Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.     

Purification, cloning, and characterization of a profibrinolytic plasminogen-binding protein, TIP49a

The plasminogen receptors responsible for enhancing cell surface-dependent plasminogen activation expose COOH-terminal lysines on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). We treated U937 cells with CpB, then subjected membrane fractions to two-dimensional gel electrophoresis followed by ligand blotting with (125)I-plasminogen. A 54-kDa protein lost the ability to bind (125)I-plasminogen after treatment of intact cells and was purified by two-dimensional gel electrophoresis and then sequenced by mass spectrometry. Two separate amino acid sequences were obtained and were identical to sequences contained within human and rat TIP49a. The cDNA for the 54-kDa protein matched the human TIP49a sequence, and encoded a COOH-terminal lysine, consistent with susceptibility to CpB. Antibodies against rat TIP49a recognized the plasminogen-binding protein on two-dimensional Western blots of U937 cell membranes. Human (125)I-Glu-plasminogen bound specifically to TIP49a protein, and binding was inhibited by epsilon-aminocaproic acid. A single class of binding sites was detected, and a K(d) of 0.57 +/- 0.14 microm was determined. TIP49a enhanced plasminogen activation 8-fold compared with the BSA control, and this was equivalent to the enhancement mediated by plasmin-treated fibrinogen. These results suggest that TIP49a is a previously unrecognized plasminogen-binding protein on the U937 cell surface.     

The effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

Chloroplast Structure and Function Is Altered in the NCS2 Maize Mitochondrial Mutant

The nonchromosomal stripe 2 (NCS2) mutant of maize (Zea mays L.) has a DNA rearrangement in the mitochondrial genome that segregates with the abnormal growth phenotype. Yet, the NCS2 characteristic phenotype includes striped sectors of pale-green tissue on the leaves. This suggests a chloroplast abnormality. To characterize the chloroplasts present in the mutant sectors, we examined the chloroplast structure by electron microscopy, chloroplast function by radiolabeled carbon dioxide fixation and fluorescence induction kinetics, and thylakoid protein composition by polyacrylamide gel electrophoresis. The data from these analyses suggest abnormal or prematurely arrested chloroplast development. Deleterious effects of the NCS2 mutant mitochondria upon the cells of the leaf include structural and functional alterations in the both the bundle sheath and mesophyll chloroplasts.     

Granzyme M has a critical role in providing innate immune protection in ulcerative colitis

Inflammatory bowel disease (IBD) is an immunoregulatory disorder, associated with a chronic and inappropriate mucosal immune response to commensal bacteria, underlying disease states such as ulcerative colitis (UC) and Crohn''s disease (CD) in humans. Granzyme M (GrzM) is a serine protease expressed by cytotoxic lymphocytes, in particular natural killer (NK) cells. Granzymes are thought to be involved in triggering cell death in eukaryotic target cells; however, some evidence supports their role in inflammation. The role of GrzM in the innate immune response to mucosal inflammation has never been examined. Here, we discover that patients with UC, unlike patients with CD, display high levels of GrzM mRNA expression in the inflamed colon. By taking advantage of well-established models of experimental UC, we revealed that GrzM-deficient mice have greater levels of inflammatory indicators during dextran sulfate sodium (DSS)-induced IBD, including increased weight loss, greater colon length reduction and more severe intestinal histopathology. The absence of GrzM expression also had effects on gut permeability, tissue cytokine/chemokine dynamics, and neutrophil infiltration during disease. These findings demonstrate, for the first time, that GrzM has a critical role during early stages of inflammation in UC, and that in its absence colonic inflammation is enhanced.Inflammatory bowel disease (IBD) is a gut-associated inflammatory disorder, which stems from a dysfunctional mucosal immune response to commensal bacteria.¹ As a multifactorial disease, IBD is the consequence of a complex interplay between environmental triggers, genetic susceptibility, and immunoregulatory defects, resulting in a pathogenesis that is still poorly understood.² These interactions result in the inability of an individual to control the normal inflammatory response to pathogens in the gut, leading to a chronic state of sustained and inappropriate inflammation. IBD underlies disease states such as ulcerative colitis (UC) and Crohn''s disease (CD), with symptoms including weight loss, abdominal pain, diarrhea, and rectal bleeding which often require intensive medical therapy and resective surgery.³ The pathogenesis of IBD, characterized by a defective mucosal immune response to microbial exposure in the gastrointestinal tract, is thought to be caused by a dysfunctional immune response to host microbiota, infection by specific pathogens, and/or a defective mucosal barrier to luminal pathogens.^{1, 2} IBD patients also have a high risk of developing colitis-associated colon cancer (CAC).⁴ Additionally, histological assessment of inflamed ileal and colonic segments from IBD patients typically shows increased infiltration of immune cells, particularly neutrophils, as well as crypt abscesses, mucin depletion, and ulcers—all correlating with the severity of small bowel and colonic tissue damage.⁵Cytotoxic pathways mediated by lymphocytes directly trigger cell death in target cells.⁶ These cytotoxic pathways are mediated by proteins such as perforin, which mediates pore formation in the target cell surface and allows granzyme (Grz)s to enter the intracellular compartment and induce cell death.⁷ To date, five different Grzs have been identified in humans (GrzA, GrzB, GrzH, GrzK, and GrzM), whereas mice express eleven Grzs (GrzA, GrzB, GrzC, GrzD, GrzE, GrzF, GrzG, GrzK, GrzL, GrzM, and GrzN).^{8, 9} Walch et al.¹⁰ recently demonstrated that Grzs (GrzA and GrzB) directly kill bacteria through granulysin-mediated delivery, suggesting that Grzs act as microbial modulating factors. Moreover, recently GrzA was shown to be increased in the colon biopsies of UC patients undergoing treatment with Etrolizumab, a monoclonal antibody targeting the β7 integrin subunit. Higher levels of GrzA could predict which patients were more likely to benefit from the therapy; however, the precise mechanism of action of GrzA in UC remains to be addressed.¹¹ GrzM was initially described as being constitutively expressed by natural killer (NK) cells,^{12, 13} and specifically associated with inflammation.¹⁴ This enzyme has been shown to preferentially cleave methionine and leucine residues in target cells, mediating direct, non-specific cell death.^{15, 16} More recently, GrzM was also shown to be an important mediator for the release of MIP-1α from NK cells, inducing NK cell and neutrophil recruitment during early microbial infection.¹⁷ We now observe that GrzM expression is increased in inflamed colon tissue samples from UC, but not CD patients. Further, GrzM-deficient (GrzM^−/−) mice are more sensitive to a mouse model of IBD and IBD-induced colorectal cancer (CRC). These findings demonstrate, for the first time, that GrzM has a critical role in mediating the early stages of the gut mucosal immune response.     

