Preliminary crystallographic data, primary sequence, and binding data for an anti-peptide Fab and its complex with a synthetic peptide from influenza virus hemagglutinin

X-ray quality crystals which diffract to high resolution (less than or equal to 1.9-2.1 A) have been grown of an anti-peptide Fab and its complex with a 9-residue peptide antigen. Both crystals are monoclinic P2(1), with unit cell dimensions a = 90.3 A, b = 82.9 A, c = 73.4 A, beta = 122.5 degrees for the native Fab and a = 63.9 A, b = 73.0 A, c = 49.1 A, beta = 120.6 degrees for the complex. The peptide sequence corresponds to residues 100-108 of all influenza virus hemagglutinins (HA1) of the H3 subtype (1968-1987). The peptide antigen has been well characterized immunologically (Wilson, I.A., Niman, H.L., Houghton, R.A., Cherenson, A.R., Connolly, M.L., and Lerner, R.A. (1984) Cell 37, 767-778; Wilson, I.A., Bergmann, K.F., and Stura, E.A. (1986) in Vaccines '86 (Channock, R.M., Lerner, R.A., and Brown, F., eds) pp. 33-37, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), structurally, as a free peptide by NMR (Dyson, J.H., Cross, K.J., Houghton, R.A., Wilson, I.A., Wright, P.E., and Lerner, R.A. (1985) Nature 318, 480-483; Dyson, J.H., Lerner, R.A., and Wright, P.E., (1988) Annu. Rev. Biophys. Chem. 17, 305-324), as part of the intact antigen by x-ray crystallography (Wilson, I.A., Skehel, J.J., and Wiley, D. C. (1981) Nature 289, 366-373) and by binding studies to the HA molecule (White, J.M., and Wilson, I.A. (1987) J. Cell Biol. 105, 2887-2896). Knowledge of the three-dimensional structure of the complex will elucidate the details of how anti-peptide antibodies recognize a small peptide antigen and provide insights into the recognition of the same sequence in the intact protein antigen. As both native Fab and the peptide-Fab complex have been crystallized, we can also determine in addition whether changes in the structure of the antibody accompany antigen binding. The nucleotide sequence of the mRNA coding region of the anti-peptide Fab has been determined to provide the amino acid sequence ultimately required for the high resolution three-dimensional structure determination.     

Anastomosis is required for virulence of the fungal necrotroph Alternaria brassicicola

A fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogen Alternaria brassicicola and two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of the A. brassicicola isolates tested, as well as A. mimicula and A. japonica, were capable of self-fusion, with two isolates of A. brassicicola being capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) in A. brassicicola resulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenic A. brassicicola mutant defective for a mitogen-activated protein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity in A. brassicicola.     

Size-dependent rheology of type-I collagen networks

We investigate the system size-dependent rheological response of branched type I collagen gels. When subjected to a shear strain, the highly interconnected mesh dynamically reorients, resulting in overall stiffening of the network. When a continuous shear strain is applied to a collagen network, we observe that the local apparent modulus, in the strain-stiffening regime, is strongly dependent on the gel thickness. In addition, we demonstrate that the overall network failure is determined by the ratio of the gel thickness to the mesh size. These findings have broad implications for cell-matrix interactions, the interpretation of rheological tissue data, and the engineering of biomimetic scaffolds.     

Pregnancy Rates in Repeat-breeder Heifers Following Multiple Artificial Inseminations during Spontaneous Oestrus

