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HVPE is an excellent and often overlooked method for obtaining objective and meaningful information about cell-wall "building blocks" and their metabolic precursors. It provides not only a means of analysis of known compounds but also an insight into the charge and/or mass of any unfamiliar compounds that may be encountered. It can be used preparatively or analytically. It can achieve either "class separations" (e.g., delivering all hexose monophosphates into a single pool) or the resolution of different compounds within a given class (e.g., ADP-Glc from UDP-Glc; or GlcA from GalA).All information from HVPE about charge and mass can be obtained on minute traces of analytes, especially those that have been radiolabeled, for example by in-vivo feeding of a

Agrocin 108 is a 3'-O-β-D-xylopyranosyl-cytidine-5'-O-phosphodiester of an ascorbate-carbocyclic cyclopentenone analogue, with bacteriocin-like properties. This bacteriocin exhibits orders of magnitude greater than the inhibition zone diameter towards the indicator strain than either ampicillin or streptomycin. It has been isolated from cultures of Rhizobium rhizogenes strain K108. The structure of the agrocin 108 without detail, has been previously published. We now report a detailed structure elucidation, including the hitherto undetermined residual 5'-phospho-diester fragment by a combination of 1D and 2D NMR studies at various pH values in H2O/D2O, high resolution MS, pKa determination, and chemical degradation.

This chapter discusses, briefly, the developments in electrophoresis of proteins from Tiselius' moving boundary electrophoresis to the modern day two-dimensional polyacrylamide gel electrophoresis. It also touches upon the staining methods used to visualize total proteins postelectrophoresis.

The incidence of idiopathic normal pressure hydrocephalus (INPH) is seen to be relatively rare, ie about two per million inhabitants per year. Five studies on the prevalence of INPH in elderly patients, from three countries, have been published between 1985 and 2009.1–5 Prevalence ranged from 0.41% to 2.94% (mean 0.8%), ie slightly less than one per 100 based on samples ranging from 170 to 982 subjects. This surprisingly high percentage was not found in a survey that attempted to identify every person with INPH in a small county in Norway with a population of 219,748. Attempts to identify all patients with INPH overlook many cases. INPH is actually a very common disease and its prevalence increases with age.

A method for the quantitative determination of plasma concentrations of ampicillin and cloxacillin in mixture is described. The antibiotics were separated by high voltage paper electrophoresis. The high affinity of cloxacillin for albumin seriously interfered with the electrophoretic separation of the antibiotics and the quantitation of cloxacillin. Therefore, prior to electrophoresis, the plasma proteins were digested with trypsin to release cloxacillin. Subsequent to the electrophoretic separation the antibiotics were developed micro-biologically by agar diffusion using the separated spots on the paper as diffusion centres. The inhibition zones were clearly visible after staining and could be quantitated. The method was evaluated statistically and showed good agreement with the paper disc diffusion method.

Aeromonas liquefaciens and Aeromonas salmonicida were both found to produce considerable amounts of extracellular proteinases, when grown on semi-solid skim milk agar at 30° C. The proteinase A, produced by Ae. liquefaciens only, and the proteinase B, produced also by Ae. salmonicida were purified approximately 100 times by precipitation twice with (NH4)2SO4, batchwise treatment with DEAE-cellulose and, finally, gel filtration on Sepha-dex G-100. The pH-optimum was estimated to be 7.9 for proteinase A and 9.0 for proteinase B. Differences between the proteinases were also found with regard to thermoresistance, and to their behaviour in the presence of some naturally occurring proteinase inhibitors. The addition of iron and cobalt ions increased the activity of both proteinases. Molecular weights were estimated to be 22 100 for the proteinase A and 43 600 for proteinase B, on the basis of gel filtration on Sephadex G-100.

Urinary metabolic studies by means of high-voltage paper electrophoresis (HVPE) and some qualitative chemical tests were performed in 40 patients in a family with hereditary macular degeneration and in 40 nonaffected members of the same family. No biochemical abnormalities were observed in the examined urine samples. The results are discussed with regard to earlier published reports of aminoaciduria in hereditary macular degeneration.

Three Danish pedigrees have been studied with a view to reduced thyroxine-binding globulin (TBG). The study confirmed that this defect is transmitted by X-chromosome-linked dominant inheritance, there being no man-to-man transmission, but also that it may occur in two types: deficient TBG and low, non-zero TBG. Definite changes of TBG and thyroid function tests were found in the hemizygous men, whereas the heterozygous women showed a marked variation in the range between the values for hemizygous men and normal persons. Determination of total thyroxine appears to afford more guidance than the protein-bound iodine suggested by others. Two of four women on oral contraception exhibited such deviating TBG values that they are assumed to be TBG heterozygous. In all persons free thyroxine proved normal or slightly reduced but all the persons tested were euthyroid. However, the propositus of one family had previously been treated for Graves' disease.

