Analysis of chromosome conservation in <Emphasis Type="Italic">Lemur catta</Emphasis> studied by chromosome paints and BAC/PAC probes

A panel of human chromosome painting probes and bacterial and P1 artificial chromosome (BAC/PAC) clones were used in fluorescence in situ hybridization (FISH) experiments to investigate the chromosome conservation of the ring-tailed lemur (Lemur catta, LCA) with respect to human. Whole chromosome paints specific for human chromosomes 7, 9, 11, 13, 14, 17, 18, 20, 21, and X were found to identify a single chromosome or an uninterrupted chromosomal region in LCA. A large set of partial chromosome paints and BAC/PAC probes were then used to refine the characterization of the rearrangements differentiating the two karyotypes. The results were also used to reconstruct the ancestral Lemuridae karyotype. Lemur catta, indeed, can be used as an outgroup, allowing symplesiomorphic (ancestral) rearrangements to be distinguished from apomorphic (derived) rearrangements in lemurs. Some LCA chromosomes are difficult to distinguish morphologically. The 'anchorage' of most LCA chromosomes to specific probes will contribute to the standardization of the karyotype of this species.     

Evolutionary history of chromosome 10 in primates

We have tracked the evolutionary history of chromosomes homologous to HSA10 (PHYL-10) in primates using appropriate panels of PCP, YAC, and BAC probes. This approach allowed us to delineate more precisely the PHYL-10 constitution in the ancestor of catarrhine, platyrrhine, and prosimians. The results suggest that (i) in the ancestor of prosimians PHYL-10 was organized in two separate PHYL-10p and PHYL-10q chromosomes; (ii) in the progenitor of New World monkeys PHYL-10p was a separate chromosome, while PHYL-10q was associated with a chromosome homologous to HSA16; (iii) in the ancestor of Old World monkeys PHYL-10 was a unique chromosome with a marker order corresponding to the orang form. We have also analyzed the cat, chosen as an outgroup for its very conserved karyotype. In agreement with published data our experiments show that the PHYL-10 in cat is structured in two blocks, PHYL-10p and PHYL-10q, both as part of larger chromosomes. The overall data indicate that, contrary to common opinion, PHYL-10p and PHYL-10q were distinct chromosomes in the primate ancestor. Analysis of the Saimiri sciureus (SSC) PHYL-10q marker order showed that it was isosequential with the Callithrix jacchus PHYL-10q, as well as with the PHYL-10q platyrrhine ancestral form. The SSC centromere, nevertheless, was located in a different chromosomal region, therefore suggesting that a centromeric repositioning event occurred in this species.     

Protein synthesis in the visual cortex is needed for ocular dominance plasticity

Experience-dependent remodelling of neural connections progresses through stages, and early phases eventually give way to later long-lasting ones. The transition from early to late stages, often associated with structural changes, depends on protein synthesis. Suppression of cortical but not geniculate protein synthesis blocks ocular dominance plasticity at its earliest stage, suggesting that structural changes occur rapidly in the visual cortex following monocular deprivation.     

Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis

Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.     

Relaxation kinetics following sudden Ca(2+) reduction in single myofibrils from skeletal muscle

To investigate the roles of cross-bridge dissociation and cross-bridge-induced thin filament activation in the time course of muscle relaxation, we initiated force relaxation in single myofibrils from skeletal muscles by rapidly (approximately 10 ms) switching from high to low [Ca(2+)] solutions. Full force decay from maximal activation occurs in two phases: a slow one followed by a rapid one. The latter is initiated by sarcomere "give" and dominated by inter-sarcomere dynamics (see the companion paper, Stehle, R., M. Krueger, and G. Pfitzer. 2002. Biophys. J. 83:2152-2161), while the former occurs under nearly isometric conditions and is sensitive to mechanical perturbations. Decreasing the Ca(2+)-activated force preceding the start of relaxation does not increase the rate of the slow isometric phase, suggesting that cycling force-generating cross-bridges do not significantly sustain activation during relaxation. This conclusion is strengthened by the finding that the rate of isometric relaxation from maximum force to any given Ca(2+)-activated force level is similar to that of Ca(2+)-activation from rest to that given force. It is likely, therefore, that the slow rate of force decay in full relaxation simply reflects the rate at which cross-bridges leave force-generating states. Because increasing [P(i)] accelerates relaxation while increasing [MgADP] slows relaxation, both forward and backward transitions of cross-bridges from force-generating to non-force-generating states contribute to muscle relaxation.     

