Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

Plasmodium cynomolgi: serum-mediated blocking and enhancement of infectivity to mosquitoes during infections in the natural host, Macaca sinica

The infectivity of Plasmodium cynomolgi in its natural host, the toque monkey, Macaca sinica, to Anopheles tessellatus mosquitoes was studied in relation to the evolution of anti-sexual-stage immunity in the host during the course of a blood-induced infection. The effects of serum on the infectivity of gametocytes and the intrinsic infectivity of gametocytes to mosquitoes on each day were assessed in membrane feeding experiments. Mosquitoes were also directly fed on the animal on each day. Our results demonstrate that during the very early patent period, before the peak of gametocytemia, the infection serum enhanced the infectivity of gametocytes up to two to three times above their infectivity in normal monkey serum. Subsequently, serum drawn post-peak of parasitemia ceased to enhance, and began to suppress, infectivity. After 2-3 months, long after parasitemias ceased patency, the serum no longer suppressed and between 3 and 4 months the serum again tended to enhance gamete infectivity before losing any significant effect. Serum infectivity enhancing effects were consistent with low indirect immunofluorescence test antibody titers against blood stage parasites first during the very early days of a blood infection before reaching blocking levels, and again during convalescence when antibodies were declining. The serum infectivity blocking effects on gametocytes were seen at the peak of antibody titers from about Days 9 to 23 of an infection. From 78 to 95% of the total infectivity of the parasite to mosquitoes during an infection occurred when infectivity enhancing activity was present in the serum. Hence, the infectivity of the parasite to mosquitoes was largely dependent on infectivity enhancing antibodies in host serum.     

Human immune responses against sexual stages of malaria parasites: considerations for malaria vaccines

Studies on the natural immune responses to the sexual stages of malaria parasites have been reviewed in the context of human malaria transmission-blocking vaccines. Antibodies against the sexual stages of the malaria parasite, gametocytes and gametes, are readily evoked by natural malaria infections. These antibodies that suppress infectivity at high concentrations can, at low concentrations, enhance the development of the parasite in the mosquito; however, because enhancing antibodies are prevalent during natural malaria infections, it is likely that a vaccine would rapidly boost these antibodies to blocking levels. The immunogenicity of sexual stage antigens appears to be constrained in the human host, probably due to T epitope polymorphism and MHC restriction in humans. These constraints apply mainly to those antigens that are sensitive targets of host immunity such as the gamete surface antigens and not to internal gamete antigens, indicating that antigenic polymorphism may have evolved in response to immune selection pressure. Evidence for immunosuppression of the host by exposure to endemic malaria is presented and its consequences on vaccine development are discussed.     

Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems

Low molecular weight peptides obtained from ultrafiltration (UF) of giant squid (Dosidicus gigas) muscle protein were studied for their antioxidative effects in different in vitro oxidative systems. The most potent two peptides, Asn-Ala-Asp-Phe-Gly-Leu-Asn-Gly-Leu-Glu-Gly-Leu-Ala (1307 Da) and Asn-Gly-Leu-Glu-Gly-Leu-Lys (747 Da), exhibited their antioxidant potential to act as chain-breaking antioxidants by inhibiting radical-mediated peroxidation of linoleic acid, and their activities were closer to highly active synthetic antioxidant, butylated hydroxytoluene. Addition of these peptides could enhance the viability of cytotoxic embryonic lung fibroblasts significantly (P<.05) at a low concentration of 50 microg/ml, and it was presumed due to the suppression of radical-induced oxidation of membrane lipids. Electron spin trapping studies revealed that the peptides were potent scavengers of free radicals in the order of carbon-centered (IC(50) 396.04 and 304.67 microM), hydroxyl (IC(50) 497.32 and 428.54 microM) and superoxide radicals (IC(50) 669.34 and 573.83 microM). Even though the exact molecular mechanism for scavenging of free radicals was unclear, unusually high hydrophobic amino acid composition (more than 75%) of giant squid muscle peptides was presumed to be involved in the observed activities.     

A novel anticoagulant purified from fish protein hydrolysate inhibits factor XIIa and platelet aggregation

A novel fish protein having anticoagulant and antiplatelet properties was enzymatically extracted from the marine fish, yellowfin sole (Limanda aspera) and purified to homogeneity producing an overall purification fold of 206.6. MALDI-TOF mass spectroscopic and SDS-PAGE analysis identified the purified protein as 12.01 kDa single-chain monomeric protein. It inhibited the activated coagulation factor XII (FXIIa) by forming an inactive complex regardless of Zn2+ mediation, and was named, yellowfin sole anticoagulant protein (YAP). In addition, YAP act to antagonize platelet membrane glycoprotein integrin, to arrest platelet aggregation. However, YAP was not able to block the adhesion of platelets to collagen, which mediate via major collagen receptors, GPIa/IIa on platelet membrane. Furthermore, YAP did not possess plasminogen activator-like activity to activate fibrinolysis. In fact, our findings indicate that YAP binds with FXIIa and platelet membrane integrins to inhibit thrombosis in vitro.     

