A temperature gradient method for lipid phase diagram construction using time-resolved x-ray diffraction.

A method that enables temperature-composition phase diagram construction at unprecedented rates is described and evaluated. The method involves establishing a known temperature gradient along the length of a metal rod. Samples of different compositions contained in long, thin-walled capillaries are positioned lengthwise on the rod and "equilibrated" such that the temperature gradient is communicated into the sample. The sample is then moved through a focused, monochromatic synchroton-derived x-ray beam and the image-intensified diffraction pattern from the sample is recorded on videotape continuously in live-time as a function of position and, thus, temperature. The temperature at which the diffraction pattern changes corresponds to a phase boundary, and the phase(s) existing (coexisting) on either side of the boundary can be identified on the basis of the diffraction pattern. Repeating the measurement on samples covering the entire composition range completes the phase diagram. These additional samples can be conveniently placed at different locations around the perimeter of the cylindrical rod and rotated into position for diffraction measurement. Temperature-composition phase diagrams for the fully hydrated binary mixtures, dimyristoylphosphatidylcholine (DMPC)/dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE)/DPPC, have been constructed using the new temperature gradient method. They agree well with and extend the results obtained by other techniques. In the DPPE/DPPC system structural parameters as a function of temperature in the various phases including the subgel phase are reported. The potential limitations of this steady-state method are discussed.     

The Uptake of NO3?, NO2?, and NH4+ by Intact Wheat (Triticum aestivum) Seedlings : I. Induction and Kinetics of Transport Systems

The inducibility and kinetics of the NO₃⁻, NO₂⁻, and NH₄⁺ transporters in roots of wheat seedlings (Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for and the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO₃⁻ and NO₂⁻ transporters. Evidence for substrate inducibility of the NH₄⁺ transporters requiring 5 hours is presented. The transport of NO₃⁻ was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO₂⁻ and NH₄⁺. The K_m values for NO₃⁻, NO₂⁻, and NH₄⁺ were, respectively, 0.027, 0.054, and 0.05 millimolar. The K_m for mechanism II of NO₃⁻ transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar.     

HLA Haplotypes with C4B5; evidence for further allelic heterogeneity

Twenty-three individuals from various disease groups and normal controls were identified by immunofixation with anti-C4, C4-dependent lysis, determination of Rg (Rodgers) and Ch (Chido) phenotypes, and immunoblotting with C4-specific mouse monoclonal antibody. We found that one haplotype predominates with the C4B ^* 5 allele, HLA-A11, B22(55), Cw3, Bf ^* S, C4A ^* 4B ^* 5, which also carries the Ch ^{1,–2, 3} haplotype. The B5 allotype was also found with HLA-1360, HLA-1335 in Caucasoids, and HLA-B18 in non-Caucasoids; these carried the Ch ^{–1, –2, –3} haplotype. Our results are in accord with an earlier report of two B5 subtypes, B5Rg⁺ and B5Rg^– (Roos et al. 1984). The specificity of the mouse monoclonal antibodies IC4 and 21312 had been previously related to C4A and C4B, respectively, but our results suggest that they relate more closely to Rg and Ch determinants.     

VNTR and microsatellite polymorphisms within the subtelomeric region of 7q.

The molecular basis of a highly polymorphic RFLP marker, HTY146c3 (D7S591), within the subtelomeric region of human chromosome 7q was determined by restriction-fragment and DNA sequence analysis. Two polymorphic systems were found--a simple base-substitution polymorphism and a GC-rich VNTR element with a core structure of C3AG2C2. In addition, a compound-imperfect CA dinucleotide-repeat element was identified approximately 10-20 kb from the telomeric sequence repeat (T2AG3), demonstrating that microsatellites can extend essentially to the ends of human chromosomes. The microsatellite marker, sAVH-6 (D7S594), is highly polymorphic, with 10 alleles and an observed heterozygosity of 84% found with the CEPH (Centre d'Etude du Polymorphisme Humain) reference pedigree collection. In combination with the RFLPs, the informativeness of the markers contained within 240 kb at the telomere approaches 100%. A unique genetic and physical STS marker, sAVH-6, defines the endpoint of the long arm of human chromosome 7.     

