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After subcutaneous or intraperitoneal injection of 14C-pentachloro-phenol (14C-PCP) in the mouse (specific activity 1.6 μci/mg, dose 15–37 mg/kg body weight) the distribution of the activity in the body was determined by whole-body autoradiography and by analysis of the individual organs after oxidation to 14CO2. The highest specific activity was found in the gall bladder and its contents, the wall of the stomach fundus, the contents of the gastro-intestinal tract and the liver. This shows that there is both a gastric and a biliary secretion of PCP and/or its metabolites, and excretion in the faeces. Most of the activity (72–83 %) was excreted in the urine in four days, about half of the dose in 24 hours. Only traces (< 0.05 %) were detected in the expired air. Identification and estimation of metabolites were performed by paper chromatography and isotope dilution techniques. All the urinary activity in the first 24 hours was due to PCP and probably tetrachlorohydroquinone. At least PCP was excreted in both the free and the conjugated form.

The study of the structure of the peptides arising from native, oxidized, reduced and alkylated or citraconylated equine growth hormone on incubation with trypsin and chymotrypsin is reported. The data obtained, combined with results already published, support the assembly of a unique sequence of amino acids for the polypeptide chain of the protein.

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).

The esterification of Ribonuclease-A in methanol/0.1 M hydrochloric acid has been studied by measuring the decrease in the number of titratable groups of the protein and estimating the amount of methanol incorporated. Esterification of nearly five of the 11 free carboxyl groups of the protein resulted in almost complete inactivation of the enzyme. The initial products of esterification have been chromatographed on Amberlite columns, and five partially active methyl ester derivatives of Ribonuclease-A have been isolated. The dimethyl ester, the initial product of esterification with reduced catalytic activity, has the carboxyl groups of Glu-49 and Asp-53 modified. Even in the non-aqueous solvent, as in the native structure of the protein in aqueous solution, these carboxyl groups are the fast reacting ones. Subsquently, the esterification reaction appears to proceed preferentially at the C-terminal region of the molecule. Comparison of the reactivities of carboxyl groups of Ribonuclease-A in acidic methanol to that known in aqueous solutions (with carbodiimides) suggests that the structure of Ribonuclease-A in the non-aqueous solvent resembles, at least in part, the structure in aqueous environment.

Hemoglobin was prepared from blood of adult animals of slow loris, and furthermore a and β polypeptide chains of the globin were separated by column chromatography. These two kinds of polypeptide chains were respectively digested with trypsin. The tryptic peptides thus obtained were isolated and purified by column chromatography and paper chromatography, and then each amino acid composition was analyzed.

Hydroxylysine was detected in hydrolysates of the inner and outer membranes of the shell of the hen's egg by ion-exchange and paper chromatography. N-ɛ-DNP-hydroxy-lysine was detected by paper chromatography of hydrolysates of dinitrophenylated membranes. Complete amino acid analysis showed hydroxylysine to be the least abundant of the known amino acids in the membranes (1-2 moles/1(fig protein).

A method for the preparation of human STH* was developed on the basis of Wilhelm's technique from 1961 which was simplified, by for example, introducing gel filtration. The purity of the preparation was established by a large number of purity criteria: zone electrophoresis on starch, zone electrophoresis in a density gradient, determination of N- and C-terminal end groups, determination of the N-terminal amino acid sequence, quantitative amino-acid analysis, determination of molecular weight by gel filtration (mol. wt.: 2.2×104), etc. Oxidation of human STH causes polymerization. The alteration of the degree of polymerization of the oxidized product was studied at varying pH levels. The molecular weight of the preparation at the highest degree of polymerization was found to be 45×104.

All component prepared from adult chicken hemoglobin was separated into α and β polypeptide chains by column chromatography. Of the two chains, the a polypeptide chain was digested with trypsin. The soluble tryptic peptides thus obtained were isolated and purified by column chromatography and paper chromatography. On the other hand, the a polypeptic chain in AH component was digested with chymotrypsin. The chymotryptic peptides thus obtained were isolated and purified by column chromatography and paper chromatography the same way as in the case of tryptic peptides. The amino acid compositions of these tryptic and chymotryptic peptides were analyzed.

BACKGROUND Some preparations of 99mTc-colloidal rhenium sulfide (Nanocis) contain excess free pertechnetate (99mTcO4) impurity (>5%). OBJECTIVES To improve the radiochemical purity of Nanocis preparations and the quality of preoperative lymphoscintigraphy in sentinel node localization of breast cancer patients, we investigated the effects of temperature on the presence of free 99mTcO4 and nanocolloid size modification. METHODS A Nanocis kit was reconstituted with sodium pertechnetate (650-850 MBq) in a final volume of 3.5 ml and heated for 30 min at 100, 115, or 130 degrees C. The radiochemical purity was determined by paper chromatography, in triplicate. The particle size was evaluated by membrane filtration through a 200-nm and 100-nm filter. The preoperative lymphoscintigraphy images were acquired about 2 h after tracer administration. RESULTS Significantly higher radiochemical purity values were observed with 28 Nanocis preparations heated at 130 degrees C (median: 99.8%, min-max: 97.0-99.9%) compared with values from 37 Nanocis preparations heated at 100 degrees C (median: 96.3%, min-max: 85.2-99.5%) or 26 Nanocis preparations heated at 115 degrees C (median: 95.1%, min-max: 85.7-99.8%). The interbatch variations of the radiochemical purity were reduced at 130 degrees C. A high temperature level (130 degrees C) did not modify the particle size. In lymphoscintigraphy, free 99mTcO4 uptake by the thyroid or stomach, which was sometimes observed with a Nanocis preparation heated at 100 or 115 degrees C, was never visualized with a Nanocis preparation heated at 130 degrees C. CONCLUSION These results indicate that increasing temperature from 100 to 130 degrees C can be used in routine clinical practice to improve the radiochemical purity of the Nanocis preparation.

