Human lymphocyte mitogenic factor: synthesis by sensitized thymus-derived lymphocytes, dependence of expression on the presence of antigen

Lymphocyte mitogenic factor (LMF) was obtained from human lymphocytes stimulated with tetanus toxoid. LMF proved to be a soluble lymphokine produced by a sensitized T lymphocyte in response to stimulation with antigen. LMF induces proliferation in cells which normally do not proliferate in response to antigen (thymocytes, B lymphocytes). This function is expressed only in the presence of antigen. LMF produced in response to stimulation with a specific antigen is able to cooperate with more than one antigen in recruiting cells into division.LMF is a nondialyzable protein different from other lymphokines as judged by the kinetics of its release. It elutes in the postalbumin fraction of Sephadex G200.     

Functional analysis of mononuclear cells infiltrating into tumors. V. A. soluble factor involved in the regulation of cytotoxic/suppressor T cell infiltration into tumors

We have analyzed the mechanisms controlling the accumulation of cytotoxic/suppressor T lymphocytes in tumor tissues. We found that tumor-infiltrating helper/inducer T cells isolated from T-9 gliosarcoma-sensitized rats between 4 and 6 days after T-9 gliosarcoma inoculation produced a lymphocyte migration factor (LMF) during in vitro culture. Four peaks of LMF activity (A through D) were detected upon fractionation of LMF by using a Mono Q anion exchange column chromatography. Peak C exhibited the strongest activity among the four peaks of LMF. The action of peak C was chemotactic, but not chemokinetic. Peak C had an isoelectric point of 8.0 and a Mr of 26,000 Da. Only cytotoxic/suppressor T cells were found to be sensitive to peak C in vitro as well as in vivo. It is thus likely that peak C is responsible for the infiltration of cytotoxic/suppressor T cells into tumor tissues. The infiltration of lymphocytes into tumor tissues might also be regulated by the expression of lymphocyte sensitivity for LMF. The target molecule for LMF at 4 days may involve an asparagine-linked oligosaccharide.     

Fibroblast mitogens in swine milk include an epidermal growth factor-related peptide

Mammary secretions obtained from lactating sows can support in vitro growth of mammalian cells when added to Dulbecco's Modified Eagle Medium. In order to identify the growth factors present within, sow milk was fractionated and fractions having mitogenic activity were identified by their ability to stimulate DNA synthesis in density-arrested, quiescent (murine) AKR-2B fibroblasts. A prominent mitogenic activity (peak III) was observed in the 3,000-5,000 Mr range. This activity was partially purified by (1) preparative Sephadex G-200 chromatography, (2) gel-filtration in Sephadex G-50 columns and (3) DEAE-cellulose anion exchange chromatography. The last step resolved peak III activity into at least two distinct components. One component was highly-purified by use of reverse-phase high-performance liquid chromatography (RP-HPLC). This activity was identified as an epidermal growth factor (EGF)-related peptide based on its inactivation by polyclonal antibody (IgG fraction) specific for murine EGF (mEGF) and proteolytic agents. The other component is unrelated to EGF. Using cloned mEGF cDNA as a probe, the presence of EGF-related mRNA in lactating mammary tissues and newborn pig small intestine was also demonstrated. These factors may contribute to the preferential growth of gastrointestinal tissues in neonatal pigs.     

Functional analysis of mononuclear cells infiltrating into tumors. III. Soluble factors involved in the regulation of T lymphocyte infiltration into tumors

We have analyzed the mechanisms controlling the accumulation of T lymphocytes in tumor tissues. Spleen cells, left or right popliteal lymph node cells, and tumor-infiltrating cells were obtained from tumor-inoculated rats and were cultured for 24 h. Culture supernatants were obtained and assessed for lymphocyte migration factor (LMF) activity with the use of a modified Boyden chamber. We found that tumor-infiltrating cells derived from T-9-sensitized rats produced LMF. Two waves of LMF production were observed. The first wave of LMF production was detected between 6 and 12 h (LMF-a) and the second wave of LMF production was detected between 4 and 6 days (LMF-4d and -6d) after tumor inoculation. The tumor-infiltrating cells consisted of heterogenous cell populations. We found that only tumor-infiltrating neutrophils of T-9-sensitized rats produced LMF-a. Five peaks of LMF (A through E) were detected upon fractionation of LMF-a using Mono Q anion exchange column chromatography. Peak D exhibited the strongest activity. The action of peak D was chemotactic, but not chemokinetic. The m.w. of peak D was 33,000 and 70,000. Only W3/25 (+) (helper/inducer) T cells were found to be sensitive to peak D. The production of LMF-a by purified tumor-infiltrating neutrophils in vitro is in agreement with the histologic observation that the infiltration of neutrophils precedes the appearance of W3/25 (+) T cells in tumor tissues of T-9-sensitized rats. It is thus likely that peak D of LMF-a is responsible for the infiltration of T lymphocytes into tumor tissues.     

