Neural differentiation of human umbilical cord matrix-derived mesenchymal cells under special culture conditions

Many neural disorders are characterized by the loss of one or several types of neural cells. Human umbilical cord-derived mesenchymal cells (hUCMs) are capable of differentiating into neuron, astroglia-like and oligodendrocyte cell types. However, a reliable means of inducing the selective differentiation of hUCMs into neural cells in vitro has not yet been established. For induction of neural differentiation, hUCMs were seeded onto sterile glass slides and six various cocktails using a base medium (DMEM/LG) supplemented with 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), epidermal growth factor (EGF) and fibroblast growth factor (FGF) were used to compare their effect on neuronal, astrocyte and oligodandrocyte differentiation. The hUCMs were positive for mesenchymal markers, while they were negative for hematopoietic markers. Differentiation to adipogenic and osteogenic lineage was detected in these cells. Our data revealed that the cocktail consisting of DMEM/LG, FBS, RA, FGF, and EGF (DF/R/Fg/E group) induced hUCM cells to express the highest percentage of nestin, ß-tubulin III, neurofilament, and CNPase. The DF/Ds/Fg/E group led to the highest percentage of GFAP expression. While the expression levels of NF, GFAP, and CNPase were the lowest in the DF group. The least percentage of nestin and ß-tubulin III expression was observed in the DF/Ds group. We may conclude that FGF and EGF are important inducers for differentiation of hUCMs into neuron, astrocyte and oligodendrocyte. RA can induce hUCMs to differentiate into neuron and oligodendrocyte while for astrocyte differentiation DMSO had a pivotal role.     

Inhibition of non-muscle myosin II leads to G0/G1 arrest of Wharton's jelly-derived mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSCs) have remarkable clinical potential for cell-based therapy. Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from umbilical cord share unique properties with both embryonic and adult stem cells. MSCs are found at low frequency in vivo, and their successful therapeutic application depends on rapid and efficient large-scale expansion in vitro. Non-muscle myosin II (NMII) has pivotal roles in different cellular activities, such as cell division, migration and differentiation. We performed this study to understand the role of NMII in proliferation and cell cycle progression in WJ-MSCs.MethodsWJ-MSCs were cultured in the presence of blebbistatin, and cell cycle analysis was performed using flow cytometry, proliferation kinetics, senescence assay and gene expression profile using polymerase chain reaction array.ResultsWhen cultured in the presence of blebbistatin, an inhibitor of NMII adenosine triphosphatase activity, WJ-MSCs exhibited dose-dependent reduction in proliferative potential along with increase in cell size and induction of early senescence. Inhibition of NMII activity also affected cell cycle progression in WJ-MSCs and led to an increase in the percentage of cells in G₀/G₁ phase with a corresponding reduction in the percentage of cells in G₂/M phase. Blebbistatin-induced G₀/G₁ arrest of WJ-MSCs was further associated with up-regulation of cell cycle inhibitory genes CDKN1A, CDKN2A and CDKN2B and down-regulation of numerous genes related to progression through S and M phases of the cell cycle.ConclusionsOur study demonstrates that inhibition of NMII activity in WJ-MSCs leads to G₀/G₁ arrest and alteration in the expression levels of certain key cell cycle-related genes.     

Molecular characteristics and physiological functions of major royal jelly protein 1 oligomer

Royal jelly contains numerous components, including proteins. Major royal jelly protein (MRJP) 1 is the most abundant protein among the soluble royal jelly proteins. In its physiological state, MRJP 1 exists as a monomer and/or oligomer. This study focuses the molecular characteristics and functions of MRJP 1 oligomer. MRJP 1 oligomer purified using HPLC techniques was subjected to the following analyses. The molecular weight of MRJP 1 oligomer was found to be 290 kDa using blue native‐PAGE. MRJP 1 oligomer was separated into 55 and 5 kDa spots on 2‐D blue native/SDS‐PAGE. The 55 kDa protein was identified as MRJP 1 monomer by proteome analysis, whereas the 5 kDa protein was identified as Apisimin by N‐terminal amino acid sequencing, and this protein may function as a subunit‐joining protein within MRJP 1 oligomer. We also found that the oligomeric form included noncovalent bonds and was stable under heat treatment at 56°C. Furthermore, MRJP 1 oligomer dose dependently enhanced and sustained cell proliferation in the human lymphoid cell line Jurkat. In conclusion, MRJP 1 oligomer is a heat‐resistant protein comprising MRJP 1 monomer and Apisimin, and has cell proliferation activity. These findings will contribute to further studies analyzing the effects of MRJP 1 in humans.     

