Enzyme immunoassay of gastrin releasing peptide (GRP)-like immunoreactivity in milk

A sensitive and specific enzyme immunoassay (EIA) for gastrin releasing peptide (GRP)-like immunoreactivity was developed using enzyme-labeled antigen. The synthetic carboxy-terminal fragment of human GRP(12-27) was conjugated with beta-D-galactosidase for EIA. The minimum amount of GRP-like immunoreactivity detectable by this method was 0.24 femtomol/well (6 picomol/liter). The level of GRP-like immunoreactive substance in bovine foremilk was about 150 nanomol/liter, the level of which was more than hundredfold higher than that in normal milk or calf serum.     

Melanocyte-Directed enzyme prodrug therapy (MDEPT): development of second generation prodrugs for targeted treatment of malignant melanoma.

Evaluation of second generation prodrugs for MDEPT, by oximetry, has highlighted structural properties that are advantageous and disadvantageous for efficient oxidation using mushroom tyrosinase. In particular, a sterically undemanding prodrug bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28 was synthesised and found to be oxidised by mushroom tyrosinase at a superior rate to tyrosine methyl ester, the carboxylic acid of which is the natural substrate for tyrosinase. The more sterically demanding phenyl mustard prodrugs 9 and 10 were oxidised by mushroom tyrosinase at a similar rate to tyrosine methyl ester. In contrast, tyramine chain elongation via heteroatom insertion was detrimental and the rate of mushroom tyrosinase oxidation of phenyl mustard prodrugs 21 and 22 decreased by 10 nanomol/min.     

Toxicology of cupric salts on honeybees IV — gluconate and sulfate action on hemolymph trehalose activity in vivo and in vitro

A biphasic increase of hemolymph glucose levels was observed following injection to bees of cupric gluconate or sulfate, both potent agents for the control of Varroa jacobsoni, a parasitic mite of hives. The simultaneous injection to bees of 0.3 μM BAYg5421 (an inhibitor of α-glucosidases) quenched the response, suggesting a direct effect of 2 nmol/bee cupric ions on trehaloses' activity. One nanomol of injected cupric gluconate increased the trehalose (Tre) activity by 233% in crude hemolymph extracts at 1 mM trehalose concentration, and exhibited biphasic dose-related effects with a maximum 15% increase at 0.5 mM cupric ion and a stabilized 20% inhibition from 4 mM, regardless of the anionic moiety. Upon partial purification of the enzyme complex, two fractions (FI = 75% and FII = 25% of total activity) were isolated that exhibited, respectively, less and more marked positive cooperativity than crude extract. Form I showed almost no susceptibility to either cupric derivatives, which indicated form II as the most likely target, with 68% and 72% increases with 0.25 mM cupric sulfate and 0.5 mM cupric gluconate, in presence of 16 mM trehalose.     

Microwave assisted synthesis of novel tetrazole/sulfonamide derivatives based on octahydroacridine,xanthene and chromene skeletons as inhibitors of the carbonic anhydrases isoforms I,II, IV and VII

The synthesis of novel tetrazole/sulfonamide derivatives based on octahydroacridine, xanthene and chromene scaffold by using microwave (MW) assisted techniques is reported in this study. These synthesized hybrid compounds were assayed for the inhibition of carbonic anhydrase (CA, EC 4.2.1.1). The inhibitory activities were determined against three cytosolic human isoforms (hCA I, II and VII) and one membrane-associated (hCA IV) isoform. Some of the newly synthesized sulfonamides showed micromolar to nanomolar inhibitory activity against these enzymes.     

Synthesis and biological evaluation of 2-(3',4',5'-trimethoxybenzoyl)-3-N,N-dimethylamino benzo[b]furan derivatives as inhibitors of tubulin polymerization

Molecules that target microtubules have an important role in the treatment of cancer. A new class of inhibitors of tubulin polymerization based on the 2-(3,4,5-trimethoxybenzoyl)-2-dimethylamino-benzo[b]furan molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. The most promising compound in this series was 2-(3,4,5-trimethoxybenzoyl)-3-dimethylamino-6-methoxy-benzo[b]furan, which inhibits cancer cell growth at nanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.     