Role of lipids in the immune response. II. An enhancer of antibody production (APS) that is a lipid closely associated with lymphocyte mitogenic factor (LMF).

Lymphocyte mitogenic factor (LMF) is a lymphokine produced by dividing T cells. An activity coeluting with LMF from a Sephadex resin is extractable from LMF with chloroform-methanol. It enhances but does not initiate antibody formation measured by generation of plaque-forming cells (PFC). It does not cause proliferation of B cells. This activity elutes from a silicic acid column and produces a pattern on thin layer chromatography that suggests that the compound(s) responsible for the activity is a polar lipid.     

Purification to homogeneity of human placental acid sphingomyelinase

Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n-octyl-beta-D-glucopyranoside. The major steps in the procedure included column chromatographies with Con A-Sepharose, sphingosylphosphorylcholine-Sepharose 4B, hexyl-agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n-octyl-beta-D-glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1-5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at -80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.     

Isoelectric focusing of rat pituitary gonadotropins]

Isoelectric focusing of rat gonadotropins has been studied using a small scale column and various pH gradients. Hormones were detected by radioimmunoassay. FSH focuses as a single peak, the pI being 2.8. It is thus slightly more acidic than the pI of FSH from other species. LH is more heterogeneous, the main activity focusing in the pH 9.0 area, whereas a second activity appears, for some samples, in the acidic part of the gradient. TSH exhibits a broad zone of activity between pH 7.0 and 10.0. The fractionation of pituitary glycoproteins using a pH 3-10 gradient followed by removal of sucrose and ampholytes through Sephadex G 50 chromatography allows the recovery with good yields of a purified rat FSH fraction devoid of LH activity as estimated by radioimmunoassay.     

Ascaris suum: human lymphocyte mitogenic factor content

Ascaris suum extract was found to contain a mitogenic factor which stimulates human lymphocytes. The extract (100 micrograms protein/ml) induced an increase in [3H]thymidine incorporation into human lymphocytes at a level similar to that obtained with pokeweed mitogen (11 micrograms/ml). Stimulation of mitosis appeared to be more effective with T lymphocytes than non-T lymphocytes. The mitogenic activity of the extract was reduced by only 27% when treated at 56 C for 30 min or when immersed into boiling water for 1 min. The extract was fractionated into four protein peaks by Sephacryl S-200 column chromatography. Lymphocyte mitogenic activity was observed in the first half of the first protein peak. Allergenic activity, assessed by the passive cutaneous anaphylaxis test in rats, was observed in the latter half of the same peak. These results suggest that A. suum contains both an allergen and a mitogenic substance.     

Isolation of three creatine kinases MM from porcine skeletal muscle

The purified creatine kinase MM of porcine skeletal muscle [Takasawa, T. & Shiokawa, H. (1981) J. Biochem. 90, 195-204] was separated into three distinct fractions by isoelectric focusing (IEF) in a sucrose gradient column, and the three active fractions were isolated by repeated IEF. There were one major fraction with isoelectric point (pI) 6.57 and two minor fractions with pI 6.74 and pI 6.34, respectively. No differences were observed in the IEF pattern of the enzyme in the presence and absence of dithiothreitol throughout the column. There was no interconversion from one form to another during IEF. The distribution of the three forms on IEF was not affected by adding protease inhibitor to the extraction medium. Of the three fractions, the major fraction had the highest specific activity. The three fractions differed from one another in their amino acid compositions. Not only porcine muscle but also rabbit muscle creatine kinase displayed this type of heterogeneity. Such microheterogeneities may occur widely in muscle creatine kinases.     

Separation of RRBC-RFC and non-rosette forming cells (non-RFC) of guinea pig T-cell populations and their functional difference. 1. Response of RRBC-RFC and non-RFC to either Con-A or LPS.

