Amicon ultrafiltration) is involved in the regulation of prostaglandin biosynthesis); (b) such a factor(s) acts by inducing rather than by activating the cyclooxygenase system.     

Characterization and partial purification of human epithelial transforming growth factor

A polypeptide growth factor has been partially purified from medium conditioned by the human adrenocortical carcinoma cell line SW13. This factor, designated h-TGFe, stimulates anchorage-independent growth of the SW13 cells. Similar activity was observed in human milk, and in conditioned media from seven of 14 epithelial cell lines. The SW13-derived activity is stable to low pH and 8M urea but labile to dithiothreitol and 2% sodium dodecyl sulfate. Human TGFe does not bind to heparin and fails to stimulate growth of endothelial cells in monolayer culture. The apparent molecular weight of h-TGFe is 59k by size exclusion chromatography in the presence of 8M urea and the activity binds strongly to cation exchangers. The activity elutes at 15-30% acetonitrile from a C18 reverse-phase column and has been partially purified by using a four-step chromatographic procedure. TGFe appears to be a novel growth factor produced by many epithelial cells and tissues.     

Purification and characterization of a colony stimulating factor from human lung.

Conditioned medium prepared from human autopsy lung tissue contains high level activity of colony stimulating factor which stimulates granulocytes and macrophage colony formation in both mouse and human bone marrow. The lung colony stimulating factor has been purified about 2250-fold by methods including hydroxylapatite chromatography, preparative gel electrophoresis, preparative isoelectric focusing, and gel filtration chromatography. The final specific activity was 2.7 X 10(6) units/mg. The purified factor has a molecular weight of 41 000 as determined by gel filtration. It is stable at the pH range of 6.5--10 and 56 degrees C for 30 min but sensitive to protease digestion and periodate oxidation. On polyacrylamide gel electrophoresis, it migrates in the alpha-globulin post-albumin region. Upon isoelectrofocusing lung colony stimulating factor appears heterogeneous with isoelectric points of 3.7--4.3. Treatment with neuraminidase did not affect its activity, but caused a change in electrophoretic mobility and isoelectric point. Antibody produced by immunizing rabbits with partially purified lung colony stimulating factor exerted strong inhibitory activity on the factor from lung as well as on colony stimulating factor from other human sources including serum, urine, and placenta.     

Growth-stimulating, cytostatic and cytotoxic activity of the secretory products of the RPMI-6410t human B-cell lymphoblastoid line

The cells of human lymphoblastoid line RPMI-6410t were shown to synthesize constitutively a factor(s) with different types of biological activity. The factor(s) stimulated the growth of both B cells 6410t, obtained from the blood of a patient with acute myeloblastic leukemia, and the human embryonic diploid fibroblasts. With B cell lines Raji and P3HR-1.G5, obtained from the patients with Burkitt's lymphoma. The growth factor(s) displayed cytotoxic and cytostatic effects, respectively. Growth-stimulating and cytotoxic activating of the factor were destroyed by a 15 hour exposure to low or high pH. The activity was stable within pH values of 6-8. With regard to heat stability, the activity destroyed at 70 degrees C within 1 hour but remained stable at 56 degrees C during 1 hour. The above factor(s) displayed biological activities similar to those of the previously known tumor necrosis factor (TNF).     

Production and properties of alpha-amylase from Penicillium chrysogenum and its application in starch hydrolysis

Fungi were screened for their ability to produce alpha-amylase by a plate culture method. Penicillium chrysogenum showed high enzymatic activity. Alpha-amylase production by P. chrysogenum cultivated in liquid media containing maltose (2%) reached its maximum at 6-8 days, at 30 degrees C, with a level of 155 U ml(-1). Some general properties of the enzyme were investigated. The optimum reaction pH and temperature were 5.0 and 30-40 degrees C, respectively. The enzyme was stable at a pH range from 5.0-6.0 and at 30 degrees C for 20 min and the enzyme's 92.1% activity's was retained at 40 degrees C for 20 min without substrate. Hydrolysis products of the enzyme were maltose, unidefined oligosaccharides, and a trace amount of glucose. Alpha-amylase of P. chrysogenum hydrolysed starches from different sources. The best hydrolysis was determined (98.69%) in soluble starch for 15 minute at 30 degrees C.     

Implications of a 5'-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer

5'-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56 degrees C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37 degrees C for 1 h and it was destroyed completely by heating at 100 degrees C for 30 min. When the heated (56 degrees C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of N-acetyl-beta-D-glucosaminidase or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5'-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35,000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggest that the previously reported undetectability of 5'-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5'-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5'-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.     