Statistical properties of the variation at linked microsatellite loci: implications for the history of human Y chromosomes [published erratum appears in Mol Biol Evol 1997 Mar;14(3):354]

It has recently been suggested that observed levels of variation at microsatellite loci can be used to infer patterns of selection in genomes and to assess demographic history. In order to evaluate the feasibility of these suggestions it is necessary to know something about how levels of variation at microsatellite loci are expected to fluctuate due simply to stochasticity in the processes of mutation and inheritance (genetic sampling). Here we use recently derived properties of the stepwise mutation model to place confidence intervals around the variance in repeat score that is expected at mutation-drift equilibrium and outline a statistical test for whether an observed value differs significantly from expectation. We also develop confidence intervals for the time course of the buildup of variation following a complete elimination of variation, such as might be caused by a selective sweep or an extreme population bottleneck. We apply these methods to the variation observed at human Y-specific microsatellites. Although a number of authors have suggested the possibility of a very recent sweep, our analyses suggest that a sweep or extreme bottleneck is unlikely to have occurred anytime during the last approximately 74,000 years. To generate this result we use a recently estimated mutation rate for microsatellite loci of 5.6 x 10(-4) along with the variation observed at autosomal microsatellite loci to estimate the human effective population size. This estimate is 18,000, implying an effective number of 4,500 Y chromosomes. One important general conclusion to emerge from this study is that in order to reject mutation-drift equilibrium at a set of linked microsatellite loci it is necessary to have an unreasonably large number of loci unless the observed variance is far below that expected at mutation-drift equilibrium.      

The small leucine-rich repeat proteoglycan biglycan binds to alpha-dystroglycan and is upregulated in dystrophic muscle

The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface. The alpha-/beta-dystroglycan subcomplex of the DAPC forms a critical link between the cytoskeleton and the extracellular matrix. A ligand blot overlay assay was used to search for novel dystroglycan binding partners in postsynaptic membranes from Torpedo electric organ. An approximately 125-kD dystroglycan-binding polypeptide was purified and shown by peptide microsequencing to be the Torpedo ortholog of the small leucine-rich repeat chondroitin sulfate proteoglycan biglycan. Biglycan binding to alpha-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant alpha-dystroglycan. The biglycan binding site was mapped to the COOH-terminal third of alpha-dystroglycan. Glycosylation of alpha-dystroglycan is not necessary for this interaction, but binding is dependent upon the chondroitin sulfate side chains of biglycan. In muscle, biglycan is detected at both synaptic and nonsynaptic regions. Finally, biglycan expression is elevated in muscle from the dystrophic mdx mouse. These findings reveal a novel binding partner for alpha-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix. These results also raise the possibility of a role for biglycan in the pathogenesis, and perhaps the treatment, of muscular dystrophy.     

Sorting out the Snakebirds: The species status,phylogeny, and biogeography of the Darters (Aves: Anhingidae)

Although the avian family Anhingidae is unequivocally monophyletic, the number and relationships of the component species within the single genus (Anhinga) have long remained unclear. Here, we use extensive mitochondrial and nuclear DNA sequence data (8,878 bp) to show that four species should be recognized. Our fully resolved and well‐supported tree shows that the American Anhinga (Anhinga anhinga) is sister to the three Old World species, with the Oriental (A. melanogaster) and African (A. rufa) Darters sister within the Old World clade, which also includes the Australian Darter (A. novaehollandiae). We estimate that the divergence between the New World and Old World branches occurred 19–22 mya, with the Australian Darter separating from its Old World congeners 14–16 mya and the Oriental and African species splitting ~10 mya. The genus is yet another example of osteological conservatism in the Suliformes, which is comparable to that shown by the cormorants and shags. Nevertheless, the relationships we infer are congruent with recent plumage studies and are biogeographically plausible. We suggest that further investigation of the variation within the African and Australian Darters would be of interest.     

Short-Term and Long-Term Survival and Virulence of Legionella pneumophila in the Defined Freshwater Medium Fraquil

Legionella pneumophila (Lp) is the etiological agent responsible for Legionnaires’ disease, a potentially fatal pulmonary infection. Lp lives and multiplies inside protozoa in a variety of natural and man-made water systems prior to human infection. Fraquil, a defined freshwater medium, was used as a highly reproducible medium to study the behaviour of Lp in water. Adopting a reductionist approach, Fraquil was used to study the impact of temperature, pH and trace metal levels on the survival and subsequent intracellular multiplication of Lp in Acanthamoeba castellanii, a freshwater protozoan and a natural host of Legionella. We show that temperature has a significant impact on the short- and long-term survival of Lp, but that the bacterium retains intracellular multiplication potential for over six months in Fraquil. Moreover, incubation in Fraquil at pH 4.0 resulted in a rapid decline in colony forming units, but was not detrimental to intracellular multiplication. In contrast, variations in trace metal concentrations had no impact on either survival or intracellular multiplication in amoeba. Our data show that Lp is a resilient bacterium in the water environment, remaining infectious to host cells after six months under the nutrient-deprived conditions of Fraquil.