Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.     

Spectrum of mutations in the Batten disease gene,CLN3.

Batten disease (juvenile-onset neuronal ceroid lipofuscinosis [JNCL]) is an autosomal recessive condition characterized by accumulation of lipopigments (lipofuscin and ceroid) in neurons and other cell types. The Batten disease gene, CLN3, was recently isolated, and four disease-causing mutations were identified, including a 1.02-kb deletion that is present in the majority of patients (The International Batten Disease Consortium 1995). One hundred eighty-eight unrelated patients with JNCL were screened in this study to determine how many disease chromosomes carried the 1.02-kb deletion and how many carried other mutations in CLN3. One hundred thirty-nine patients (74%) were found to have the 1.02-kb deletion on both chromosomes, whereas 49 patients (41 heterozygous for the 1.02-kb deletion) had mutations other than the 1.02-kb deletion. SSCP analysis and direct sequencing were used to screen for new mutations in these individuals. Nineteen novel mutations were found: six missense mutations, five nonsense mutations, three small deletions, three small insertions, one intronic mutation, and one splice-site mutation. This report brings the total number of disease-associated mutations in CLN3 to 23. All patients homozygous for mutations predicted to give rise to truncated proteins were found to have classical JNCL. However, a proportion of the patients (n = 4) who were compound heterozygotes for a missense mutation and the 1.02-kb deletion were found to display an atypical phenotype that was dominated by visual failure rather than by severe neurodegeneration. All missense mutations were found to affect residues conserved between the human protein and homologues in diverse species.     

N2 Fixation (C2H2-Reducing Activity) and Leghaemoglobin Content during Nitrate- and Water-Stress-Induced Senescence of Medicago sativa Root Nodules

Nitrate and water stress were used to induce senescence in rootnodules of alfalfa (Medicago sativa L. cv. Aragon). Nodule senescencewas assessed by determinations of the nitrogenase (C₂H₂-reducing)activity, and the leghaemoglobin (LHb) and total soluble proteincontents of the nodules. Nodules responded similarly to and water stress in many respects, but there was a significant difference.All parameters of nodule activity, expressed on the basis ofnodule dry weight (DW), consistently decreased following treatmentwith or during drought; there was a significant interaction (synergism) between the inhibitory effects of and water stress on nitrogenase activity, but sucheffects were merely additive in the case of LHb content or LHb/solubleprotein ratio. However, caused the selective decay of LHb with respect to other nodular soluble proteins,whereas the decrease of LHb during water stress was due to ageneral inhibition of protein synthesis and to an increasedproteolytic activity in the nodule cytosol rather than to aspecific proteolysis of LHb. Key words: Leghaemoglobin, Medicago saliva, nitrogen fixation, root nodule senescence, water stress     

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments.

Isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli were compared with their parent strain in temperature shift experiments. To improve detection of phenotypic differences in division behavior and cell shape, the strains were grown in glucose-minimal medium with a decreased osmolality (about 100 mosM). Already at the premissive temperature, all mutants, particularly the pbpB and ftsQ mutants, showed an increased average cell length and cell mass. The pbpB and ftsQ mutants also exhibited a prolonged duration of the constriction period. All strains, except ftsZ, continued to initiate new constrictions at 42 degrees C, suggesting the involvement of FtsZ in an early step of the constriction process. The new constrictions were blunt in ftsQ and more pronounced in ftsA and pbpB filaments, which also had elongated median constrictions. Whereas the latter strains showed a slow recovery of cell division after a shift back to the permissive temperature, ftsZ and ftsQ filaments recovered quickly. Recovery of filaments occurred in all strains by the separation of newborn cells with an average length of two times LO, the length of newborn cells at the permissive temperature. The increased size of the newborn cells could indicate that the cell division machinery recovers too slowly to create normal-sized cells. Our results indicate a phenotypic resemblance between ftsA and pbpB mutants and suggest that the cell division gene products function in the order FtsZ-FtsQ-FtsA, PBP3. The ftsE mutant continued to constrict and divide at 42 degrees C, forming short filaments, which recovered quickly after a shift back to the permissive temperature. After prolonged growth at 42 degree C, chains of cells, which eventually swelled up, were formed. Although the ftsE mutant produced filaments in broth medium at the restrictive temperature, it cannot be considered a cell division mutant under the presently applied conditions.     

