Mitogenic Substance Research Articles

Insulin-like growth factor binding protein (IGFBP)-mediated hair cell survival on the mouse utricle exposed to neomycin: the roles of IGFBP-4 and IGFBP-5

Conclusion. This study suggests for the first time that 1) IGF-I, IGFBP-4, and -5 alone and IGF-I+IGFBP-5 mixture stimulated hair cell survival and prevented neomycin-induced hair cell loss in the sensory epithelial culture of mouse utricles, 2) When administered together, IGFBP-4 diminished the effect of IGF-I, 3) In P3-5 mice utricle, IGF-I, IGFBP-4, and IGFBP-5 are expressed in the cytoplasm of hair cells. And Insulin/IGF-I Receptor is expressed in the nucleus of hair cells. Objectives. Several growth factors have been demonstrated to protect auditory sensory cells in vitro and in vivo from aminoglycoside toxicity. IGF-I is one of the most well-known mitogenic and protective substance working in the inner ear. However, there are no reports available regarding the function of IGFBPs in the inner ear. In the present study, the effects of IGFBP-4 and -5 on hair cell survival were investigated in mouse utriclular organ cultures. Materials and Methods. The amount of cellular damage and cell viability in vestibular organs were assessed by counting hair cells stained with a rhodamine-phalloidin probe. The expressions of IGFBP-4, IGFBP-5, IGF-IR, and IGF-I were localized by immunohistochemistry. Results. When treated with IGF-I, IGFBP-4, or IGFBP-5 for 24 h, explant culture showed hair cell survival rates of 136±18%, 140±15%, and 133±6%, respectively, compared to controls. Neomycin (1 mM) induced hair cell loss resulted in 45±17% of hair cell survival. However, pre-treatment of IGF-I, IGFBP-4, or -5 before neomycin insult showed survival rates of 113±14%, 98±8%, and 73±24%, respectively. Similar to IGF-I, IGFBP-4 and IGFBP -5 were significantly protective. IGFBP-4 and -5 immunoreactivities were observed in the cytoplasm of normal explanted vestibular hair cells as well as in the P3 mouse utricular hair cells in vivo.

Protein kinase C modulation of cyclic GMP in rat neonatal pulmonary vascular smooth muscle.

Protein kinase C (PKC) has been implicated in the control of vascular tone and mitogenesis in the adult pulmonary vasculature, but little is known about the role of PKC in the neonatal pulmonary vasculature. In addition, the vasodilator nitric oxide (NO) is important in the transition of the pulmonary circulation from fetal to postnatal life, and it is thought that attenuated production of NO and therefore, cGMP may contribute to the pathophysiology of a variety of forms of neonatal pulmonary vascular disease states. Although evidence exists for an interaction between PKC and NO in the adult pulmonary vasculature, the identification of specific roles for PKC in neonatal pulmonary vascular smooth muscle (NPVSM) has not been determined, and no studies have been done on the modulation of cGMP by PKC in NPVSM. Accordingly, immunoblot analysis revealed the expression of the alpha, delta, epsilon, and iota PKC isozymes in NPVSM. Treatment of NPVSM with 10 nM 4-beta phorbol myristate acetate (PMA), a PKC activator, induced translocation of PKCalpha, and PKCdelta from the soluble to the particulate fraction, while exposure to 10 nM endothelin-1 (ET-1), a potent vasoconstrictor and mitogenic substance, caused translocation of PKCdelta and PKCiota from the soluble to the particulate fraction. Sodium nitroprusside (SNP) significantly increased intracellular cGMP levels, an effect attenuated by PMA but not by ET-1. In addition, pretreatment with the specific PKC isozyme antagonist Go 6983 blocked the effect of PMA on cGMP levels. Collectively, these data demonstrate the expression and activation of multiple PKC isozymes in NPVSM, and indicate that PKC inhibits SNP-stimulated cGMP production in NPVSM. These data also suggest that complex intracellular signaling pathways by specific PKC isozymes may be important in the development of neonatal pulmonary vascular function.

Streptococcus dysgalactiae-derived mitogen (SDM), a novel bacterial superantigen: characterization of its biological activity and predicted tertiary structure.

