Conserved DNA Motifs,Including the CENP-B Box-like,Are Possible Promoters of Satellite DNA Array Rearrangements in Nematodes

Tandemly arrayed non-coding sequences or satellite DNAs (satDNAs) are rapidly evolving segments of eukaryotic genomes, including the centromere, and may raise a genetic barrier that leads to speciation. However, determinants and mechanisms of satDNA sequence dynamics are only partially understood. Sequence analyses of a library of five satDNAs common to the root-knot nematodes Meloidogyne chitwoodi and M. fallax together with a satDNA, which is specific for M. chitwoodi only revealed low sequence identity (32–64%) among them. However, despite sequence differences, two conserved motifs were recovered. One of them turned out to be highly similar to the CENP-B box of human alpha satDNA, identical in 10–12 out of 17 nucleotides. In addition, organization of nematode satDNAs was comparable to that found in alpha satDNA of human and primates, characterized by monomers concurrently arranged in simple and higher-order repeat (HOR) arrays. In contrast to alpha satDNA, phylogenetic clustering of nematode satDNA monomers extracted either from simple or from HOR array indicated frequent shuffling between these two organizational forms. Comparison of homogeneous simple arrays and complex HORs composed of different satDNAs, enabled, for the first time, the identification of conserved motifs as obligatory components of monomer junctions. This observation highlights the role of short motifs in rearrangements, even among highly divergent sequences. Two mechanisms are proposed to be involved in this process, i.e., putative transposition-related cut-and-paste insertions and/or illegitimate recombination. Possibility for involvement of the nematode CENP-B box-like sequence in the transposition-related mechanism and together with previously established similarity of the human CENP-B protein and pogo-like transposases implicate a novel role of the CENP-B box and related sequence motifs in addition to the known function in centromere protein binding.     

Inborn errors of complex II--unusual human mitochondrial diseases.

The succinate dehydrogenase consists of only four subunits, all nuclearly encoded, and is part of both the respiratory chain and the Krebs cycle. Mutations in the four genes encoding the subunits of the mitochondrial respiratory chain succinate dehydrogenase have been recently reported in human and shown to be associated with a wide spectrum of clinical presentations. Although a comparatively rare deficiency in human, molecularly defined succinate dehydrogenase deficiency has already been found to cause encephalomyopathy in childhood, optic atrophy or tumor in adulthood. Because none of the typical housekeeping genes encoding this respiratory chain complex is known to present tissue-specific isoforms, the tissue-specific involvement represents a quite intriguing question, which is mostly addressed in this review. A differential impairment of electron flow through the respiratory chain, handling of oxygen, and/or metabolic blockade possibly associated with defects in the different subunits that can be advocated to account for tissue-specific involvement is discussed.     

Archaeal complex II: 'classical' and 'non-classical' succinate:quinone reductases with unusual features.

Reversible succinate dehydrogenase (SDH) activities have been ubiquitously detected in organisms from the three domains of life. They represent constituents either of respiratory complexes II in aerobes, or of fumarate dehydrogenase complexes in anaerobes. The present review gives a survey on archaeal succinate:quinone oxidoreductases (SQRs) analyzed so far. Though some of these could be studied in detail enzymologically and spectroscopically, the existence of others has been deduced only from published genome sequences. Interestingly, two groups of enzyme complexes can be distinguished in Archaea. One group resembles the properties of SDHs known from bacteria and mitochondria. The other represents a novel class with an unusual iron-sulfur cluster in subunit B and atypical sequence motifs in subunit C which may influence electron transport mechanisms and pathways. This novel class of SQRs is discussed in comparison to the so-called 'classical' complexes. A phylogenetic analysis is presented suggesting a co-evolution of the flavoprotein-binding subunit A and subunit B containing the three iron-sulfur clusters.     

Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.     

Effect of lidocaine and pyromecaine on the size of an experimental myocardial infarct

The experiments on rats with a 3-day myocardial infarction caused by the left coronary artery ligation have shown that multiple lidocaine and pyromecaine injections according to a given scheme decrease the size of the necrosis area. Drug effects were not related to their action on the blood supply of the ischemic area.     

Reasons for disorders of fatty acid oxidation in isolated heart mitochondria during ischemia

The influence of malate and cytochrome c on fatty acid oxidation under control and ischemic conditions was investigated. In the medium without malate, cytochrome did not make fatty acid oxidation decreased during ischemia return to normal. Oxidation in the media containing malate and cytochrome did not differ from control only when it was measured after preliminary oxidation of endogenous substrates. The ratio of palmitoyl-CoA and palmitoyl carnitine to the respiration rates at state 3 was unchanged at 60 min ischemia. Apparently, no changes in carnitine acyltransferase playing a role in oxidation of palmitoyl-CoA took place. Thus, the decrease of fatty acid oxidation at early periods of ischemia is largely caused by a reduction in the content of cytochrome c and intermediates of Krebs cycle in the mitochondria.     

Diel emigration and colonization responses of blackfly larvae (Diptera: Simuliidae) to ultraviolet radiation

1. Total counts of blackfly larvae densities over 30- and 57-h periods in experimental channels during May of 1996 & 1997 indicate that ultraviolet radiation (UV; 290–400 nm) may be important in stimulating emigration.
2. Under experimentally controlled solar UV exposure, larval densities at dawn in UV-shielded channels were 161% and 168% higher than in the UV-exposed channels. Larval densities in UV-exposed channels then decreased by 68.2% and 81.1% between dawn and early afternoon of the two days; density decreases in UV-shielded channels were slight, and not statistically significant, during the same periods.
3. Larvae within UV-exposed channels occupied shaded microhabitats during hours of intense solar radiation, suggesting that simuliid larvae can detect and respond to UV radiation over very short periods of time.
4. A cyclical pattern of UV-induced emigration during hours of increasing solar flux (06.30–13.30) and net immigration in the hours of decreasing solar flux and at night emerged. Thus stream invertebrates may be very sensitive to environmental changes, resulting in either increased UV flux or decreased shading of streams. Diel cycles in invertebrate densities should be taken into account in research designs and sampling protocols in order to identify and interpret correctly results of both periodic surveys and experiments.     

The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K–adenosine triphosphatase (ATPase) α subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane’s electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis α1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain–sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 µM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump–induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the binding pocket. Gating measurements of palytoxin-opened Na/K pump channels additionally imply that the C-terminal contacts also help stabilize pump conformations with occluded K ions.     

Purification and various properties of asialoglycoprotein receptors from the mouse liver

The receptor for asialoglycoproteins was isolated from murine liver and was purified by means of biospecific chromatography on sepharose-Asialo-orosomucoid. The obtained receptor with an absorption maximum at 277 nm binds to the nonreducing terminal galactosyl residues of glycoproteins similar to the receptors from liver of other mammalians. The interaction between this receptor and desialylated glycoproteins requires the presence of calcium. The dependence of specific binding on the concentration of [125I]acialo-orosomucoid used as a ligand gives a saturating curve. The dissociation constant for the receptor-ligand complex is 0.4 X 10(-9) M. Similar to asialo-orosomucoid, the receptor binds the p-aminophenyl-beta-D-galactopyranoside derivatives of bovine serum albumin, ovalbumin and acid alpha-glucosidase synthesized by us earlier. Possible use of the asialoglycoprotein receptor as a highly specific carrier transporting the modified acid alpha-glucosidase to hepatocyte lysosomes is discussed.