Synthetic substrates of vertebrate collagenase

The active site specificity of vertebrate collagenase was mapped with the synthesis of a variety of peptides, peptolides, and peptide esters. The enzyme was found to prefer very lipophilic sequences, and it was also found to be an esterase. The thio peptolide Ac-Pro-Leu-Gly-SCH[CH2CH(CH3)2]CO-Leu-Gly-OC2H5 was found to be an exceptional substrate. High-performance liquid chromatography and tandem mass spectrometry were used to unambiguously establish the cleavage site in several peptide substrates.     

Cleavage site specificity of vertebrate collagenases

A series of synthetic peptide substrates for vertebrate collagenase having the structure Ac-Pro-Leu-Gly-X-Leu-Gly-OC2H5, where X is Leu, Ile, Val, Phe and Ala, have been prepared. Collagenolytic enzymes from various sources cleave these substrates with differing relative rate patterns. This series of peptides should be valuable for characterization of collagenases.     

DNA restriction fragment analysis of the proopiomelanocortin gene in schizophrenia and bipolar disorders.

The method of DNA restriction fragment analysis using gene probes for the proopiomelanocortin (POMC) gene was employed to detect possible molecular variation in the POMC gene in schizophrenia and bipolar illness. No gross structural abnormalities in restriction fragments were observed with the set of restriction enzymes used. Two allelic restriction sites were observed giving rise to fragment length polymorphisms. One of these is a new polymorphism, not previously reported, which will be of value as a linkage marker. The associations between the two DNA polymorphisms that are closely linked to the POMC gene and both schizophrenia and bipolar disorder were investigated. No association was found, thus adding weight to the evidence that there are no alterations in the POMC gene in schizophrenia and bipolar illness.     

Effect of Soil Dose on Bioavailability of Lead from Mining Waste Soil in Rats

Abstract

The purpose of this study was to determine the extent of absorption of lead (Pb) in mining waste soil from Butte, Montana. It is the first study to fully investigate the bioavailability of lead in soils containing mine waste using a soil dose response approach. Young 7–8 week-old male and female Sprague-Dawley rats (5 animals/sex/group) were given mining waste soil [810 ppm lead (Test Soil I) or 3,908 ppm lead (Test Soil III)] mixed in a purified diet (AIN—76?) at four different dose levels (0.2, 0.5, 2 and 5% dietary soil) for 30 consecutive days. The test soil dose levels at 2 and 5% were chosen to bracket a pica-for-soil child's soil exposure levels. A pica-for-soil child is a young child who eats large quantities of soil (10 g day^?1). Standard groups included untreated controls and dosed feed soluble lead acetate groups (1, 10, 25, 100 and 250 μg Pb g^?1 feed). The concentrations of lead acetate were chosen to bracket the test soil dose levels of lead. Liver, blood and femur, representing the three compartments in which lead is distributed in the body, were analyzed for total lead concentration using graphite furnace atomic absorption spectroscopy. Clinical signs, body weight, food consumption and liver weights for treated and standard groups were similar to control. Tissue lead concentrations from test soil animals were significantly lower than the tissue concentrations for the dosed feed lead acetate group. Group mean whole blood, bone and liver lead concentrations increased with increasing dose levels for most treatment groups. The increases in blood, bone and liver lead concentrations were not proportional with increasing dose levels and plateaued at the high dose levels. Relative percent bioavailability values, based on dosed feed soluble lead as the standard, were independent of the two different test soils, dose levels or sex, and only slightly dependent on the tissue (blood > bone, liver). Overall relative percent bioavailability values were 20% based on the blood data; 9% based on the bone data; and 8% based on the liver data (2 and 5% dose levels only). The results of this study will provide the scientific validity needed to determine the significance of lead exposure from Butte soils in assessing human health risks as part of the Superfund Remedial Investigation/Feasibility Study process.     

Plasminogen activator and MIF-like activities in Kirsten virus transformed mouse NIH culture fluids.

Kirsten virus transformed mouse NIH cells produce both a macrophage migration inhibition activity for guinea pig and mouse peritoneal exudate cells and a plasminogen activator. The migration inhibition factor activity exhibited thermal stability up to 80°C while the plasminogen activator was inactivated after 15 minutes at 70°C. Separation of these activities was achieved by absorption of the migration inhibition activity on agarose-fucosamine or high speed centrifugation.     

Photon correlation spectroscopy of human IgG

The translational diffusion coefficient D _20,w ⁰ , of monomeric human immunoglobulin G (IgG) has been studied by photon-correlation spectroscopy as a function of pH and protein concentration. At pH 7.6, we find D _20,w ⁰ =3.89×10^–7±0.02 cm²/sec, in good agreement with the value determined by classic mehods. This value corresponds to an effective hydrodynamic radius R, of 55.1±0.3 Å. As pH is increased to 8.9; with the same ionic strength, the molecule appears to expand slightly (3.5% increase in hydrodynamic radius). The concentration dependence of the IgG diffusion constant is interpreted in terms of solution electrostatic effects and shows that long-range repulsive interactions are negligible in the buffer used. The diffusion coefficient for dimeric IgG has also been determined to be D_20,w=2.81×10^–7±0.04 cm²/sec at 1.6 mg/ml, which corresponds to a hydrodynamic radius of 75 Å. For light-scattering studies of protein molecules in the dimension range of 5–10 nm (M_r=10⁵–10⁷) we find monomeric horse spleen ferritin well suited as a reference standard. Ferritin is a spherical molecule with a hydrodynamic radius R of 6.9±0.1 nm and is stable for years in our standard Tris-HCl-NaCl buffer even at room temperature.     

Standing geographic variation in eclosion time and the genomics of host race formation in Rhagoletis pomonella fruit flies

Taxa harboring high levels of standing variation may be more likely to adapt to rapid environmental shifts and experience ecological speciation. Here, we characterize geographic and host‐related differentiation for 10,241 single nucleotide polymorphisms in Rhagoletis pomonella fruit flies to infer whether standing genetic variation in adult eclosion time in the ancestral hawthorn (Crataegus spp.)‐infesting host race, as opposed to new mutations, contributed substantially to its recent shift to earlier fruiting apple (Malus domestica). Allele frequency differences associated with early vs. late eclosion time within each host race were significantly related to geographic genetic variation and host race differentiation across four sites, arrayed from north to south along a 430‐km transect, where the host races co‐occur in sympatry in the Midwest United States. Host fruiting phenology is clinal, with both apple and hawthorn trees fruiting earlier in the North and later in the South. Thus, we expected alleles associated with earlier eclosion to be at higher frequencies in northern populations. This pattern was observed in the hawthorn race across all four populations; however, allele frequency patterns in the apple race were more complex. Despite the generally earlier eclosion timing of apple flies and corresponding apple fruiting phenology, alleles on chromosomes 2 and 3 associated with earlier emergence were paradoxically at lower frequency in the apple than hawthorn host race across all four sympatric sites. However, loci on chromosome 1 did show higher frequencies of early eclosion‐associated alleles in the apple than hawthorn host race at the two southern sites, potentially accounting for their earlier eclosion phenotype. Thus, although extensive clinal genetic variation in the ancestral hawthorn race exists and contributed to the host shift to apple, further study is needed to resolve details of how this standing variation was selected to generate earlier eclosing apple fly populations in the North.