J. H. David Wu

Cellulase, Clostridia, and Ethanol

SUMMARY Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the worlds dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.

Localization of androgen receptor expression in human bone marrow

Androgens have been shown to modulate the haematopoietic and immune systems and have been used clinically for stimulating haematopoiesis in bone marrow failure conditions. To identify the bone marrow cell types as potential targets of androgens, an androgen receptor (AR)‐specific antibody was used to localize the AR in normal human bone marrow biopsies. The results show that AR was ubiquitously expressed in the bone marrow of both males and females. Furthermore, the AR expression pattern did not change with age. Stromal cells, macrophages, endothelial cells, myeloblasts, myelocytes, neutrophils, and megakaryocytes expressed AR. In contrast, AR was not detected in the lymphoid and erythroid cells, or in eosinophils. These results indicate that androgens may exert direct modulating effects on a wide spectrum of bone marrow cell types via AR‐mediated responses. Copyright

The crystal structure and catalytic mechanism of cellobiohydrolase CelS, the major enzymatic component of the Clostridium thermocellum Cellulosome.

Cellobiohydrolase CelS plays an important role in the cellulosome, an active cellulase system produced by the thermophilic anaerobe Clostridium thermocellum. The structures of the catalytic domain of CelS in complex with substrate (cellohexaose) and product (cellobiose) were determined at 2.5 and 2.4 A resolution, respectively. The protein folds into an (alpha/alpha)(6) barrel with a tunnel-shaped substrate-binding region. The conformation of the loops defining the tunnel is intrinsically stable in the absence of substrate, suggesting a model to account for the processive mode of action of family 48 cellobiohydrolases. Structural comparisons with other (alpha/alpha)(6) barrel glycosidases indicate that CelS and endoglucanase CelA, a sequence-unrelated family 8 glycosidase with a groove-shaped substrate-binding region, use the same catalytic machinery to hydrolyze the glycosidic linkage, despite a low sequence similarity and a different endo/exo mode of action. A remarkable feature of the mechanism is the absence, from CelS, of a carboxylic group acting as the base catalyst. The nearly identical arrangement of substrate and functionally important residues in the two active sites strongly suggests an evolutionary relationship between the cellobiohydrolase and endoglucanase families, which can therefore be classified into a new clan of glycoside hydrolases.

Induction of the celC operon of Clostridium thermocellum by laminaribiose

Clostridium thermocellum is an anaerobic, thermophilic, cellulolytic, and ethanogenic bacterium. It produces an extracellular multiprotein complex termed the cellulosome, which consists of >70 subunits, most of them glycosyl hydrolases. It also produces many free glycosyl hydrolases. How the organism commands such a large number of genes and proteins for biomass degradation is an intriguing yet unresolved question. We identified glyR3, which is cotranscribed with the cellulase/hemicellulase genes celC and licA, as a potential cellulase transcription regulator. The gel-shift assay (EMSA) revealed that the recombinant GlyR3 bound specifically to the celC promoter region. GlyR3 was also identified from the lysate of the lichenan-grown cells, which bound to the same sequence. DNase I footprinting and competitive EMSA showed the binding site to be an 18-bp palindromic sequence with one mismatch. The DNA-binding activity was specifically inhibited by laminaribiose, a β-1-3 linked glucose dimer, in a dose-dependent manner. In in vitro transcription analysis, celC expression was repressed by rGlyR3 in a dose-dependent manner. The repression was relieved by laminaribiose, also in a dose-dependent manner. These results indicate that GlyR3 is a negative regulator of the celC operon consisting of celC, glyR3, and licA, and inducible by laminaribiose. Thus, the bacterium may modulate the biosynthesis of its enzyme components to optimize its activity on an available biomass substrate, in this case, β-1-3 glucan, because both CelC and LicA are active on the substrate. The results further indicate that, despite the insolubility of the biomass substrate, regulation of the degradative enzymes can be accomplished through soluble sugars generated by the action of the enzymes.

Long-term immunologically competent human peripheral lymphoid tissue cultures in a 3D bioreactor.

