Cathepsin D from horse spleen. I. Purification and study of certain physicochemical properties

Horse spleen cathepsin D (3.4.23.5.) was purified from crude extract by sodium chloride and ethanol precipitation, column chromatography fractionation on DEAE cellulose and CM Sephadex, re-chromatography on DEAE cellulose and gel filtration. The enzyme has been purified about 3.000 folds with a yield of 30 per cent. The purified enzyme seems to be homogeneous on Sephadex G100, one protein band is apparent on disc electrophoresis. Determined by dansylation the N-terminal amino acid is glycine. A molecular weight of 42,500 +/- 3,000 was obtained with Sephadex G100 gel filtration and light scattering measurements. Amino acid analysis and chemical determinations were performed: cathepsin D is a glycoprotein (2 or 3 osamine residues) including 344 amino acids and 4 disulfide bonds. Spectrophotometric data show that E1cm/1 mg/ml = 1.01 at lambda = 280 nm. ORD measurements indicate about 20 per cent of helicoidal content in the molecule.     

Presterilization interviewing: An evaluation.

The role of interviewing in diffusing possible harmful side effects of sterilization operations was evaluated in an acute general hospital. This hospital required an interview with a chaplain before either a vasectomy or a tubal-ligation operation. Two simultaneous field experiments were conducted with 50 vasectomy couples and 50 tubal-ligation couples. Couples applying for either operation were alternately assigned to either the interview or the control group. Questionnaires were mailed to all couples 6 mo after the operation. Results are based on the responses of 67 couples who followed through with the sterilization procedure. There were no significant differences between the interview and control groups. The vasectomy couples scored significantly higher than the tubal-ligation couples on 6 of 9 measures in the questionnaire. The wives in the vasectomy group had a significantly lower level than the wives in the tubal-ligation group. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Nitrogenase activity and respiration of cultures of Rhizobium spp. with special reference to concentrations of dissolved oxygen

Studies of nitrogenase in cultures of the cowpea rhizobia (Rhizobium spp.) strains 32H1 and CB756 are reported. Preliminary experiments established that, even when agar cultures were grown in air, suspensions of bacteria prepared anaerobically from them were most active at low concentrations of free dissolved O2. Consequently, assays for activity used low concentrations of O2, stabilized by adding the nodule pigment leghaemoglobin. In continuous, glutamine-limited cultures of 32H1, nitrogenase activity appeared only when the concentration of dissolved O2 in the cultures approached 1 muM. Lowering the glutamine concentration in the medium supplied to the culture from 2 to 1 mM halved the cell yield and nitrogenase activity was also diminished. Omitting succinate from the medium caused the concentration of dissolved O2 to rise and nitrogenase activity was lost. Upon restoration of the succinate supply, the O2 concentration immediately fell and nitrogenase was restored. The activity doubled in about 8 h, whereas the doubling time of this culture was 14 h. Sonic extracts of 32H1 cells from continuous cultures with active nitrogenase contained components reacting with antiserum against nitrogenase Mo-Fe protein from soybean bacteroids. Continuous cultures grown at higher O2 concentration, with only a trace of active nitrogenase, contained less of these antigens and they were not detected in highly aerobic cultures. Nitrogenase activity of a continuous culture was repressed by NH+4; the apparent half-life was about 90 min. Cells of 32H1 from a continuous culture growing at between 30 and 100 muM dissolved O2 possessed a protective mechanism which permitted respiration to increase following exposure to a rapid increase in O2 concentration from low levels (O2 shock). This effect disappeared as the O2 concentration for growth was reduced towards 1 muM.     

Substrate inhibition of beta-lactamases, a method for predicting enzymatic stability of cephalosporins

Selected cephalosporins, including cefamandole, cephaloridine, cephaloglycin, and cefoxitin, were examined for their ability to inhibit the enzymatic activity of and act as substrates for beta-lactamases produced by Enterobacter cloacae and Staphylococcus aureus. Enzyme inhibition was determined by Michaelis-Menten kinetic measurements and by a spot plate assay using a chromogenic substrate (Glaxo compound 87/312). These two methods provide comparable estimates of kinetic parameters. Inhibition of beta-lactamase, as measured by these two methods, was generally found to correlate with resistance to hydrolysis and is proposed as a preliminary method of assessing susceptibility of cephalosporins to beta-lactamase hydrolysis. Four 7-alphaOCH(3), 7-alphaH cephalosporin analogue pairs were also examined. The presence of the 7-alphaOCH(3) substituent invariably resulted in reduced susceptibility to enzymatic hydrolysis, regardless of the other C7 substituent. The 7-alphaOCH(3) compounds were also better inhibitors than were their 7-alphaH analogues, with the exception that 7-alphaOCH(3) compounds having C7 adipic acid substituents were less inhibitory to the S. aureus enzyme than were the corresponding 7-alphaH analogues. Response of these two enzymes to 7-alphaOCH(3) and 7-alphaH cephalosporins suggests that beta-lactamase hydrolysis of these compounds involves attack at the alpha side of the betalactam ring.     