Characterization and applications of lymphocytic clonal growth factor in human plasma

Human plasma was found to contain a macromolecular protein which can grow even a single cell of human lymphocytic cell lines (B-lymphoblastoid cell line HO-323-3 and T-lymphoblastoid cell line CCRF-CEM) and human-human hybridoma clones (SH-9, SU-1 and HB4C5) in a dish, but it has no effect on the growth of epithelial cell lines (lung cancer cell lines PC-8, QG-56 and QG-90). The proliferating activity for lymphocytic cell lines was gradually decreased at 4 or -20°C and dramatically decreased by heating at more than 60°C for 15 min. From human plasma, active fractions were purified by a successive application of Ca²⁺ treatment, ammonium sulfate fractionation, DEAE-5PW column chromatography (FPLC) at pH 7.6. These active fractions were divided into at least three proteins by DEAE-5PW chromatography at pH 8.5 and chromatofocusing. These purified factors, named lymphocytic clonal growth factors (LCGFs), had similar molecular weights of about 600 K and each factor consisted of a 180 K and two 210 K subunits associated with hydrogen bondings. By the addition of 5 g/ml of each factor into culture media, incidences of human-human hybridomas and cloning efficiencies of the hybridomas increased several-fold.     

IL-2 production in human T lymphotropic virus I-infected leukemic T lymphocytes analyzed by in situ hybridization

In situ hybridization studies were performed with 35S-labeled anti-sense RNA probes to study IL-2 mRNA expression in three human T lymphotropic virus I-infected T cell lines at the single cell level. In HuT 102, MT-2, and MT-4 cells, IL-2 mRNA-expressing cells were identified, occurring at frequencies of 2 x 10(-2), 8 x 10(-3), and 5 x 10(-3), respectively. In these cell lines, IL-2 mRNA was not detectable in RNA extracted from whole adult T cell leukemia cell populations because of dilution by other RNA species from the vast majority of cells that do not contain IL-2 mRNA. The data indicate the possibility of paracrine growth stimulation via IL-2 and its receptor even in those human T lymphotropic virus I-infected T cell populations that apparently lack IL-2 activity when analyzed by conventional assay procedures.     

Identification and characterization of a B cell growth inhibitory factor (BIF) on BCGF-dependent B cell proliferation

The culture supernatants of Con A-activated human peripheral blood mononuclear cells (PBM) contained at least two regulatory factors upon B cell proliferation. One was B cell growth factor (BCGF), which activated antigen-stimulated B cells to proliferation and clonal expansion, and the other was its inhibitory factor, arbitrarily named B cell growth inhibitory factor (BIF). This BIF inhibited the effect of BCGF on anti-mu-stimulated B cells or the monoclonal mature B cell line (CLL-T.H.) obtained from the peripheral blood lymphocytes of B cell-type chronic lymphocytic leukemia patients, which were activated only with BCGF and without adding other proliferating stimuli (e.g., anti-mu). BIF activity was detected in the 24 hr culture supernatants of Con A-activated human PBM in FCS containing medium and also in serum-free RPMI 1640 medium. This substance with BIF activity could not be derived from FCS. Con A-induced BIF (m.w. of 80,000 and an isoelectric point of pH 5.4) was analyzed by Sephadex G-200 gel filtration and chromatofocusing. BIF was stable at pH 2.0 and at 56 degrees C for 30 min. Partially purified BIF had no effect on cell viability and almost no interferon activity (less than 1 IU/ml). BIF with high titer had a slight but significant inhibition on TCGF-dependent T cell growth and on PHA or Con A responses, but the extent of these inhibitions was far less than that of BCGF-dependent B cell growth. Absorption of BIF with Con A blasts made its inhibition on T cell growth even less. On the other hand, BIF activity could not be absorbed with Con A blasts but was almost absorbed with large numbers of CLL-T.H. cells. BIF had almost no inhibitory effect on the proliferation of a mouse fibroblast cell line (NIH 3T3), a mouse myeloma cell line (NS-1), human lymphoid cell lines (MOLT-4, HSB-2, and Daudi), or a human myeloid cell line (K-562). BIF-producing cells were estimated to be T cells and were identified as T8+ T cells. On the other hand, Con A-induced BCGF was demonstrated to be produced predominantly by T4+ T cells. These results show that human B cell proliferation is regulated by interaction between T4+ and T8+ cells via soluble factors, namely BCGF and BIF, respectively.     

Production of a low molecular weight growth inhibitory factor by adenovirus 12-transformed cells.