Guinea pig lymph node cells suspension (LNC-O) was filtered through a glass wool column and the effluent (LNC-G) was further filtered through a nylon column. In this effluent (LNC-NE) about 30 per cent of the lymphocytes was identified as non-rosette forming cells (non-RFC). The non-RFC fraction was separated from LNC-NE fraction by Ficol-Conray specific gravity centrifugation or effluent cells reacted previously to rabbit red blood cells (RRBC). The upper layer after centrifugation, designated non-RFC fraction, was separated. In this fraction 96% of the cells were lymphocytes and about 95% of them were non-RFC, which lacked receptors for rabbit red blood cells (RRBC) or EAC and detectable surface immunoglobulin by conventional techniques. Though the response of the lymphocytes in the non-RFC fraction to mitogenic (Con-A, LPS) or antigenic stimulation was lower in comparison with that in RFC-rich fraction, the response of non-RFC to ConA exceeded the response to LPS. These facts suggest that at least a portion of the non-RFC may be cells from the T-cell line.     

Biological Activities of Guinea Pig Mitogenic Factor from Immune Lymphocytes Stimulated with Antigen

Mitogenic factor was produced by sensitized guinea pig lymph node cells stimulated with a specific antigen. Both T lymphocytes and macrophages were required for the production of this factor. The culture supernatant of lymphocytes containing the mitogenic factor exhibited a strong helping effect on the proliferative response of T lymphocytes to phytohemagglutinin (PHA). Mitogenic factor and the factor with the helping activity coeluted in the molecular weight range of 25,000-35,000 daltons in gel filtration. Furthermore the fraction containing mitogenic factor was found to support the proliferation of lymphoblasts induced by PHA or antigen, suggesting that the mitogenic factor may be the guinea pig equivalent of T cell growth factor (TCGF) reported in the mouse, rat, and human. On the other hand, the T cell-activating monokine of the guinea pig, possessing the helping activity for the proliferative response of T lymphocytes to PHA, never exhibited TCGF-like activity.     

Purification and characterization of a third glucosyltransferase from Streptococcus mutans serotype g

Streptococcus mutans strain AHT (serotype g) secretes at least two glucosyltransferases with different pI values. A novel glucosyltransferase with a pI of 5.8 was purified 244-fold from the ammonium sulphate fraction by DEAE-cellulose chromatography, FPLC (Mono Q column, Pharmacia) and hydrophobic chromatography. The enzyme preparation gave a single protein band on analysis by both PAGE and SDS-PAGE, and did not form multiple protein bands detectable by IEF. The Mr was estimated to be about 130,000 by SDS-PAGE and about 135,000 by ultracentrifugal analysis. The apparent Km value and pH optimum of the enzyme were 3.9 +/- 0.2 mM (mean +/- SD) and about 4.7, respectively. The enzyme synthesized water-soluble glucan from sucrose, and the glucan consisted of over 90 mol% 1,6-alpha-D-glucosidic linkages. The enzyme activity was not stimulated by primer dextran. Anti-enzyme serum produced a single precipitin band with the purified enzyme preparation, whereas it did not react with either of the other two known glucosyltransferases.     

Purification and properties of a mammary-uterine-pituitary tumor cell growth factor from pregnant sheep uterus

A mammary-uterine-pituitary tumor cell growth factor has been purified from lyophilized powders of pregnant sheep uteri by a five-step procedure. Uterine-derived growth factor (UDGF) was extracted from the powders with 0.1 M acetic acid, heated at 95 degrees C, and further purified by sulfopropyl-Sephadex C-25, Sephadex G-50, and carboxymethyl-Sephadex C-25 chromatography. From 500 g of uterine powder, 40 to 50 mg of UDGF can be isolated at an overall yield of 33%. The degree of homogeneity of the final preparations was estimated by 8 M urea, 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), and by PAGE under nondissociating conditions at either pH 8.5 or 4.5. In all PAGE experiments, the purified UDGF preparation showed a single Coomassie blue-stained band that directly corresponded to the only area of elution of UDGF activity from duplicate unstained gels. Molecular sieve high performance liquid chromatography HPLC, reverse phase HPLC on an octylsilyl (C8) column, and hydrophobic chromatography on octyl-Sepharose CL-4B all confirm a similar degree (i.e. greater than 90%) of homogeneity. The Mr of UDGF estimated by urea/sodium dodecyl sulfate-PAGE was 4200 +/- 500 and, by molecular sieve HPLC, 6200 +/- 1000. The isoelectric point of UDGF was estimated as pI = 7.3. The UDGF isolated showed marked cell-type specificity for established cell lines that were derived from estrogen-responsive tumors; purified sheep UDGF was mitogenic for MTW9/PL rat mammary tumor cells (at 10(-10) to 10(-9) M concentrations) while showing no mitogenic activity toward normal rat diploid fibroblasts. UDGF also promoted growth of uterine-derived tumor cells and the GH3/C14 rat pituitary line. Measuring growth as an increase in cell number, UDGF supported the logarithmic growth of the MTW9/PL rat mammary tumor cells over 6 days; other known hormones and growth factors were not able to substitute for the UDGF mitogenic action on MTW9/PL cells. It is concluded that a rapid, high-yield method of purification of a new uterine-derived growth factor activity has been developed.     

