Leukocyte lipid bodies: inflammation-related organelles are rapidly detected by wet scanning electron microscopy

Leukocyte lipid bodies are dynamic, functionally active organelles with central roles in inflammation. Here, we report that leukocyte lipid bodies are facilely detected by a versatile, potent technique, termed wet scanning electron microscopy (SEM), which combines the rapid preparation of light microscopy with the resolution of SEM. Using as leukocyte models resting and agonist-stimulated human eosinophils, cells that generate prominent numbers of lipid bodies in inflammatory conditions, we demonstrated that lipid bodies can be rapidly imaged as bright, highly contrasted structures under wet SEM and scored by computerized image processing. Critical advantages of this approach are that it permits cell observation in a fully hydrated system and facilitates lipid preservation. These attributes are especially important because lipid bodies are degraded during routine dehydration processes. Moreover, this technology is advantageous over lipophilic fluorescent probes because it allows sustained detection of lipid bodies in contrast to short-lived fluorescent labeling of these organelles. The value of wet SEM in enabling rapid and large-scale lipid body imaging and scoring within leukocytes is particularly important because lipid bodies are organelles underlying the heightened functions of inflammatory cells. Wet SEM technology provides new approaches and opportunities for delineations of lipid bodies in inflammatory diseases, including allergic inflammation.     

Preliminary results on interleukin-4 and interleukin-10 cytokine production in malnourished, inducible nitric oxide synthase-deficient mice with schistosomiasis mansoni infection

Schistosoma mansoni infected C57Bl/6 inducible nitric oxide synthase (iNOS)-deficient and non-deficient malnourished mice, both fed a balanced controlled diet were studied. Interleukins, IL-4 and IL-10 responses to soluble egg antigens (SEA) 90 days after infection, were determined. Our results suggest that in iNOS deficient, malnourished mice, 90 days after of infection, nitric oxide has a downregulating effect on IL-4 and IL-10 production. We are currently investigating the biological significance of these findings.     

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.     

High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow

Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.     

Orpotrin: a novel vasoconstrictor peptide from the venom of the Brazilian stingray Potamotrygon gr. orbignyi

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97–105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms.     

Evaluation of GnRH treatment 12 days after AI in the reproductive performance of dairy cows

The objective of this study was to evaluate the effects of GnRH administered at Day 12 post-AI on the reproductive performance of dairy cows. Holstein-Friesian dairy cows (n=103) on a large Hungarian dairy farm were allocated randomly to treated (n=54) or control (n=49) groups. Twelve days after AI, treated cows received a GnRH agonist i.m., while the control group received a placebo (physiological saline). Progesterone radioimmunoassay was used to determine the correct timing of artificial insemination (Day 0) and the incidence of luteal insufficiency on Day 12. Ultrasonography and radioimmunoassay for pregnancy-associated glycoprotein were used to detect pregnancy and late embryonic/fetal mortality between Days 32 and 55 after AI. Three cows from each group were inseminated when progesterone concentrations were >1.0 ng/mL, and six cows (four from the treated and two from the control group) had luteal insufficiency (progesterone<1.0 ng/mL) on Day 12. Late embryonic/fetal mortality occurred in three treated cows and in two control cows. When these cows were removed from the model, calving rates after first service were 59.6% (28/47) and 59.1% (26/44) for treated and control cows, respectively (P>0.05). There was no significant difference between treated and control cows when they were inseminated before or after Day 100 from calving. In summary, administration of a GnRH agonist on Day 12 after AI did not improve reproductive performance in dairy cows. However, our approach may be used for the field evaluation of different treatment protocols.     