A mitogenic substance, designated Streptococcus dysgalactiae-derived mitogen (SDM), was purified from S. dysgalactiae culture supernatant, and the gene encoding the mitogen was cloned. Both native and recombinant SDM expressed in Escherichia coli significantly activated human V beta 1+ and V beta 23+ T cells in association with major histocompatibility complex (MHC) class II molecules on accessory cells, indicating that SDM possesses superantigenic properties. The sdm gene consists of two segments encoding a signal peptide and a mature 25 kDa protein composed of 212 amino acids. Three of 34 S. dysgalactiae strains but none of 28 Streptococcus pyogenes strains examined carried sdm. Phylogenetic analysis indicated that SDM belongs to a family distinct from established bacterial superantigens. SDM showed around 30% homology with other superantigens at the amino acid sequence level. The tertiary structure of SDM was predicted by modelling onto streptococcal pyrogenic exotoxin C and streptococcal mitogenic exotoxin Z-2, both of which share highly homologous structure-determining regions. SDM showed overall structural similarity to both these superantigens. This is the first study to characterize fully a bacterial superantigen from S. dysgalactiae.

Does GH replacement therapy in adult GH-deficient patients result in recurrence or increase in size of pituitary tumours?

Hypopituitary GH-deficient patients have an increased cardiovascular mortality and GH replacement in this population has resulted in considerable therapeutic benefit. GH replacement involves administration of a potentially mitogenic substance to patients with a previous or residual pituitary tumour. Our objective was to evaluate whether GH replacement results in an increase in the size of pituitary tumours. This was a non-randomised observational study on patients recruited from the endocrine clinic. All subjects had GH deficiency, proven on an insulin tolerance test and were divided into those who were or were not receiving long-term GH replacement. Comparison of change in pituitary size was made with interval radiological imaging of the pituitary. Seventy-five patients (40 men and 35 women) were in the study, 47 were on long-term GH replacement and there were 28 controls. The average length of treatment for the treated group was 3.6 patient years. Thirty-nine patients in the treated group had at least 2 years of GH treatment between imaging studies of the pituitary. Two patients in the treated group had an increase in pituitary size (non-functioning adenomas) and two in the control group (one functioning and one non-functioning adenoma adenoma). None of these four patients required further treatment. There was no statistically significant difference between the two groups. Using a representative cohort of hypopituitary patients attending an endocrine clinic, GH replacement was not associated with an increased pituitary tumour recurrence rate. Although the results are not conclusive, in the period of observation GH had little adverse effect but longer studies are required to be certain.

Control of human vascular smooth muscle cell proliferation by sera derived from 'experimentally stressed' individuals.

Recently we reported that individuals with high scores in standardized hostility evaluation tests, when placed in a environment, may have an association with increases in a blood-borne mitogenic substance(s) for arterial smooth muscle cells. To further investigate the molecular basis for such an association, PDS [plasma derived serum with platelet derived growth factor (PDGF) removed] from individuals showing the greatest differential pre/post-stress mitogenic activity, were tested for ability to modulate changes in the steady state of the c-myc mRNA in cultured VSMC (vascular smooth muscle cells) by Northern blot analysis. Post-stress PDS resulted in a significant increase in c-myc mRNA, when compared with pre-stress PDS of the same individual. These results give further experimental support for the notion that stress (even in the form of transient, episodic psychological challenges) may affect the cardiovascular system via rapid elicited rises in serum mitogenic activity for VSMC.

Colonic endocrine cells in patients with carcinoma of the colon.

To investigate the colonic endocrine cells in patients with colon carcinoma in order to establish a possible abnormality. Twelve patients with colon adenocarcinoma (eight women and four men; mean age 69 years; range 52-88 years) were studied. As controls, macroscopically and histologically normal tissues from the colon of 12 patients (eight women and four men; mean age 66 years; range 34-86 years) were examined. These patients suffered from polyps, prolapsus, chronic diverticulitis, volvulus or haemorrhoids. Macroscopically and histologically normal tissues from the colon of the patients, about 10 cm from the tumour, and of the controls were examined. Endocrine cells were immunostained with the avidin-biotin-complex method, and were quantified by computer image analysis using an automatic standard sequence analysis operation. Three parameters were used: (1) the number of endocrine cells per mm3 of epithelial cells; (2) the cell secretory index (CSI); and (3) the nuclear volume. The numbers of somatostatin- and serotonin-immunoreactive cells were significantly reduced in patients with colon carcinoma (P = 0.03 and 0.009, respectively). There was no difference between patients and controls regarding the numbers of PYY-, PP-, and enteroglucagon-immunoreactive cells. The CSI of somatostatin- and serotonin-immunoreactive cells were significantly decreased. There was no difference in the CSI between the patients and controls regarding PYY-, PP- or enteroglucagon-immunoreactive cells. The nuclear volumes of PYY- and enteroglucagon-immunoreactive cells increased significantly in the patients. The nuclear volume of PP-, somatostatin- and serotonin cells did not differ from those of the controls. The present results support the assumption that a disturbance in the colonic neuroendocrine system occurs in patients with colon carcinoma, which might affect the development of the tumour. The decrease in the number and CSI of somatostatin cells may account for the decrease of the colonic content of this peptide observed previously in these patients. The decrease in the number and CSI of serotonin-immunoreactive cells in patients with colon adenocarcinoma might be a method by which the body defends itself against this mitogenic substance.

