Characterization of golimumab,a human monoclonal antibody specific for human tumor necrosis factor α

We prepared and characterized golimumab, a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p = 0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2-fold; p = 0.017) and adalimumab (3.3-fold; p = 0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8°C vs. 69.5°C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.Key words: TNF, golimumab, neutralization, affinity, bioassay, arthritis, stability, solubility     

Location of the origin of replication for the 7.5-kbChlamydia trachomatis plasmid

The hypothetical origin of replication for the 7.5-kb plasmid common toChlamydia trachomatis is believed to be in a region of the plasmid that contains four 22-bp tandem repeats preceded by an A-T-rich region. To test this hypothesis, replication of plasmid DNA in metabolically active reticulate bodies of the Lymphogranuloma venereum biovar ofC. trachomatis was examined by electron microscopy. The results presented show that the origin of replication appears to be near the tandem repeats of pCHL2. In addition, replication of the 7.5-kb plasmid is unidirectional, and the copy number during replication is 7–10. The evidence presented suggests thatC. trachomatis has a homologue to theEscherichia coli dnaA gene and that this homologue might be involved in replication of theC. trachomatis 7.5-kb plasmid.     

Synthesis of deoxy and methoxy analogs of octyl alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside as probes for mycobacterial lipoarabinomannan biosynthesis

A panel of analogs of the disaccharide alpha-D-Manp-(1-->6)-alpha-D-Manp-OOctyl, a known acceptor substrate for a polyprenol monophosphomannose-dependent alpha-(1-->6)-mannosyltransferase involved in the assembly of the alpha-(1-->6)-linked mannan core of mycobacterial lipoarabinomannan, has been synthesized. Described are synthetic routes to the target deoxy and methoxy analogs in which one of the hydroxyl groups of the parent disaccharide has been modified. All glycosylation reactions involved the use of octyl glycoside acceptors and thioglycoside donors using iodonium-ion activation, and the stereochemistry of the mannopyranoside bond formed was established by measurement of the 1J(C-1,H-1). Depending on the target, the key methylation or deoxygenation reactions were carried out on either mono- or disaccharide substrates. This series of analogs will be useful for probing the substrate specificity of the enzyme, in particular, its steric and hydrogen-bonding requirements.     

Modulation of bone marrow mesenchymal stem cell secretome by ECM-like hydrogels

It has been demonstrated that bone marrow mesenchymal stem cell (BM-MSCs) transplantation has beneficial effects on several central nervous system (CNS) debilitating conditions. Growing evidence indicate that trophic factors secreted by these cells are the key mechanism by which they are acting. These cells are frequently used in combination with 3D artificial matrices, for instance hydrogels, in tissue engineering-based approaches. However, so far, no study has been reported on the influence of such matrices, namely the presence or absence of extracellular matrix motifs, on BM-MSCs secretome and its effects in neuronal cell populations. In this sense, we herein studied the impact of a hydrogel, gellan gum, on the behavior and secretome of BM-MSCs, both in its commercial available form (commonly used in tissue engineering) and in a fibronectin peptide-modified form. The results showed that in the presence of a peptide in the gellan gum hydrogel, BM-MSCs presented higher proliferation and metabolic activity than in the regular hydrogel. Moreover, the typical spindle shape morphology of BM-MSCs was only observed in the modified hydrogel. The effects of the secretome of BM-MSCs were also affected by the chemical nature of the extracellular matrix. BM-MSCs cultured in the modified hydrogel were able to secrete factors that induced higher metabolic viabilities and neuronal cell densities, when compared to those of the unmodified hydrogel. Thus adding a peptide sequence to the gellan gum had a significant effect on the morphology, activity, proliferation and secretome of BM-MSCs. These results highlight the importance of mimicking the extracellular matrix when BM-MSCs are cultured in hydrogels for CNS applications.     

Application of mitochondrial control region in population genetic studies of the shrimp Penaeus

This study reports a primer set for amplifying a partial fragment of about 610 bp in the fast mutating mitochondrial control region in shrimps of the genus Penaeus (Decapoda: Penaeidae). The utility of this amplified fragment for studying population differentiation and structuring, compared with more conservative mitochondrial genes (16S rRNA and COI), was explored in P. merguiensis populations over a vast geographical range based on sequence and RFLP analyses. The results indicate that the mitochondrial control region provides more informative sites and reveals more haplotypes, making it most useful for evaluating genetic variations within and between populations of Penaeus species.     

Association of polymorphisms in the Chr18q11.2 locus with tuberculosis in Chinese population

A GWAS study has reported that two single nucleotide polymorphisms (SNPs) were associated with predisposition to tuberculosis (TB) in African populations. These two loci represented the long-waited GWAS hits for TB susceptibility. To determine whether these two SNPs are associated with TB in Chinese population, we attempted an replication in a cohort of over one thousand Chinese TB patients and 1,280 healthy controls using melting temperature shift allele-specific genotyping analysis. We found that only SNP rs4331426 was significantly associated with TB in Chinese population (p = 0.011). However, the effect was opposite. The G allele of the SNP in Chinese population is a protective allele (OR = 0.62, 95 % CI 0.44–0.87), while it was the risk allele for African population (OR = 1.19, 95 % CI 1.12–1.26). No significance was found for SNP rs2335704. The results provided an independent support for a role in susceptibility to TB for SNP rs4331426. However, it also indicated that direct predisposition element to TB and the association effects may vary across ethnic groups.     

Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its ∼ 200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites. No individual site is essential but phosphorylation of increasing numbers of sites enables progressively greater channel activity. In spite of numerous studies of the role of the R domain in CFTR regulation, its mechanism of action remains largely unknown. This is because neither its structure nor its interactions with other parts of CFTR have been completely elucidated. Studies have shown that the R domain lacks well-defined secondary structural elements and is an intrinsically disordered region of the channel protein. Here, we have analyzed the disorder pattern and employed computational methods to explore low-energy conformations of the R domain. The specific disorder and secondary structure patterns detected suggest the presence of molecular recognition elements (MoREs) that may mediate phosphorylation-regulated intra- and inter-domain interactions. Simulations were performed to generate an ensemble of accessible R domain conformations. Although the calculated structures may represent more compact conformers than occur in vivo, their secondary structure propensities are consistent with predictions and published experimental data. Equilibrium simulations of a mimic of a phosphorylated R domain showed that it exhibited an increased radius of gyration. In one possible interpretation of these findings, by changing its size, the globally unstructured R domain may act as an entropic spring to perturb the packing of membrane-spanning sequences that constitute the ion permeability pathway and thereby activate channel gating.     

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca²⁺ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca²⁺-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca²⁺ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca²⁺-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca²⁺.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

CDKL5 regulates flagellar length and localizes to the base of the flagella in Chlamydomonas

The length of Chlamydomonas flagella is tightly regulated. Mutations in four genes—LF1, LF2, LF3, and LF4—cause cells to assemble flagella up to three times wild-type length. LF2 and LF4 encode protein kinases. Here we describe a new gene, LF5, in which null mutations cause cells to assemble flagella of excess length. The LF5 gene encodes a protein kinase very similar in sequence to the protein kinase CDKL5. In humans, mutations in this kinase cause a severe form of juvenile epilepsy. The LF5 protein localizes to a unique location: the proximal 1 μm of the flagella. The proximal localization of the LF5 protein is lost when genes that make up the proteins in the cytoplasmic length regulatory complex (LRC)—LF1, LF2, and LF3—are mutated. In these mutants LF5p becomes localized either at the distal tip of the flagella or along the flagellar length, indicating that length regulation involves, at least in part, control of LF5p localization by the LRC.