Role of physical activity in preventing and treating obesity.

There is an inverse relationship between physical activity and weight gain. However, additional research is needed to quantify the amount of physical activity required to prevent weight gain in different populations, improve the way we convey physical activity recommendations to the public, and help the individuals increase their physical activity. Although physical activity does not appear to contribute significantly to weight loss, it is critical for maintenance of weight loss. Available data are consistent in that 60-90 min/day of moderate-intensity physical activity is required to maintain a significant weight loss. Although there is agreement about the need for high levels of physical activity to maintain weight loss, there is a need for more research to understand why physical activity is critical for weight loss maintenance. Finally, additional research is needed to determine whether there is an optimal level of physical activity below which it is difficult for most people to achieve a balance between energy intake and expenditure at a healthy body weight. The increasing prevalence of obesity may reflect the fact that the majority of the population has fallen below such a level of physical activity.     

Properties of diacylglycerol kinase in adult and fetal rat lung

Diacylglycerol kinase activity is found in both adult and fetal lung. Approximately 27 and 52% of the total activity is found in microsomes and cytosol, respectively. The activity is maximal at pH 7.4. The apparent Km for ATP is 0.11 mM and 0.21 mM for cytosol and microsomes, respectively. The apparent Km for dioleoylglycerol is 0.05 mM for cytosol and 0.14 for microsomes. Maximal activity in cytosol and microsomes is obtained with 2.0 mM dexoycholate. Other detergents cannot substitute for deoxycholate. Phosphatidylglycerol stimulates activity in the absence and in the presence of deoxycholate. Phosphatidylserine also stimulates activity, whereas phosphatidylethanolamine was inactive and phosphatidylcholine inhibited the reaction. Linoleic acid produced inhibition. The general properties of the enzyme were similar for fetal and adult lung. Diacylglycerol kinase from microsomes and cytosol fraction from both fetal and adult lung was most active with dioleoylglycerol and diacylglycerol from egg phosphatidylcholine. Significantly lower activity was obtained with dipalmitoylglycerol. Phosphatidylglycerol did not alter the relative substrate preferences. The activity in microsomes increased with development from 19 days gestation to a maximal activity at 21 days gestation. Maximal activity was about 2-fold higher than the adult. The activity dropped rapidly reaching adult values prior to birth (22 days gestation). The activity in cytosol fractions increased gradually from 19 days gestation, reaching adult values by 22 days gestation.     

Myocardial aminoacyl-transfer-ribonucleic acid synthetase and aminoacyl-transferring enzyme activity

The properties of cytoplasmic aminoacyl-tRNA synthetase and aminoacyl-transferring enzymes in the myocardium were examined and methods for the assay of the activity of these enzyme systems were developed. Aminoacyl-tRNA synthetase activity was measured from the rate of incorporation of ¹⁴C-labelled amino acid into aminoacyl-tRNA. Transferase activity was measured from the rate of incorporation of amino[¹⁴C]acyl-tRNA into protein in the presence of a standard preparation of hepatic ribosomes. Aminoacyl-tRNA synthetase activity is labile once the heart has been homogenized, whereas transferase activity is stable. The source of energy for synthetase activity is ATP; that for transferase is GTP. Transferase activity was inhibited by puromycin and stimulated by dithiothreitol, whereas synthetase activity was unaffected.     

Diisopropylfluorophosphate-sensitive aryl acylamidase activity of fatty acid free human serum albumin

Butyrylcholinesterase in human plasma and acetylcholinesterase in human red blood cells have aryl acylamidase activity toward o-nitroacetanilide, hydrolyzing the amide bond to produce o-nitroaniline and acetate. People with a genetic variant of butyrylcholinesterase that had no detectable activity with butyrylthiocholine, nevertheless had aryl acylamidase activity in their plasma. To determine the source of this aryl acylamidase activity we tested fatty acid free human albumin for activity. We found that albumin had aryl acylacylamidase activity and that this activity was inhibited by diisopropylfluorophosphate. Since the esterase activity of albumin is also inhibited by diisopropylfluorophosphate, and since it is known that diisopropylfluorophosphate covalently binds to Tyr 411 of human albumin, we conclude that the active site for aryl acylamidase activity of albumin is Tyr 411. Albumin accounts for about 10% of the aryl acylamidase activity in human plasma.     

Exploratory activity in Drosophila requires the kurtz nonvisual arrestin

When Drosophila adults are placed into an open field arena, they initially exhibit an elevated level of activity followed by a reduced stable level of spontaneous activity. We have found that the initial elevated component arises from the fly's interaction with the novel arena since: (1) the increased activity is independent of handling prior to placement within the arena, (2) the fly's elevated activity is proportional to the size of the arena, and (3) the decay in activity to spontaneous levels requires both visual and olfactory input. These data indicate that active exploration is the major component of elevated initial activity. There is a specific requirement for the kurtz nonvisual arrestin in the nervous system for both the exploration stimulated by the novel arena and the mechanically stimulated activity. kurtz is not required for spontaneous activity; kurtz mutants display normal levels of spontaneous activity and average the same velocities as wild-type controls. Inhibition of dopamine signaling has no effect on the elevated initial activity phase in either wild-type or krz(1) mutants. Therefore, the exploratory phase of open field activity requires kurtz in the nervous system, but is independent of dopamine's stimulation of activity.     