The sub-cellular feruloylation and oxidative coupling sites of cell wall polysaccharides were investigated in planta by monitoring the kinetics of appearance of arabinosyl- and feruloyl-radiolabelled polysaccharides in the protoplasmic compartment and their secretion in the wall either in the presence or absence of brefeldin A (BFA). By using root apical segments excised from wheat seedlings (Triticum durum Desf.), incubated with trans-[U-(14)C]cinnamic acid, we demonstrated that [14C]ferulate, likely [14C]diferulate, as well as trimers and larger products of ferulate are incorporated into the protoplasmic polysaccharides very rapidly within 1-3 min of [14C]cinnamate feeding. This agrees with the assumption that (glucurono)arabinoxylans [(G)AX] feruloylation and oxidative coupling occur intracellularly, likely in the Golgi apparatus. Simultaneously, polymer bound radioactive hydroxycinnamic acids appeared to be incorporated into the cell wall of root apical segments as early as 2 min after trans-[U-(14)C]cinnamic acid feeding. On the contrary, starting from L-[1-(14)C]arabinose as tracer, the secretion of the pentose-containing polymers into the wall was between 5 to 10 min. These results indicated that (G)AX feruloylation and oxidative coupling occur both intra-protoplasmically and in muro. The occurrence of in muro feruloylation and oxidative coupling was confirmed by the use of BFA a well known inhibitor of secretion. The drug caused a strong inhibition of the synthesis and secretion into the wall of the 14C-pentosyl-labelled polymers as well as of 14C-feruloyl-polymers. In spite of this, the total amount of 14C-feruloyl-polymers incorporated into the wall was only slightly affected by BFA. This indicates the existence of a mechanism involved into secretion of the activated hydroxycinnamoyl precursors to the wall, alternative to that involved in polysaccharide secretion.

We present the results of a study of the porphyrin-forming enzymes in the erythrocytes of a recently detected case of CBP, in the immediate family, and in the family of a second cousin who died in infancy with a clinical picture similar lo that seen in the present patient. Except for a moderate increase in the activity of the uroporphyrinogen synthetase in the patient's lysates no patological changes have been found either in the patient or in any of the other persons studied. Our findings do not exclude the possibility that impaired activity of the isomerase (uroporphyrinogen-III-cosynthetase) plays a role, but they do indicate that this is not the only or even the most important metabolic error in the present type of erythropoictic porphyria. A study of the kinship between the two families seems to make a simple autosomal recessive in. heritance unlikely, hut does not exclude dominant inheritance by a rare single gene with low penetrance carried by the fathers. It is possible that the present case may represent a type of porphyria variegata in which the metabolic defect in porphyrin biosynthesis is located not in the liver hut in the bone marrow.

Aspartylglucosaminuria (AGU) is a genetic lysosomal storage disorder which probably affects the metabolism of glycoproteins. Earlier studies have shown a deficiency of a lysosomal hydrolase, N-aspartyl-β-glucosaminidase in the serum and seminal fluid, as well as in the brain and liver tissues of the patients. The present studies demonstrated a very low activity of N-aspartyl-β-glucosaminidase in cultured skin fibroblasts from AGU patients. The fibroblasts of the parents of the patients had a moderately low enzyme activity when conipared with control cultures. Thus, demonstration of the enzyme defect in fibroblasts offers possibilities both for detection of heterozygotes and for prenatal diagnosis of AGU.

We have prepared a new substrate from a keratan sulfate-derived-oligosaccharide (2-acetamido-2-deoxyglucosyl-(1--3)-[1-3H] Galactitol), which is necessary to measure beta-N-acetylhexosaminidase activity. This substrate was prepared from a cornea keratan sulfate by digestion with endo-beta-galactosidase, followed by isolation of disaccharide on gel filtration chromatography and chemical desulfation. Using this substrate, we found that a striking deficiency of beta-N-acetylhexosaminidase activity was present in the skin fibroblasts of patients with Sandhoff disease but not in Tay-Sachs disease. Both beta-N-acetyl-hexosaminidase A & H contributed to the catabolism of keratan sulfate.

In an optimalised radiochemical procedure for simultaneous measurement of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) and adenine phosphoribosyl transferase (A-PRT) from individual human hair roots, the HG-PRTIA-PRT activity ratio was used as an index for heterozygote detection in the Lesch-Nyhan syndrome. Hair roots of female carriers of HG-PRT deficiency could be distinguished from those of normal and mutant individuals. Evidence is presented that temperature at which hair roots are frozen prior to the enzyme assays affects the HG-PRTIA-PRT activity ratio The ratio tends to be higher after lower freezing temperatures.