Serum γ-globulin levels have been determined by paper electrophoresis in 54 patients with cirrhosis of the liver before and after establishment of a portocaval shunt. As a control group 23 patients operated on for cancer of the lung were studied in the same way. A significant average increase from 18.6 to 23.0 g/1 in the cirrhotics was found, whereas no increase in the control group was observed. Serum immunoglobulins and antibodies to E. coli 0 group antigens were studied in 31 patients with cirrhosis and a portocaval shunt and 128 patients with cirrhosis without portocaval shunt. Significantly higher levels of IgG and E. coli antibodies were found in the patients with a portocaval shunt. It is proposed that these observations can be explained by the change in liver circulation brought about by the operation. If the cirrhotic liver has retained some of the ability of the normal liver to inhibit immunogens as previously proposed by us, the observed changes in immunoglobulins and E. coli antibodies should be expected.

In a patient with chronic lymphocytic leukaemia, two M components of the IgGkappa and IgGlambda classes were demonstrated in the serum at the time of diagnosis. The patient was first followed without treatment; after 18 months multiple myeloma developed. At that time, immunofluorescence study of lymphocytes of the peripheral blood showed mainly membrane-bound immunoglobulins (S-Ig) of the IgGkappa class. The bone marrow disclosed a definite predominance of plasma cells with cytoplasmic IgGlambda, suggesting that the two B cell-derived diseases had arisen from two different cell clones. During the development of multiple myeloma, the serum concentration of the M component IgGlambda increased. Concurrently, the M component of IgGkappa gradually disappeared from the serum, and the concentrations of the normal immunoglobulins IgA and IgM declined. Following cytostatic treatment, the concentration of the myeloma-derived M component IgGlambda was halved and simultaneously the M component IgGkappa reappeared in the serum. To our knowledge, this case is the second reported of chronic lymphocytic leukaemia and multiple myeloma indicating development of the two diseases from different cell clones, and the first reported cases with myeloma-induced suppression of M-component secretion from malignant cells.

Nucleolar and nuclear size of bone marrow plasma cells has been studied in 26 cases of myeloma, 19 of benign essential monoclonal gammapathy (BEMG) and 9 without an M-component. Bone marrow smears were Feulgen-stained to visualize nucleoli and nuclei. Nucleolar area, nuclear area and the ratio nucleolar area/nuclear area were calculated. The myeloma group differed from the other two groups in having plasma cells with larger nucleoli, larger nuclei and an increased ratio nucleolar area/nuclear area. No difference was found between the BEMG group and the cases without an M-component. In some cases--in which the absence of osteolytic lesions at X-ray examination, a low plasma cell percentage in bone marrow smears or a low M-component concentration initially made the diagnosis of myeloma uncertain--the observation of enlarged nucleoli could help to establish the diagnosis. Three cases were observed in which the onset of myeloma was preceded by a long period of essentially unchanged M-component concentration. In these cases a sudden increase of M-component concentration was accompanied by an increase of mean nucleolar size of bone marrow plasma cells.

A series of 71 consecutive patients with myelomatosis formed the basis of determining survival in relation to sex, type of M-component, and its initial magnitude. For the entire material the median survival was 29 months. It was essentially shorter for IgA (15 months) than for IgG myelomatosis (37 months), whereas the survival was the same for both types among patients who were still alive 5–6 years after diagnosis. The magnitude of the M-component at diagnosis appears to play no prognostic role. 49 patients were treated with cytostatics, and 21 also with prednisone, for a minimum of 2 months. 21 patients responded within 2 months by a decrease in the serum concentration of the M-component exceeding 0.6 g/100 ml. On the other hand, 28 patients showed no significant change in the magnitude of the M-component during the first 2 months' medication. However, the latter patients had a median survival of 62 months as compared to only 13 months for the quickly responding patients. Thus, the quick responders had a significantly poorer prognosis.

This is the first case, hitherto described, of benign essential gammopathy, presenting symptoms and histological evidence of peripheral neuropathy. Furthermore, in no previous case, has a specific localization of immunoglobulin (type IgM) to the areas of nerves involved by the lymphocytes been demonstrated. After six months' treatment with chlorambucil the neurological symptoms disappeared and in a biopsy from the right n saphenus no endoneural lymphocytes or local IgM could be found.