Solution conformation of d-CTCGAGCTCGAG by two-dimensional NMR: conformational heterogeneity at XhoI cleavage site

Nonexchangeable proton resonances in the 500-MHz NMR spectrum of d-CTCGAGCTCGAG have been assigned by using two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). 1H-1H coupling constants (J) in the deoxyribose rings have been measured by analyzing intensity and multiplet patterns in the phase-sensitive omega 1-scaled COSY spectra. A modification of the J-resolved technique, called amplitude-modulated J-resolved spectroscopy, has been described and used to increase the accuracy of J measurements. Absorption mode omega 1-scaled NOESY spectra at mixing times in the range 50-200 ms have been analyzed to monitor spin diffusion. A 50-ms spectrum has been used to estimate several interproton distances. The coupling constant and distance data have been used to arrive at sequence-specific sugar geometries and glycosidic torsion angles. The backbone structure has been refined by model building using the FRODO program, employing the sugar geometries and glycosidic torsion angles discussed above. The molecule shows interesting sequence-dependent variations in the structure. The cleavage site of the restriction enzyme XhoI exhibits unique differences in the sugar geometry and backbone torsion angles.     

Projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the moth Manduca sexta

Octopod (Octo) is a mutation of the moth Manduca sexta, which transforms the first abdominal segment (A1) in the anterior direction. Mutant animals are characterized by the appearance of homeotic thoracic-like legs on A1. We exploited this mutation to determine what rules might be used in specifying the fates of sensory neurons located on the body surface of larval Manduca. Mechanical stimulation of homeotic leg sensilla did not cause reflexive movements of the homeotic legs, but elicited responses similar to those observed following stimulation of ventral A1 body wall hairs. Intracellular recordings demonstrated that several of the motoneurons in the A1 ganglion received inputs from the homeotic sensory hairs. The responses of these motoneurons to stimulation of homeotic sensilla resembled their responses to stimulation of ventral body wall sensilla. Cobalt fills revealed that the mutation transformed the segmental projection pattern of only the sensory neurons located on the ventral surface of A1, resulting in a greater number with intersegmental projection patterns typical of sensory neurons found on the thoracic body wall. Many of the sensory neurons on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons: an anteriorly directed projection terminating in the third thoracic ganglion (T3). Once this projection reached T3, however, it mimicked the projections of the thoracic leg sensory neurons. These results demonstrate that the same rules are not used in the establishment of the intersegmental and leg-specific projection patterns. Segmental identity influences the intersegmental projection pattern of the sensory neurons of Manduca, whereas the leg-specific projections are consistent with a role for positional information in determining their pattern. © 1995 John Wiley & Sons, Inc.