Purification and properties of the catalytic subunit of the branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney mitochondria

The catalytic subunit of the branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase (Damuni, Z., Merryfield, M.L., Humphreys, J.S., and Reed, L.J., (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4335-4338) has been purified over 50,000-fold from extracts of bovine kidney mitochondria. The apparently homogeneous protein consists of a single polypeptide chain with an apparent Mr = approximately 33,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BCKDH phosphatase, with an apparent Mr = 460,000, was dissociated to its catalytic subunit with no apparent change in activity, at an early stage in the purification procedure by treatment with 6 M urea. The specific activity of the catalytic subunit was 1,500-2,500 units/mg. The catalytic subunit exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex but was inactive with phosphorylase a and with p-nitrophenyl phosphate. The catalytic subunit, like the Mr = 460,000 species, was inhibited by nanomolar concentrations of BCKDH phosphatase inhibitor protein, was unaffected by protein phosphatase inhibitor 1 and inhibitor 2, and was inhibited by nucleoside tri- and diphosphates but not by nucleoside monophosphates.     

The isolation and characterization of sphingomyelinase from human placental tissue.

Human placental sphingomyelinase activity was eluted as a single symmetrical peak from Sephadex G-200 with a molecular weight of 290000; however, the enzyme behaved heterogeneously on ion exchange chromatography. A specific species of sphingomyelinase was purified approx. 10 000-fold to a constant specific activity of 274 000 nanomol of sphingomyelin hydrolyzed per mg protein per h. When the purified enzyme was examined on sodium dodecyl sulfate disc gel electrophoresis, two distinct protein bands in approximately equal proportions with molecular weights of 36 800 and 28 300 were found. The specificity of the enzyme is directed towards both the hydrophilic phosphocholine and the hydrophobic ceramide moieties of sphingomyelin. Possible interrelationships between the heterogenous forms of placental sphingomyelinases are discussed.     

Design of amphiphilic protein maquettes: enhancing maquette functionality through binding of extremely hydrophobic cofactors to lipophilic domains

We demonstrate coordination of the extremely hydrophobic 13(2)-OH-Ni-bacteriochlorophyll (Ni-BChl) to the lipophilic domain of a novel, designed amphiphilic protein maquette (AP3) dispersed in detergent micelles [Discher et al. (2005) Biochemistry 44, 12329-12343]. Sedimentation velocity and equilibrium experiments and steady-state absorption spectra indicate that Ni-BChl-AP3 is a four-helix bundle containing one Ni-BChl axially ligated by one or two histidines. The nature of the ligation was pursued with ultrafast visible spectroscopy. While it is well established that light excitation of axially ligated mono- and bisimidazole Ni-BChl in solution leads to rapid imidazole dissociation and nanosecond recombination, there is no evidence of axial ligand dissociation in the light-excited Ni-BChl-AP3. This indicates that Ni-BChl is confined within the AP3 protein, ligated to histidines with severely restricted mobility. Dissociation constants show that Ni-BChl binding to AP3 is considerably weaker than the nanomolar range usual for heme and hydrophilic (HP) maquettes; moreover, there is a tendency for the Ni-BChl-AP3 four-helix bundles to dimerize into eight-helix bundles. Nevertheless, the preparation of the Ni-BChl-AP3 four-alpha-helix maquettes, supported by time-resolved spectroscopic analysis of the nature of the ligation, provides a viable new approach to AP maquette designs that address the challenges involved in binding extremely hydrophobic cofactors.     