As a first step for the development of biosensing inks for inexpensive paper-based biodetection, we prepared paper strips printed with carboxylic poly( N-isopropylacrylamide) microgels that were modified either with an antibody or with a DNA aptamer. We found that the antibody and the DNA aptamer retained their recognition capabilities when coupled to microgel. The printed microgel remains stationary during chromatographic elution while the microgel-supported molecular recognition elements are accessible to their intended targets present in the elution solution. Our work indicates that microgels, large enough to isolate the biosensors from the paper surface, are sufficiently hydrophilic to be wetted during chromatographic elution, exposing the gel-supported affinity probes to their targets.

This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper- PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.

Many nucleic acid enzymes and aptamers have modular architectures that allow them to retain their functions when combined with other nucleotide sequences. This modular function facilitates the engineering of RNAs and DNAs that have more complex functions. We sought to create new DNA aptamers that bind cellulose to provide a module for immobilizing DNAs. Cellulose has been used in a variety of applications ranging from coatings and films to pharmaceutical preparations, and therefore DNA aptamers that bind cellulose might enable new applications. We used in vitro selection to isolate aptamers from a pool of random-sequence DNAs and subjected two distinct clones to additional rounds of mutagenesis and selection. One aptamer (CELAPT 14) was chosen for sequence minimization and more detailed biochemical analysis. CELAPT 14 aptamer variants exhibit robust binding both to cellulose powder and paper. Also, an allosteric aptamer construct was engineered that exhibits ATP-mediated cellulose binding during paper chromatography.

Paper spray (PS), as a new ambient ionization method, has been applied for direct qualitative and quantitative analysis. The high sensitivity and minimum internal energy (low spray voltage) with optimized paper spray conditions is a significant request for real application in POCT. In this study, a simple and efficient ambient ionization method is developed by spraying from a mesoporous graphene foams (MGFs)-modified paper surface. The good electrical conductivity of MGFs results in obvious spray voltage decrease. Meanwhile, the MGFs-paper substrate has a well improvement in separation and elution efficiency ascribing to ultrahigh specific surface area and π-π electrostatic stacking property of graphene. In combination a commercial triple quadrupole mass spectrometer, the paper spray is successfully used for analysis of amphetamine in saliva. The linear dynamic ranges expand 10 fold in comparison with unmodified chromatography papers and the low limit of quantitation (LOQ) is as low as 1 pg/mL. A small sample volume (0.5 μL) could be analyzed immediately after spotting, without any pretreatment. The performance of this method was demonstrated for application in fast point-of-care mass spectrometry.

The transpeptidase (TP) activity of penicillin-binding proteins (PBPs), target of the β-lactam antibiotics, is a well-validated antibacterial drug target. The TP activity of PBP1b converts un–cross-linked peptidoglycan to the cross-linked form. Directly measuring TP activity is difficult because cross-linked and un–cross-linked peptidoglycan have very similar chromatographic properties. The authors report a microdilution plate method to directly measure the TP enzyme activity, uncoupled from the transglycosylase (TG), for detection of TP inhibitors. Escherichia coli membranes were incubated with 100 mM ampicillin, followed by removal of unbound ampicillin. The substrate for the TP, un–cross-linked peptidoglycan, was prepared by incubating these membranes with peptidoglycan sugar precursors, 1 of which was radiolabeled. Subsequently, solubilized PBP1b was added and TP activity assayed. The cross-linked peptidoglycan formed was monitored by addition of wheat germ agglutinin scintillation proximity assay beads plus N-laurylsarcosine, which selectively captures cross-linked peptidoglycan. The PBP1bcatalyzed activity was inhibited by penicillin G but not by cephalexin or cephradine, which have higher affinity for PBP1a. Moenomycin, a TG inhibitor, also inhibited TP activity. Because this is a true enzyme assay, it has the potential to detect novel, non-β-lactam TP inhibitors and could lead to the discovery of new antibacterial agents. (Journal of Biomolecular Screening 2006:1005-1014)

A cytoplasmic peptide: N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where its active site resides. This cytoplasmic enzyme was first detected in 1993 by a simple, sensitive assay method using 14C-labeled glycopeptide as a substrate. The deglycosylation reaction by PNGase brings about two major changes on substrate the peptide; one is removal of the N-glycan chain and the other is the introduction of a negative charge into the core peptide by converting the glycosylated asparagine residue(s) into an aspartic acid residue(s). The assay method we developed monitors these major changes in the core peptide, and the respective changes were detected by distinct analytical methods: i.e., paper chromatography and paper electrophoresis. This chapter will describe the simple, sensitive in vitro assay method for PNGase.

In order to find the conditions under which Tc-99m-methylenediphosphonate (Tc-99m-MDP) and Tc-99m(V)-dimercaptosuccinate (Tc-99m(V)-DMSA) may become tumour-seeking agents, leaving healthy organs free from the radionuclide uptake, the solution chemistry of these radiopharmaceuticals was studied by paper chromatography and paper electrophoresis in distilled water, in physiological saline, in NAHCO3, and ascorbic acid solutions. Both radiopharmaceuticals are anionic in the radiopharmaceutical solution but get easily hydrolysed to form cationic Tc-99m species which concentrates in healthy bone and in some bone metastases. Tc-99m (V)-DMSA being more stable remains long in the blood pool giving undesirable presence of the radionuclide in lung, heart and kidneys, in addition to its reduced uptake in bone metastases and in some primaries. We are trying to eliminate these drawbacks of healthy organ uptake of Tc-99-m(V)-DMSA not only to get a clean scintigraphic image of the tumour with this radiopharmaceutical but to extend its formulation, thus obtained, to prepare radiopharmaceutical with Re-188, which is the higher homologue of Tc-99m, for systemic therapy of cancer. Essentially similar solution chemistry of both radiopharmaceuticals suggests that like Tc-99m-MDP, technetium-99m-dimercaptosuccinate is also a complex of tetravalent Tc-99m and not of pentavalent Tc-99m as so far supposed to be.