Isolation and characteristics of micromycetes--producers of neutral phenol oxidase from trophic soil with a high level of dioxins

Samples of South Vietnamese soils intensely treated with Agent Orange defoliant were tested for the presence of fungi and actinomycetes with elevated phenol oxidase activity. As a result, fast-growing non-sporulating strain producing neutral phenol oxidases was isolated and identified as Mycelia sterilia INBI 2-26. The strain formed extracellular phenol oxidases during surface growth on liquid medium in the presence of guayacol and copper sulfate, as well as during submerged cultivation in liquid medium containing wheat bran and sugar beet pulp. Isoelectric focusing of cultural liquid has revealed two major catechol oxidases (PO1 and PO2) with pI 3.5 and 8, respectively. The enzymes were purified by ultrafiltration, ion exchange chromatography and exclusion HPLC. Both were stable between pH 3 and 8. At pH 8 and 40 degrees C they retained at least 50% of activity after incubation for 50 h. At 50 degrees C PO2 was more stable and retained 40% of activity after 50 h, whereas PO1 was inactivated in 3-6 h. The pH optimums for PO1 and PO2 towards catechol were equal to 6 and 6.5, and the Km values were 1.5 +/- 0.35 and 1.25 +/- 0.2 mM, respectively. PO1 and PO2 most optimally oxidized 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) at pH 3 with Km values 1.6 +/- 0.18 and 0.045 +/- 0.01 mM, respectively, but displayed no activity towards tyrosine. The PO2 absorbance spectrum had a peak at 600 nm, thus indicating the enzyme to be a member of the laccase family.     

Polyclonal antibody-forming cell activation and immunomodulation of the in vitro immune response induced by streptococcal extracellular products

The capacity of three different extracellular streptococcal products to induce polyclonal activation of precursors of plaque-forming cells (PFC) was investigated. The gamma fraction (pI = 4.2), previously shown to be only weakly mitogenic, was the most potent activator of rabbit and mouse immunoglobulin-secreting cells. The polyclonal stimulation induced by the two other fractions (kappa: pI = 4.8 and epsilon: pI = 10.3), shown to be mitogenic in both systems, was only observed in the rabbit system. Using these fractions, the in vitro immunomodulation of the anti-sheep red blood cell immune response was also investigated. Both gamma and epsilon fractions were shown to possess adjuvant properties, whereas the kappa fraction was a suppressor of the specific immune response. It appears, therefore, that the diversified immunological activities observed with extracellular streptococcal products can be dissociated and belong to different entities.     

An assay for human lymphocyte mitogenic factor: B-lymphocyte stimulation by a T-lymphocyte product.

Human lymphocyte mitogenic factor (LMF) is produced only by E-rosette positive T-lymphocytes when purified cell populations are used. Production is antigen specific and requires the presence of monocytes during the first 24 hr of culture. However, LMF production or regulation of production by cells other than T-lymphocytes may occur as more LMF activity is produced when B-lymphocytes are added to a primary culture of T-lymphocytes. The molecular weight of LMF is estimated by gel filtration to be between 20,500 and 28,000. LMF is heat stable.The response to LMF by B- or T-lymphocytes without monocytes is non-specific and is independent of and unaffected by antigen. The B-lymphocyte response is quantitatively greater that the response of T-lymphocytes. B-lymphocytes are much more sensitive to small concentrations of LMF.     

