PURIFICATION OF PROTEIN CARBOXYMETHYLASE FROM OX BRAIN

Abstract— The enzyme protein carboxymethylase from the soluble fraction of ox brain was purified to electrophoretic homogeneity. Brain protein carboxymethylase activity was also detected in a membrane-bound form which could only be solubilized by treatment with detergent. The solubilized membrane-bound form differed from the 'native' soluble form in that the former irreversibly lost activity on removal of the detergent. The two forms, however, have several similarities, having a molecular weight of 35,000, a K _m of 2.7 × 10⁻⁶ M for S -adenosyl-L-methionine, and a pH optimum of 6.2 when ovalbumin was used as the methyl acceptor.     

A sensitive enzyme-linked immunosorbent assay for dihydrofolate reductase.

A competitive enzyme-linked immunosorbent assay (ELISA) has been developed for the enzyme, dihydrofolate reductase. The sensitivity of the assay system approximates 3 ng of immunoreactive enzyme protein which is comparable to the sensitivity achieved in the radioimmunoassay (RIA) for this enzyme. The mean coefficient of variation for within--and between--assay precision was less than 13%. The assay appears to be specific and valid as the concentration of the enzyme is the same whether measured by ELISA or [3H]-methotrexate binding. Since this method, like the RIA, measures the mass concentration of enzyme protein, in conjunction with a [3H]-methotrexate binding assay it will be useful for quantitating functional as well as non-functional immunoreactive forms of the enzyme.     

Species diversity of freshwater hyphomycetes in some streams of Pakistan. Comparison of sampling techniques

Freshwater hyphomycete communities in four streams were sampled by filtration of water samples, trapping conidia in artificial foam, and examination of alder leaf pack baits and random sampling of naturally occurring submerged leaves. No two communities of freshwater hyphomycetes detected by different sampling techniques used singly in a stream showed 100% similarity. Based on relative frequency values, the same 10 top-ranking species in a stream were detected by artificial foam trap and on the baited leaves. These species differed in ranking, and the community in artificial foam was riche than on baited leaves. Of the various communities detected by different sampling techniques used singly or in combination, those detected by artificial foam trap or a combination of this technique with leaf baiting or with leaf baiting and randomly sampled leaves or filtration showed the highest similarity to the total community based on data generated using four sampling techniques simultaneously.This work was conducted under Grant No. PSF Project Environment 3 from the Pakistan Science Foundation.     

1H N.M.R. studies of protected alpha-aminoisobutyric acid containing peptides. Chemical shift nonequivalence of benzyloxycarbonyl methylene protons

The benzylic methylene protons in a large number of benzyloxycarbonyl alpha-aminoisobutyric acid (Z-Aib) containing peptides, show chemical shift nonequivalence. The magnitude of the geminal nonequivalence is correlated with the involvement of the urethane carbonyl group, in an intramolecular hydrogen bond. Studies of the model compounds Z-Aib-Aib-Ala-NHMe, and Z-Aib-Aib-Aib-Pro-OMe clearly establish the presence of intramolecular hydrogen bonds, involving the urethane CO group. In both compounds marked anisochrony of the benzylic methylene protons is demonstrated. In Z-Aib-Aib-Pro-OMe, where a 4 leads to 1 hydrogen bonded beta-turn is not possible, the benzylic-CH2-protons appear as a singlet in CDCl3 and have a very small chemical shift difference in (CD3)2SO. The observation of such nonequivalence is of value in establishing whether the amino terminal Aib-Pro beta-turn is retained in large peptide-fragments of alamethicin.     

Regeneration of whole limbs from shank stumps in toad tadpoles treated with vitamin A

Summary Young tadpoles of the toad,Bufo melanostictus (Schneider), were immersed in 15 IU/ml vitamin-A palmitate solution for 3 days, only prior to amputation through the shank. In more than 65% of cases the resultant regenerates were whole limbs containing the skeletal elements from femur to phallanges; in several of them a new girdle had also differentiated. In others regenration had progressed only up to the blastema stage and postblastemic development was inhibited. Opposite results were obtained when treatment was extended to another 3 days after amputation. A normal control-type regenerate consisting of parts distal to amputation level was not obtained in any case treated in either manner. The removed distal part of the shank was not restored in any treated case. It appears that, if suitably administered, vitamin A can make the limb regeneration blastema of amphibians completely equivalent to the original limb bud, probably by intensifying dedifferentiation of its cells. It is suggested that this chemical can be a useful tool to investigate the biochemical and genetic changes which occur during dedifferentiation and also whether through this process differentiated cells can really revert to a pluripotent state.     

