Phage display of ScFv peptides recognizing the thymidine(6–4)thymidine photoproduct

Solar ultraviolet (UV) radiation induces DNA photoproducts in skin cells and is the predominant cause of human skin cancers. To understand human susceptibility to skin cancer and to facilitate the development of prevention measures, highly specific reagents to detect and quantitate UV-induced DNA adducts in human skin will be needed. One approach towards this end is the use of monoclonal antibody-based molecular dosimetry methods. To facilitate the development of photoproduct-specific antibody reagents we have: (i) cloned and sequenced a single chain variable fragment (ScFv) gene coding for one such high affinity monoclonal antibody, αUVssDNA-1 (mAb C3B6), recognizing the thymidine(6–4)thymidine photoproduct; (ii) expressed and displayed the cloned ScFv gene on the surface of phage; (iii) selected functional recombinant phage by panning; (iv) purified the ScFv peptide; (v) shown that the purified ScFv peptide binds to UV-irradiated polythymidylic acid but not unirradiated polythymidylic acid. This is the first demonstration of the use of phage display to select a ScFv recognizing DNA damage. In addition, this is the initial step towards immortalizing the antibody gene for genetic manipulation, structure–function studies and application to human investigations.     

Late Quaternary paleoenvironments in the southwestern Kalahari

Landforms of the southwestern Kalahari in southern Africa include linear dune systems, pans and associated lunette dunes and the valleys of ephemeral rivers. Present day climates in the region are transitional from arid to semi-arid, making the area sensitive to climatic change.

The linear dunes are part of an extensive system of fixed dunes developed in the surface Kalahari sands. The dunes were in existence prior to 32,000 yr B.P., but have been reactivated on a number of occasions since then in conditions of increased wind velocities, more NW winds and reduced rainfall, especially in northern and eastern parts of the region. Periods of dune activity probably occurred at 19,000-17,000, 10,000-6000 and 4000-3000 yr B.P.

The development of pans in the region has been strongly influenced by changes in groundwater conditions. Deflation from pans and formation of lunettes occurred in periods when groundwater levels were higher and fluctuated seasonally more than they do today. Such periods probably occurred 25,000–20,000 yr B.P., around 10,000 yr B.P. and just prior to 1000 yr B.P. Still higher groundwater discharges, promoted by increased regional rainfall, resulted in the formation of spring tufas and seasonal or permanent lakes in the pans, which were also fed by surface runoff, during the intervals between 33,000–28,000; 23,000–21,000; 17,000–15,000 and 12,000–11,000 yr B.P.

The headwaters of the major ephemeral streams lie outside the region, but within the same climatic zone. Increased flow in the Molopo River occurred prior to 19,000, and between 15,000 and 13,000 yr B.P.     

Mimicking the membrane-mediated conformation of dynorphin A-(1-13)-peptide: circular dichroism and nuclear magnetic resonance studies in methanolic solution.

The structural requirements for the binding of dynorphin to the kappa-opioid receptor are of profound clinical interest in the search for a powerful nonaddictive analgesic. These requirements are thought to be met by the membrane-mediated conformation of the opioid peptide dynorphin A-(1-13)-peptide, Tyr1-Gly2-Gly3-Phe4-Leu5-Arg6-Arg7-Ile8-Arg9-Pro10- Lys11-Leu12-Lys13. Schwyzer has proposed an essentially alpha-helical membrane-mediated conformation of the 13 amino acid peptide [Schwyzer, R. (1986) Biochemistry 25, 4281-4286]. In the present study, circular dichroism (CD) studies on dynorphin A-(1-13)-peptide bound to an anionic phospholipid signified negligible helical content of the peptide. CD studies also demonstrated that the aqueous-membraneous interphase may be mimicked by methanol. The 500- and 620-MHz 1H nuclear magnetic resonance (NMR) spectra of dynorphin A-(1-13)-peptide in methanolic solution were sequence-specifically assigned with the aid of correlated spectroscopy (COSY), double-quantum filtered phase-sensitive COSY (DQF-COSY), relayed COSY (RELAY), and nuclear Overhauser enhancement spectroscopy (NOESY). 2-D CAMELSPIN/ROESY experiments indicated that at least the part of the molecule from Arg7 to Arg9 was in an extended or beta-strand conformation, which agreed with deuterium-exchange and temperature-dependence studies of the amide protons and analysis of the vicinal spin-spin coupling constants 3JHN alpha. The results clearly demonstrated the absence of extensive alpha-helix formation. chi 1 rotamer analysis of the 3J alpha beta demonstrated no preferred side-chain conformations.     