Mechanistic studies on protopanaxadiol, Rh2, and ginseng (Panax quinquefolius) extract induced cytotoxicity in intestinal Caco-2 cells

Certain ginsenosides, also known as triterpene glycosides, have been recently reported to have a characteristic effect on cultured intestinal and leukemia cell growth. Ginsenoside aglycones 20(S)-protopanaxadiol (PD), 20(S)-protopanaxatriol (PT), and ginsenoside Rh2 have been identified as having a strong effect on reducing cell viability. Furthermore, ginsenoside Rh2 is thought to be a rare ginsenoside not found in all ginseng products. Rather, Rh2 has been recently reported to be a breakdown product of thermal processing of North American ginseng. In this study, pure ginsenosides PD, PT, Rh2 standards and an enriched Rh2 fraction derived from ginseng leaf were tested in cultured Caco-2 cells for relative cytotoxic potency. PD and Rh2 LC50 were similar after 24 to 72 h, whereas a drop in PT LC50 occurred later at 48 and 72 h. Furthermore, PD and Rh2 affected membrane integrity as indicated by LDH secretion earlier than PT and the enriched Rh2 fraction (P < or = 0.05). Ginsenoside Rh2 showed the greatest (P < or = 0.05) build up of necrotic cells (18.3 +/- 0.1%) at the respective LC50 after 24 h and PD (21.3 +/- 0.3%) showed the largest effect after 44 h of exposure. The effect on apoptotic cells at 44 h of treatment were significantly different (P < or = 0.05) for Rh2 (21 +/- 0.4%), PD (14.6 +/- 0.1%), enriched Rh2 leaf fraction (9.9 +/- 0.6%), and PT (2.3 +/- 0.1%) treatments. Caco-2 caspase-3 activity was different between ginsenoside exposure; Rh2 (10.6 +/- 0.3 nM pNA) had the greatest (P < or = 0.05) activity followed by the enriched Rh2 leaf fraction (8.3 +/- 0.2 nM pNA), PT (7.3 +/- 0.3 nM pNA). The PD (4.8 +/- 0.04 nM pNA) treatment was similar to untreated cells (4.3 +/- 0.05 nM pNA) in caspase-3 activity. These results show variable bioactive response in cultured intestinal cell to specific ginsenosides and an enriched Rh2 North American ginseng extract which may be explained on basis of hydrophobic/hydrophilic balance.     

Isolation by isoelectric focusing of a mitogenic substance released by stimulated human lymphocytes

Supernatants of human lymphocytes incubated for 4 hr with extracellular products of group A streptococci (streptococcal filtrate) and then washed and reincubated had a mitogenic activity for homologous lymphocytes. Fractionation by isoelectric focusing of the lymphocyte supernatants and of the streptococcal filtrates showed mitogenic activity in fractions with different isoelectric points. Moreover, human lymphocytes stimulated either with a mitogenic fraction of the lymphocyte supernatant or with a mitogenic fraction of the streptococcal filtrate, after treatment with bromodeoxyuridine (BUdR) and reciprocal restimulation, were responsive only to the heterologous stimulant.     

A rapid four column purification of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human liver

2-Deoxy-D-glucoside-2-sulphamate sulphohydrolase was extracted from human liver and purified 40 000-fold by a simple four column procedure. The purification was followed using a specific substrate isolated from an acid hydrolysate of heparin, O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid. Only one form of the enzyme was seen on either ion exchange chromatography or isoelectric focussing, with a pI of 6.8. The apparent Mr of the holoenzyme as determined by gel filtration was 190 000 +/- 20 000. Two other larger Mr protein peaks observed on gel filtration appear to be an inactive dimer of the 190 000 dalton peak and a larger aggregate near the exclusion limit of the column. On polyacrylamide disc gel electrophoresis in sodium dodecyl sulphate, with or without prior reduction, each protein peak from the gel filtration column electrophoresed as a single major band with an apparent Mr corresponding to 55 000 +/- 6000.     

Characterization and Functional Properties of Sub-Fractions of Soluble Soybean Polysaccharides

Soluble soybean polysaccharide (SSPS) was fractionated into two sub-fractions, a high-molecular-weight fraction (HMF) and a low-molecular-weight fraction (LMF) by the ethanol-extraction method. Characterization of the sub-fractions, that is, analysis of chemical composition, gel filtration, and SDS–PAGE, revealed that the main component of HMF was a large polysaccharide molecule with covalently-attached peptides, possibly corresponding to the intact SSPS molecule. LMF consisted of free peptides and saccharides of small size, which might have occurred as by-products during the production process of SSPS. HMF exhibited high ability to emulsify oil droplets and stabilize α-casein dispersions in an acidic pH region, but this ability of LMF was inferior to HMF. On the other hand, LMF had higher activity to prevent the oxidation of emulsified lipids than HMF. These results suggest that HMF and LMF had different characteristics and functional properties, and that the combination of the two sub-fractions generates the multi-functions of commercial SSPS.