Peripheral lymphoid organs (PLOs), the primary sites of development of adaptive immune responses, display a complex structural organization reflecting separation of cellular subsets (e.g., T and B lymphocytes) and functional compartments which is critical for immune function. The generation of in vitro culture systems capable of recapitulating salient features of PLOs for experimental, biotechnological, and clinical applications would be highly desirable, but has been hampered so far by the complexity of these systems. We have previously developed a three‐dimensional bioreactor system for long‐term, functional culture of human bone marrow cells on macroporous microspheres in a packed‐bed bioreactor with frequent medium change. Here we adapt the same system for culture of human primary cells from PLOs (tonsil) in the absence of specific exogenous growth factors or activators. Cells in this system displayed higher viability over several weeks, and maintain population diversity and cell surface markers largely comparable to primary cells. Light microscopy showed cells organizing in large diverse clusters within the scaffold pores and presence of B cell‐enriched areas. Strikingly, these cultures generated a significant number of antibody‐producing B cells when challenged with a panel of diverse antigens, as expected from a lymphoid tissue. Thus the three‐dimensional tonsil bioreactor culture system may serve as a useful model of PLOs by recapitulating their structural organization and function ex vivo. Biotechnol. Bioeng. 2011; 108:1430–1440.

Interactions and synergism between the recombinant CelD, an endoglucanase, and the cellulosome-integrating protein (CipA) of Clostridium thermocellum

Abstract The Clostridium thermocellum cellulosome is a multicomponent cellulase complex. Its largest subunit, CipA, contains nine repeated domains (Rs), each of which binds to a cellulosomal catalytic subunit, and a cellulose binding domain (CBD). We have previously reported that the activity of CelS, an exoglucanse subunit, is enhanced by the anchorage function of CipA. In this work, we examined the effect of anchorage on the activity of CelD, an endoglucanase subunit, using recombinant CelD (rCeiD) and the CipA functional domains, R3 (a repeat next to CBD) and CBD R3 , expressed in Escherichia coli, rCelD formed a stable complex with CBD R3 as analyzed by a gel-shift assay on a nondenaturing polyacrylamide gradient gel. Binding of rCelD to crystalline cellulose, as its activity toward both phosphoric acid-swollen and crystalline cellulose, was dependent on CBD R3 . These results indicate that the activity of an endoglucanase subunit of the cellulosome, as that of the exoglucanase subunit, is enhanced by the anchorage function of CipA. Such anchorage function of CipA may thus augment the potential endo-exo synergism in the cellulosome.

A cDNA from human bone marrow encoding a protein exhibiting homology to the ATP1γ1/PLM/MAT8 family of transmembrane proteins

A cDNA clone, IWU-1, was cloned from human bone marrow. Its putative open reading frame encoded a protein of 115 amino acids with a calculated molecular mass of 12.9 kDa. The deduced amino acid sequence exhibited high homology (>68%) to members of the ATP1gamma1/PLM/MAT8 family of single transmembrane proteins, primarily in the region containing the putative transmembrane domain. The sequence at the amino-terminal side exhibited high homology (>61%) to the cytoplasmic region of the angiotensin II type 1 receptors.

Molecular cloning and sequencing of the human heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase from bone marrow culture

The cDNA encoding human heme-regulated eukaryotic initiation factor-2 f (eIF-2 f ) kinase was cloned from a human bone marrow culture. Its deduced amino acid sequence comprised of 629 amino acids with a calculated molecular weight of 71,031 Da. RT-PCR analysis revealed that the gene was also expressed in heart, kidney, spleen, muscle, and stomach.

Cell density-dependent proliferation of the murine bone marrow-derived stromal cell lines

Proliferation of three murine marrow-derived stromal cell lines, LC1, LC2, and LC3, depended on initial cell density. For LC2 and LC3, the cell density-dependence was negated by conditioned-media, indicating growth dependence on a soluble growth factor. For LC1, conditioned-media failed to stimulate proliferation, suggesting growth dependence on direct cell-cell contact.

A Novel Three-Dimensional Long-Term Bone Marrow Bioreactor Culture System

Hemopoiesis occurs in the extravascular space between bone marrow sinuses where a high cell density facilitates cell-cell and cell-matrix interactions. The bone marrow stromal cells and the extracellular matrices produced by the stromal cells form a three-dimensional scaffolding upon which the hemopoietic cells are lodged. To simulate this marrow microenvironment, we have developed a long-term bone marrow culture bioreactor in which an artificial three-dimensional porous matrix is provided for the lodging and growth of the marrow cells. Unlike the two-dimensional growth configuration of the traditional flask culture, the marrow cells grow in the bioreactor in three-dimensional clusters or colonies, thus simulating the in vivo growth configuration. More importantly, although the flask long-term bone marrow cultures give rise only to neutrophils and monocyte-macrophages, multilineal hemopoiesis is observed in the bioreactor. The cell output includes all stages/subtypes of granulocytes, erythrocytes, monocyte-macrophages and megakaryocytes. In addition, lymphoid cells are produced. Therefore, the bioreactor, which provides a microenvironment different than that of the flask culture, can be used as a hemopoiesis model for delineating the bone marrow microenvironment and optimized for treating various blood-related diseases.