Modulation of the bilayer to hexagonal phase transition of phosphatidylethanolamines by acylglycerols

The effect of mono-, di- and triacylglycerols on the bilayer to hexagonal phase (HII) transition was studied by differential scanning calorimetry and 31P-NMR spectroscopy. The acylglycerols were mixed with either dielaidoylphosphatidylethanoline or with 1-palmitoyl-2-oleoylphosphatidylethanolamine. Acylglycerols of lauric, oleic and stearic acids were utilized. All of the acylglycerols lowered the bilayer to HII phase transition temperature. Diacylglycerols were much better HII phase promoters than monoacylglycerols while triacylglycerols were the most potent bilayer phase destabilizers. Fatty acid composition generally had less of an effect except for the monoacylglycerols where bilayer destabilization increased from monolaurin to monostearin to monoolein. The most marked difference in behaviour resulting from changes in the fatty acid composition of the acylglycerol occurred with tristearin. This was the only acylglycerol which decreased the bilayer to HII phase transition temperature only below a mol fraction of 0.005. Above this mol fraction, further addition of tristearin had no effect on the bilayer to HII phase transition. These results suggest that the tristearin has limited solubility in phosphatidylethanolamine.     

Beta-thalassemia disease prevention: genetic medicine applied

We report here an evaluation of a program for thalassemia-disease prevention, comprising education, population screening for heterozygotes, and reproductive counseling; the evaluation includes cost analysis. A preprogram survey in 1978 of 3,247 citizens in the high-risk communities (85% were high-school students) showed that 88% favored a program but that only 31% considered fetal diagnosis as an acceptable option. Screening in high school or before marriage was preferred by 56%. In a 25-month period (December 1979-December 1982), we screened 6,748 persons, including 5,117 senior high-school students, using MCV/HbA2 indices. The participation rate was 80% in the high-school group. The frequency for beta-thalassemia heterozygosity was 4.7% with 10-fold variation among ethnic groups at risk; the overall frequency for all variants found was 5.4%. We surveyed 60 carriers and 120 noncarriers after screening high-school students (response rate 77%): most carriers told parents (95%) and friends (67%) the test result; and 38% of the carriers' parents (vs. 18% of the noncarriers' parents) were also screened. Carriers would ascertain their spouses' genotype (91%) and approved uniformly (95%) the high-school screening experience and its goal. We performed 11 fetal diagnoses in a 25-month interval (greater than 75% participation in target population) either by fetoscopy and globin-chain analysis or by amniocentesis and genomic DNA analysis; two of three affected fetuses were aborted at parental request, there was one spontaneous abortion (after fetoscopy), and seven live births. The at-risk couples claimed pregnancy would not be contemplated without the fetal-diagnosis option. We analyzed economic costs of the program: cost per case prevented is approximately equal to $ 6,700, slightly less than cost-per-patient-treatment-year or about 4% of undiscounted treatment cost incurred in the first 25 years of life for an affected individual. These findings indicate: collective acceptance of the program, appropriate attitudes among carriers, general acceptance and efficacy of fetal diagnosis, and global cost-effectiveness.     

A comparison between formol saline and Mirsky's fluid as fixatives for nematodes

In order to evaluate the fixing properties of Mirsky's fluid on nematodes, a comparison was made with formaldehyde. On two occasions, nematodes recovered from the abomasa of calves were fixed in each of three of the following fluids: 5% formol saline, 10% formol saline, 5% Mirsky's fluid and 10% Mirsky's fluid. Based on measurements of length and microscopical examination of over 800 nematodes on each occasion, the authors concluded that Mirsky's fluid conferred no advantages over 5% formol saline or 10% formol saline for fixing nematodes.     

Prolidase deficiency: biochemical classification of alleles

Prolidase (E.C.3.4.13.9) is a homodimeric enzyme encoded at a locus on chromosome 19. Prolidase deficiency is an autosomal recessive disorder with a highly variable clinical phenotype. We purified prolidase to homogeneity from normal human fibroblasts, raised a monospecific rabbit antiserum, and studied biosynthesis of the subunit in normal and prolidase--deficient fibroblasts. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized and retained in cytosol as a 58-KDa polypeptide. Three types of mutations were identified in six prolidase-deficient cell strains; half conferred a CRM-negative phenotype, while the CRM-positive mutations were of two types, one mutation encoding an enlarged subunit (60 KDa) and the others associated with subunits of normal size. Complementation analysis indicated that these mutations map to one locus. Normal subjects and obligate heterozygotes expressing CRM-negative mutations had thermostable prolidase activity at 50 degrees C in cell extracts, whereas heterozygotes expressing CRM-positive mutations had thermolabile activity under the same condition, implying negative allelic complementation in the putative heterodimer. The occurrence of prolidase-like activity about 5% of normal in amount but with a preference for substrate different from normal, in cells homozygous (or compound) for CRM-negative mutations, identified an alternative cleavage activity not encoded at the prolidase locus. Allelic heterogeneity at the major locus and the amount of alternative peptidase activity encoded elsewhere appear to be determinants of the associated and heterogeneous clinical phenotype.