Hormonal asynchronies during oestrus, related to the presence of suprabasal plasma-progesterone (P4) concentrations and a delayed ovulation, interfere with the fertility of repeat-breeder heifers (RBH). Since tubal dysfunction can occur in connection with hormonal asynchronies and constrained availability of fertile spermatozoa at the time of ovulation, the present study tested the hypothesis that frequent sperm deposition from onset of oestrus to ovulation may improve pregnancy rates in RBH. Five RBH and five virgin heifers (VH; controls) were repeatedly artificially inseminated (AI) at 6 h intervals from onset of oestrus to spontaneous ovulation. Hormone analyses revealed suprabasal P₄ concentrations and a delay in the occurrence of the luteinising hormone (LH) surge, but a normal cortisol profile in RBH. Compared with controls, RBH presented longer interval from onset of oestrus to ovulation, and therefore, received more AIs. Pregnancy rates in RBH reached control levels (60%; NS), indicating that the hypothesis might be correct. Pregnancy rates in VH were below the expected range, presumably attributed to a deleterious influence of the frequent handling. The study suggests that pregnancy rates can be improved in RBH by frequent AI in relation to spontaneous ovulation. However, this practice of repeated manipulations, while seeming not to show adverse effects, lacks practicality for routine use.     

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.     

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.     

Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide

Background: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6‐glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6‐glucan and characterize their binding properties and functional activity. Materials and Methods: BALB/c mice were injected intraperitoneally with 10⁸ formalin‐fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti‐α1,6‐glucan‐producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole‐cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild‐type and mutant strains of H. pylori. Results: The generated anti‐α1,6‐glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6‐linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD₄₅₀ ≥ 0.2). Bactericidal activity was observed against selective wild‐type and mutant H. pylori strains exhibiting OD₄₅₀ values of ≥0.45 in WCE. Conclusions: Anti‐α1,6‐glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS‐based vaccine against H. pylori infections.     

Influence of extender,temperature, and addition of glycerol on post-thaw sperm parameters in ram semen

Using a two-step extension methodology, two experiments were conducted using a split-sample design to compare the effect on post-thaw ram sperm parameters of a milk-based extender (Experiment 1) containing four different egg yolk concentrations (5% [M5], 10% [M10], 15% [M15], and 20% [M20]), and a commercially available extender (Bioexcell); IMV, L'Aigle, France) free from additives of animal origin, containing two different final glycerol concentrations (3.2% [B] and 6.4% [BB]) (Experiment 2). In both experiments, glycerol was added either at 5 degrees C or at 15 degrees C together with the second fraction of each extender. The sperm characteristics assessed were motility (measured subjectively [SM] and by means of cell motion analysis (CASA), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline (CTC)/EthD-1). Results of Experiment 1 showed no significant positive effect of increasing the concentration of egg yolk above 10% on post-thaw motility, membrane integrity, or induction of sperm capacitation-like changes. In Experiment 2, Bioexcell (BB) yielded similar post-thaw results as did the milk extender (control). In both experiments, post-thaw sperm parameters were better preserved when glycerol was added at 5 degrees C, although the results were not always statistically significant for all variables studied. In conclusion, when using milk-based extenders for freezing ram semen, low (5-10%) concentrations of egg yolk and the addition of glycerol at 5 degrees C are recommended. Furthermore, the results indicate that when freezing ram semen, Bioexcell containing 6.4% glycerol may be used as an alternative extender to the conventional milk extender containing 5% egg yolk.     

Intraaortic administration of protamine: Method for heparin neutralization after cardiopulmonary bypass

In neutralizing heparin with intravenous protamine sulfate, hypotension may be prevented by administering the drug intraarterially. Forty patients underwent cardiac surgery with extracorporeal circulation in our hospital; each received a rapid injection of nondiluted protamine sulfate in the aortic root to reverse the effects of heparin. To maintain the blood volume at a constant level, volume expanders and inotropic drugs were avoided. The intraaortic injections ranged in duration from 0.2 min to 2.8 min, with a mean of 1.1 min. The mean systolic pressure only dropped from 92 mm Hg (SD +/- 21) before protamine injection to 85 mm Hg (SD +/- 23) after injection (p < 0.0001). In seven patients (18%), no hypotension was evident; in the remaining patients, the systolic pressure returned to preinjection values within a mean of 2.2 min. Coagulation was observed within 3 to 4 min (mean = 2.2 min) after the initiation of injection. This study indicates that intraaortic administration of protamine is a rapid and safe technique for heparin reversal after cardiopulmonary bypass.