GABA as a trophic factor during development

The process of synaptogenesis has been studied by many investigators to determine the factors which regulate synapse formation. We have used neonatal rabbit retina to investigate the role of the gamma-aminobutyric acid (GABA) neurotransmitter system during development. By utilizing an in vitro incubation treatment of isolated eyecups we found that treatment with nipecotic acid, a GABA uptake blocker, resulted in a 4-fold increase in the amount of specific 3H-muscimol binding. In addition, incubation of the tissue in the presence of the GABA agonists muscimol, 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (THIP), or GABA itself led to similar increases in specific 3H-muscimol binding. The findings support the conclusion from previous studies that the induction of GABA receptors observed after in vivo treatment of 1-day-old rabbits with nipecotic acid resulted from an increase in the extracellular concentration of GABA. A possible role for GABA in the regulation of GABAergic synapse formation is presented in this report.     

Is transferrin a neurotrophic factor controlling myosin composition of the skeletal muscle?]

After transferrin injection to the slow m. soleus of guinea pig no immunohistochemical changes in myosin composition with monoclonal antibodies (AB) against fast myosin heavy chain (HCf) have been found. All muscles fibers in intact and experimental animals were identified as slow ones (type I of muscle fibers). After colchicine treatment of nerve trunk innervating slow muscle, some muscle fibers reacting with monoclonal AB against myosin HCf have been found. However, after colchicine treatment of nerve trunk and transferrin injection also some muscle fibers in slow muscle reacting with monoclonal AB against myosin HCf have been found. In appears that transferrin probably can not be the factor of neurotrophic control for myosin composition in skeletal muscle.     

Hepatocyte growth factor in human breast milk acts as a trophic factor.

To evaluate the significance of hepatocyte growth factor (HGF) in milk in the perinatal period, we examined immunoreactive HGF levels and bioactivity in human milk. Human milk samples were obtained from women at various postpartum ages, and the levels of HGF were measured by ELISA. In the cross sectional study, the concentration of milk HGF from term deliveries showed a significant inverse correlation with progress of lactation, whereas in cases of preterm delivery concentrations, levels remained high after a long period of lactation. In the longitudinal analysis, the contents of HGF in colostrum, transitional, and mature milk from preterm deliveries were significantly be higher than those from term deliveries. Although mature milk from term and preterm deliveries contained significantly lower levels of HGF than colostrum, high levels of HGF persisted in mature milk from preterm deliveries. After partial purification, immunoblotting analysis showed the presence of both alpha- and beta-chains of HGF. HGF in milk stimulated proliferation of rat hepatocytes in primary culture, which was inhibited by supplementation with anti-HGF antibody. Thus, a high concentration of bioactive HGF is present in human milk in the postpartum period. Our results suggest that HGF in milk acts as a trophic factor for the gastrointestinal tract in neonates.     

VIP as a trophic factor in the CNS and cancer cells

The effects of vasoactive intestinal peptide (VIP) on the proliferation of central nervous system (CNS) and cancer cells were investigated. VIP has important actions during CNS development. During neurogenesis, VIP stimulates the proliferation and differentiation of brain neurons. Addition of VIP to embryonic mouse spinal cord cultures increases neuronal survival and activity dependent neurotrophic factor (ADNF) secretion from astroglial cells. VIP is an integrative regulator of brain growth and development during neurogenesis and embryogenesis. Also, VIP causes increased proliferation of human breast and lung cancer cells in vitro. VIP binds with high affinity to cancer cells, elevates the cAMP and increases gene expression of c-fos, c-jun, c-myc and vascular endothelial cell growth factor. The effects of VIP on cancer cells are reversed by VIPhybrid, a synthetic VPAC(1) receptor antagonist. VIPhyb inhibits the basal growth of lung cancer cells in vitro and tumors in vivo and potentiates the ability of chemotherapeutic drugs to kill cancer cells. Due to the high density of VPAC(1) receptors in cancer cells, VIP has been radiolabeled with 123I, 18F and 99mTc to image tumors. It remains to be determined if radiolabeled VIP analogs will be useful agents for early detection of cancer in patients.     

Characterization of transferrin binding and specificity of the placental transferrin receptor

