Chemical modifications of actin: effects on interaction with deoxyribonuclease I

Maleylation of lysine residues, nitration of tyrosine residues or modification with 2,3-butanedione or 1,2-cyclohexanedione of arginine residues on actin resulted in a loss of polymerizability of the modified actin. However, only lysine modification produced a complete loss of the deoxyribunuclease I inhibitory ability of actin at low degrees of modification. By the level of one modified lysine per actin monomer, the samples completely lost polymerizability and lost 65% of their inhibitory power against deoxyribonuclease I-catalysed hydrolysis of DNA. By two lysines modified per actin, all inhibitory activity was lost. One lysine residue on actin apparently overlaps both an actin action contact site and an actin-deoxyribnuclease 1 contact site, offering a suggestion as to how deoxyribonuclease I blocks actin polymerization.     

Urinary excretion of a novel hexasaccharide and glycopeptide analogue in fucosidosis.

Repeated Biogel P6 chromatography of the urine from a patient with fucosidosis yielded several fractions containing fucosyloligosaccharides and glycopeptides. Two of these were shown by ¹H nuclear magnetic resonance (¹H-n.m.r.) spectroscopy and permethylation analysis to have the following structures respectively: (I) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) glcNAc and (II) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) βglcNAc (1→4) [αfuc ($\text{1→}\text{3}\text{6}$)] βglcNAc-Asn.     

A spectrofluorimetric investigation of calf thymus DNA modified by BP diolepoxide and 1-pyrenyloxirane

7β,8α-Dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP diolepoxide, $\text{1}$) and 1-pyrenyloxirane ($\text{2}$) bind chemically to calf thymus DNA. The fluorescence efficiency of pyrenyl groups in mutagen modified DNA varies appreciably with its conformation and decreases in the order: pyrenees, modified denatured DNA and modified native DNA. A particularly interesting observation is that the fluorescence efficiency of mutagen modified DNA intensifies substantially upon denaturation. Our results suggest that the pyrenyl groups in mutagen modified DNA are intercalated between the base pairs of DNA. Since both $\text{1}$ and $\text{2}$ are powerful frame-shifting mutagens for S. typhimurium TA-98, the intercalative covalent binding of these compounds to DNA may provide a molecular basis for their mutagenic activity.     

Halogenated monoterpenes of the red alga Microcladia

The red marine algae Microcladia borealis, M. californica and M. coulteri produce several unusual halogenated monoterpenes including violacene, plocamene-B, plocamene-C, and plocamane-D. The isolation of these terpenes along with a study of their variation in each Microcladia at different locations are described.     

Death of Staphylococcus aureus in Liquid Whole Egg Near pH 8

Incubating and shaking Staphylococcus aureus in liquid whole egg causes a decline in viability. During the period of agitation, the natural pH of the egg rises from about 7.2 to between 8.0 and 8.2 as a result of a loss of carbon dioxide. However, if the pH of the egg is prevented from rising, either by not shaking or by addition of a buffer, S. aureus will grow. The cause of death is traced to the presence of lysozyme of egg white. Interestingly, the action of lysozyme is not attributable to its bacterial lytic property but, instead, to the basicity of the lysozyme molecule. This conclusion is supported by the fact that the lytic property of lysozyme is known to have its optimal activity near neutrality and by the finding that protamine sulfate, a nonenzymatic basic polypeptide, also caused death of S. aureus at pH 8.0 but not at 7.0. It was postulated that the rise in pH renders the bacterial cells more negatively charged, so that in the presence of positively charged molecules like lysozyme or protamine sulfate a complex is formed, agglutinating the cells.     

Purification and regulatory properties of pyruvate kinase from Veillonella parvula.

The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations o-f phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg-2+ ions with S0.5 values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 m PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg-2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (Ki = 3.4 mM), and this inhibition was only slightly affected by increasing concentration of Mg-2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP greater than fructose-1,6-P2 greater than dihydroxyacetone-P greater than glucose-6-P greater than fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was significantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M4 plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP.     

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 μg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 μg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.     

Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.     

Ex vivo expansion of umbilical cord blood

The efficacy of cord blood (CB) transplantation is limited by the low cell dose available. Low cell doses at transplant are correlated with delayed engraftment, prolonged neutropenia and thrombocytopenia and elevated risk of graft failure. To potentially improve the efficacy of CB transplantation, approaches have been taken to increase the cell dose available. One approach is the transplantation of multiple cord units, another the use of ex vivo expansion. Evidence for a functional and phenotypic heterogeneity exists within the HSC population and one concern associated with ex vivo expansion is that the expansion of lower 'quality' hematopoietic progenitor cells (HPC) occurs at the expense of higher 'quality' HPC, thereby impacting the reserve of the graft. There is evidence that this is a valid concern while other evidence suggests that higher quality HPC are preserved and not exhausted. Currently, ex vivo expansion processes include: (1) liquid expansion: CD34+ or CD133+ cells are selected and cultured in medium containing factors targeting the proliferation and self-renewal of primitive hematopoietic progenitors; (2) co-culture expansion: unmanipulated CB cells are cultured with stromal components of the hematopoietic microenvironment, specifically mesenchymal stem cells (MSC), in medium containing growth factors; and (3) continuous perfusion: CB HPC are cultured with growth factors in 'bioreactors' rather than in static cultures. These approaches are discussed. Ultimately, the goal of ex vivo expansion is to increase the available dose of the CB cells responsible for successful engraftment, thereby reducing the time to engraftment and reducing the risk of graft failure.