Neuroregulation of nonexercise activity thermogenesis and obesity resistance

High levels of spontaneous physical activity in lean people and the nonexercise activity thermogenesis (NEAT) derived from that activity appear to protect lean people from obesity during caloric challenge, while obesity in humans is characterized by dramatically reduced spontaneous physical activity. We have similarly demonstrated that obesity-resistant rats have significantly greater spontaneous physical activity than obesity-prone rats, and that spontaneous physical activity predicts body weight gain. Although the energetic cost of activity varies between types of activity and may be regulated, individual level of spontaneous physical activity is important in determining propensity for obesity. We review the current status of knowledge about the brain mechanisms involved in controlling the level of spontaneous physical activity and the NEAT so generated. Focus is on potential neural mediators of spontaneous physical activity and NEAT, including orexin A (also known as hypocretin 1), agouti-related protein, ghrelin, and neuromedin U, in addition to brief mention of neuropeptide Y, corticotrophin releasing hormone, cholecystokinin, estrogen, leptin, and dopamine effects on spontaneous physical activity. We further review evidence that strain differences in orexin stimulation pathways for spontaneous physical activity and NEAT appear to track with the body weight phenotype, thus providing a potential mechanistic explanation for reduced activity and weight gain.     

Characterisation of the role of zinc in the hepatitis C virus NS2/3 auto-cleavage and NS3 protease activities

Cleavage of the hepatitis C virus polyprotein between the non-structural NS2 and NS3 proteins is mediated by a poorly characterised auto-proteolytic activity that maps to the C terminus of NS2 and the N terminus of NS3, but is distinct from the NS3 protease activity responsible for downstream cleavages in the polyprotein. We have exploited the fact that the minimal precursor (residues 904-1206 of the HCV polyprotein) can be expressed as an insoluble protein in Escherichia coli and subsequently refolded into a form active for both auto-cleavage and NS3 protease activity, to further characterise the NS2/3 auto-cleavage activity. We show that both activities are zinc-dependent and show an absolute requirement for cysteine residues 1123, 1125 and 1171 within NS3. In contrast cysteine 922 (within NS2) is only required for NS2/3 auto-cleavage activity and histidine 1175 is only required for NS3 activity. Although the complete NS3 protease domain (including the C-terminal alpha-helix) is required for NS2/3 auto-cleavage, the activity of the NS3 protease is not essential. Lastly we show that the NS2/3 auto-cleavage activity is more sensitive to zinc chelation by 1,10-phenanthroline than the NS3 protease activity. This observation is consistent with different conformations of the precursor competent for either NS2/3 auto-cleavage or NS3 protease activity; these two conformations can be distinguished by their relative strength and geometry of zinc coordination.     

Production of chemotactic activity in mixed leukocyte cultures: maximum effect caused by H-2I region disparity.

Primary mixed mouse leukocyte culture supernatants contain an activity chemotactic for mouse peritoneal exudate cells and it can be detected within 72 h after initiation of the culture. Disparity for H-21 region leads to maximum production of chemotactic activity whereas H-2K or H-2D region differences result in the production of significantly less activity. The rate of production of chemotactic activity follows closely the rate of incorporation of 3H-thymidine, and both attain the peak on day 4 after initiation of the culture. But whereas proliferation is sensitive to gamma-irradiation, chemotactic activity production is not. It is our hypothesis that proliferating cells are primarily responsible for the production of chemotactic activity. The possible relevance of chemotactic activity production to graft rejection is discussed.     

Native thrombocidin-1 and unfolded thrombocidin-1 exert antimicrobial activity via distinct structural elements

Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.     

DNA-binding activity associated with the v-myb oncogene product is not sufficient for transformation.

The product of the v-myb oncogene of avian myeloblastosis virus is a nuclear protein with an associated DNA-binding activity. We demonstrated that the highly conserved amino-terminal domain of p48v-myb is required for its associated DNA-binding activity. This activity is not required for the nuclear localization of p48v-myb. Furthermore, the associated DNA-binding activity and nuclear localization of p48v-myb together are not sufficient for transformation.     

Herpesvirus proteins: induction of nucleoside phosphotransferase activity after herpes simplex virus infection.

After herpes simplex virus infection of hamster kidney cells there is an induction of nucleoside phosphotransferase activity which can utilize AMP as phosphate donor. The activity is immunologically specific for the infected cell and is induced concomitantly with the virus-coded pyrimidine deoxynucleoside kinase activity. Phosphotransferase activity is not induced in cells lacking both thymidine and deoxycytidine kinase activity.     

Method for real-time detection of inorganic pyrophosphatase activity

A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.     