Developmental Biochemistry of Cottonseed Embryogenesis and Germination: VIII. Free Amino Acid Pool Composition during Cotyledon Development

The composition of the free amino acid pool in embryonic cotton (Gossypium hirsutum) cotyledons is quite distinct from that of endosperm, and that of germinated, greened cotyledons is quite distinct from that of leaves. During germination (including the precocious germination of immature seeds), the pool expands considerably showing a pronounced accumulation of asparagine. The high level of asparagine found in seedling roots and in the cotyledon vascular exudate indicates that this is the major transported amino acid in germination. There is no pool expansion in the presence of abscisic acid. In the presence of actinomycin D, the pool expands, but an enormous accumulation of glutamine takes place. The composition of the pool at any stage is not related to the composition of the isoacceptor transfer RNA pool, nor to the composition of the storage protein. Anaerobiosis leads to an accumulation of aspartate, alanine, and glycine at the expense of asparagine; however, desiccation does not result in an accumulation of proline. Conspicuously high levels of arginine are maintained through embryogenesis and germination. The levels of individual amino acids are presented as nanomol per cotyledon pair and as per cent of total pool.     

Serum carboxypeptidase B: A spectrophotometric assay using protamine as substrate

A quantitative colorimetric assay for serum carboxypeptidase B (SCPB, anaphylatoxin inactivator, kininase I) is described. SCPB is known to possess an enzymatic specificity for cleaving COOH-terminallysyl and arginyl residues which is similar to the specificity of bovine pancreatic carboxypeptidase B. One function of SCPB involves the inactivation of C3a and C5a, the two complement derived anaphylatoxins. Since cobalt markedly enhances the activity of the enzyme, serum is treated with CoCl₂ before the SCPB assay is performed. Salmine, a protamine from salmon sperm, was selected as the substrate because it contains multiple COOH-terminal arginyl residues and is digested more rapidly by SCPB than other common substrates of carboxypeptidase B, including hippuryl-arginine and benzyl-glycylarginine. The kinetics for arginine release from salmine were first-order throughout the course of the assay and the colorimetric values obtained were related to micromols of arginine released. A unit of SCPB is defined as one nanomol of arginine released per minute per milliliter of serum. The range of SCPB activity in serum from healthy individuals was found to be 318 to 466 units. The medians of SCPB activity in sera obtained from patients with Dengue shock syndrome and with shock following intravenous dextran infusion were both lower than the mean SCPB activity of healthy individuals. SCPB levels in patients homozygous and heterozygous for cystic fibrosis were within the normal range.     

A novel form of dependency of hepatic extraction ratio of opioids in vivo upon the portal vein concentration of drug: Comparison of morphine,diamorphine, fentanyl,methadone and buprenorphine in the chronically cannulated cow

In the chronically cannulated cow, the hepatic extraction ratio for intravenous boluses of morphine, diamorphine, fentanyl, methadone and buprenorphine increased towards a plateau value as portal vein drug concentration increased. An extraction ratio close to zero for morphine was observed at a portal vein plasma drug concentration of about 200 nanomol per litre, which is within the range for significant pharmacodynamic effects. The similar concentrations extrapolated for the other narcotics would be of less pharmacodynamic importance. The phenomenon did not depend with morphine on the history of drug delivery to the liver; measurement of hepatic blood flow showed the effect was not an artifact of unrepresentative blood sampling, and was not related to any action of the narcotics on hepatic blood flow. The existence of this novel type of concentration dependent hepatic extraction ratio in vivo can explain a number of anomalous observations on narcotic pharmacokinetics, especially for morphine. Furthermore, similar behaviour may be expected for non-opioid drugs having similar pharmacokinetic properties.     

Mechanism of DNA binding by the ADR1 zinc finger transcription factor as determined by SPR

