Mass spectral analyses of the two major apolipoproteins of great ape high density lipoproteins

The two major apolipoproteins associated with human and chimpanzee (Pan troglodytes) high density lipoproteins (HDL) are apoA-I and dimeric apoA-II. Although humans are closely related to great apes, apolipoprotein data do not exist for bonobos (Pan paniscus), western lowland gorillas (Gorilla gorilla gorilla) and the Sumatran orangutans (Pongo abelii). In the absence of any data, other great apes simply have been assumed to have dimeric apoA-II while other primates and most other mammals have been shown to have monomeric apoA-II. Using mass spectrometry, we have measured the molecular masses of apoA-I and apoA-II associated with the HDL of these great apes. Each was observed to have dimeric apoA-II. Being phylogenetically related, one would anticipate these apolipoproteins having a high percentage of invariant sequences when compared with human apolipoproteins. However, the orangutan, which diverged from the human lineage between 16 and 21 million years ago, had an apoA-II with the lowest monomeric mass, 8031.3 Da and the highest apoA-I value, 28,311.7 Da, currently reported for various mammals. Interestingly, the gorilla that diverged from the lineage leading to the human–chimpanzee branch after the orangutan had almost identical mass values to those reported for human apoA-I and apoA-II. But chimpanzee and the bonobo that diverged more recently had identical apoA-II mass values that were slightly larger than reported for the human apolipoprotein. The chimpanzee A-I mass values were very close to those of humans; however, the bonobo had values intermediate to the molecular masses of orangutan and the other great apes. With the already existing genomic data for chimpanzee and the recent entries for the orangutan and gorilla, we were able to demonstrate a close agreement between our mass spectral data and the calculated molecular weights determined from the predicted primary sequences of the respective apolipoproteins. Post-translational modification of these apolipoproteins, involving truncation and oxidation of methionine, are also reported.     

A food safety control low mass-range proteomics platform for the detection of illicit treatments in veal calves by MALDI-TOF-MS serum profiling

Performance enhancing agents (PEAs) are illegally used in cattle and other meat producing species to increase food conversion and lean meat production. Due to the very short breeding cycle, veal calves represent the meat producing bovine category mostly subjected to illicit treatments. These chemical agents are difficult to detect by conventional analytical approaches due to the employment of synergistic formulations at very low dosage and given the use of uncharacterized novel compounds. Such a scenario has fostered a strong interest in the discovery of functional molecular biomarkers for the detection of growth promoting agents in meat producing species. A multivariate MALDI-TOF-MS proteomics platform has been developed using bovine serum samples. Analytical performances have been thoroughly evaluated in order to enable reproducible profiles from 10 μL sera samples. We propose univariate and multivariate discrimination models capable to identify calves undergoing illicit treatments. In particular, we found a strong discrimination power associated with a polypeptide fragment from β2-glycoprotein-I. We provide a fundamental proof of concept in the potential application of MALDI-TOF-MS proteomics profiling in the food safety control.     

Patterns of style polymorphism in five species of the South African genus Nivenia (Iridaceae)

Background and Aims

Heterostylous plants have been characterized by the presence of two or three discrete morphs that differ in their sex organ position within populations. This polymorphism is widely distributed among the angiosperms, but detailed studies are limited to few taxonomic groups. Although a small representation, evolutionary meaningful variations of the heterostylous syndrome have been reported when precise measurements of the sexual whorls were taken. A thorough exploration of groups where heterostyly has been reported should offer new opportunities to further testing the evolutionary hypotheses explaining heterostyly. Here, the traits defining heterostyly were explored in half of the species in Nivenia, the only genus of Iridiaceae where heterostyly has been reported.

Methods

Detailed morphometric analysis of the flower sexual whorls and some traits considered as ancillary are supplied to determine for each population (a) the kind of stylar polymorphism, (b) the morph ratio and (c) the degree of reciprocity between sexual whorls. Also the rates of assortative (within morph) versus disassortative (between morphs) pollen transfer were estimated by analysing pollen loads on stigmas. The association between floral phenotypic integration and the reciprocity between sexual whorls was estimated; both characteristics have been quoted as dependent on the accuracy of the fit between pollinators and flowers and therefore related to the efficiency of pollen transfer.

Key Results

Different types of polymorphism, differing in their degree of reciprocity, were found in Nivenia. Effective disassortative mating appears to be common, since (a) all dimorphic populations show equal morph-ratios (isoplethy), and (b) the pollen placed on the stigmas of each morph is likely to be coming from the other (complementary) morph. The most reciprocal populations of the heterostylous species have also the highest values of phenotypical integration.

Conclusions

Stigma height dimorphism, as opposed to distyly, is proven for the first time in Nivenia. The presence of different types of polymorphism within the genus is consistent with hypotheses of the evolution of heterostyly. The role of the pollinators as the leading force of the transition seems to be apparent, since floral integration is related to reciprocity.     

Setting of Methods for Analysis of Mucosal Antibodies in Seminal and Vaginal Fluids of HIV Seropositive Subjects from Cambodian and Italian Cohorts

Background

Genital mucosae play a key role in protection from STD and HIV infection, due to their involvement in both horizontal and vertical disease transmission. High variability of published observations concerning IgA isolation and quantification underlies the strong requirement of specific methods able to maximize investigation on HIV-specific IgA.

Methodology

Genital fluids from 109 subjects, including male and female cohorts from Italy and Cambodia, were collected, aliquoted and processed with different techniques, to assess optimal conditions maximizing mucosal antibody recovery. Three sampling techniques, up to sixteen preservation conditions, six ELISA methods and four purifications protocols were compared.