Rapamycin inhibits substance P-induced protein synthesis and phosphorylation of PHAS-I (4E-BP1) and p70 S6 kinase (p70(S6K)) in human astrocytoma cells

The mitogenic substance P receptor (NK-1 subtype) is expressed in many primary human tumors with the highest frequency of expression appearing in astrocytomas and glioblastomas (75% and 100%, respectively). Recently, we showed that substance P neuropeptide induces DNA synthesis in the human astrocytoma U-373MG cells by activating the mitogen-activated protein (MAP) kinase pathway leading to the induction of c-Fos and c-Myc expression. The induction of these immediate early genes is necessary for the progression of cells form G1 to S phase of the cell cycle. In this study, we demonstrate that U-373MG cells are highly sensitive to the growth-inhibitory action of rapamycin at nanomolar concentrations (IC50 <1 ng/ml). We also show that SP peptide stimulates protein synthesis in the U-373MG cell line by activating a rapamycin-sensitive signaling pathway. Further, we demonstrate that SP is potent in stimulating PHAS-I protein (also known as 4E-BP1) phosphorylation and p70 S6 kinase (p70(S6K)) phosphorylation and enzymatic activity, and that this stimulation is inhibited by subnanomolar concentrations of rapamycin. In contrast, rapamycin was not at all effective in repressing SP-induced activation of MAP kinase pathway, c-Fos phosphoprotein expression, and DNA synthesis in U-373MG astrocytoma cells.

Epidermal growth factor suppresses renal tubular apoptosis following ureteral obstruction

Objectives. Acute unilateral ureteral obstruction (UUO) results in ipsilateral hydronephrosis characterized by a decrease in epidermal growth factor (EGF) mRNA expression and EGF protein levels in the distal renal tubules. UUO results in programmed cell death with increases in the characteristic markers of apoptosis. To suppress the apoptotic response during UUO, recombinant EGF was administered during renal obstruction and the ensuing molecular and histologic changes were studied. Methods. Mature Sprague-Dawley rats underwent left ureteral obstruction and the kidneys were harvested at 24, 48, and 72 hours. Markers of apoptosis included DNA laddering pattern on agarose gel electrophoresis, in situ gap labeling of fragmented DNA for quantitative apoptotic body determination, polyadenylated mRNA expression of SGP-2, and in situ hybridization for sulfated glycoprotein-2 (SGP-2) mRNA. Studies were repeated in rats following administration of 10, 20, and 40 μg of subcutaneous recombinant EGF on a daily basis after UUO. Results. Subcutaneous injection of EGF into unilaterally obstructed rats promotes renal tubular epithelial cell regeneration, as demonstrated by increased cortical mitotic activity. Systemic EGF supplementation in these unilaterally obstructed rats also resulted in a decrease in the intensity of the DNA laddering pattern associated with renal tubular apoptosis. An in situ labeling procedure to identify apoptotic nuclei in the ureterally obstructed kidneys revealed a 50% reduction in apoptosis after EGF administration. Northern blot analysis and in situ hybridization for SGP-2 mRNA or clusterin gene product also revealed a decreased expression in the obstructed and EGF-treated renal parenchyma. Conclusions. These data suggest that EGF, apart from its known role as a mitogenic substance for renal tubular epithelial cells, is also a critical in vivo renal cell survival factor for the developmentally mature kidney.

Analysis of mitogenic substances in Bupleurum chinense by ESR spectroscopy.