Ion-exchange chromatography used to isolate a spermadhesin-related protein from domestic goat (Capra hircus) seminal plasma

Mammalian seminal plasma contains among others, proteins called spermadhesins, which are the major proteins of boar and stallion seminal plasma. These proteins appear to be involved in capacitation and sperm-egg interaction. Previously, we reported the presence of a protein related to spermadhesins in goat seminal plasma. In the present study, we have further characterized this protein, and we propose ion-exchange chromatography to isolate this seminal protein. Semen was obtained from four adult Saanen bucks. Seminal plasma was pooled, dialyzed against distilled water and freeze-dried. Lyophilized proteins were loaded onto an ion-exchange chromatography column. Dialyzed-lyophilized proteins from the main peak of DEAE-Sephacel were applied to a C2/C18 column coupled to an RP-HPLC system, and the eluted proteins were lyophilized for electrophoresis. The N-terminal was sequenced and amino acid sequence similarity was determined using CLUSTAL W. Additionally, proteins from DEAE-Sephacel chromatography step were dialyzed and submitted to a heparin-Sepharose high-performance liquid chromatography. Goat seminal plasma after ion-exchange chromatography yielded 6.47 +/- 0.63 mg (mean +/- SEM) of the major retained fraction. The protein was designated BSFP (buck seminal fluid protein). BSFP exhibited N-terminal sequence homology to boar, stallion and bull spermadhesins. BSFP showed no heparin-binding capabilities. These results together with our previous data indicate that goat seminal plasma contains a protein that is structurally related to proteins of the spermadhesin family. Finally, this protein can be efficiently isolated by ion-exchange and reverse-phase chromatography.     

Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies

Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E ⁰=455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the k _cat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15–0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.     

Cactophilic Drosophila in South America: A Model for Evolutionary Studies

The Drosophila buzzatii cluster is composed of seven cactophilic species and their known geographical distribution encompasses the open vegetation diagonal, which includes the morphoclimatic Domains of the Caatinga, Chaco and Cerrado, which are situated between the Amazon and the Atlantic forests. Besides these areas, these cactophilic species are also found in a narrow strip along the Atlantic coast from northeastern Brazil to the southern tip of the country. The hypothesis of vicariant events, defining the core areas of each species, is proposed to explain the historical diversification for the cluster. The intraspecific analysis for the cluster shows a population structure with gene flow restricted by distance, range expansion with secondary contact resulting in introgression and simpatry, especially in the limits of the species distribution, polytypic populations and assortative mating in inter population experiments. There is a variation related to these events that depends on the species and geographic origin of the population analyzed. These events are, hypothetically, described as the results of expansion and retraction of the population ranges, as a consequence of their association with cacti, which theoretically follow the expansion and retraction of dry areas during the paleoclimatic oscillations in South America, as that promoted by the glacial cycles of the Quaternary. The Drosophila buzzatii cluster is divided into two groups. The first one is composed of D. buzzatii, a species that has a broad geographic distribution and no significant differentiation between its populations. The second is the Drosophila serido sibling set, which encompasses the others species and is characterized by a significant potential for differentiation.     

pH-dependent aggregation of cutinase is efficiently suppressed by 1,8-ANS

We have studied the thermal stability of the triglyceride-hydrolyzing enzyme cutinase from F. solani pisi at pH values straddling the pI (pH 8.0). At the pI, increasing the protein concentration from 5 to 80 microM decreases the apparent melting temperature by 19 degrees C. This effect vanishes at pH values more than one unit away from pI. In contrast to additives such as detergents and osmolytes, the hydrophobic fluorophore 1,8-ANS completely and saturably suppresses this effect, restoring 70% of enzymatic activity upon cooling. ANS binds strongly to native cutinase as a noncompetitive inhibitor with up to 5 ANS per cutinase molecule. Only the first ANS molecule stabilizes cutinase; however, the last 4 ANS molecules decrease Tm by up to 7 degrees C. Similar pI-dependent aggregation and suppression by ANS is observed for T. lanuginosus lipase, but not for lysozyme or porcine alpha-amylase, suggesting that this behavior is most prevalent for proteins with affinity for hydrophobic substrates and consequent exposure of hydrophobic patches. Aggregation may be promoted by a fluctuating ensemble of native-like states associating via intermolecular beta-sheet rich structures unless blocked by ANS. Our data highlight the chaperone activity of small molecules with affinity for hydrophobic surfaces and their potential application as stabilizers at appropriate stoichiometries.