Immunological Determination of Labeling Index on Human Tumor Tissue Sections Using Monoclonal Anti-Brdurd Antibody

The use of a monoclonal antibody against the thymidine analogue bromodeoxyuridine together with an in vitro labeling technique allowed rapid determination of the labeling index in human tumors. The labeling index estimated by these relatively simple immunofluorexence or immunoenzymatic staining methods was equivalent to that obtained by autoradiography. The interpretation of the preparations is easy since there is a minimum of background staining. This immunohistochemical technique combined with in vitro labeling provides a suitable alternative for determining the labeling index of human tumors.     

A Study on Erythrocyte Membrane Plasmalogen in Myotonic Dystrophy

Phospholipid classes that included plasmalogens of erythrocyte membranes in seven myotonic dystrophy (MyD) patients and seven normal controls were analyzed by HPLC. No significant difference in phospholipid classes was found between patients with MyD and normal controls, but there was a visible difference in peak profiles of compounds of the phosphatidylethanolamine class. In the study of plasmalogens, we used two preparation methods: exposure to HCl and deacylation with mild alkaline. The area ratio of the plasmalogen form to the diacyl form in the phosphatidylethanolamine class of MyD erythrocyte membranes was significantly lower than that of normal controls. Fatty acid analyses showed that fatty acids of both phosphatidylethanolamine subclasses have less unsaturation in MyD.     

Reaction of some D-glucobioses with 2,2-dimethoxypropane

Laminarabiose, cellobiose, and gentiobiose were acetonated with 2,2-dimethoxy-propane under various conditions. Two isopropylidene acetals in which the reducing D-glucose residue had the furanoid form were obtained from laminarabiose, and two, in which the reducing D-glucose residue formed the acyclic dimethyl acetal, from cellobiose. Gentiobiose gave both types of isopropylidene compound.     

Roles of MAPKKK ASK1 in stress-induced cell death

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein (MAP) kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase signaling cascades. Recent findings from analyses of ASK1-deficient mice have revealed that ASK1 is required for apoptosis induced by oxidative stress, TNF and endoplasmic reticulum (ER) stress. In addition, several lines of evidence have suggested that ASK1 has diverse functions in the decision of cell fate beyond its pro-apoptotic activity. Thus, ASK1 appears to be a pivotal component not only in stress-induced cell death but also in a broad range of biological activities in order for cells to adapt to or oppose various stresses.     

Caspase-7 mediates caspase-1-induced apoptosis independently of Bid

Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.     

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.     

Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.     

Overexpression of mutant cell division cycle 25 homolog B (CDC25B) enhances the efficiency of selection in Chinese hamster ovary cells

The effects of mutant cell division cycle 25 homolog B (CDC25B) overexpression on the generation of cells producing a monoclonal antibody were investigated in Chinese hamster ovary (CHO) cells. Mutant CDC25B (m-CDC25B) expression plasmids were transfected into CHO DG44-derived cells producing a monoclonal antibody, and the frequency of highly producing cells was assessed following gene amplification in the presence of 250 nM methotrexate. Most of the clones obtained from the m-CDC25B-overexpressing cells had higher antibody titers than did mock-transfected control cells. This arose from either higher transgene copy numbers or higher mRNA expression levels for the antibody. However, the high mRNA expression levels were not always accompanied by increases in transgene copy numbers. Our results suggest that cells producing high levels of a monoclonal antibody can be selected efficiently using m-CDC25B overexpression.     

Impaired Neural Differentiation of Induced Pluripotent Stem Cells Generated from a Mouse Model of Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.