The polyphenolic substance(s) in the hot water extract of Bupleurum chinense (PSF) showed strong mitogenic activity. In this paper, we analyzed PSF by using ESR spectroscopy, and found that i) PSF showed a strong ESR signal on g = 2.005 which was similar to the commercially available lignin; ii) Sho-saiko-to, which contains an extract of B. chinense, also showed similar signals on ESR; iii) Powdered B. chinense also showed similar signals on g = 2.005. Peroxidase activity, essential for producing polyphenolic substances, was detected in the cold water extract of B. chinense. In addition, the signal intensity of the ESR spectrum of B. chinense was increased after boiling. The data of the ESR spectra of the model reactions using lignin, arginine, proline and maltose also strongly suggested that a certain chemical modification proceeded during the hot water extraction to increase the percentage of the stable free radical. These facts strongly suggested that the mitogenic substance in B. chinense is a polyphenolic substance extracted by hot water, and the structure was modified during the extraction to increase the stable free radical components.

Wide range of molecular weight distribution of mitogenic substance(s) in the hot water extract of a Chinese herbal medicine, Bupleurum chinense.

In this study, we examined the contribution of lignin-like materials in lower molecular weight (MW) fractions from the hot water extract of Bupleuri Radix (Bupleurum chinense) (HWE-BR) for their immunopharmacological activities. Mitogenic activity was detected in all the fractions of MW ranges: lower than 1.0 kDa, 1.0-3.5 kDa, 3.5-10 kDa, and 10-50 kDa. After NaClO2 treatment of these subfractions, UV spectra, ESR spectra, mitogenic activities on murine B-cells, and the activity of inducing nitric oxide in RAW 264.7 cells were significantly reduced, suggesting that lignin-like polyphenolic substance(s) of various MW might take part in these activities. The intensity of ESR spectra and mitogenic activities were stronger in higher MW subfractions, thus the content of stable radical species and/or the degrees of polymerization would be important for their immunopharmacological activities.

Isolation of a new superantigen with potent mitogenic activity to murine T cells from Streptococcus pyogenes

A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28 000 and p I 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H HeN spleen cells began at 1 ng ml −1 and reached a maximal response at 100 ng ml −1 of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class I mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing Vβ13 T-cell receptor was observed up to 73% among the Thy1.2 + cells in cultures stimulated with SPM, indicating expansion in a Vβ-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of p I 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand Vβ8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and Vβ21 + T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.

Isolation of a new superantigen with potent mitogenic activity to murine T cells from Streptococcus pyogenes.

A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28,000 and pI 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H/HeN spleen cells began at 1 ng ml-1 and reached a maximal response at 100 ng ml-1 of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class 1 mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing V beta 13 T-cell receptor was observed up to 73% among the Thy 1.2+ cells in cultures stimulated with SPM, indicating expansion in a V beta-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of pI 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand V beta 8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and V beta 21+ T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.

Role of platelets in progressive glomerular diseases.

There is increasing evidence that platelets are involved in the pathogenesis of glomerulonephritis. Intraglomerular platelets or their degradation products are observed in biopsies from patients with lupus nephritis, mesangioproliferative, membranous or IgA nephropathy. Moreover shortened platelet survival in patients with various glomerular diseases has also been described. In models of experimental glomerulonephritis, platelets may participate in glomerular injury, together with other mediators, by complex mechanisms. As extensively documented, platelets release within the glomerulus vasoactive, chemotactic and mitogenic substance that interact with a number of soluble mediators generated by renal resident or inflammatory cells and contribute to amplify glomerular injury. Thus platelet-activating factor and other platelet secretory products, polycationic macromolecules, platelet factor 4 and beta-thromboglobulin, alter glomerular permeability to proteins and enhance immune-mediated glomerular injury. Platelet-derived factors, like platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta) mediate renal disease progression in experimental and human glomerulonephritis via their chemotactic activity for infiltrating leucocytes and their effect of promoting extracellular matrix synthesis by resident renal cells. In these settings increased renal expression of PDGF and TGF beta has correlated with clinical features. Specific PDGF and TGF beta inhibitors ameliorated experimental glomerular disease. A wide variety of therapies to inhibit platelet function have been employed over the years, however the results of clinical studies are controversial and do not allow conclusions to be drawn about the efficacy of anti-platelet agents in progressive renal disease. Identification of specific platelet inhibitors or interventions specific for platelet secretory products and their target cells will be crucial for understanding the exact role of platelets and their products in glomerular disease.

Response of peripheral blood leucocytes to mitogenic factor(s) in porcine seminal plasma.