The ADR1 protein recognizes a six base-pair consensus DNA sequence using two zinc fingers and an adjacent accessory motif. Kinetic measurements were performed on the DNA-binding domain of ADR1 using surface plasmon resonance. Binding by ADR1 was characterized to two known native binding sequences from the ADH2 and CTA1 promoter regions, which differ in two of the six consensus positions. In addition, non-specific binding by ADR1 to a random DNA sequence was measured. ADR1 binds the native sites with nanomolar affinities. Remarkably, ADR1 binds non-specific DNA with affinities only approximately tenfold lower than the native sequences. The specific and non-specific binding affinities are conferred mainly by differences in the association phase of DNA binding. The association rate for the complex is strongly influenced by the proximal accessory region, while the dissociation reaction and specificity of binding are controlled by the two zinc fingers. Binding kinetics of two ADR1 mutants was also examined. ADR1 containing an R91K mutation in the accessory region bound with similar affinity to wild-type, but with slightly less sequence specificity. The R91K mutation was observed to increase binding affinity to a suboptimal sequence by decreasing the complex dissociation rate. L146H, a change-of-specificity mutation at the +3 position of the second zinc finger, bound its preferred sequence with a slightly higher affinity than wild-type. The L146H mutant indicates that beneficial protein-DNA contacts provide similar levels of stabilization to the complex, whether they are hydrogen-bonding or van der Waals interactions.     

Effect of adenosine receptor blockade with caffeine on sympathetic response to handgrip exercise in heart failure

Adenosine (Ado) increases muscle sympathetic nerve activity (MSNA) reflexively. Plasma Ado and MSNA are elevated in heart failure (HF). We tested the hypothesis that Ado receptor blockade by caffeine would attenuate reflex MSNA responses to handgrip (HG) and posthandgrip ischemia (PHGI) and that this action would be more prominent in HF subjects than in normal subjects. We studied 12 HF subjects and 10 age-matched normal subjects after either saline or caffeine (4 mg/kg) infusion during isometric [30% of maximal voluntary contraction (MVC)] and isotonic (10%, 30%, and 50%) HG exercise, followed by 2 min of PHGI. In normal subjects, caffeine did not block increases in MSNA during PHGI after 50% HG. In HF subjects, caffeine abolished MSNA responses to PHGI after both isometric and 50% isotonic exercise (P < 0.05) but MSNA responses during HG were unaffected. These findings are consistent with muscle metaboreflex stimulation by endogenous Ado during ischemic or intense nonischemic HG in HF and suggest an important sympathoexcitatory role for endogenous Ado during exercise in this condition.     

Fruit production of shrub, Caragana korshinskii, following above-ground partial shoot removal: mechanisms underlying compensation

A number of studies have showed that under some conditions plant may partially, fully or overcompensate for tissue loss, however, the mechanisms underlying compensation are not well understood and still need to be researched. We examined the ability of Caragana korshinskii to compensate for fruit production after above-ground partial shoot removal. Fruit production of 30% main shoot length removal (30% RSL) and 25 and 50% main shoot number removal (25% RSN, 50% RSN) resulted in overcompensation and the response of 60% main shoot length removal (60% RSL) was full compensation. Plants’ responses associated with compensation included (1) greater reproduction efficiency (RA); (2) increased fruit set; (3) decreased fruit abortion; (4) increased seed number per pod; and (5) higher individual seed biomass. These responses may have resulted from more nectar production per flower, more sucrose flux per pod and more sucrose flux per seed of clipped plants, which may in turn have resulted from (1) drawing upon more non-structural carbohydrate (TNC) from roots to supply flower bud development and the flush of new foliage; (2) supplying more photosynthetic assimilation to fruit development owing to increases in leaf-level photosynthetic rates. Increases in leaf-level photosynthetic rates may be caused by more nutrient (nitrogen) and water availability per unit area of resource leaves after clipping.     

Elevated levels of polyneuronal innervation persist for as long as two years in reinnervated frog neuromuscular junctions

When the nerve to an adult frog sartorius muscle is crushed, and axons are allowed to regenerate, the level of polyneuronal innervation at reinnervated neuromuscular junctions is higher than normal. With time, much of this polyneuronal innervation is reduced by the process of synapse elimination (Werle and Herrera, 1988). Using intracellular recording, we estimated the level of polyneuronal innervation in adult frog (Rana pipiens) sartorius muscles 2 years (range: 1.7-2.4 years) after crushing the sartorius nerve. We found that 27% (S.E. = 1.4%) of the junctions in muscles 2 years after reinnervation were polyneuronally innervated, whereas only 10% (S.E. = 1.2%) of the junctions in normal frog muscles were polyneuronally innervated. Thus, the synapse elimination that occurs following reinnervation does not restore the normal level of polyneuronal innervation. Histological comparisons of junctional structure between muscles 2 years after reinnervation and normal muscles revealed substantial differences. Reinnervated junctions had a greater length of synaptic gutter apposed by nerve terminal processes, more axonal inputs, more empty synaptic gutter, more instances of single synaptic gutters innervated by more than one axon, and longer lengths of nerve terminal processes that connect synaptic gutters within a junction. On the basis of this physiological and anatomical evidence, we conclude that nerve injury causes persistent changes in the pattern of muscle innervation.     