This study systematically examined the characteristics of specific binding of adult diferric transferrin to its receptor using a Triton X-100 solubilized preparation from human placentas as the receptor source. The following information was obtained. The ionic strength for maximal binding is in the range of 0.1-0.3 M NaCl. The pH optimum for specific binding extends over the range, from pH 6.0-10.0. Specific binding of diferric transferrin is not affected by 2.5 approximately 50 mM CaCl2 or by 10 mM EDTA. Triton X-100 in the concentration range of 0.02-3.0% does not affect specific binding. Specific binding is saturated within 10 min at 25 or 37 degrees C in the presence of excess amounts of diferric transferrin. The binding is reversible and the dissociation of diferric transferrin from the transferrin receptor is complete within 40 min at 25 degrees C. Apotransferrin, both adult and fetal, showed less binding than the holotransferrin species by competitive binding assay in the presence of 10 mM EDTA independent of up to 20 mM CaCl2. A 1500-fold molar excess of adult and fetal apotransferrin is required to give 40% inhibition for 125I-labeled diferric transferrin binding. Since calcium ion is not a factor, and since apotransferrin has such high binding affinity for iron (Ka = 1 X 10(24], this experiment suggests that the EDTA was necessary to prevent conversion of apotransferrin to holotransferrin from available iron in the reaction system. The specificity of the transferrin receptor for transferrin was examined by competitive binding studies in which 125I-diferric transferrin binding was measured in the presence of a series of other proteins. The proteins tested in the competitive binding studies were classified into three groups; in the first group were human serum albumin and ovalbumin; in the second group were proteins containing iron ions, such as hemoglobin, hemoglobin-haptoglobin complex, heme-hemopexin complex, ferritin, and diferric lactoferrin; in the third group were the metal-binding serum proteins, ceruloplasmin and metallothionein. None of these proteins except ferritin showed inhibition of diferric transferrin binding to the receptor. The effect of ferritin was small since a 700- to 1500-fold molar excess of ferritin is required for 50% inhibition of binding of diferric transferrin to the receptor.     

Identification of transferrin as a progression factor for ML-1 human myeloblastic leukemia cell differentiation

We have previously demonstrated (Guan X.-P., Hromchak, R. A., and Bloch, A. (1989) Cancer Commun. 1, 111-115) that ML-1 human myeloblastic leukemia cells differentiate to monocyte/macrophage-like cells by the sequential action of competence and progression factors. Tumor necrosis factor-alpha, transforming growth factor-beta, and the phorbol ester tetradecanoylphorbol acetate were found to induce competence, whereas a 77-kDa glycoprotein (DF77) isolated from mitogen-stimulated human leukocyte-conditioned medium initiated progression. In this communication we show DF77 to be an isoform of human transferrin. Hemin or soluble iron complexes did not induce differentiation progression, suggesting that the participation of transferrin in ML-1 cell differentiation may not be related to its iron-carrying capacity.     

The specificity of fumarate as a switching factor of the bacterial flagellar motor

Fumarate restores to flagella of cytoplasm-free, CheY- containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo-taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.     

Interleukin 3 as a trophic factor for central cholinergic neurons in vitro and in vivo

We have found that interleukin 3 (IL-3), a growth factor for hematopoietic cells, is a novel trophic factor for mouse and rat central cholinergic neurons. It enhanced neurite outgrowth and elevated choline acetyltransferase activity. The effect seems to be specific for cholinergic neurons, since somatostatin release and glutamic acid decarboxylase and 2',3'-cyclic nucleotide 3'-phosphodiesterase activities were not significantly influenced by IL-3. In vivo, IL-3 was infused into the lateral ventricles of rats after unilateral axotomy of the septohippocampal pathways. Two weeks later, the IL-3-treated animals showed significant numbers of acetylcholinesterase-positive neurons remaining in the septal region.     

Identification of alpha transforming growth factor as a possible local trophic agent for the mammary gland

Biologically active alpha-transforming growth factor (alpha-TGF) has been identified in medium conditioned by rat mammary myoepithelial and, to a lesser extent, by epithelial cell lines in culture and in the rat mammary gland. The alpha-TGF has been identified by its wide spectrum of activity in promoting growth of mammary-derived cells in vitro, by its chromatographic behaviour on reversed-phase high-performance liquid chromatography (HPLC), by its competition with epidermal growth factor (EGF) for the EGF receptor, and by the presence of messenger RNA for alpha-TGF in the secreting cells. In vivo the amount of alpha-TGF isolated is sixfold greater from the mammary glands of lactating than from those of virgin female rats. It is proposed that alpha-TGF is produced by the myoepithelial cells of the mammary gland, as a local trophic agent that stimulates growth of the various cell types of the gland.     

Dedifferentiation of stromal smooth muscle as a factor in prostate carcinogenesis