Evidence that phosphate release is the rate-limiting step on the overall ATPase of psoas myofibrils prevented from shortening by chemical cross-linking

It has been suggested that the mechanical condition determines the rate-limiting step of the ATPase of the myosin heads in fibers: when fibers are isometrically contracting, the ADP release kinetics are rate-limiting, but as the strain is reduced and the fibers are allowed to shorten, the ADP release kinetics accelerate and P(i) release becomes rate-limiting. We have put this idea to the test with myofibrils as a model because with these both mechanical and chemical kinetic measurements are possible. With relaxed or rapidly shortening myofibrils, P(i) release is rate-limiting and (A)M.ADP.P(i) states accumulate in the steady state [Lionne, C., et al. (1995) FEBS Lett. 364, 59]. We have now studied the kinetics of P(i) release with chemically cross-linked myofibrils that, when adequately cross-linked, appear to be a good model for isometric contraction. By using a method that is specific for free P(i) and rapid quench flow that measures the amount of (A)M.ADP.P(i) states and free P(i), we show that (A)M.ADP.P(i) states predominate which suggests that the overall ATPase is limited by P(i) release kinetics. Therefore, under our experimental conditions with myofibrils prevented from shortening, the concentration of (A)M.ADP states is low, as with rapidly shortening and relaxed myofibrils. This result is difficult to reconcile with the sensitivity of force development in fibers and myofibrils to P(i) which implies interaction of P(i) with an (A)M.ADP state. We discuss two models for accommodating the mechanical and chemical kinetics with reference to the duty cycle in skeletal muscle.     

Evolution of chromosome Y in primates

We have investigated, by fluorescence in situ hybridization (FISH), the cytogenetic evolution of the Y chromosome in primates using 17 yeast artificial chromosomes, representative of the Y-specific euchromatic region of the human chromosome Y. The FISH experiments were performed on great apes (Homo sapiens, Pan troglodytes, Gorilla gorilla and Pongo pygmaeus pygmaeus), and on two Old World monkeys species as an outgroup (Cercopitecidae Macaca fascicularis and Papio anubis). The results showed that this peculiar chromosome has undergone rapid and unconstrained evolution both in sequence content and organization. Received: 16 January 1998; in revised form: 29 May 1998 / Accepted: 24 June 1998     

New 4H-chromen-4-one and 2H-chromene derivatives as anti-picornavirus capsid-binders

Substituted (E)-3-styryl-4H-chromen-4-ones 1a–d, 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-4H-chromen-4-ones 2a–d, (E)-3-styryl-2H-chromenes 3a–d and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chromenes 4a–d were designed and synthesized to improve the anti-picornavirus activity of previously tested analogues. The new compounds were evaluated in vitro against human rhinovirus (HRV) serotypes 1B and 14 and enterovirus (EV) 71. All the compounds interfered with the replication of picornaviruses, although considerable differences were observed in the sensitivity of viruses to each compound. Generally, both HRVs were more susceptible than EV71 and their sensitivity was dependent upon the linker chain length as well as upon the oxidation state of the heterocyclic ring. (E)-3-Styryl-2H-chromene (3a) emerged as the most effective inhibitor of both HRVs showing IC₅₀ values of 0.20 μM and 1.38 μM towards serotype 1B and 14, respectively. The potent activity was also coupled with low cytotoxicity resulting in high therapeutic indexes (250 and 36, respectively). Mechanism of action studies indicated that 3a, like structurally related compounds, behaves as a capsid binder interfering with the early stages of rhinovirus infection, probably at the adsorption and/or uncoating level.