Serendipity in discovery of proteasome inhibitors

Among its various catalytic activities, the 'chymotrypsin-like' activity of the proteasome, a large multicatalytic proteinase complex has emerged as the focus of drug discovery efforts in cancer therapy. Herein, a series of first generation (2S, 3R)-2-amino-3-hydroxybutyric acid derived proteasome inhibitors that were discovered serendipitously en route to original goal of generating a series of sterically constrained oxazoline derivatives has been reported.     

Wake-promoting agents: search for next generation modafinil: part I

In search of a next generation molecule to the novel wake promoting agent modafinil, a series of bi-phenyl derived wakefulness enhancing agents (in rat) was developed. From this work, compound 17 has been selected for additional studies.     

Anticoccidial activity of herbal complex in broiler chickens challenged with Eimeria tenella

The objective of the present study was to evaluate the anticoccidial effect of different concentrations of the herbal complex of 4 plants (leaves of Azadirachta indica and Nicotiana tabacum, flowers of Calotropis procera and seeds of Trachyspermum ammi) in broiler chickens in comparison with amprolium anticoccidial. Three concentrations (2 g, 4 g and 6 g) of herbal complex were given to the experimental groups once a day and amprolium (at the dose rate of 125 ppm) was given orally in drinking water from the 14th to the 21st days of age. One group was kept as infected, non-medicated control and one as non-infected, non-medicated control. All groups were inoculated orally with 75,000 sporulated oocysts on the 14th day of age except the non-infected, non-medicated control. Among herbal complex medicated groups, the maximum anticoccidial effect was seen in the group medicated with 6 g herbal complex followed by 4 g and 2 g herbal complex medicated groups. Treatment with 6 g of the herbal complex significantly reduced the negative performance and pathogenic effects associated with Eimeria tenella challenge at a level that was comparable with amprolium when using a largely susceptible recent field isolate. In summary, concentration-dependent anticoccidial activity of the studied herbal complex suggests its use as an alternative anticoccidial agent to chemotherapeutic drugs for Eimeria tenella control.     

Vitamin C Prevents Hypogonadal Bone Loss

Epidemiologic studies correlate low vitamin C intake with bone loss. The genetic deletion of enzymes involved in de novo vitamin C synthesis in mice, likewise, causes severe osteoporosis. However, very few studies have evaluated a protective role of this dietary supplement on the skeleton. Here, we show that the ingestion of vitamin C prevents the low-turnover bone loss following ovariectomy in mice. We show that this prevention in areal bone mineral density and micro-CT parameters results from the stimulation of bone formation, demonstrable in vivo by histomorphometry, bone marker measurements, and quantitative PCR. Notably, the reductions in the bone formation rate, plasma osteocalcin levels, and ex vivo osteoblast gene expression 8 weeks post-ovariectomy are all returned to levels of sham-operated controls. The study establishes vitamin C as a skeletal anabolic agent.     

Luminal iron levels govern intestinal tumorigenesis after Apc loss in vivo

It is clear from epidemiological studies that excess iron is associated with increased risk of colorectal cancer; however, questions regarding the mechanism of how iron increases cancer risk, the source of the excess iron (circulating or luminal), and whether iron reduction represents a potential therapeutic option remain unanswered. In this study, we show that after Apc deletion, the cellular iron acquisition proteins TfR1 and DMT1 are rapidly induced. Conversely, restoration of APC reduces cellular iron due to repression of these proteins. To test the functional importance of these findings, we performed in vivo investigations of the impact of iron levels on intestinal tumorigenesis. Strikingly, depletion of luminal (but not systemic) iron strongly suppressed murine intestinal tumorigenesis, whereas increased luminal iron strongly promoted tumorigenesis. Taken together, our data definitively delineate iron as a potent modifier of intestinal tumorigenesis and have important implications for dietary iron supplementation in patients at high risk of colorectal cancer.