Porcine seminal plasma (PSP) contains a potent mitogenic substance capable of causing proliferation and extensive agglutination in cultured porcine peripheral blood leucocytes (PBL). In order to determine the specific leucocyte cell population affected by this mitogen, lymphocyte, monocyte and polymorphonuclear (PMN)-enriched cell fractions were separated from PBL and treated with increasing concentrations of PSP (1-8%, v/v). For monocyte cell populations enriched through elutriation and adherence to plastic, cell proliferation in response to PSP treatment was consistently higher than PSP-treated unfractionated PBL. Monocyte-depleted PBL and lymphocyte populations enriched through elutriation demonstrated a decrease in proliferation compared to PSP-treated unfractionated PBL. PMN populations separated from PBL by discontinuous Percoll gradient centrifugation were unresponsive to PSP. Agglutination was observed for the unfractionated PBL and each enriched cell population. These results demonstrate that PSP contains a potent mitogen which induces proliferation in monocyte-enriched cell populations and may reflect the potential of PSP to act as an immune regulator in the uterine environment during early embryo development and implantation.

Superantigenic properties of a novel mitogenic substance produced by Yersinia pseudotuberculosis isolated from patients manifesting acute and systemic symptoms.

A novel product from Yersinia pseudotuberculosis exhibiting mitogenic activity for human PBMC (designated Y. pseudotuberculosis-derived mitogen, YPM) was examined for its biologic activities for human lymphocytes. YPM induced a substantial proliferative response and IL-2 production at 0.1 ng/ml or more in whole PBMC but not in T-depleted PBMC. IL-2 production occurred within 12 h after YPM stimulation. T cells from PBMC produced IL-2 in the presence of L cells transfected with HLA DR genes or DP genes but not in the presence of control L cells used as accessory cells. Paraformaldehyde fixation did not abolish the AC activity of the DR+ L cells. The results suggest that YPM bind directly to HLA class II molecules. Analysis of the TCR V beta element using the polymerase chain reaction revealed that YPM selectively activated human T cells bearing V beta 3, V beta 9, V beta 13.1, and V beta 13.2 in TCR. These results indicate that YPM is a potent T cell activator and has superantigenic properties (abilities to bind directly to MHC class II molecules and selectively stimulate T cell populations bearing particular V beta elements in TCR). The role of YPM in the mechanism of pathogenesis of Y. pseudotuberculosis infections manifesting acute and systemic clinical symptoms is discussed.

B-Cell Mitogen Produced by Slime-Forming, Encapsulated Lactococcus lactis ssp. cremoris Isolated from Ropy Sour Milk, Viili

A substance, active as a B-cell mitogen, was isolated from the slime products produced by Lactococcus lactis ssp. cremoris KVS20. The mitogenic substance was prepared by anion-exchange chromatography and gel filtration chromatography and then purified by proteinase digestion and HPLC. Chemical analysis determined that the mitogenic substance was a phosphopolysaccharide and consisted of rhamnose, glucose, galactose, and phosphorus. The activity of the mitogenic substance was higher than that of the slime products. The optimal concentration for the activity was approximately 120μg/ml. The mitogenic substance also had substantial mitogenic activity to spleen cells from C3H/HeJ mice, which are resistant to lipopolysaccharide. The findings indicated that a B-cell mitogen different from lipopolysaccharide is produced from L. lactis ssp. cremoris KVS20.

Germ cell mitogenic activity is associated with nerve growth factor‐like protein(s)

The mitogenicity of germ cell proteins released from round spermatids (RS) and pachytene spermatocytes (PS) was investigated. Germ cells were isolated by centrifugal elutriation from 90-day-old rat testes and incubated in a supplement enriched culture media that lacked exogenous proteins. The conditioned culture media of RS and PS were dialysed/concentrated and lyophilized to prepare RS protein (RSP) and PS protein (PSP). Mitogenic activity of RSP and PSP was determined by 3H-thymidine incorporation into Swiss 3T3 fibroblasts. RSP and PSP stimulated 3H-thymidine incorporation by fibroblasts in a dose-dependent manner. At a higher concentration of RSP (300 micrograms/ml), fibroblast proliferation was stimulated from 6- to 20-fold of control cultures, whereas PSP (300 micrograms/ml) stimulated fibroblast proliferation 2.5-fold of control cultures. Since RSP exhibited substantially greater mitogenic activity than PSP we further investigated the RSP mitogenic substance(s) by immunoneutralization with antibodies against several growth factors. The mitogenic activity of RSP was significantly reduced by treatment with nerve growth factor (NGF) antibody, while neither the treatment of RSP with acidic fibroblast growth factor (aFGF) antibody, nor basic fibroblast growth factor (bFGF) antibody significantly modified the mitogenic activity of RSP. Interestingly, murine NGF-beta, recombinant human NGF-beta, and bovine serum albumin (BSA) did not exhibit mitogenic activity on 3T3 fibroblasts. Nevertheless, the presence of a NGF-like protein in RS and PS was confirmed by indirect immunofluorescence staining with a murine NGF antibody. Subsequently, a Western blot analysis with the NGF antibody identified two immunoreactive bands of 41 +/- 2 kDa and 51 +/- 1 kDa in both RSP and PSP under reduced conditions. These germ cell NGF-like proteins were apparently different from similarly prepared murine and human NGFs (13 kDa) in their molecular weight. Furthermore, neurite outgrowth from pheochromocytoma cells (PC-12), a functional bioassay for NGF-like activity, was stimulated by addition of RSP and PSP to the culture media of the PC-12 cells. These results demonstrate mitogenic activity in germ cell proteins (RSP and PSP) and identify a NGF-like protein(s) which is associated with most of this activity.