UV-absorbing substances were isolated from the translucent bracts of Rheum nobile, which grows in the alpine zone of the eastern Himalayas. Nine kinds of the UV-absorbing substances were found by high performance liquid chromatography (HPLC) and paper chromatography (PC) surveys. All of the five major compounds are flavonoids, and were identified as quercetin 3- O-glucoside, quercetin 3- O-galactoside, quercetin 3- O-rutinoside, quercetin 3- O-arabinoside and quercetin 3- O-[6"-(3-hydroxy-3-methylglutaroyl)-glucoside] by UV, 1H and 13C NMR, mass spectra, and acid hydrolysis of the original glycosides, and direct PC and HPLC comparisons with authentic specimens. The four minor compounds were characterised as quercetin itself, quercetin 7- O-glycoside, kaempferol glycoside and feruloyl ester. Of those compounds, quercetin 3- O-[6"-(3-hydroxy-3-methylglutaroyl)-glucoside] was found in nature for the first time. The translucent bracts of R. nobile accumulate a substantial quantity of flavonoids (3.3-5 mg per g dry material for the major compounds). Moreover, it was clarified by quantitative HPLC survey that much more of the UV-absorbing substances is present in the bracts than in rosulate leaves. Although the flavonoid compounds have been presumed to be the important UV shields in higher plants, there has been little characterisation of these compounds. In this paper, the UV-absorbing substances of the Himalayan R. nobile were characterised as flavonol glycosides based on quercetin.

The alginates from five species of brown algae from the Egyptian Red Sea coast, namely: Cystoseira trinode, Cystoseira myrica, Sargassum dentifolium, Sargassum asperifolium, and Sargassum latifolium, were isolated and their compositions and structures studied by 1H NMR spectroscopy. All the alginates studied contain more guluronic acid (G) than mannuronic acid (M) and have a homopolymeric block-type structure (eta

A procedure for the synthesis of a selective adsorbent for phosphate-containing biomolecules is described. The sorbent is based on Whatman chromatography paper, which is activated with epichlorohydrine, followed by the coupling of iminodiacetic acid to the active surface of the sorbent. The immobilized complex-forming chelating groups are saturated with ferric ions. The synthesized adsorbent is a counterpart to Chelating Sepharose and makes it possible to extend the use of immobilized ferric chelating groups for analytical purposes. It displays a high affinity towards compounds containing free terminal phosphate groups (phosphopeptides, nucleotides). The results of the binding experiments are compared to the corresponding data obtained with Chelating Sepharose gels.

A novel washing scheme following antigen-antibody reactions with analyte was used during construction of a fluorescent immunosensor to resolve the background problem in the lateral flow assay with human serum. An immuno-membrane strip was devised to simultaneously measure cardiac troponin I (cTnI), creatinine kinase-MB isoform (CK-MB), and myoglobin to diagnose acute myocardial infarction. This strip was then installed within a cartridge containing a built-in washing solution tank, which was used to supply the solution across the signal generation pad of the strip after the immune reactions. Such cross-flow washing was initiated by onset-signaling from the internal control and began to run automatically upon sample addition. Under optimal conditions, the immunosensor displayed a stably suppressed background baseline, enabling us to attain a low detection limit for cTnI (0.05 ng/mL) as well as favorable reproducibility for repetitive measurements (relative standard deviation10%). No interference was observed among the different complex formations at the respective analyte sites, and no artifacts were caused by sample matrices. We tested the performance relationship with the Pathfast reference system for positive serum samples (36 for cTnI, 58 for CK-MB, and 17 for myoglobin), and the correlation coefficients were0.98. This result suggests that the new immunosensor system based on two-dimensional chromatography can be used for clinical testing.

The structure of a hemicellulose B fraction (B-1) isolated from grape skins (Vitis vinifera) of the Palomino variety has been studied by methylation analysis, (1)H NMR and (13)C NMR spectroscopy, and partial acid hydrolysis. Hemicellulose B-1 appeared to be homogeneous by gel filtration with a weight-average molecular weight of 22 600. This polysaccharide is a linear xyloglucan chain composed of xylopyranosyl and glucopyranosyl residues linked by beta-(1--4) glycosidic bonds. Attached to this backbone, 4-O-methyl-D-glucuronopyranosyl acid, D-glucopyranosyl, and L-fucopyranosyl residues occur at position 2 in a ratio of one residue for every five units of xylose in the main chain, with D-xylopyranosyl residues attached at position 6 of glucose units in a ratio of one residue for every two glucose-derived moieties in the main chain.