Towards functional proteomics of minority component of honeybee royal jelly: The effect of post‐translational modifications on the antimicrobial activity of apalbumin2

This study illustrates multifunctionality of proteins of honeybee royal jelly (RJ) and how their neofunctionalization result from various PTMs of maternal proteins. Major proteins of RJ, designated as apalbumins belong to a protein family consisting of nine members with M_r of 49–87 kDa and they are accompanied by high number of minority homologs derived from maternal apalbumins. In spite of many data on diversity of apalbumins, the molecular study of their individual minority homologous is still missing. This work is a contribution to functional proteomics of second most abundant protein of RJ apalbumin2 (M_r 52.7 kDa). We have purified a minority protein from RJ; named as apalbumin2a, differ from apalbumin2 in M_r (48.6 kDa), in N‐terminal amino acids sequences – ENSPRN and in N‐linked glycans. Characterization of apalbumin2a by LC‐MALDI TOF/TOF MS revealed that it is a minority homolog of the major basic royal jelly protein, apalbumin2, carrying two fully occupied N‐glycosylation sites, one with high‐mannose structure, HexNAc2Hex9, and another carrying complex type antennary structures, HexNAc4Hex3 and HexNAc5Hex4. We have found that apalbumin2a inhibit growth of Paenibacillus larvae. The obtained data call attention to functional plasticity of RJ proteins with potential impact on functional proteomics in medicine.     

Ethanol‐soluble proteins from the royal jelly of Xinjiang black bees

Royal jelly is a nutritious food that has beneficial effects to human health. However, the functional substances remain unclear. Herein, we fractioned the royal jelly proteins of Xinjing black bees according to the Osboren method. Two main proteins from the ethanol‐soluble fraction were purified and identified. RJG‐1 was determined as glucosylceramidase, and RJG‐2 was major royal jelly protein 1 (MRJP1). RJG‐1 showed potent cytotoxicity toward various mammalian cells, and caused quick disruption of cell membranes. With glucosylceramidase activity, RJG‐1 may degrade the glucosylceramide of the cell membranes and disrupt the membrane structure, thereby resulting in cell necrosis. This study extends our knowledge about the composition and function of royal jelly, and is significant for the application of royal jelly.     

Pollen analysis as a means to determine the geographical origin of royal jelly

In this study we investigated the use of pollen analysis as a method to determine the geographical origin of royal jelly. We recorded the pollen flora sampled by bees using pollen traps for two consecutive years and we also collected and examined royal jelly samples from the same apiary. For royal jelly production, bees mainly used the freshly collected pollen. All major pollen types that were recorded in the area using pollen traps were also detected in the royal jelly samples. Thus, pollen analysis can be used as a method to determine the geographical origin of royal jelly.     

Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera

Analysis of natural host-parasite relationships reveals the evolutionary forces that shape the delicate and unique specificity characteristic of such interactions. The accessory long gland-reservoir complex of the wasp Leptopilina heterotoma (Figitidae) produces venom with virus-like particles. Upon delivery, venom components delay host larval development and completely block host immune responses. The host range of this Drosophila endoparasitoid notably includes the highly-studied model organism, Drosophila melanogaster. Categorization of 827 unigenes, using similarity as an indicator of putative homology, reveals that approximately 25% are novel or classified as hypothetical proteins. Most of the remaining unigenes are related to processes involved in signaling, cell cycle, and cell physiology including detoxification, protein biogenesis, and hormone production. Analysis of L. heterotoma's predicted venom gland proteins demonstrates conservation among endo- and ectoparasitoids within the Apocrita (e.g., this wasp and the jewel wasp Nasonia vitripennis) and stinging aculeates (e.g., the honey bee and ants). Enzyme and KEGG pathway profiling predicts that kinases, esterases, and hydrolases may contribute to venom activity in this unique wasp. To our knowledge, this investigation is among the first functional genomic studies for a natural parasitic wasp of Drosophila. Our findings will help explain how L. heterotoma shuts down its hosts' immunity and shed light on the molecular basis of a natural arms race between these insects.     