Linking phosphorus sequestration to carbon humification in wetland soils by 31P and 13C NMR spectroscopy

Phosphorus sequestration in wetland soils is a prerequisite for long-term maintenance of water quality in downstream aquatic systems, but can be compromised if phosphorus is released following changes in nutrient status or hydrological regimen. The association of phosphorus with relatively refractory natural organic matter (e.g., humic substances) might protect soil phosphorus from such changes. Here we used hydrofluoric acid (HF) pretreatment to remove phosphorus associated with metals or anionic sorption sites, allowing us to isolate a pool of phosphorus associated with the soil organic fraction. Solution (31)P and solid state (13)C NMR spectra for wetland soils were acquired before and after hydrofluoric acid pretreatment to assess quantitatively and qualitatively the changes in phosphorus and carbon functional groups. Organic phosphorus was largely unaffected by HF treatment in soils dominated by refractory alkyl and aromatic carbon groups, indicating association of organic phosphorus with stable, humified soil organic matter. Conversely, a considerable decrease in organic phosphorus following HF pretreatment was detected in soils where O-alkyl groups represented the major fraction of the soil carbon. These correlations suggest that HF treatment can be used as a method to distinguish phosphorus fractions that are bound to the inorganic soil components from those fractions that are stabilized by incorporation into soil organic matter.     

Enhanced efficacy and sensory properties of an anti‐dandruff shampoo containing zinc pyrithione and climbazole

Dandruff is a common complaint and is suffered by as much as half of the population at some time post puberty. The condition is characterized by the presence of flakes on the scalp and in the hair, and is often accompanied by itch. The most common treatment for dandruff is the use of shampoo formulations that contain fungistatic agents such as zinc pyrithione (ZPT) and octopirox. Whilst most antidandruff shampoos are effective in resolving the symptoms of dandruff these shampoos can often result in hair condition that is less than acceptable to consumers which can lead to a tendency for them to revert to use of a non‐antidandruff shampoo. This can result in a rapid return of dandruff symptoms. The aim of this investigation was to study the impact of using a combination of antidandruff actives and silicones on the resolution of dandruff and to deliver superior sensory properties to the hair. We have demonstrated that shampoo containing the dual active system of ZPT/Climbazole deposits both active agents onto a model skin surface (VitroSkin) and reduces Malassezia furfur regrowth in vitro. Clinical evaluation of the dual active shampoo demonstrated superior efficacy and retained superiority during a regression phase where all subjects reverted to using a non‐antidandruff shampoo. We have also demonstrated that it is possible to deposit silicone materials from antidandruff shampoo uniformly over both virgin and damaged hair fibres that results in smoother hair fibres (as evidenced by reduced dry friction). This combination of antidandruff agents and conditioning silicones delivered from a shampoo provides subjects with superior antidandruff efficacy and desired end sensory benefits ensuring compliance and longer term dandruff removal.     

Meat goat kids finished on alfalfa,red clover or orchardgrass pastures: Carcass merit and meat quality

This experiment was conducted in 2005–2007 to evaluate carcass and chevon (goat meat) quality parameters when meat-goat kids (n = 72) were finished on alfalfa (Medicago sativa L; ALF); red clover (Trifolium pratense L.; RCG); or orchardgrass (Dactylis glomerata L.; OGR) pastures. Carcass conformation score was greater (P = 0.08) when meat-goat kids were finished on ALF compared to OGR with RCG intermediate. Chevon meat samples from goats finished on the three pasture treatments did not differ in ash, intramuscular fat, or crude protein content or in concentrations of omega6 and omega3 fatty acids, or the omega6 to omega3 ratio. Goats finished on OGR had higher (P < 0.001) 18:1 trans-11 fatty acids (FA) compared to ALF or RCG. Overall, meat-goat kids finished on ALF, RCG, or ORG produced desirable carcass weights for most niche markets in the USA. Chevon is a low-fat meat option with high desirable fatty acids for human diets.     

Relationship between proteolysis and angiotensin-I-converting enzyme inhibition in different cheeses

The inhibition of angiotensin-I-converting enzyme (ACE) by the ethanol (70%)-soluble fraction (ESF) from different cheeses was analysed with an extract from rabbit lung acetone powder as enzyme source and 2-furanacryloyl-1-phenylalanylglycylglycine (FAPGG) as substrate. Proteolysis was assessed by a spectroscopic o-phthaldialdehyde (OPA) assay of the pH 4.6-soluble fraction and ESF. Peptides in the ESF were separated by reverse phase-HPLC. The traditional Norwegian cheese Gamalost had per unit cheese weight higher ACE inhibition potential than Brie, Roquefort and Gouda-type cheese, likely due to the combination of highest protein content and most extensive proteolysis providing a high content of ACE inhibitory peptides. However, ACE-inhibition expressed as IC₅₀ per unit peptide concentration from ESF assessed by the OPA-assay was highest for Kesam, a Quark-type cheese with a low degree of proteolysis.