188Re is one of the radioisotopes expected to emerge as useful for therapy. Development of new radiopharmaceuticals based on 188Re depends on the radiolabeling methods used, which would give stable complexes having predefined radiochemical properties and in vitro and in vivo stability. This paper has attempted to provide a perspective of 188Re-labeled monoclonal antibodies, their radiolabeling characteristics, methods for quality control of radioimmunoconjugates and in vitro stability for radioimmunotherapy of solid tumors. The direct method of 188Re radiolabeling of antibodies by reductive attachment of 188Re in which free sulfhydryl groups have been generated by reduction of the intramolecular S–S disulfide bonds has been shown to be a promising approach in particular. Moreover, excellent methods have been developed to test the radionuclide, radiochemical purity and stability of 188Re-radioimmunoconjugates using high performance liquid chromatography (HPLC) and paper chromatography.

Although intentional and unintentional rodenticide poisoning is common, most readily available agents are of relatively low acute toxicity. A four-year long epidemic of severe toxicity from rodenticide exposure continues among patients predominantly of Dominican descent living in New York City. This study characterizes the ongoing epidemic of acute cholinesterase inhibitor poisoning due to an illicit rodenticide and identifies its etiology.A prospectively collected case series of poisoned patients referred to the New York City Poison Control Center. The main outcome measures include the clinical characteristics upon presentation, antidotal and other therapeutic requirements, and patient outcome. Product analysis was performed with paper chromatography, gas chromatography/mass spectrometry, and high-performance liquid chromatography. A murine model assessing both clinical effect and cholinesterase activity was also performed.Thirty-five patients were referred following exposure to Tres Pasitos. Patients developed signs of cholinergic hyperactivity and many required high doses of atropine (10 mg) to control these symptoms. The source was identified as a rodenticidal compound sold illicitly in local groceries primarily within the Dominican community. Murine cholinesterase activity fell significantly following exposure to the rodenticide. High-performance liquid chromatography identified aldicarb, an extremely potent carbamate-type cholinesterase inhibitor, not licensed for rodenticidal use in this country.Illicit sale of undocumented compounds poses a substantial public health threat. Despite several public health interventions, the epidemic continues.

The aim of the study is to screen patients for homocystinuria with and without cataract and analyse for homocystine and methionine. Fifty-eight samples from 29 patients, i.e., plasma and urine collected after overnight fasting were analysed by the screening test for homocystine, and paper chromatography for homocystine and methionine. Out of 29 homocystinuric patients, 24 had cataract. Only one had appreciable amounts of methionine in his serum. He also had mental retardation as expected and belongs to Type I. The other types did not have methionine but had only homocystine. There was no mental retardation or ectopia lentis. So they belonged to Types II, III or IV. As there is excess methionine in Type I, with low cystine, cataract may be due to deficiency of cysteine and reduced glutathione and might be averted by suitable therapy, i.e., high cystine-low methionine diet with B6. In other types with low methionine, cataract may be due to decreased availability of amino acids for the synthesis of lens proteins; the treatment of choice should be B12, and folate with methionine.

Prior studies have shown that Onchocerca volvulus DNA can be detected in skin snips and in black flies after polymerase chain reaction (PCR) with primers specific for repeated "O-150" DNA sequences. We have adapted a paper chromatography hybridization assay (PCHA) to detect amplified O-150 DNA and compared this method to two established methods, namely agarose gel electrophoresis (AGE) and hybridization enzyme-linked immunosorbent assay (ELISA). The minimum amounts of purified O-150 DNA detected by PCHA, AGE, and ELISA were 5, 10, and 2 ng, respectively. The three methods had similar estimated sensitivities for detecting O. volvulus DNA amplified from skin snips from African subjects with onchocerciasis (88%, 84%, and 91%, respectively). No false positive results were observed with skin snips from uninfected control subjects. The paper chromatography hybridization assay detects PCR products in 30 minutes without electricity or special equipment. This technology brings DNA detection a step closer to widespread use in field settings.

Summary This study investigated the influence of zinc bacitracin on the intestinal flora of horses. The functionally active intestinal flora was examined in 6 horses during treatment with zinc bacitracin. Utilising gas chromatography, spectrophotometry, gel electrophoresis and paper chromatography, samples were analysed on biochemical markers reflecting the action of parts of the intestinal flora. The following 5 flora-related functions were studied in faecal samples and intestinal samples from different sections of the hindgut: conversion of cholesterol to coprostanol and of bilirubin to urobilinogens, degradation of mucin and of β-aspartylglycine and inactivation of tryptic activity. Conversion to coprostanol, conversion to urobilinogens and degradation of mucin were affected by treatment of zinc bacitracin and conversion to coprostanol was most sensitive. All functions were normalised in a short time, in contrast to man and rats. Differences in environmental exposures are probably the reason for a more rapid normalisation of the intestinal flora functions in horses.