Sperm penetration in vitro into ovarian and tubal oocytes from mice of the inbred KE and C57 strains

The method of in vitro fertilization was applied to test a previous suggestion that the lowered fertilizability of the tubal oocytes of female KE strain mice and the high resistance of their zona pellucida to proteolytic enzymes, are due to the premature cortical reaction taking place near the time of ovulation. Therefore higher fertilizability of ovarian oocytes is expected. The effectiveness of F₁ hybrid sperm penetration into ovarian and tubal KE oocytes confirmed these assumptions. The ovarian KE oocytes recovered 9–10 hours after administration of human chorionic gonadotropin (HCG) showed significantly higher penetrability (70–83%) than did the tubal oocytes recovered 12 hours after HCG (about 50%) and 14–16 hours after HCG (20%). Similar results were obtained with C57 oocytes. Sperm penetration into ovarian oocytes (10 hours after HCG) was much more effective (67%) than into tubal oocytes (18%); this finding correlated with more rapid zona dissolution by chymotrypsin. On the basis of these results one might speculate that premature cortical reaction takes place also in the C57 strain.     

Removing of Disinfection By-Product Precursors from Surface Water by Using Magnetic Graphene Oxide

The magnetic graphene oxide (MGO) was successfully synthesised by the in situ chemical co-precipitation method with Fe³⁺, Fe²⁺ and graphene oxide (GO) in laboratory and, was used as an adsorbent for disinfection by-product (DBP) precursors removing from four natural surface water samples. The results indicate that various DBPs formation significantly decreased by 7–19% to 78–98% for the four samples after MGO treatment and, the treatment process was rapidly reached equilibrium within 20 minutes. The DBP precursors removal efficiency decreased with the increasing pH value from 4 to 10. Hydrophobic compounds (humic acid and fulvic acid) are more sensitive to MGO, whereas hydrophilic and nitrogenous compounds (aromatic proteins) are more insensitive. MGO could be regenerated by using 20% (v/v) ethanol and, the DBP precursors removal efficiency can stay stable after five cycles. These results indicate that MGO can be utilized as a promising adsorbent for the removal of DBP precursors from natural surface water.     

Cleavage of proteins of reproductive secretions by extracellular proteinases of Tritrichomonas foetus

Cleavage of host defense proteins from reproductive secretions was investigated as a potential virulence mechanism for Tritrichomonas foetus extracellular proteinases. Three categories of susceptibility to digestion were found among the defense proteins tested. Cleavage of fibrinogen, fibronectin, and albumin occurred rapidly with more than 50% of these digested within 30 min. Lactoferrin, immunoglobulin G1, and immunoglobulin G2 were more than 50% digested after 4 h. Transferrin, immunoglobulin M, and immunoglobulin A were the most resistant to the Tritrichomonas foetus extracellular proteinases, since 50% or more of the parent molecule remained after 24 h. The responsible proteinases were classified as cysteine (thiol) proteinases because cleavage was inhibited by the cysteine proteinase specific inhibitor, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and not by the serine proteinase specific inhibitor, phenylmethylsulfonyl fluoride. In addition, alpha 2-macroglobulin, but not alpha 1-antitrypsin, inhibits the action of the proteinases. The ratio of this naturally occurring inhibitor to the quantity of proteinases released may determine whether the above substrates are cleaved in vivo. Since these substrates are implicated in iron acquisition, cell adherence, and acquired immunity, Tritrichomonas foetus proteinases are likely to play a role in host-parasite interactions.