Principal Findings

The optimal method here described took advantage of Weck-Cel sampling of female mucosal fluids. Immediate processing of genital fluids, with the addition of antibiotics and EDTA, improved recovery of vaginal IgA, while the triple addition of EDTA, antibiotics and protease inhibitors provided the highest amount of seminal IgA. Due to low amount of IgA in mucosal fluids, a high sensitive sandwich ELISA assay was set; sensitivity was enhanced by milk-based overcoating buffer and by a two-step biotin-streptavidin signal amplification. Indeed, commercial antisera to detect human immunoglobulins showed weak cross-reactivity to different antibody types. Three-step affinity purification provided reproducible immunoglobulin recovery from genital specimens, while conventional immuno-affinity IgA purification was found poorly manageable. Affinity columns were suitable to isolate mucosal IgA, which are ten-fold less concentrated than IgG in genital specimens, and provided effective separation of IgA monomers, dimers, and J-chains. Jacalin-bound resin successfully separated IgA1 from IgA2 subfraction.

Conclusions/Significance

Specific, effective and reliable methods to study local immunity are key items in understanding host mucosal response. The sequence of methods here described is effective and reliable in analysing humoral local responses, and may provide a solid advance to identify and measure the effective mucosal responses to HIV.     

Histochemical study of cardiac mast cells degranulation and collagen deposition: interaction with the cathecolaminergic system in the rat

Although their role in the cardiovascular system is still largely unknown, mast cells are present in the myocardium of both experimental animals and humans. Interestingly, cathecolaminergic nerve fibres and mast cells are often described in close morphological and functional interactions in various organs. In the present study we investigated the effects of chronic interference with beta-adrenergic receptors (via either sympathectomy or beta-blockade) on cardiac mast cell morphology/activation and on interstitial collagen deposition. In rats subjected to chemical sympathectomizy with the neurotoxin 6-hydroxydopamine (6-OHDA) we observed a significant increase of mast cell density, and in particular of degranulating mast cells, suggesting a close relationship between the cardiac catecholaminergic system and mast cell activation. In parallel, chronic 6-OHDA treatment was associated with increased collagen deposition. The influence of the beta-adrenergic receptor component was investigated in rats subjected to chronic propranolol administration, that caused a further significant increase in mast cell activation associated with a lower extent of collagen deposition when compared to chemical sympathectomy. These data are the first demonstration of a close relationship between rat cardiac mast cell activation and the catecholaminergic system, with a complex interplay with cardiac collagen deposition. Specifically, abrogation of the cardiac sympathetic efferent drive by chemical sympathectomy causes mast cell activation and interstitial fibrosis, possibly due to the local effects of the neurotoxin 6-hydroxydopamine. In contrast, beta-adrenergic blockade is associated with enhanced mast cell degranulation and a lower extent of collagen deposition in the normal myocardium. In conclusion, cardiac mast cell activation is influenced by beta-adrenergic influences.     

Purification and characterization of two recombinant human granulocyte colony-stimulating factor glycoforms

Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line. The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133. The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GalNAc. The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM). In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli. The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively. The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination. The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life. Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c. route either singularly or as a mixture. Detectable levels of rhG-CSF persisted for more than 8 h in the i.v. and more than 24 h in the s.c. route of administration. All three substances induced early neutrophilia in mice. All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection. Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v. and s.c. administration, respectively.     

The coronary vasculature and the myocardium

The effect of bradykinin (BK) on myocardial inotropic state was tested on 5 isolated rat heart preparations, in which a proper ballon was placed to record left ventricular pressure, whose developed systolic value was taken as an index of contractility. A reduction of developed left ventricular pressure was observed when BK was added to the perfusion oxygenated Tyrode solution. However, when BK was given after 1-amino-benzotriazole, an inhibitor of Cytochrome P-450 (Cyt P-450), developed pressure did not change. Since Cyt P-450 is known to act on arachidonic acid inducing the production of epoxiecocistrienoic acids (EETs) which hyperpolarizes myocardial fibres, it was argued that the reduction in contractility by bradykinin was the result of the hyperpolarizing effect of EETs. The fact that the concentration of Cyt P-450 is higher in the vascular endothelial cells than in the sarcolemma of the myocytes and the observation that the coronary resistance decreases together with the contractility suggest that the endothelium plays a pivotal role in mediating the negative inotropic effect of BK.     

Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice

Constitutive expression of gamma-glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N-terminal amino acid sequences of two of the generated endo-proteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37 degrees C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37 degrees C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His-tagged-GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro-GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT-encoding gene of a mouse-adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co-infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.     

B cell response after MMTV infection: extrafollicular plasmablasts represent the main infected population and can transmit viral infection

The immune response to mouse mammary tumor virus (MMTV) relies on the presentation of an MMTV-encoded superantigen by infected B cells to superantigen-specific T cells. The initial extrafollicular B cell differentiation involved the generation of B cells expressing low levels of B220. These B220low B cells corresponded to plasmablasts that expressed high levels of CD43 and syndecan-1 and were CD62 ligand- and IgD-. Viral DNA was detected nearly exclusively in these B220low B cells by PCR, and retroviral type-A particles were observed in their cytoplasm by electron microscopy. An MMTV transmission to the offspring was also achieved after transfer of B220low CD62 ligand- CD43+ plasmablasts into noninfected females. These data suggest that B220low plasmablasts, representing the bulk of infected B cells, are capable of sustaining viral replication and may be involved in the transmission of MMTV.