A "quality control" system associated with the endoplasmic reticulum (ER) that discriminates between misfolded proteins and correctly folded proteins is present in a variety of eukaryotic cells, including yeast. Recently, it has been shown that misfolded proteins that are N -glycosylated in the lumen of the ER are transported out of the ER, de-N-glycosylated by a soluble peptide: N -glycanase (PNGase) and degraded by action of the proteasome. It also has been shown that small N -glycosylatable peptides follow a fate similar to that of misfolded proteins, i.e., glycosylation in the lumen of the ER, transport out of the ER, and de- N -glycosylation in the cytosol. These processes of retrograde glycopeptide transport and de- N -glycosylation have been observed in mammalian cells, as well as in yeast cells. However, little is known about the mechanism involved in the movement of glycopeptides from the ER to the cytosol. Here we report a simple method for assaying N -glycosylation/de- N -glycosylation by simple paper chromatographic and electrophoretic techniques using an N -glycosylatable(3)H-labeled tripeptide as a substrate. With this method, we confirmed the cytosolic localization of the de- N -glycosylated peptide, which supports the idea that de- N -glycosylation occurs after the export of the glycopeptide from the lumen of the ER to the cytosol. Further, we found that the variations in the structure of the oligosaccharide chain on the glycopeptide did not cause differences in the export of the glycopeptide. This finding suggests that the mechanism for the export of small glycopeptides may differ from that of misfolded (glyco)proteins.

Strains of Mucor pusillus and M. miehei were found to synthesize β-1,4-glucan glucanohydrolase (cellulase) in a complex medium. The inducible enzyme complex hydrolysed carboxymethylcellulose, acid-swollen cellulose and unmodified cellulose.

We have already reported that raw nori (Porphyra tenera) contains cobalamin (Cbl) but not Cbl analogues (J. Nutr. Sci. Vitaminol., 42, 497, 1996). It seems, therefore, that it is an excellent natural vegetable source of Cbl. On the other hand, it has been reported that the Cbl nutritional status of vegetarian children deteriorated as estimated by the hematological index, mean corpuscular volume (MCV), after they had dried nori as a source of Cbl. Such a discrepancy between raw and dried nori as a source of Cbl led us to investigate whether Cbl in dried nori had different properties from that in raw nori. We found that contents of Cbl homologues determined by a bioassay method in both raw and dried nori were similar. The urinary methylmalonic acid excretion increased when human female volunteers were given 40 g of dried nori daily during the test period. On the other hand, the urinary methylmalonic acid excretion did not change when volunteers were daily given 320 g of raw nori, which was equivalent to 40 g of the dried one on the basis of dehydrated weight, during the test period. By paper chromatography, 65% of the Cbl homologues were found to be comprised of Cbl analogues in dried nori, while 73% of the Cbl homologues in the raw nori were genuine Cbl. These results were confirmed by the finding that the bioassay method gave higher values for Cbl homologues than those obtained by a competitive binding assay method using an intrinsic factor as a Cbl-binding protein. Our present data demonstrated that Cbl in raw nori can be changed into harmful Cbl analogues by the drying process.

Recently the genes encoding the human and Escherichia coli GDP-mannose dehydratase and GDP-fucose synthetase (GFS) protein have been cloned and it has been shown that these two proteins alone are sufficient to convert GDP mannose to GDP fucose in vitro. GDP-fucose synthetase from E. coli is a novel dual function enzyme in that it catalyzes epimerizations and a reduction reaction at the same active site. This aspect separates fucose biosynthesis from that of other deoxy and dideoxy sugars in which the epimerase and reductase activities are present on separate enzymes encoded by separate genes. By NMR spectroscopy we have shown that GFS catalyzes the stereospecific hydride transfer of the ProS hydrogen from NADPH to carbon 4 of the mannose sugar. This is consistent with the stereospecificity observed for other members of the short chain dehydrogenase reductase family of enzymes of which GFS is a member. Additionally the enzyme is able to catalyze the epimerization reaction in the absence of NADP or NADPH. The kinetic mechanism of GFS as determined by product inhibition and fluorescence binding studies is consistent with a random mechanism. The dissociation constants determined from fluorescence studies indicate that the enzyme displays a 40-fold stronger affinity for the substrate NADPH as compared with the product NADP and utilizes NADPH preferentially as compared with NADH. This study on GFS, a unique member of the short chain dehydrogenase reductase family, coupled with that of its recently published crystal structure should aid in the development of antimicrobial or anti-inflammatory compounds that act by blocking selectin-mediated cell adhesion.

A reported paper chromatographic technique has been extended to delineate the hydrolysed components in 201Tl radiopharmaceuticals for the determination of radiochemical purity. Detailed radiochemical purity analysis of 201TlCl has been demonstrated using mini paper chromatographic systems. The technique is also useful in evaluation of the radiochemical purity of 67Ga and 111In radiopharmaceuticals.

A highly specific and sensitive diazotized sulfanilamide reagent is synthesized for determination of tea catechins. The reagent is employed both as spray reagent for selective visualization of tea catechins on two-dimensional paper chromatograms (sensitivity