Effect of nitrate pulses on the nitrate-uptake rate,synthesis of mRNA coding for nitrate reductase,and nitrate-reductase activity in the roots of barley seedlings

Using pulses of nitrate, instead of the permanent presence of external nitrate, to induce the nitrate-assimilating system in Hordeum vulgare L., we demonstrated that nitrate can be considered as a trigger or signal for the induction of nitrate uptake, the appearance of nitratereductase activity and the synthesis of mRNA coding for nitrate reductase. Nitrate pulses stimulated the initial rate of nitrate uptake, even after subsequent cultivation in N-free medium, and resulted in a higher acceleration of the uptake rate in the presence of nitrate than in its absence.Abbreviations NR nitrate reductase     

Structurally defined synthetic cancer vaccines: analysis of structure, glycosylation and recognition of cancer associated mucin, MUC-1 derived peptides

Translation of an immune response into therapy is probably the toughest task in designing vaccines for cancer due to the heterogeneity of the cell surface antigens which display tremendous variations in glycoforms. Consequently, a small segment (antigen) of the cancer-associated mucin, in spite of generating antigen-specific immune responses, may be limited in therapeutic value. It is important that the synthetic segment resembles the native cancer-associated mucin in both structure and conformation. Synthetic cancer associated mucin derived 16 amino acid peptide GVTSAPDTRAPAPGSTA and its partially glycosylated forms have demonstrated specific binding to two monoclonal antibodies, B27.29 and BCP8, raised against the native cancer associated mucin, MUC-1 and a MUC-1 derived synthetic peptide, respectively. In spite of the structural similarities at the core peptide level of both glycosylated and unglycosylated peptides, it appears that partial glycosylation does not inhibit and even slightly enhances binding to the MAb B27.29 indicating that the glycosylated synthetic peptide more closely resembles the native mucin epitope recognized by MAb B27.29. From molecular dynamic simulations using NMR derived distance constraints, both glycosylated and unglycosylated peptides have shown a type I turn involving the same amino acids in both glycosylated and unglycosylated peptides. The GalNAc attached to the threonine (T³) and serine (S⁴) in the 16 amino acid sequence has not imposed any conformational changes to the peptide backbone nor has offered severe steric resistance to the binding of either antibody to the glycopeptides as indicated by hapten inhibition studies. Nevertheless, all peptides have displayed glycosylation dependent specificities in binding to these antibodies, i.e. the glycosylated peptides demonstrated relative higher affinities to the native mucin antibody B27.29 while the unglycosylated peptide is more specific to the MAb BCP8. Immune responses generated by these synthetic glycopeptides are highly specific in recognizing the native cancer associated mucin.     

Peripheral chemoreceptor modulation of the pulmonary vasculature in the cat.

The present study was undertaken to determine whether stimulation of the carotid and aortic bodies (cb and ab) could affect the pulmonary vasculature. Our hypothesis was that each promoted vasodilation and thus could modulate the pulmonary vasoconstrictor response to hypoxia. The experimental design of the first set of experiments took advantage of the facts that 1) the ab, but not the cb, increases its neural output in response to CO, whereas both respond to a decreased arterial PO2 (hypoxic hypoxia, HH) and 2) the aortic nerves in cats are easily transected. Hence, both cb and ab sent neural activity to the brain stem when the intact cat was exposed to 10% O2 in N2. Only the ab sent information during CO hypoxia (COH intact). Only the cb did so during HH in the cat in which the aortic nerves had been transected, removing the aortic body (HH abr); neither ab nor cb did so during COH abr. Fifteen anesthetized paralyzed artificially ventilated cats were fit with catheters in the femoral artery and vein, right and left atria, left ventricle, and pulmonary artery and with an aortic flow probe. In the HH intact and HH abr conditions, there was a significant rise in cardiac output, whereas pulmonary arterial pressure (Ppa) rose initially but then leveled off while cardiac output continued to rise. During the 15-min exposure to HH, pulmonary vascular resistance [PVR = (Ppa - Pla)/cardiac output, where Pla is left atrial pressure] rose initially and then decreased significantly at 2-3 min. In response to COH, PVR showed only a significant decrease. In the second set of experiments, seven cats were instrumented as above and had loops placed in the common carotid arteries for selectively perfusing the cbs. In response to a brief infusion of venous blood mixed with 0.3-0.5 micrograms NaCN, which selectively stimulated only the cb, aortic flow remained relatively constant while heart rate and Ppa - alveolar pressure difference decreased significantly; so also did PVR. These data are consistent with the hypothesis that stimulation of the ab and cb singly or together can provoke a significant pulmonary vasodilation in the anesthetized paralyzed artificially ventilated cat.     

Gene Network Analysis of Candidate Loci for Human Anorectal Malformations

Anorectal malformations (ARMs) are birth defects that require surgery and carry significant chronic morbidity. Our earlier genome-wide copy number variation (CNV) study had provided a wealth of candidate loci. To find out whether these candidate loci are related to important developmental pathways, we have performed an extensive literature search coupled with the currently available bioinformatics tools. This has allowed us to assign both genic and non-genic CNVs to interrelated pathways known to govern the development of the anorectal region. We have linked 11 candidate genes to the WNT signalling pathway and 17 genes to the cytoskeletal network. Interestingly, candidate genes with similar functions are disrupted by the same type of CNV. The gene network we discovered provides evidence that rare mutations in different interrelated genes may lead to similar phenotypes, accounting for genetic heterogeneity in ARMs. Classification of patients according to the affected pathway and lesion type should eventually improve the diagnosis and the identification of common genes/molecules as therapeutic targets.