Cloning and sequence analysis of two embryonic beta-like globin cDNAs (y and z) of hamster.

A cDNA library was prepared from poly(A) mRNA extracted from 9-day hamster-yolk-sac erythroid cells. Two clones containing inserts coding for embryonic beta-like z or y globin-chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNAs and (b) nucleotide sequence analysis of the inserts and comparison to the embryonic beta-like globin genes of Balb/c mouse. Availability of sequences for embryonic and adult globin cDNAs will aid in investigations of the molecular mechanisms of the globin switch in hamster YSEC.     

Blocking of acidosis-mediated apoptosis by a reduction of lactate dehydrogenase activity through antisense mRNA expression.

Lactic acid produced from the cells is a potential cause of extra- and intracellular acidification. Due to scarce technical tools, lactic acid that leads to acidification could not be reduced and direct evidence of the relationship between metabolic lactate and apoptosis has not yet been elucidated. In this study, we designed a cellular pH regulation system in CHO cells by a reduction of lactate dehydrogenase (LDH) activity through LDH antisense mRNA expression. This inhibited lactate production and, therefore, acidification of the cytosol. Under HCO3(-)-buffered growth conditions, both the parent CHO cells and the engineered CHO cells maintained their extracellular pH and intracellular pH fairly well. However, upon acidification of the cytosol, only the parent CHO cells underwent apoptosis under HCO3(-)-free conditions. In fact, we observed a number of apoptosis-related events only in control cells, including mitochondrial dysfunction, cytochrome c release, and an increase in caspase-3 enzymatic activity.     

CD40 ligand-activated human monocytes amplify glomerular inflammatory responses through soluble and cell-to-cell contact-dependent mechanisms.

Monocytes/macrophages play a critical role in the initiation and progression of a variety of glomerulonephritides. We sought to define the interactions between physiologically activated human monocytes and glomerular mesangial cells (MC) by employing a cell culture system that permits the accurate assessment of the contribution of soluble factors and cell-to-cell contact. Human peripheral blood monocytes, primed with IFN-gamma and GM-CSF, were activated with CD40 ligand (CD40L) or TNF-alpha and cocultured with MC. CD40L-activated monocytes induced higher levels of IL-6, monocyte chemoattractant protein-1 (MCP-1) and ICAM-1 synthesis by MC. Separation of CD40L-activated monocytes from MC by a porous membrane decreased the mesangial synthesis of IL-6 by 80% and ICAM-1 by 45%, but had no effect on MCP-1. Neutralizing Abs against the beta 2 integrins, LFA-1 and Mac-1, decreased IL-6 production by 40 and 50%, respectively. Ligation of mesangial surface ICAM-1 directly enhanced IL-6, but not MCP-1, production. Simultaneous neutralization of soluble TNF-alpha and IL-1 beta decreased MCP-1 production by 55% in membrane-separated cocultures of MC/CD40L-activated monocytes. Paraformaldehyde-fixed CD40L-activated monocytes (to preserve membrane integrity but prevent secretory activity), cocultured with MC at various ratios, induced IL-6, MCP-1, and ICAM-1 synthesis by MC. Plasma membrane preparations from activated monocytes also induced mesangial IL-6 and MCP-1 synthesis. The addition of plasma membrane enhanced TNF-alpha-induced mesangial IL-6 production by approximately 4-fold. Together, these data suggest that the CD40/CD40L is essential for optimal effector function of monocytes, that CD40L-activated monocytes stimulate MC through both soluble factors and cell-to-cell contact mediated pathways, and that both pathways are essential for maximum stimulation of MC.     

Electrical injury mechanisms: dynamics of the thermal response

The thermal response of the human upper extremity to large electric currents was examined using an axisymmetric unidimensional model containing bone, skeletal muscle, fat, and skin in coaxial cylindrical geometry. Appropriate thermal and electrical properties were assigned to each tissue, and the tissue response to joule heating was determined by a finite-element numerical technique. We found that when the tissues are electrically in parallel, skeletal muscle sustained the largest temperature rise and then heated adjacent tissues. Thus, when bone is not in series with other tissues, joule heating of bone is unlikely to be responsible for thermal damage to adjacent tissue. In addition, the effect of tissue perfusion on the thermal response was found to be essential for rapid cooling of the centrally located tissues.     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

Unbalanced ribosome assembly in Saccharomyces cerevisiae expressing mutant 5 S rRNAs.

Mutant 5 S rRNA genes were expressed in Saccharomyces cerevisiae to further define the function of the ribosomal 5 S RNA. RNA synthesis and utilization were assayed using previously constructed markers which have been shown to be functionally neutral and easily detected by gel electrophoresis. Most mutations were found not to affect the growth rate because they were poorly expressed or could be accommodated effectively in the ribosomal structure. Two of the mutants, Y5A99U56U57 and Y5U90i5 adversely affected cell growth as well as protein synthesis in vitro. Polyribosome profiles in both of these mutants were substantially shorter, and an analysis of the ribosomal subunit composition revealed a significant imbalance with a 25-35% excess in 40 S subunits. Kinetic analyses of RNA labeling indicated very low cellular levels of mutant RNA either because it was poorly expressed (Y5U90i5) or rapidly degraded before being incorporated into mature 60 subunits (Y5A99U56U57). The results suggest that the 5 S RNA is required for the assembly of stable ribosomal 60 S subunits and raise the possibility that this RNA or, more likely, its corresponding ribonucleoprotein complex is critical for subunit assembly or even RNA processing.     

Characterization of ribonucleolytic activity of angiogenin towards tRNA

Yeast tRNA is a convenient substrate for the assay of the ribonucleolytic activity of human angiogenin. The optimal pH, [NaCl], and temperature for tRNA cleavage by angiogenin are approximately 6.8, 15-30 mM, and approximately 55 degrees C, respectively, as compared with approximately 8.0, 100-200 mM, and approximately 65 degrees C, respectively, for RNase A. Polyanions and metals both inhibit angiogenin and RNase A but to different extents.