Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

A Pleistocene Clone of Palmer's Oak Persisting in Southern California

Background

The distribution of Palmer''s oak (Quercus palmeri Engelm.) includes numerous isolated populations that are presumably relicts of a formerly larger range that has contracted due to spreading aridity following the end of the Pleistocene.

Principal Findings

We investigated a recently discovered disjunct population of Palmer''s oak in the Jurupa Mountains of Riverside County, California. Patterns of allozyme polymorphism, morphological homogeneity, widespread fruit abortion, and evidence of fire resprouting all strongly support the hypothesis that the population is a single clone. The size of the clone and estimates of annual growth from multiple populations lead us to conclude that the clone is in excess of 13,000 years old.

Conclusions

The ancient age of the clone implies it originated during the Pleistocene and is a relict of a vanished vegetation community. Range contraction after climate change best explains the modern disjunct distribution of Q. palmeri and perhaps other plants in California.     

Myosin5a tail associates directly with Rab3A-containing compartments in neurons

Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation velocity analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mm GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotide-free Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles.     

Global health policies that support the use of banked donor human milk: a human rights issue

Background

The past ten years have witnessed a rising trend in the prevalence and duration of breastfeeding in Italy, but breastfeeding rates increase in an unequal way; they are higher in the North of Italy than in the South. The purpose of this study was to describe the experiences, expectations and beliefs of a sample of mothers, and to identify differences, if any, between the North and the South of Italy.

Methods

The study was conducted in two regions of Italy, Friuli Venezia Giulia in the Northeast and Basilicata in the South. Two hundred and seventy-nine mothers of infants and children 6 to 23 months of age were interviewed using an 85-item questionnaire including closed and open questions on infant feeding experiences and beliefs, sources of information and support, reasons for intended and actual choices and practices, and some demographic and social variables. Face-to-face interviews were conducted between May 2001 and September 2002. Quantitative and qualitative methods were used for data analysis.

Results

The distribution of the mothers by age, education, employment and parity did not differ from that of the general population of the two regions. The reported rates of initiation and duration of breastfeeding were also similar: 95% started breastfeeding, exclusive breastfeeding was 32% at three and 9% at six months, with 64% and 35% of any breastfeeding, respectively. Some differences were reported in the rates of full breastfeeding, reflecting different ages of introduction of non-nutritive fluids. These, as well as nutritive fluids – including infant formula – and complementary foods, were introduced far too early. Advice on infant feeding was generally provided by health professionals and often was not based on up-to-date recommendations. Mothers were generally aware of the advantages of breastfeeding, but at the same time reported problems that they were not able to solve alone or through social and health system support. Most mothers would welcome the support of a peer counsellor. More mothers in Basilicata than in Friuli Venezia Giulia reported difficulties with breastfeeding related to returning to work and were not familiar with their rights on breastfeeding and maternity leave.

Conclusion

Programmes for the protection, promotion and support of breastfeeding in these and similar regions of Italy should concentrate on better training of health professionals with regards to lactation management, communication, and counselling skills. The addition of trained peer counsellors could reinforce the work done by the health system and, through community involvement, could help change social prejudice in the mid- and long-term. The differences between regions should be taken into account in formulating these programmes to avoid increasing, and possibly to decrease, the current gaps.     

Melanophilin, the product of the leaden locus, is required for targeting of myosin-Va to melanosomes

The formation of complex subcellular organelles requires the coordinated targeting of multiple components. Melanosome biogenesis in mouse melanocytes is an excellent model system for studying the coordinated function of multiple gene products in intracellular trafficking. To begin to order events in melanosome biogenesis and distribution, we employed the classical coat-color mutants ashen , dilute , and leaden , which affect melanosome distribution, but not melanin synthesis. The loci have been renamed Rab27a , Myo5a , and Mlph for their gene products. While each of the three loci has been shown to be required for melanosome distribution, the point(s) at which each acts is unknown. We have utilized primary melanocytes to examine the interdependencies between rab27a, myosin-Va, and melanophilin. The localization of rab27a to melanosomes did not require the function of either myosin-Va or melanophilin, but leaden function was required for the association of myosin-Va with melanosomes. In leaden melanocytes permeabilized before fixation, myosin-Va immunoreactivity was greatly attenuated, suggesting that myosin-Va is free in the cytoplasm. Finally, we have complemented both the leaden and ashen phenotypes by cell fusion and observed redistribution of mature melanosomes in the absence of both protein and melanin synthesis. Together, our data suggest a model for the initial assembly of the machinery required for melanosome distribution.