Formation of 5-Hydroxy-4-Ketohexanoic Acid by Yeast

When grown on medium containing ethanol as the sole carbon source, three of five strains of yeast tested produced a keto acid which was demonstrated by paper chromatography. This compound was isolated and its structure was examined by elementary analysis, infrared spectrum, nuclear magnetic resonance and periodate oxidation. The compound was proved to be identical with 5-hydroxy-4-ketohexanoic acid. Formation of this compound by cell suspensions of Hansenula miso IFO 0146 was achieved by addition of acetaldehyde, although the presence of α-ketoglutaric acid enhanced the formation of the keto acid from acetaldehyde during a short incubation period. Added 5-hydroxy-4-ketohexanoic acid was exhausted by the cell suspension.     

Immunomodulatory effect of human umbilical cord Wharton's jelly-derived mesenchymal stem cells on lymphocytes

Studies have shown that mesenchymal stem cells (MSCs) have low immunogenicity and immune regulation. Human umbilical cord Wharton’s jelly provides a new source for MSCs that are highly proliferative and have multi-differentiation potential. To investigate immunomodulatory effects of human Wharton’s jelly cells (WJCs) on lymphocytes, we successfully isolated MSCs from human umbilical cord Wharton’s jelly. WJCs expressed MSC markers but low levels of human leukocyte antigen (HLA)-ABC and no HLA-DR. These results indicate that WJCs have low immunogenicity. Both WJCs and their culture supernatant could inhibit the proliferation of phytohemagglutinin-stimulated human peripheral blood lymphocytes and mouse splenocytes. Additionally, WJCs suppressed secretion of transforming growth factor-β1 and interferon-γ by human peripheral blood lymphocytes. We conclude that the immunomodulatory effect of WJCs may be related to direct cell contact and inhibition of cytokine secretion by human peripheral blood lymphocytes.     

Trace and mineral elements in royal jelly and homeostatic effects

Royal jelly from Apis mellifera is a highly active natural biological substance and is probably one of the most interesting raw substances in natural product chemistry. Trace elements play a key role in the biomedical activities associated with royal jelly, as these elements have a multitude of known and unknown biological functions. For this reason concentrations of 28 trace (Al, Ba, Sr, Bi, Cd, Hg, Pb, Sn, Te, Tl, W, Sb, Cr, Ni, Ti, V, Co, Mo) and mineral (P, S, Ca, Mg, K, Na, Zn, Fe, Cu, Mn) elements were systematically investigated in botanically and geologically defined royal jelly samples. In addition, concentrations of 14 trace elements were measured in the associated honey samples--honey being the precursor of royal jelly. Concentrations of K, Na, Mg, Ca, P, S, Cu, Fe, Zn, Al, Ba and Sr in royal jelly were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES), while concentrations of Bi, Cd, Hg, Pb, Sn, Te, Tl, W, Sb, Cr, Mn, Ni, Ti, V, Co and Mo in royal jelly were determined by double focusing magnetic sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). In the honey samples, trace and mineral element concentrations strongly depended on botanical and geological origin, and substantial variation was found. In contrast, the concentrations of trace and mineral elements were highly constant in the associated royal jelly samples. The most important results were the homeostatic adjustments of trace and mineral element concentrations in royal jelly. This effect was evidently produced in the endocrine glands of nurse bees, which are adapted for needs of bee larvae. In conclusion, this research yielded a surprising and completely new finding--that royal jelly, as a form of lactation on the insect level, shows the same homeostatic adjustment as mammalian and human breast milk.