Malignant rodent cells transformed by human adenovirus 12 produce a potent cell growth inhibitory factor. The cell growth inhibitory factor inhibits the growth of and DNA synthesis in normal fibroblasts in vitro. Extent of the production of the cell growth inhibitory factor appears to be proportional to that of the malignancy of the transformed cells. C57AT1-AB cells, an adenovirus 12-transformant of C57BL/6 mouse origin, are highly tumorigenic in the syngeneic and allogeneic mice. The cell growth inhibitory factor produced by these cells was characterized for the physicochemical properties; the cell growth-inhibitory activity was quantitatively recovered in the filtrates of YM-2 membrane (M(r) less than 1,000), resistant to the heat treatments at 56 degrees C for 30 min and 100 degrees C for 5 min, and extractable by ethyl acetate under acid-condition. These results suggest that the cell growth inhibitory factor may be lipid or oligopeptides.     

Complement-induced histamine release from human basophils. I. Generation of activity in human serum.

The incubation of zymosan, endotoxin, or immune aggregates with normal human serum activates a factor which induces release of histamine from autologous basophils. The reaction can be divided into two steps: in the first, complement must be activated and in the second, the histamine-releasing factor interacts with basophils. The generation of histamine-releasing activity in serum occurs at 17 to 37 degrees C but not at 0 degrees C, is inhibited by heating the serum at 56 degrees C for 30 min, or by the addition of EDTA to the serum. Once generated, the histamine-liberating activity is stable to heating at 56 degrees C for 30 min. Gel filtration of the activated serum demonstrated that this factor eluted in the same region as a factor with chemotactic activity. Both factors have a molecular weight of about 16,000 daltons and their activities were inhibited by antibody to human C5. This is therefore a pathway for histamine release by C5a where the activation of the basophil is unrelated to the membrane bound IgE.     

Regulation of lymphocyte blastogenesis and antibody production by soluble factor released by a human B-lymphoblastoid cell line

Yam 1B, a human B lymphoblastoid cell line, spontaneously produced an immunoregulatory factor, which suppresses blastogenesis and antibody formation by human lymphocytes. The Yam 1B cells, which were derived from the peripheral blood of an adult T-cell leukemia patient, have been established and maintained in our laboratory since 1985. This cell line expressed mature B-cell surface antigens including surface immunoglobulin M (IgM), CD23, and HLA-DR; had cytoplasmic IgM; and secreted small amounts of IgM in the culture supernatants. Yam 1B was positive for Epstein-Barr virus-associated antigen (EBNA) but negative for adult T-cell-associated antigen (ATLA). The serum-free Yam 1B culture supernatants (SN) inhibited the expression of transferrin R, but neither the expression of interleukin 2 (IL-2) R(CD25) nor the production of IL-2 in the lymphocytes stimulated with phytohemagglutin. Yam 1B SN also inhibited DNA synthesis by human T and B lymphocytes and immunoglobulin generation by normal B cells as well as by Epstein-Barr virus-transformed human B lymphoblastoid cell lines. The inhibitory activity of Yam 1B SN was inactivated at 56 degrees C and at pH 10 but was relatively stable at pH 2. It was abrogated by digestion with pronase and was partially stable by digestion with trypsin. Fractions collected from a Sephacryl S-300 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden) were found to have a peak of inhibitory activity of cell proliferation associated with molecules of apparent MWr of 43,000 to 67,000. The inhibitory activity of Yam 1B SN was not blocked by the anti-transforming growth factor beta antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal stability of beta-xylanases produced by different Thermomyces lanuginosus strains

The thermostability of beta-xylanases produced by nine thermophilic Thermomyces lanuginosus strains in a coarse corn cob medium was assessed. The xylanase produced by T. lanuginosus strain SSBP retained 100% of its activity after 6 h at temperatures up to 65 degrees C. In comparison seven ATCC strains and the DSM 5826 strain of T. lanuginosus only retained 100% xylanase activity at temperatures up to 60 degrees C. Culture filtrates of T. lanuginosus strain SSBP grown on coarse corn cobs, oatspelts xylan, birchwood xylan, wheatbran, locust beangum, and sugar cane bagasse, retained 100% xylanase activity at temperatures up to 60 degrees C. The xylanase produced on corn cobs was the most thermostable and showed an increase of approximately 6% from 70 degrees C to 80 degrees C. The T(1/2) of all strains at 70 degrees C at pH 6.5 varied greatly from 63 min for strain ATCC 28083 to 340 min for strain SSBP. The xylanase of strain SSBP was much less thermostable at pH 5.0 and pH 12.0 with T(1/2) values of 11.5 min and 15 min, respectively at 70 degrees C. At 50 degrees C, the enzyme of T. lanuginosus strain SSBP produced on coarse corn cobs was stable within the pH range of 5.5-10.0. Furthermore, the enzyme retained total activity at 60 degrees C for over 14 days and at 65 degrees C for over 48 h. The xylanase of T. lanuginosus strain SSBP possesses thermo- and pH stability properties that may be attractive to industrial application.