Large‐scale production of pharmaceuticals by marine sponges: Sea,cell, or synthesis?

Marine sponges are known to produce an overwhelming array of secondary metabolites with pharmaceutical potential. The technical and economical potential of using marine sponges for large-scale production of these compounds was assessed for two cases: the anticancer molecule halichondrin B from a Lissodendoryx sp., and avarol from Dysidea avara for its antipsoriasis activity. An economic and technical analysis was done for three potential production methods: mariculture, ex situ culture (in tanks), and cell culture. We concluded that avarol produced by mariculture or ex situ culture could become a viable alternative to currently used pharmaceuticals for the treatment of psoriasis. Production of halichondrin B from sponge biomass was found to not be a feasible process, mainly due to the extremely low concentration of the compound in the sponge. Technical feasibility was also analyzed for five alternatives: chemical synthesis, wild harvest, primmorph culture, genetic modification and semi-synthesis. It was concluded that the latter two approaches could prove to be valuable methods for the production of pharmaceuticals, based on chemical structures of secondary metabolites present in trace amounts in marine sponges.     

Methylation of promoter region of <Emphasis Type="Italic">RAR</Emphasis>-β2 gene in renal cell,breast, and ovarian carcinomas

The protein encoded by RAR-β2 (retinoic acid receptor) gene is a member of the superfamily, of nuclear receptors of retinoids which are involved in regulation of cell differentiation and proliferation. The level of RAR-β2 mRNA is downregulated in a number of cell lines derived from human epithelial tumors. Inactivation of the RAR-β2 gene is associated with methylation of its promoter region, which is observed in various carcinomas at a frequency of 30–70%. In renal and ovarian tumors, methylation at this region is poorly studied, the data being contradictory. We report a high methylation frequency in the gene promoter region in RCC (59%, 36/61) and a somewhat lower frequency in EOC (30%, 15/50). Methylation frequency in BC (46%, 26/56) is consistent with the published data. Significant correlation of methylation frequency in promoter region of RAR-β2 gene with RCC progression (P ≤ 0.005 by Fisher’s exact test) was established.     

Karyotype of the human insulinoma CM cell line—Beta cell model in vitro?

The CM cell line is derived from a human pancreatic insulinoma and is used as a beta cell model for the study of the pathogenesis of diabetes, as it appears to maintain the characteristics of beta cells. However, a karyotype study of the CM cell line was not previously performed. We aimed at karyotyping the CM cell line to confirm its human origin, diploid karyotype, and chromosomal structure. We karyotyped the CM cells at earlier passages with the standard Giemsa technique. The karyotyping procedure confirmed the human origin of the CM cell line. However, the karyotype showed 64 chromosomes with structural abnormalities, including chromosome 11, in which the insulin gene is located. Our Medline search of other existing insulinoma cell lines of rodent, mouse and hamster origin did not show any karyotype performed. As the CM cell karyotype reveals significant structural and numerical chromosomal abnormalities, we question the use of such a cell line as an in vitro beta cell model. We suggest that insulinoma cell lines established in vitro to study beta cell function should have a karyotype performed to exclude chromosomal aberrations.     

The control of δ-crystallin gene expression during lens cell development: Dissociation of cell elongation,cell division, δ-crystallin synthesis,and δ-crystallin mRNA accumulation

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of δ-crystallin, and accumulate δ-crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved 6-day-old and freshly explanted 19-day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of δ-crystallin gene expression (as measured by δ-crystallin synthesis and δ-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether δ-crystallin gene expression is increased in lens epithelial cells stimulated to divide by treatment with fetal calf serum, as it is in those stimulated to elongate by treatment with serum. Three new findings were made in this study. First, the stimulation of δ-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of δ-crystallin synthesis is observed in lens epithelial cells during normal development and during serum starvation; in neither case is this decrease associated with a reduction in the number of δ-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of δ-crystallin synthesis, but can promote the accumulation of δ-crystallin mRNA. Thus, the relative proportion of δ-crystallin synthesized during chick lens development is not solely a function of the number of δ-crystallin mRNA sequences in the lens cells.     

Sickle cell anemia,sickle cell β-thalassemia,and thalassemia major in Albania: characterization of mutations

We have analyzed the hemoglobin abnormalities in nearly 50 Albanian patients with a significant hemoglobinopathy and included 37 relatives in this study. Sickle cell anemia (SS) is a common disorder; all 15 sickle cell anemia patients had the complications expected for this disease. The ^s haplotype was type 19 (Benin); -thalassemia-2 was rare. Three -thalassemia alleles (IVS-I-110, GA; codon 39, CT; IVS-I-6, TC) were present in nearly 85% of the -thalassemia alleles; their frequencies were intermediate between those observed in the populations of neighboring countries. A few rare mutations were also found, which might have originated in India, Turkey, Macedonia, and Greece. Nearly all patients with Hb S--thalassemia had the IVS-I-110 (GA) mutation. The frequencies of 11 -thalassemia mutations in 17 mostly Mediterranean countries have been reviewed.     