Role of water activity in ethanol fermentations

A separate role for water activity in the conversion of sugars to ethanol by two strains of yeast is identified. During fermentation of both single and mixed sugar substrates, the water activity was shown to remain constant during the logarithmic growth phase. This is despite the changes in concentration of substrates and product, the constancy reflecting the fact that the greater influence of ethanol on the solution activity is counterbalanced, in the early stages of the fermentation, by its low yield. The end of the log phase of growth coincides with the start of a period of gradually decreasing water activity. For the more ethanol-tolerant strain UQM66Y, growth was found to cease at a constant value of water activity while that for the less tolerant strain UQM70Y depended on both ethanol concentration and water activity. It is argued that water activity is a more appropriate variable than ethanol concentration for describing some of the nonspecific inhibitory effects apparent in ethanol fermentations. A straightforward method for the calculation of water activity during such fermentations based on the use of solution osmolality is presented.     

On hepatic and extrahepatic postheparin serum lipase activities and the influence of experimental hypercortisolism and diabetes on these activities.

This paper shows that the palmitoyl-CoA hydrolase activity of postheparin serum of the rat is mainly derived from the liver. The identity of this activity with the heparin-releasable hepatic triacylglycerol hydrolase activity is established. The consequences of the different substrate specificities of the hepatic and extrahepatic enzymes for the measurement of the overall postheparin serum lipase activity are discussed. Treatment of the rats with either a corticosteroid or with streptozotocin was found to lower the lipolytic activity from the liver and to enhance the extrahepatic activity. Also in human postheparin serum, palmitoyl-CoA hydrolase activity is shown to behave identical with hepatic triacylglycerol hydrolase activity. The possible function of the liver in the serum triacylglycerol metabolism is discussed in connection with the proposed mechanism for the role of extrahepatic lipoprotein lipase in atherogenesis.     

The inhibition of peroxidase and indole-3-acetic acid oxidase activity by British Anti-Lewisite

British Anti-Lewisite (BAL) binds to horseradish peroxidase in a manner which results in inhibition of both peroxidatic and oxidative functions of the enzyme. BAL competes with hydrogen peroxide for binding on peroxidase, and the inhibition of peroxidatic activity is irreversible. Solutions of purified horseradish peroxidase and individually resolved peroxidase isozymes show a gradual loss of peroxidatic activity with time when incubated with BAL. In these same treatments, however, the inhibition of indole-3-acetic acid (IAA) oxidase activity is immediate. With increasing amounts of enzyme in the incubation mixture, IAA oxidase activity is not completely inhibited and is observed following a lag period in the assay which shortens with longer incubation times. Peroxidase activity during this same time interval shows a lag period which increases with longer incubation times. Lowering the pH removed the lag period for oxidase activity, but did not change the pattern of peroxidase activity. These results suggest that the sites for the oxidation of indole-3-acetic acid and for peroxidatic activity may not be identical in horseradish peroxidase isozymes.     

Purine nucleoside phosphorylase (NP) of bovine erythrocytes: genetic control of electrophoretic variants

A genetic polymorphism of the purine nucleoside phosphorylase is described in red cells of cattle. Electrophoresis distinguishes two types, one associated with a high activity (H), the other with a low activity (L). Breeding tests suggest the segregation of two alleles: a dominant allele for high activity ( H ) and a recessive allele for low activity (L). On the basis of quantitative estimation of enzyme activity, the individuals of the H phenotype can be separated in two classes, the enzyme activity in one class being twice as high as the activity in the other class. It is suggested that the class with the highest enzymatic activity corresponds to the homozygote for the H allele, the other class to the heterozygote. In fact, an A.I. bull (H phenotype and with the highest enzymatic activity) mated with cows of the L phenotype, gave offspring of the H phenotype, only. The decline of activity, with age, is stressed. Four breeds have been typed. The Charolais breed is characterized by a high frequency of the H allele.     

A novel enhancing mechanism for hydrogen sulfide-producing activity of cystathionine beta-synthase

H2S is produced from cysteine by cystathionine beta-synthase (CBS) in the brain and functions as a neuromodulator. Although the production of H2S is regulated by Ca2+ and calmodulin in response to neuronal excitation, little is known about the molecular mechanism for the regulation in CBS activity. Here we show that four cysteine residues of CBS are involved in the regulation of its activity in the presence of Ca2+ and calmodulin. Sodium nitroprusside (SNP), a modifying agent for cysteine residues, enhances CBS activity, whereas N-ethylmaleimide, an alkylating agent for cysteine residues, completely abolished the effect of SNP. Site-directed mutagenesis of the 13 cysteine residues of CBS identified four cysteine residues that are involved in the regulation of CBS activity by SNP, and two of the four residues are involved in the regulation of the basal CBS activity. The enhancement of CBS activity by SNP is independent of nitric oxide production. In the presence of Staphylococcus aureus alpha-hemolysin, which permeabilizes the cell membrane, exogenously applied SNP enhances the activity of CBS in intact cells. The present study demonstrates a novel mechanism for the regulation of CBS activity and provides a possible therapeutic application of SNP for the diseases in which CBS activity is deficient.     

Defying the activity–stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability

The biotechnological applications of enzymes are limited due to the activity–stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity–stability trade-off can be overcome. In this review, the origin of the activity–stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity–stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel–Danson equilibrium model, catalytic antibodies, and life on cold planets.     

Binding of a cellular protein to the gibbon ape leukemia virus enhancer.

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.