The assessment of the hypothalamic-pituitary-adrenal axis in patients with chronic renal failure (CRF) on hemodialysis is often hampered by abnormal responses to the standard 1-mg dexamethasone suppression test. Various mechanisms have been proposed to explain this lack of suppressibility. The present study was designed to look into the mechanisms possible for these findings in patients with CRF. We studied 6 patients with CRF on hemodialysis and 5 healthy subjects utilizing the 1-mg dexamethasone suppression test as well as the 50-mg hydrocortisone suppression test. Samples were assayed for dexamethasone, adrenocorticotropic hormone, corticosterone, and cortisol by both direct radioimmunoassay (RIA) and RIA after paper chromatography. Utilizing the direct cortisol RIA, 4 of 6 patients with CRF exhibited blunted dexamethasone suppression, while all 6 patients showed normal suppressibility after dexamethasone when cortisol was measured after paper chromatography. In contrast, all controls showed normal suppressibility regardless of the cortisol assay procedure used. The hydrocortisone suppression test was unreliable in the setting of CRF. Mean dexamethasone levels were similar in both groups. Plasma adrenocorticotropic hormone levels were significantly higher in the CRF patients, possibly indicative of an underlying hypothalamic-pituitary-adrenal axis abnormality. Abnormalities in dexamethasone suppression testing in patients with CRF may be explained by the overestimation of cortisol levels by direct RIA rather than by alteration of dexamethasone absorption or metabolism. Measurement of cortisol after paper chromatography is superior to direct RIA of cortisol in patients with CRF.

Substitution of the oxidation-sensitive thiol function of mercaptoacetyltriglycine (MAG3) by a hydroxyl group yields a tetraligand (hydroxyacetyltriglycine or HAG3) which is almost insensitive to oxidation and has the advantage over MAG3 that it can be stored safely without protection of the alcohol function. We found that deprotected HAG3 could be directly labelled at alkaline pH (pH/=11.5) and room temperature in high yield (95%). Results of electrophoresis experiments suggested a comparable structure for 99mTc-HAG3 and 99mTc-MAG3, namely binding of an oxotechnetium(V)core via three deprotonated amides and a deprotonated hydroxyl group. Biodistribution studies in mice at 10 min and 30 min p.i. showed a slightly higher urinary excretion, a faster renal transit and a significantly lower hepatobiliary handling for 99mTc-HAG3 than for 99mTc-MAG3. In a baboon, the 1-h plasma clearance of 99mTc-HAG3 was clearly higher than that of 99mTc-MAG3. Its plasma protein binding was in the same order as that of Hippuran and much lower than that of 99mTc-MAG3. Evaluation in a human volunteer confirmed the favourable biological characteristics of 99mTc-HAG3, namely a rapid renal excretion, a high 1-h plasma clearance and a negligible hepatobiliary handling. The results indicate that 99mTc-HAG3 may be an easy-to-prepare and practical substitute for 99mTc-MAG3 with improved renal excretion characteristics.

Branched polylactosamines on animal cell surfaces are believed to contribute to multivalent interactions in cell adhesion and cell signalling. Their biosynthesis proceeds via linear precursors that become branched by beta1,6-GlcNAc transferases (IGnT6, GlcNAc to Gal). Previous work has identified the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (1) and the hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc (4) as acceptors for a rat serum enzyme activity (cIGnT6), which transfers GlcNAcbeta1-6 units to the midchain galactose residues. Thereby, 1 is converted to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc and 4 to the doubly branched octasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3(GlcNAcb eta1-6)Galbeta1-4GlcNAc [Leppänen, A., Salminen, H., Zhu, Y., Maaheimo, H., Helin, J., Costello, C. E.,Renkonen, O. (1997) Biochemistry 36, 7026-7036]. Here we report that neither the alpha1, 3-fucose-containing derivatives of 1 [Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)G lcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Gl cNAc] nor a similar derivative of 4 [Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)+ ++GlcNAcbeta1-3Galbeta1- 4GlcNAc] were acceptors for the rat serum cIGnT6 activity. Hence, the enzyme's branch-forming action was completely prevented at sites in the immediate neighborhood of the fucosylated loci of the polylactosamines. In Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, the inhibition of the branch-forming reaction was restricted to the fucose-carrying LacNAc unit; at the middle LacNAc, the branching proceeded normally. However, in the isomeric Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, the fucose residue prevented branching completely at the middle LacNAc and almost completely at the reducing end LacNAc. In summary, alpha1,3-fucose residues in polylactosamine chains inhibited the cIGnT6 reaction in a site-specific manner, at the fucosylated LacNAc unit itself and also at sites one and two LacNAc units upstream, but not at the LacNAc units downstream from the fucosylated locus. These data imply that site-directed branching in polylactosamines is possible in vitro with the aid of specifically positioned alpha1,3-fucosyl units, that can be removed afterward without harming the branched backbones.

The culture supernatant of a strain of Propionibacterium acnes was investigated for its phospholipase (PL) activity. The microorganism was isolated from a periodontal pocket of a patient with periodontal disease. Supernatants from cultures of this microorganism were used as a source to obtain enzymes. Proteins from the supernatants were concentrated, and their enzymatic activity was partially purified through molecular sieving. The procedure yielded two peaks of activity. This activity was shown to hydrolyze phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) and was effective in a pH range of 5 to 9, with an optimal activity at pH 7.0. Divalent cations were not required for activity of the enzymes. Analysis of the products obtained from the hydrolysis of PC labeled in the choline, phosphoryl, or acyl moieties and PE containing labeled oleic acid indicated that the supernatants' activity was mostly phospholipase C (PL-C). Phospholipase C can act synergistically with other factors to produce tissue damage, and hence may contribute to the pathogenesis of periodontal disease.