Structure peculiarities of cell walls of <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>—an autotroph of cephalosporin C

Alterations of cell walls of Acremonium chrysogenum occurring at intensive synthesis of cephalosporin C has been studied. It is shown, using electron microscopy, that the cell wall of the cells of ATCC 11550 strain (“wild” type) became looser and thicker during growth. The cell wall of the cells of strain 26/8 (hyperautotroph of cephalosporin C) considerably degraded by the end of the stationary phase. Biochemical analysis has shown that these alterations entailed decrease of the proteins’ content covalently or noncovalently linked with the polysaccharides of cell walls of both strains. An increase of sensitivity of cell walls of the strain-hyperautotroph to an activity of lytic enzymes of chitinase, laminarinase, proteinase K, and lyticase preparation has been observed during the growth, but this increase has not been found in the case of “wild” type strain. The obtained results evidence to the structure failure of the cell wall of A. chrysogenum entailing the intensive creation of antibiotic.     

A risk-based approach for cell line development,manufacturing and characterization of genetically engineered,induced pluripotent stem cell–derived allogeneic cell therapies

Advances in cellular reprogramming and gene-editing approaches have opened up the potential for a new class of ex vivo cell therapies based on genetically engineered, induced pluripotent stem cell (iPSC)-derived allogeneic cells. While these new therapies share some similarities with their primary cell-derived autologous and allogeneic cell therapy predecessors, key differences exist in the processes used for generating genetically engineered, iPSC-derived allogeneic therapies. Specifically, in iPSC-derived allogeneic therapies, donor selection and gene-editing are performed once over the lifetime of the product as opposed to as part of the manufacturing of each product batch. The introduction of a well-characterized, fully modified, clonally derived master cell bank reduces risks that have been inherent to primary-cell derived autologous and allogeneic therapies. Current regulatory guidance, which was largely developed based on the learnings gained from earlier generation therapies, leaves open questions around considerations for donor eligibility, starting materials and critical components, cell banking and genetic stability. Here, a risk-based approach is proposed to address these considerations, while regulatory guidance continues to evolve.     

Effects of hydroxypropyl-β-cyclodextrin on cell growth,activity, and integrity of steroid-transforming <Emphasis Type="Italic">Arthrobacter simplex</Emphasis> and <Emphasis Type="Italic">Mycobacterium</Emphasis> sp.

A comparative investigation was performed on the effects of hydroxypropyl-β-cyclodextrin (HP-β-CD) on the growth, biocatalytic activity, and cell integrity of Arthrobacter simplex TCCC 11037 (ASP) and Mycobacterium sp. NRRL B-3683 (MSP). The addition of HP-β-CD to ASP medium improved its cell wall permeability for lipophilic compounds but significantly inhibited its growth and biocatalytic activity. On the other hand, the addition of HP-β-CD to MSP broth had lesser effects. Atomic force microscopy scanning analysis revealed that HP-β-CD damaged the cell integrity in ASP, especially the outermost cell surface structure, but not in MSP, which remained intact, owing to the differences in their cell wall and cell membrane composition. Protein leaking and lipid content in ASP increased with increased HP-β-CD concentration, indicating possible alterations in ASP cell membrane features caused by HP-β-CD. These alterations may also explain the slow cell growth and decreased cell ΔΨm in ASP upon the addition of HP-β-CD.     

α<Subscript>1</Subscript>-Thymosin, α<Subscript>2</Subscript>-interferon,and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The ¹²⁵I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (K _d = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α₂-interferon (IFN-α₂), α₁-thymosin, (TM-α₁), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α₁ and the 131–135 sequence of human IFN-α₂ (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (K _i > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α₁, IFN-α₂, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.     

Organization of multiprotein complexes at cell–cell junctions

The formation of stable cell-cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell-cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell-cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell-cell contact formation and maintenance.     

<Emphasis Type="Italic">Ignicoccus hospitalis</Emphasis> and <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis>: ultrastructure,cell–cell interaction,and 3D reconstruction from serial sections of freeze-substituted cells and by electron cryotomography

Ultrastructure and intercellular interaction of Ignicoccus hospitalis and Nanoarchaeum equitans were investigated using two different electron microscopy approaches, by three-dimensional reconstructions from serial sections, and by electron cryotomography. Serial sections were assembled into 3D reconstructions, for visualizing the unusual complexity of I. hospitalis, its huge periplasmic space, the vesiculating cytoplasmic membrane, and the outer membrane. The cytoplasm contains fibres which are reminiscent to a cytoskeleton. Cell division in I. hospitalis is complex, and different to that in Euryarchaeota or Bacteria. An irregular invagination of the cytoplasmic membrane is followed by separation of the two cytoplasms. Simultaneous constriction of cytoplasmic plus outer membrane is not observed. Cells of N. equitans show a classical mode of cell division, by constriction in the mid-plane. Their cytoplasm exhibits two types of fibres, elongated and ring-shaped. Electron micrographs of contact sites between I. hospitalis and N. equitans exhibit two modes of interaction. One is indirect and mediated by thin fibres; in other cells the two cell surfaces are in direct contact. The two membranes of I. hospitalis cells are frequently seen in direct contact, possibly a prerequisite for transporting metabolites or substrates from the cytoplasm of one cell to the other. Rarely, a transport based on cargo vesicles is observed between I. hospitalis and N. equitans.     