We had earlier established an animal model of prostate carcinogenesis using a combination of testosterone (T) and 17beta-estradiol benzoate (E2) on Noble rats (Wang and Wong, 1998). In the present study we examined the changes in a number of smooth muscle differentiation markers including smooth muscle alpha-actin and myosin, vinculin, desmin, laminin and vimentin as well as changes in fine structure by electron microscopy. Our immunohistochemical (IHC) studies revealed that smooth muscle cells (SMCs) subjacent to dysplastic (precancerous) sites and carcinoma usually exhibited a preferential loss of myosin, desmin and laminin. However, the expression of alpha-actin and vinculin appeared to be more persistent in most dysplastic or neoplastic sites. The study reaffirmed our earlier observation that there was a concurrent dedifferentiation of surrounding SMCs during the development and progression of prostate carcinogenesis. The structural study revealed that SMC subjacent to epithelial dysplasia displayed a spectrum of derangements. These included the loosening of muscular layers with SMC characterized by their highly irregular external contours with numerous spine-like cytoplasmic projections. There was also a reduction in density of myofilaments and presence of many enlarged caveolae in muscle cells. Additionally, focal discontinuity or disruptions of muscular layer were often observed together with an increase in abundance of fibrous connective tissue. Moreover, the amount of smooth muscle appeared to be inversely correlated with the histologic grade of prostate tumors. In most instances, SMCs were totally absent in the moderately or poorly differentiated tumors and in metastatic tumors in the lung and the small intestine. Stromal muscular deformity was associated with concurrent changes in epithelial cells. Dysplastic epithelial cells were characterized by a reduction in abundance of secretory organelles such as reduction in size of Golgi apparatus, paucity of granular endoplasmic reticulum and secretory vesicles. The nuclei showed typical deformity characterized by deep nuclear membrane foldings. The basal lamina of dysplastic or tumor cells was present although focal structural abnormalities such as reduplication, disruption and smearing were sometimes observed. The present data indicate that derangements of epithelial cells during prostate carcinogenesis are associated with a reduction or dedifferentiation of stromal SMCs. Our results lend support to the hypothesis that transformed epithelium is incapable of maintaining normal differentiation of adjacent muscle. In turn, abnormal stromal, resulting from dedifferentiation or reduction of SMC, may lead to loss of stromal control over epithelial proliferation and differentiation. Consequently, a loss of differentiation in both epithelium and stromal SMCs may be critically involved in hormone-induced prostate carcinogenesis.     

Quorum sensing molecules as a novel microbial factor impacting muscle cells

Skeletal muscle makes up the largest part of human body mass and a good maintenance of this organ is essential for general health. In accordance, muscle wasting, a frequent phenomenon in many diseases, is associated with functional decline and a decrease in quality of life. Unfortunately, due to a lack of knowledge of the underlying pathophysiology, no targeted therapies exist today to encounter muscle wasting. Recent studies suggest a role for the gut microbiome in muscle wasting, without the mediators of this gut-muscle axis being identified.Here we evaluated the possible effects of 75 quorum sensing molecules (QSM), traditionally only seen as intra-bacterial communication molecules, on C2C12 muscle cells, studying viability, differentiation, inflammation, mitochondrial changes and protein degradation as biological outcomes. The responses were evaluated using different approaches: median absolute deviation, quartiles, strictly standardized mean difference and robust strictly standardized mean difference.This study resulted in 30 QSM, with effects observed on C2C12 cells. Known producers of the 27 peptide QSM belong to species of the genus Staphylococcus, Streptococcus, Enterococcus, Bacillus, Lactobacillus and Escherichia, while the 3 non-peptide QSM are produced by a broad range of Gram-positive and Gram-negative bacteria. Altogether, these proof-of-concept findings provide the first evidence that QSM produced by microbiota play a role in the gut-muscle axis, opening new perspectives for diagnostic and therapeutic targets in muscle wasting diseases.     

Vascular endothelial growth factor (VEGF) as a key therapeutic trophic factor in bone marrow mesenchymal stem cell-mediated cardiac repair

We recently demonstrated a novel effective therapeutic regimen for treating hamster heart failure based on injection of bone marrow mesenchymal stem cells (MSCs) or MSC-conditioned medium into the skeletal muscle. The work highlights an important cardiac repair mechanism mediated by the myriad of trophic factors derived from the injected MSCs and local musculature that can be explored for non-invasive stem cell therapy. While this therapeutic regimen provides the ultimate proof that MSC-based cardiac repair is mediated by the trophic actions independent of MSC differentiation or stemness, the trophic factors responsible for cardiac regeneration after MSC therapy remain largely undefined. Toward this aim, we took advantage of the finding that human and porcine MSCs exhibit species-related differences in expression of trophic factors. We demonstrate that human MSCs when compared to porcine MSCs express and secrete 5-fold less vascular endothelial growth factor (VEGF) in conditioned medium (40 ± 5 and 225 ± 17 pg/ml VEGF, respectively). This deficit in VEGF output was associated with compromised cardiac therapeutic efficacy of human MSC-conditioned medium. Over-expression of VEGF in human MSCs however completely restored the therapeutic potency of the conditioned medium. This finding indicates VEGF as a key therapeutic trophic factor in MSC-mediated myocardial regeneration, and demonstrates the feasibility of human MSC therapy using trophic factor-based cell-free strategies, which can eliminate the concern of potential stem cell transformation.     

Identification of a new serum protein polymorphism as transferrin

Summary By isoelectric focusing at pH 3.5–9.5, Kühnl and Spielmann (1977) recently demonstrated a new genetically determined serum protein polymorphism designated Hp^a because of an apparently specific reaction with antihaptoglobin. In this study the polymorphism was reproduced, but the components were found to focus at pH 5.8, which is different from the pI of haptoglobin, and immunologic relation to haptoglobin could not be comfirmed. Using pure transferrin as a reference, the results of isoelectric focusing, crossed immunoelectrophoresis, and immunofixation indicated that the polymorphic components were identical to transferrin. This polymorphism does not correspond to the already known transferrin polymorphism, as the two usual genes, tentatively designated Tf¹ and Tf², in my population sample (n=132) were 0.19 and 0.81, and, further, all individuals except three in the sample belong to type Tf-C.