Isolation of Mitogenic Substance from Sclerotia of Sclerotinia sclerotiorum IFO 9395 Extracted with Phosphate Buffer.

The buffer extracts (3S) of sclerotia of Sclerotinia sclerotiorum IFO 9395 contained mitogenic substance(s) to murine splenocytes (Shinohara et al. Chem. Pharm. Bull., 38, 2219 (1990)). Although the native 3S was slightly mitogenic, heating of 3S induced significant mitogenic activity. To isolate the mitogen, we separated 3S by ion-exchange and gel filtration chromatographies. The isolated mitogen, named sclerogen, has a molecular mass of 32 kilodaltons (kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the isoelectric point (pI) of 5.9 by chromatofocusing. Sclerogen was significantly mitogenic in vitro against murine splenocytes after heat denaturation, and also showed the augmentation of the primary mixed lymphocyte reaction (MLR) in vitro. However, sclerogen did not show the activation of an alternative pathway of complement and hemagglutination activity. These results suggest that sclerogen is a unique mitogen which differs from lectins and shows mitogenicity after heat denaturation.

Heat induced generation of the mitogenic substance(s) responding to murine splenocytes obtained from sclerotia of Sclerotinia sclerotiorum IFO 9395.

We have demonstrated that hot water extracts of sclerotia of Sclerotinia sclerotiorum IFO 9395 (TSHW) show various immunomodulating activities and mitogenic substance(s) were recovered from the beta-1,3-glucanase resistant-fraction (EDP) (Shinohara et al. Chem. Pharm. Bull., 37, 2174 (1989]. In this paper, we examined whether or not the mitogenic substance(s) were also obtained from the other methods, phosphate buffer extraction. Although the native extracts (3S-M) sterilized with a membrane filter showed a slight mitogenicity to murine splenocytes, 3S-M denatured in boiling water (3S-MB) showed significant activity. Treatment of 3S-M for only 1 min in boiling water or 10 min at 70 degrees C was sufficient to show significant mitogenic activity. After heat treatment of 3S-M in boiling water for 30 s, the main band corresponding to that of 3S-M was not clearly observed. Instead, new bands appeared at the top of the gel in normal-polyacrylamide gel electrophoresis (normal-PAGE), suggesting that many physicochemical changes occurred during the heat treatment. These findings suggest that heat denaturation of the substance(s) from sclerotia was one of the triggering mechanisms expressing mitogenic activity to murine splenocytes.

Ontogeny of Rat Testicular Mitogenic Activity Studied with Transformed and Nontransformed Mouse 3T3 Fibroblasts

The ontogeny of the testicular mitogenic activity of Sprague Dawley rats was evaluated by stimulation in vitro of [3H]thymidine uptake and cell number in Swiss 3T3 and transformed NIH 3T3 fibroblasts. Addition of various amounts of testicular cytosolic protein resulted in a dose-dependent increase in cell number in both cell lines. The [3H]thymidine responses to testicular cytosolic proteins was also dose dependent but in a nonparametric manner. In addition, the responses of 3T3 cells to testicular cytosol was apparently dependent on the developmental stage of the testis, the time of stimulation, and the proliferative nature of the fibroblast cell line. These results suggest the presence of multiple forms of mitogenic substance in testicular cytosol and imply that the relative amounts of these substances may depend on the age of the donor animal. These substances may be important in the proliferation or differentiation of cell types at various stages of testicular development.