The effects of dehydroaltenusin,a novel mammalian DNA polymerase α inhibitor,on cell proliferation and cell cycle progression

As described previously, a natural product isolated from fungus (Acremonium sp.), dehydroaltenusin, is an inhibitor of mammalian DNA polymerase α in vitro [Y. Mizushina, S. Kamisuki, T. Mizuno, M. Takemura, H. Asahara, S. Linn, T. Yamaguchi, A. Matsukage, F. Hanaoka, S. Yoshida, M. Saneyoshi, F. Sugawara, K. Sakaguchi, Dehydroaltenusin, a mammalian DNA polymerase α inhibitor, J. Biol. Chem. 275 (2000) 33957_33961]. In this study, we investigated the interaction of dehydroaltenusin with lipid bilayers using an in vitro liposome system, which is a model of the cell membrane, and found that approximately 4% of dehydroaltenusin was incorporated into liposomes. We also investigated the influence of dehydroaltenusin on cultured cancer cells. Dehydroaltenusin inhibited the growth of HeLa cells with an LD₅₀ value of 38 μM, and as expected, S phase accumulation in the cell cycle. The total DNA polymerase activity of the extract of incubated cells with dehydroaltenusin was 23% lower than that of nontreated cells. Dehydroaltenusin increased cyclin E and cyclin A levels. In the analysis of the cell cycle using G1/S synchronized cells by employing hydroxyurea, the compound delayed both entry into the S phase and S phase progression. In a similar analysis using G2/M synchronized cells by employing nocodazole, the compound accumulated the cells at G1/S and inhibited entry into the S phase. Thus, the pharmacological abrogation of cell proliferation by dehydroaltenusin may prove to be an effective chemotherapeutic agent against tumors.     

Influence of production media on Cinchona cell cultures; spontaneous formation of β-carbolines from L-tryptophan

Evidence is presented that the β-carboline alkaloids norharman and harman are artifacts formed from L-tryptophan. Transfer of Cinchona ledgeriana suspension cultured cells to Zenk's alkaloid production medium (ZAP medium) resulted in cell death. No quinoline or terpenoid indole alkaloids were formed. However, the L-tryptophan present in the production medium was transformed to the β-carboline alkaloids, harman and norharman. It was demonstrated that norharman was also formed in ZAP medium without cells and in ZAP medium containing frost-killed cell material.     

Effects of curvature and cell–cell interaction on cell adhesion in microvessels

It has been found that both circulating blood cells and tumor cells are more easily adherent to curved microvessels than straight ones. This motivated us to investigate numerically the effect of the curvature of the curved vessel on cell adhesion. In this study, the fluid dynamics was carried out by the lattice Boltzmann method (LBM), and the cell dynamics was governed by the Newton’s law of translation and rotation. The adhesive dynamics model involved the effect of receptor-ligand bonds between circulating cells and endothelial cells (ECs). It is found that the curved vessel would increase the simultaneous bond number, and the probability of cell adhesion is increased consequently. The interaction between traveling cells would also affect the cell adhesion significantly. For two-cell case, the simultaneous bond number of the rear cell is increased significantly, and the curvature of microvessel further enhances the probability of cell adhesion.     

Sulforaphene attenuates multinucleation of pre-osteoclasts by suppressing expression of cell–cell fusion-associated genes DC-STAMP,OC-STAMP,and Atp6v0d2

We assessed the effect of sulforaphene (SFE) on osteoclast differentiation. SFE significantly decreased the number of RANKL-induced tartrate-resistant acid phosphatase-positive cells and suppressed pre-osteoclast multinucleation. Furthermore, SFE downregulated mRNA expression of DC-STAMP, OC-STAMP, and Atp6v0d2, which encode cell–cell fusion molecules. Our data suggest that SFE attenuates pre-osteoclast multinucleation via suppression of cell–cell fusion.     

The skeletal muscle satellite cell: stem cell or son of stem cell?

The concept of the adult tissue stem cell is fundamental to models of persistent renewal in functionally post-mitotic tissues. Although relatively ignored by stem cell biology, skeletal muscle is a prime example of an adult tissue that can generate terminally differentiated cells uniquely specialized to carry out tissue-specific functions. This capacity is attributed to satellite cells, a population of undifferentiated, quiescent precursors that become activated to divide and differentiate in response to the demands of growth or damage. The aim of this review is to discuss the role of the satellite cell as an adult tissue-specific stem cell. We examine evidence for the presence of behaviourally and phenotypically distinct subpopulations of precursor within the satellite cell pool. Further, we speculate on the possible identity, origins and relevance of multipotent muscle stem cells, a population with both myogenic and hematopoietic potentials that has been isolated from whole muscle. Taken together, current evidence suggests the possibility that the regenerative compartment of adult skeletal muscle may conform to an archetypal stem cell-based hierarchy, maintained within a stem cell niche. It therefore remains to be seen whether all satellite cells are skeletal muscle-specific stem cells, or whether some or all are the progeny of an as yet unidentified muscle stem cell.