Semen cryopreservation in golden‐headed lion tamarin,Leontopithecus chrysomelas

Wild animal genetic resource banking (GRB) represents a valuable tool in conservation breeding programs, particularly in cases involving endangered species such as the golden‐headed lion tamarin (Leontopithecus chrysomelas). Thus, we aimed to assess a sperm freezing protocol for golden‐headed lion tamarins using two different exenders: BotuBOV® (BB) and Test Yolk Buffer® (TYB). Ejaculates were collected by penile vibrostimulation from animals housed at São Paulo Zoological Park Foundation, São Paulo, Brazil, and after immediate analysis, two aliquots were diluted in BB and TYB. Postthawing samples were evaluated for total and progressive motility, plasma membrane and acrosome integrities, mitochondrial activity, susceptibility to oxidative stress, and sperm–egg‐binding. No differences between BB and TYB were found for most seminal parameters, except for acrosome integrity and susceptibility to oxidative stress (in both cases BB showed higher values). However, in spite of these differences and regardless of the extender used, postthaw sperm motility and viability with the described protocol were encouraging (on average >50% and >80%, respectively), indicating that sperm cryopreservation may be a short‐term measure for the conservation of golden‐headed lion tamarins.     

The control of flower initiation by gibberellin in Helianthus annuus L. (sunflower), a non-photoperiodic plant

The involvement of gibberellins in the control of flowering of sunflower was studied by direct application of GA₃ to the apex of the plants, analysis of the endogenous levels of gibberellin-like substances at different plant ages, and indirectly by the application of paclobutrazol, an inhibitor of gibberellin synthesis. GA₃ speeded-up flower initiation and floral apex development. The time of GA₃ application was more critical than the amount of GA₃ applied. The endogenous levels of gibberellin-like compounds increased significantly by day 15 after sowing. The application of paclobutrazol markedly delayed floral initiation and this effect was also depedent on plant age. Both GA₃ and paclobutrazol had their greatest effects between 10 and 20 days after sowing suggesting that an increase in gibberellins in that time period plays a role in floral initiation.     

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

In Silico Insights into Protein-Protein Interactions and Folding Dynamics of the Saposin-Like Domain of Solanum tuberosum Aspartic Protease

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.     

Experimental infections of simians with human malaria: attempts to infect Callithrix penicillata with Plasmodium falciparum

After reviewing the use of non-human primates of the Old and New Worlds for human malaria research, we concluded that another experimental animal which is easily available to use and possible to rear indoors is needed. Thus, we studied the susceptibility of the marmoset Callithrix penicillata to Plasmodium falciparum erythrocytic infections. The marmosets received various P. falciparum human isolates, directly from a patient and from continuous cultures. The Palo Alto strain, which has been adapted to the night monkey Aotus trivirgatus and further maintained in the squirrel monkey Saimiri sciureus was also used. In a total of 20 marmosets we performed 31 inoculations, with 10(5) to 10(9) parasites, intraperitoneally, intracardiacly or intravenously. Blood samples from each animal were examined daily up to day 90 post-inoculation. None of the intact marmosets developed patent infections. Four out of 19 C. penicillata, previously splenectomized, showed circulating parasites for up to five days after intravenous inoculation with the Palo Alto strain, becoming negative thereafter. Neither the addition to the simian diet of p-aminobenzoic acid, essential for the parasite metabolism, nor drug-immunosuppression, improved the marmoset susceptibility to P. falciparum.     

Arterial effects of aldosterone and antimineralocorticoid compounds mechanism of action

The aim of our work was to study the mechanism of action of aldosterone and antialdosterone compounds on Na+ and K+ fluxes in vascular smooth muscle. In the long term, regulation of salt metabolism depends on aldosterone effects on Na+, K+, H+ and H2O transport by the renal tubules. Furthermore, it has been shown that aldosterone modifies several epithelial transports, inducing a positive sodium balance. The chronic in vivo administration of aldosterone modifies transmembrane ionic fluxes in vascular smooth muscle. Garwitz and Jones suggested that aldosterone may enhance net Na+ transport through the stimulation of the sodium pump. The results obtained in our laboratory indicate that aldosterone has a direct stimulatory action on ouabain-dependent and on ouabain-independent Na efflux. Furthermore, the mineralocorticoid enhances passive K permeability, as well as the Na pump dependent K influx. Both effects are blocked by antimineralocorticoid compounds. Recent experiments have shown that vasopressin potentiates some of the in vivo effects of aldosterone.     

Quantitative functional anatomy of the lower limb with application to human gait

A scheme was developed to classify muscles according to their primary, secondary and tertiary functions, e.g. a muscle which produces primarily a flexion moment may also produce secondary abduction and tertiary internal rotation moments. The functions of muscles crossing the hip and knee joints were computed based upon the changing relative positions of joint centers and muscle origins and insertions during one gait cycle. The function of several of the major muscles crossing the hip and knee joints is reported for the different limb positions corresponding to normal gait. It was found that the amount of force necessary to produce a given moment about a joint was dependent upon the limb position. In addition, the muscle functions changed significantly with limb position. Electrical stimulation of muscles of a paralyzed subject gave qualitative support to the results.     

Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin

Yeast submitochondrial particles, in a P_i- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5–10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.