Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of Escherichia coli Possesses Chitinase Functions Comparable to CHO-Expressed Protein

Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)₆ tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N′-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.     

Thrombopoietic-mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.     

The mechanism of nucleotide‐binding domain dimerization in the intact maltose transporter as studied by all‐atom molecular dynamics simulations

The Escherichia coli maltose transporter MalFGK₂‐E belongs to the protein superfamily of ATP‐binding cassette (ABC) transporters. This protein is composed of heterodimeric transmembrane domains (TMDs) MalF and MalG, and the homodimeric nucleotide‐binding domains (NBDs) MalK₂. In addition to the TMDs and NBDs, the periplasmic maltose binding protein MalE captures maltose and shuttle it to the transporter. In this study, we performed all‐atom molecular dynamics (MD) simulations on the maltose transporter and found that both the binding of MalE to the periplasmic side of the TMDs and binding of ATP to the MalK₂ are necessary to facilitate the conformational change from the inward‐facing state to the occluded state, in which MalK₂ is completely dimerized. MalE binding suppressed the fluctuation of the TMDs and MalF periplasmic region (MalF‐P2), and thus prevented the incorrect arrangement of the MalF C‐terminal (TM8) helix. Without MalE binding, the MalF TM8 helix showed a tendency to intrude into the substrate translocation pathway, hindering the closure of the MalK₂. This observation is consistent with previous mutagenesis experimental results on MalF and provides a new point of view regarding the understanding of the substrate translocation mechanism of the maltose transporter.     

Caspases and matrix metalloproteases facilitate collective behavior of non-neural ectoderm after hindbrain neuropore closure

Background

Mammalian brain is formed through neural tube closure (NTC), wherein both ridges of opposing neural folds are fused in the midline and remodeled in the roof plate of the neural tube and overlying non-neural ectodermal layer. Apoptosis is widely observed from the beginning of NTC at the neural ridges and is crucial for the proper progression of NTC, but its role after the closure remains less clear.

Results

Here, we conducted live-imaging analysis of the mid-hindbrain neuropore (MHNP) closure and revealed unexpected collective behavior of cells surrounding the MHNP. The cells first gathered to the closing point and subsequently relocated as if they were released from the point. Inhibition of caspases or matrix metalloproteases with chemical inhibitors impaired the cell relocation.

Conclusions

These lines of evidence suggest that apoptosis-mediated degradation of extracellular matrix might facilitate the final process of neuropore closure.

     

Use of a human immunodeficiency virus type 1 Rev mutant without nucleolar dysfunction as a candidate for potential AIDS therapy.

Applications of transdominant mutants of human immunodeficiency virus type 1 (HIV-1) regulatory proteins, especially Rev mutant, have been attempted for gene therapy against AIDS, because the Rev protein is essential for viral replication. We have previously reported that a mutant Rev protein (dRev) lacking its nucleolar targeting signal remained out of nuclei in expressed cells and strongly inhibited the function of Rev. To investigate the effects of dRev on HIV-1 replication, we established several dRev-expressing human cell lines with two different vector systems and examined virus production in these cells. An HIV-1-derived vector containing drev cDNA was constructed and introduced into CD4-positive HeLa cells and cells of the human T-cell line CCRF-CEM (CEM). In dRev-expressing HeLa cells, virus replication, syncytium formation, and cell death caused by HIV-1 infection were remarkably suppressed, and the same vector also conferred a resistant phenotype on CEM cells. The production was also suppressed in CEM cells containing the drev gene driven by a cytomegalovirus promoter. In addition, we found that dRev did not cause nucleolar dysfunction in a transient assay, in contrast to other transdominant mutants and wild-type Rev. Since dRev cannot migrate into the nuclei, it is expected not to interfere with nuclear/nucleolar functions of the host cell. We conclude that dRev is one promising candidate as an antiviral molecule for gene therapy against AIDS.     

LckBP1, a proline-rich protein expressed in haematopoietic lineage cells, directly associates with the SH3 domain of protein tyrosine kinase p56lck.

The Lck tyrosine kinase molecule plays an essential role in T cell activation and T cell development. Using the expression cloning technique, we have isolated a gene that encodes a molecule, LckBP1, able to associate with murine Lck. Analysis of full-length LckBP1 cDNA indicates at least four potentially important segments: a four tandem 37 amino acid repeat motif with a potential helix-turn-helix DNA binding motif; a proline-rich region; a proline-glutamate repeat; and an SH3 domain. These four regions are very similar to the human haematopoietic-specific protein 1 (HS1). Deletion mutant analysis of LckBP1 revealed two proline-rich regions that permit association with Lck SH3. One region contains prolines conserved among HS1 and cortactin, and the other region contains a potential MAP kinase recognition site. In vivo association between Lck and LckBP1 was confirmed by immunoprecipitation of lysates from a pre-T cell line and adult thymocytes using antibodies specific for Lck and LckBP1. LckBP1 is tyrosine phosphorylated after T-cell receptor stimulation. The SH3 domain and the potential helix-turn-helix motif in LckBP1 suggest that this molecule may associate with various molecules and function as a DNA binding molecule. The data also suggest that LckBP1 mediates intracellular signalling through Lck in T cells.     

Stretch-induced enhancement of mechanical work production in frog single fibers and human muscle

Takarada, Yudai, Hiroyuki Iwamoto, Haruo Sugi, YuichiHirano, and Naokata Ishii. Stretch-induced enhancement ofmechanical work production in frog single fibers and human muscle.J. Appl. Physiol. 83(5):1741-1748, 1997.The relations between the velocity of prestretchand the mechanical energy liberated during the subsequent isovelocityrelease were studied in contractions of frog single fibers and humanmuscles. During isometric contractions of frog single fibers, a rampstretch of varied velocity (amplitude, 0.02 fiber length; velocity,0.08-1.0 fiber length/s) followed by a release (amplitude, 0.02 fiber length; velocity, 1.0 fiber length/s) was given, and the amountof work liberated during the release was measured. For human muscles,elbow flexions were performed with a prestretch of variedvelocity (range, 40°; velocity, 30-180°/s) followed by anisokinetic shortening (velocity, 90°/s). In both frog single fibersand human muscles, the work production increased with both the velocityof stretch and the peak of force attained before the release up to acertain level; thereafter it declined with the further increases ofthese variables. In human muscles, the enhancement of work productionwas not associated with a significant increase in integratedelectromyogram. This suggests that changes in intrinsic mechanicalproperties of muscle fibers play an important role in thestretch-induced enhancement of work production.