Germinal angiotensin I-converting enzyme is totally shed from the rodent sperm membrane during epididymal maturation

Acquisition of sperm fertilizing ability is due, in part, to the reorganization of plasma membrane proteins that occurs during epididymal sperm transit. Using polyclonal antibodies against angiotensin I-converting enzyme (ACE), we showed that this enzyme is immunolocalized mainly on the middle piece of rat and mouse testicular sperm and with less intensity along the initial part of the principal piece of the flagellum. In both species, only some sperm from the caput epididymis were still reactive, whereas no labeling was observed on cauda epididymal sperm. The 105- to 110-kDa germinal ACE was absent from the rat testicular fluid but appeared in the fluid of the anterior epididymis. Thereafter, its molecular weight shifted to 94 kDa in the corpus epididymal fluid and remained at this weight in the caudal region. The 105- to 110-kDa immunoreactive protein was present in testicular rat sperm extract but was completely absent from epididymal sperm extracts. Western blot analysis of testicular and epididymal tissue extracts from the rat and mouse also confirmed that the germinal enzyme was absent from the epididymal sperm cell. Our results demonstrated that the rodent germinal ACE is released from the testicular sperm membrane when sperm enter the epididymis, a process similar to that observed in domestic mammals. This result is discussed in view of the suggested role for this enzyme in sperm fertility.     

The motion of a single molecule,the lambda-receptor,in the bacterial outer membrane

Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo. The efficiency of this in vivo biotinylation is very low, thus enabling the attachment of a streptavidin-coated bead binding specifically to a single biotinylated lambda-receptor. The bead was used as a handle for the optical tweezers and as a marker for the single particle tracking routine. We propose a model that allows extraction of the motion of the protein from measurements of the mobility of the bead-molecule complex; these results are equally applicable to analyze bead-protein complexes in other membrane systems. Within a domain of radius approximately 25 nm, the receptor diffuses with a diffusion constant of (1.5 +/- 1.0) x 10(-9) cm(2)/s and sits in a harmonic potential as if it were tethered by an elastic spring of spring constant of ~1.0 x 10(-2) pN/nm to the bacterial membrane. The purpose of the protein motion might be to facilitate transport of maltodextrins through the outer bacterial membrane.     

Electrostatic interactions at the C-terminal domain of nucleoplasmin modulate its chromatin decondensation activity

The chromatin decondensation activity, thermal stability, and secondary structure of recombinant nucleoplasmin, of two deletion mutants, and of the protein isolated from Xenopus oocytes have been characterized. As previously reported, the chromatin decondensation activity of recombinant, unphosphorylated nucleoplasmin is almost negligible. Our data show that deletion of 50 residues at the C-terminal domain of the protein, containing the positively charged nuclear localization sequence, activates its chromatin decondensation ability and decreases its stability. Interestingly, both the decondensation activity and thermal stability of this deletion mutant resemble those of the phosphorylated protein isolated from Xenopus oocytes. Deletion of 80 residues at the C-terminal domain, containing the above-mentioned positively charged region and a poly(Glu) tract, inactivates the protein and increases its thermal stability. These findings, along with the effect of salt on the thermal stability of these proteins, suggest that electrostatic interactions between the positive nuclear localization sequence and the poly(Glu) tract, at the C-terminal domain, modulate protein activity and stability.     

Dermatophyte infection and colonization in dogs from South Santiago, Chile

Our main aim was to determine the dermatophyte infection and colonization prevalence in canines from South Santiago, Chile. We studied 241 dogs, 121 of them presented cutaneous lesions suggestive of dermatophytosis and the other 120 were free from lesions and were considered clinically healthy. Dermatophytes were isolated from the lesions of 48.8% of animals with cutaneous diseases and from 5% of healthy dogs. Microsporum canis was the principal species isolated with a frequency of 98.3% from dermatophytosis and from all healthy carriers. The direct microscopic test showed a sensibility of 85% with a positive and negative predictive value of 74 and 79%, respectively. The highest prevalence of dermatophytosis were detected in animals up to one year old (p <= 0.05) and in dogs with high level of irritation, scaly skin, itching and alopecia (p < or = 0.05). The lesions were detected more frequently in head-neck and anterior members (p < or = 0.05). This study shows some clinical characteristics and a frequency of dermatophytes in canines that can be interesting for laboratory professionals and veterinarians.     

Characterization of the Adhesive from Cuvierian Tubules of the Sea Cucumber <Emphasis Type="Italic">Holothuria forskali</Emphasis> (Echinodermata,Holothuroidea)

Abstract Sea cucumbers possess a peculiar specialized defense system: the so-called Cuvierian tubules. The system is mobilized when the animal is mechanically stimulated, resulting in the discharge of a few white filaments, the tubules. Their great adhesivity, combined with their high tensile strength, allows Cuvierian tubules to entangle and immobilize potential predators. The cellular origin and composition of the Cuvierian tubule adhesive were investigated in the species Holothuria forskali by studying prints left on the substratum after mechanical detachment of the tubule. Polyclonal antibodies raised against tubule print material were used to locate the origin of tubule print constituents in the tubules. Extensive immunoreactivity was detected in the secretory granules of mesothelial granular cells, suggesting that their secretions make up the bulk of the adhesive material. Tubule print material consists of 60% proteins and 40% carbohydrates, a composition that is unique among the adhesive secretions of marine invertebrates. Although it is highly insoluble, a small fraction of this material can be extracted using denaturing buffers. Electrophoretic analysis of the extracts revealed that it contains about 10 proteins with apparent molecular masses ranging from 17 to 220 kDa and with closely related amino acid compositions, rich in acidic and in small side-chain amino acids. The adhesive from the Cuvierian tubules of H. forskali shares these characteristics with many marine bioadhesives and structural biomaterials.     

Molecular mechanisms by which dietary isoflavones potentially prevent atherosclerosis

Dietary isoflavones are currently receiving much attention because of their potential role in preventing coronary artery disease and other chronic diseases. Accumulating evidence from cell culture and laboratory animal experiments indicates that isoflavones have the potential to prevent or delay atherogenesis. Suggested mechanisms of action include: a reduction in low-density lipoprotein (LDL) cholesterol and a potential reduction in the susceptibility of the LDL particle to oxidation; (2) an improvement in vascular reactivity; (3) an inhibition of pro-inflammatory cytokines, cell adhesion proteins and nitric oxide (NO) production; and (4) an inhibition of platelet aggregation. These mechanisms are consistent with the epidemiological evidence that a high consumption of isoflavone-rich soy products is associated with a reduced incidence of coronary artery disease. Biological effects of isoflavones are dependent on many factors, including dose consumed, duration of use, protein-binding affinity, and an individual's metabolism or intrinsic oestrogenic state. Further clinical studies are necessary to determine the potential health effects of isoflavones in specific population groups as we currently know little about age-related differences in exposure to these compounds and there are few guidelines on optimal dose for cardiovascular health benefits.     

Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis

Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.     

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.