Studies on a leukocyte elastase inhibitor present in the culture medium of inflamed synovial tissue

The spent medium of cultured inflamed synovial tissue contains a potent inhibitor of leukocyte elastase. This leukocyte elastase inhibitor has no effect on leukocyte cathepsin G and pancreatic elastase is only marginally affected. The inhibitor is a glycoprotein, stable to heat, acid and reductive alkylation. Pretreatment of the inhibitor with either trypsin or chymotrypsin results in its inactivation.     

T cell recognition of HIV synthetic peptides in a natural infection

Because T cell responses are critical for defense against viral infections, a series of synthetic peptides derived from the predicted sequence for HIV-1 proteins gp41, pg120, gag, and viral polymerase were used to test the T cell proliferative response of HIV-1 seropositive individuals. Of HIV-1-infected donors from various clinical categories 90% (27/30) had sensitized cells that proliferated in response to at least one of 21 HIV peptides tested. Cells from HIV seronegative controls did not proliferate (0/9) in response to these HIV peptides. Individuals with fewer clinical manifestations of HIV-1 disease responded to a greater number of peptides (average for asymptomatic seropositives = 8.1 peptides; AIDS patients averaged 2.0). The number of peptides recognized also correlated with absolute number of CD4+ cells, but not with delayed cutaneous hypersensitivity to a (non-HIV) battery of Ag. However, clinical stage at no time correlated with the response to any particular peptide. Response patterns differed considerably among individuals, and some peptides stimulated proliferation in many (48%) HIV-infected donors (peptides gp41-2 and pol-3), whereas another peptide elicited no T cell response in any donor tested (peptide gp120-8). We have also begun to investigate the basis for individual heterogeneity of T lymphocyte proliferative responses of HIV-infected donors to the 21 HIV synthetic peptides. Peptide structure and HLA class II determinants both influenced patterns of lymphocyte responses. Reactivity correlated with peptide size, the presence of alpha and beta secondary structure and lack of reverse turn potential. Hydropathy and charge had no predictive value. Peptides derived from HIV sequences that vary highly among strains tended to be recognized less frequently. HIV-infected lymphocyte donors were HLA typed to examine the influence of the MHC on T lymphocyte proliferation. Analysis of the frequencies of individuals reacting to specific peptides, when compared to the allele frequencies in the population at large, indicated association of some responses to DR alleles. More DR association was observed with peptides that showed "moderate" reactivity than with those that were "highly" reactive. We suggest that highly reactive peptides are capable of forming a structure closer to an "ideal" T cell epitope that can associate with many DR alleles. In contrast, "moderately" reactive determinants have less favorable structures for interaction, are more limited in their ability to interact and therefore show more restriction to specific class II alleles.     

Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH).

Herein we report the results of the first major prospective study directly comparing aneuploidy detection by fluorescence in situ hybridization of interphase nuclei with the results obtained by cytogenetic analysis. We constructed probes derived from specific subregions of human chromosomes 21, 18, 13, X, and Y that give a single copy-like signal when used in conjunction with suppression hybridization. A total of 526 independent amniotic fluid samples were analyzed in a blind fashion. All five probes were analyzed on 117 samples, while subsets of these five probes were used on the remaining samples (because of insufficient sample size), for a total of over 900 autosomal hybridization reactions and over 400 sex chromosome hybridization reactions. In this blind series, 21 of 21 abnormal samples were correctly identified. The remaining samples were correctly classified as disomic for these five chromosomes. The combination of chromosome-specific probe sets composed primarily of cosmid contigs and optimized hybridization/detection allowed accurate chromosome enumeration in uncultured human amniotic fluid cells, consistent with the results obtained by traditional cytogenetic analysis.     

Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation

Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I + III₂ + IV_n but also decreased amounts of individual complexes I and IV and supercomplexes I + III and III + IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.     

Female reproduction bears no survival cost in captivity for gray mouse lemurs

The survival cost of reproduction has been revealed in many free‐ranging vertebrates. However, recent studies on captive populations failed to detect this cost. Theoretically, this lack of survival/reproduction trade‐off is expected when resources are not limiting, but these studies may have failed to detect the cost, as they may not have fully accounted for potential confounding effects, in particular interindividual heterogeneity. Here, we investigated the effects of current and past reproductive effort on later survival in captive females of a small primate, the gray mouse lemur. Survival analyses showed no cost of reproduction in females; and the pattern was even in the opposite direction: the higher the reproductive effort, the higher the chances of survival until the next reproductive event. These conclusions hold even while accounting for interindividual heterogeneity. In agreement with aforementioned studies on captive vertebrates, these results remind us that reproduction is expected to be traded against body maintenance and the survival prospect only when resources are so limiting that they induce an allocation trade‐off. Thus, the cost of reproduction has a major extrinsic component driven by environmental conditions.     

Electrophysiology and pathology evaluation of the Yucatan pig as a non-rodent animal model for regulatory and mechanistic toxicology studies

Six male and six female Yucatan pigs were utilized to investigate the feasibility of this species as a non-rodent model for routine regulatory and mechanistic toxicology studies. This study evaluated disease surveillance and computerized electrophysiology, along with possible gross and micropathology changes. Two pigs were used as sentinel animals to evaluate the microbiological status of the vendor upon arrival; the other pigs were maintained as biomonitors and to provide baseline clinical chemistry, urinalysis, pathology and electrophysiology data. The electrophysiology tests conducted included electrocardiography (ECG), electroretinography (ERG) and quantitative electroencephalography (qEEG), which achieved consistent baseline values with acceptable intrasubject variation. Tissue cholinesterase and histochemical staining were done to determine their suitability for testing cholinesterase compounds. Evaluation of the serum chemistry profile demonstrated increased CPK and LDH, which was likely associated with slight haemolysis or minor subclinical muscle stress during handling. There were no additional clinical chemistry changes or findings in haematology, urinalysis parameters or gross pathology. Micropathology found an absence of background lesions which would interfere with routine toxicology studies, except for a mild rhinitis. The aetiological agent was identified by electron microscopy as being consistent with inclusion body rhinitis of swine, previously unreported in miniature swine. This would most notably interfere with inhalation studies. The anatomical and physiological similarities of the Yucatan pig, along with its ability to accept the performance of electrophysiology tests allow this species to be considered as a suitable model for organ system testing in toxicology studies.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.