In this work, the high affinity iron-acquisition systems displayed by virulent and avirulent strains of Vibrio damsela have been investigated. This species is an autochthonous member of marine ecosystems that can behave as an opportunistic pathogen for fish and mammals. All strains tested (i) were able to grow under the restricted conditions imposed by the iron chelators transferrin (Tf) and EDDHA, (ii) secreted siderophores of hydroxamic type, other than aerobactin and desferal, that were able to stimulate the growth of the auxotroph mutant Arthrobacter flavescens JG9, and (iii) expressed common iron-regulated outer membrane proteins (IROMPs). No change in LPS patterns was observed in response to iron restriction. Results from the assays with transferrin suggest that these siderophores could be utilized to sequester iron from Tf, a protein for which no surface receptor was detected in any strain. In summary, the overall data demonstrate that V. damsela expresses siderophore-mediated iron-uptake systems. These systems are probably involved in the survival of the species in the different environments that it can colonize, i.e. water and several vertebrate hosts.

[(99m)Tc]pentetate ([(99m)Tc]DTPA) is the most commonly used radiopharmaceutical renography agent. The aim of this work was to validate an alternative method for assessing [(99m)Tc]DTPA radiochemical purity (RCP), according to the ICH Q2(R1) guidance: "Validation of Analytical Procedures". The proposed method is composed of two chromatographic systems. System A is a miniaturized system of thin layer chromatography (TLC) silica gel impregnated aluminum strips as stationary phase (SP) and distilled water as mobile phase (MP). System B consists of Whatman 1 paper strips as SP and methyl ethyl ketone as MP. Results indicate that the proposed RCP method has been validated, as it is specific, precise, accurate, linear and robust. Therefore, it can be used as an alternative method for RCP quality control purposes and as stability indicator as well.

Several Chinese hamster V79 cell line variants resistant to arsenite and one arsenite-hypersensitive variant have been isolated. The basis for the variation in arsenite sensitivity was studied by transport experiments using radiolabeled arsenite. Two arsenite-resistant variants (As/R7 and As/R27) exhibited decreased accumulation of arsenite, and the hypersensitive variant (As/S5) exhibited increased arsenite accumulation compared with the parental line. Cells depleted of endogenous energy reserves were loaded with radiolabeled arsenite, and the rate of arsenic efflux was measured. Arsenite-resistant variants exhibited an increased rate of efflux, while the hypersensitive variant exhibited a decreased efflux rate. Efflux was decreased in cells incubated with the protonophore carbonyl cyanide m-chlorophenylhydrazine, demonstrating its energy dependence. Two inhibitors of glutathione S-transferase also decreased arsenite efflux, suggesting the involvement of an arsenite-glutathione complex. However, separation of the products of extrusion and the intracellular arsenic species by paper chromatography followed by autoradiography failed to show the appearance of an arsenite-glutathione complex in either case. Rather, all label in the product of the transport reaction appeared to be arsenite whether cells were loaded with arsenate or arsenite, indicating first that intracellular reduction of As(V) to As(III) had occurred and second that the arsenite was transported as an unconjugated species. All intracellular label was associated with high-molecular-weight material, possibly protein. Our results demonstrate the existence of an energy-dependent arsenical efflux pump in mammalian cells and show that arsenic is extruded as arsenite.

We have previously found that the purified chondroitin 6-sulfotransferase (C6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine in chondroitin, catalyzed the sulfation of keratan sulfate, and that both the C6ST activity and the keratan sulfate sulfotransferase (KSST) activity were expressed in COS-7 cells when C6ST cDNA was transfected. In this report we describe some properties of the KSST activity contained in the purified C6ST, and characterize the sulfated products formed from keratan sulfate and partially desulfated keratan sulfate. Optimal pH, requirement for cationic activators, and Km value for PAPS of the KSST activity were very similar to those of the C6ST activity. 35S-Labeled glycosaminoglycans formed from keratan sulfate and partially desulfated keratan sulfate were N-deacetylated by treatment with hydrazine/hydrazine sulfate and then cleaved with HNO2 at pH 4, and the resulting products were reduced with NaB3H4. Analysis of the degradation products with paper chromatography and high performance liquid chromatography provided evidence that C6ST transferred sulfate to position 6 of galactose residue which was glycosidically linked to N-acetylglucosamine 6-sulfate residue or to N-acetylglucosamine residue. Northern blot analysis using poly (A)+ RNA from 12-d-old chick embryos indicated that the message of C6ST was expressed not only in the cartilage but also in the cornea in which keratan sulfate is actively synthesized.

A noninvasive or minimally invasive optical approach for theranostics, which would reinforce diagnosis, treatment, and preferably guidance simultaneously, is considered to be major challenge in biomedical instrument design. In the present work, we have developed an evanescent field-based fiber optic strategy for the potential theranostics application in hyperbilirubinemia, an increased concentration of bilirubin in the blood and is a potential cause of permanent brain damage or even death in newborn babies. Potential problem of bilirubin deposition on the hydroxylated fiber surface at physiological pH (7.4), that masks the sensing efficacy and extraction of information of the pigment level, has also been addressed. Removal of bilirubin in a blood-phantom (hemoglobin and human serum albumin) solution from an enhanced level of 77 μM/l (human jaundice >50 μM/l) to ∼30 μM/l (normal level ∼25 μM/l in human) using our strategy has been successfully demonstrated. In a model experiment using chromatography paper as a mimic of biological membrane, we have shown efficient degradation of the bilirubin under continuous monitoring for guidance of immediate/future course of action.