Morphology agrees with molecular data: phylogenetic affinities of Euptychiina butterflies (Nymphalidae: Satyrinae)

Euptychiina is the most species‐rich subtribe of Neotropical Satyrinae, with over 450 known species in 47 genera (14 monotypic). Here, we use morphological characters to examine the phylogenetic relationships within Euptychiina. Taxonomic sampling included 105 species representing the majority of the genera, as well as five outgroups. A total of 103 characters were obtained: 45 from wing pattern, 48 from genitalia and 10 from wing venation. The data matrix was analysed using maximum parsimony under both equal and extended implied weights. Euptychiina was recovered as monophyletic with ten monophyletic genera, contrasting previous DNA sequence‐based phylogenies that did not recover the monophyly of the group. In agreement with sequence‐based hypotheses, however, three main clades were recognized: the ‘Megisto clade’ with six monophyletic and three polyphyletic genera, the ‘Taygetis clade’ with nine genera of which three were monophyletic, and the ‘Pareuptyhia clade’ with four monophyletic and two polyphyletic genera. This is the first morphology‐based phylogenetic hypothesis for Euptychiina and the results will be used to complement molecular data in a combined analysis and to provide critical synapomorphies for clades and genera in this taxonomically confused group.     

Characterizing pathogenic processes in Batten disease: use of small eukaryotic model systems

The neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative disorders. Nevertheless, small model organisms, including those lacking a nervous system, have proven invaluable in the study of mechanisms that underlie the disease and in studying the functions of the conserved proteins associated to each disease. From the single-celled yeast, Saccharomyces cerevisiae and Schizosaccharomyces pombe, to the worm, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster, biochemical and, in particular, genetic studies on these organisms have provided insight into the NCLs.     

RNA aptamers binding the double-stranded RNA-binding domain

Specific RNA recognition of proteins containing the double-strand RNA-binding domain (dsRBD) is essential for several biological pathways such as ADAR-mediated adenosine deamination, localization of RNAs by Staufen, or RNA cleavage by RNAse III. Structural analysis has demonstrated the lack of base-specific interactions of dsRBDs with either a perfect RNA duplex or an RNA hairpin. We therefore asked whether in vitro selections performed in parallel with individual dsRBDs could yield RNAs that are specifically recognized by the dsRBD on which they were selected . To this end, SELEX experiments were performed using either the second dsRBD of the RNA-editing enzyme ADAR1 or the second dsRBD of Xlrbpa, a homolog of TRBP that is involved in RISC formation. Several RNA families with high binding capacities for dsRBDs were isolated from either SELEX experiment, but no discrimination of these RNAs by different dsRBDs could be detected. The selected RNAs are highly structured, and binding regions map to two neighboring stem-loops that presumably form stacked helices and are interrupted by mismatches and bulges. Despite the lack of selective binding of SELEX RNAs to individual dsRBDS, selected RNAs can efficiently interfere with RNA editing in vivo.     

Purification and crystal structure of human ODA16: Implications for ciliary import of outer dynein arms by the intraflagellar transport machinery

Motile cilia protrude from cell surfaces and are necessary to create movement of cells and fluids in the body. At the molecular level, cilia contain several dynein molecular motor complexes including outer dynein arms (ODAs) that are attached periodically to the ciliary axoneme, where they hydrolyse ATP to create the force required for bending and motility of the cilium. ODAs are preassembled in the cytoplasm and subsequently trafficked into the cilium by the intraflagellar transport (IFT) system. In the case of the green alga Chlamydomonas reinhardtii, the adaptor protein ODA16 binds to ODAs and directly to the IFT complex component IFT46 to facilitate the ciliary import of ODAs. Here, we purified recombinant human IFT46 and ODA16, determined the high‐resolution crystal structure of the ODA16 protein, and carried out direct interaction studies of IFT46 and ODA16. The human ODA16 C‐terminal 320 residues adopt the fold of an eight‐bladed β‐propeller with high overall structural similarity to the Chlamydomonas ODA16. However, the small 80 residue N‐terminal domain, which in Chlamydomonas ODA16 is located on top of the β‐propeller and is required to form the binding cleft for IFT46, has no visible electron density in case of the human ODA16 structure. Furthermore, size exclusion chromatography and pull‐down experiments failed to detect a direct interaction between human ODA16 and IFT46. These data suggest that additional factors may be required for the ciliary import of ODAs in human cells with motile cilia.     

Carbon and nitrogen content based estimation of the fat content of animal carcasses in various species

The objective of this study was to explore whether the C and N content can be used to estimate the fat content of animal carcasses. Considering the mean C and N contents of body fat and body protein, the fat content (EE) [%] can be predicted from C and N values [%] according to the generally valid equation EE=1.3038·C – 4.237·N. The application of this equation to estimate the total fat content of all animal carcasses results in significant differences in fat content between predicted and measured values. Therefore, we derived specific equations for rats, pigs, cattle, sheep, broilers and mice to predict the fat content by dual linear regression analysis (y=EE [% DM], x₁=C [% DM], x₂=N [% DM]) based on measured fat, C, and N contents of animal body samples. The specific equations for different animals showed residual standard deviations of 1.55, 1.63, 1.12, 1.35, 1.85 and 0.92% fat for rats, pigs, cattle, sheep, broilers and mice, respectively.