An immunological study has been made of 12 patients with myelofibrosis. The study included determinations of serum protein and immunoglobulin concentrations, serial tuberculin skin tests, and in vitro stimulation of peripheral lymphocytes with phyto-haemagglutinin (PHA). In two patients, very low gammaglobulin values were found (low IgG and IgA concentrations; low IgM in one of them). Four other patients had subnormal IgA values. Depressed delayed-type hypersensitivity reactions were observable in four of eight patients tested. The immuno-deficiency noted was probably secondary to the bone marrow damage. Nevertheless, a disturbed immune response may contribute to the development of bone marrow fibrosis and myeloid metaplasia.

Three new hydroxamamide derivatives with different lipophilicity, N ′-hydroxy- N -methyl-3-(4-nitro-1H-imidazol-1-yl)propanamidine, N -butyl- N ′-hydroxy-3-(4-nitro-1H-imidazol-1-yl)propanamidine, and N ′-hydroxy-3-(4-nitro-1H-imidazol-1-yl)- N -octadecylpropanamidine were easily synthesized and labeled with technetium-99m as markers for hypoxic cells. The results of in vitro experiment indicate that the accumulation of three 99m Tc-complexes steadily increases with time in hypoxic cells, but fluctuates with time and has no fixed trend in aerobic cells; the complex with higher lipophilicity shows more uptake in cells; and the effect of lipophilicity of the 99m Tc-complexes on the hypoxic/aerobic difference can be neglected.

The transfer of selenite via the erythrocytes to the plasma was investigated using Se 75-bodium selenite and cow blood. The selenite was initially fluxed into the erythrocytes by a cyanide sensitive mechanism. During the uptake period (within 1 min.) the erythrocytes consumed oxygen and it can be concluded that selenium is reduced and catalyzes the electron flow to the oxygen. The lack of electrons thus produced is balanced by the oxygenation of GSH. The release is dependent on GSH and it is possible that selenium is ejected from the erythrocytes as a glutathione complex which is fixed to the SH of plasma albumin.

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 peptide [15, 16-D-lysine, 17, 18-D-arginine]-adrenocorticotropin-(1-19) and an all-D-retropeptide related to the amino terminal octadecapeptide of adrenocorticotropin have been synthesized by the solid-phase method. The nonadecapeptide was shown to possess 10-15% of the steroidogenic activity and 3% of the lipolytic activity of adrenocorticotropin-(1-19). The all-D-retropeptide showed no activity and exhibited no inhibitory activity in steroidogenesis and lipolysis.

Digestion of a commercial mixture of the peptides polymyxin B1 and B2 with subtilopeptidase A yielded a peptide whose amino acid composition corresponded to the ring fragment common to both antibiotics. However, in contrast to the earlier chemical studies of Suzuki and collaborators, our high resolution nmr studies indicated that the ring had been split. Edman degradation, hydrazinolysis and exopeptidase digestion showed the rupture to have occurred between positions 8 and 9.

Ten new nonapeptide and decapeptide analogues of bradykinin were prepared by solid phase synthesis. Analogues of proline (3,4-dehydroproline; azetidine-2-carboxylic acid; cis,trans-4-methylproline; thiazolidine-4-carboxylic acid and trans-4-hydroxyproline) were successfully incorporated into the peptides in place of proline at position 3. In vivo and in vitro pharmacological studies revealed that the analogues were in general less active than bradykinin. Several structure-activity relationships were postulated from rat blood pressure, rat uterus and guinea pig ileum studies.

A nonapeptide, Arg-Glu-Leu-Glu-Asp-Gly-Thr-Pro-Arg, corresponding to the 123–131 sequence of bovine pituitary growth hormone, was prepared by a modified solid phase synthesis. To avoid an acid- or base-catalysed rearrangement at the Asp-Gly bond, which gives rise to an aspartimide-containing peptide, a new combination of protecting groups was introduced. The 2-(4-biphenylyl)-2-propyloxycarbonyl group was used for Nα-protection, a t-butyl ester for the β-carboxyl of aspartic acid, and a p-alkoxybenzyl alcohol ester for the anchoring bond to the resin support. The nonapeptide showed growth hormone activity in the hypophysectomized rat tibia epiphyseal width test, but was inactive in the hypophysectomized rat body weight gain test.

The binding of pyridoxal 5′-phosphate (PLP) to bovine serum albumin (BSA), and to large BSA fragments obtained after proteolytic hydrolysis, was investigated in order to study the structure of these fragments in relation to the albumin structure itself, and to get information about the PLP binding sites on albumin. From absorbance and circular dichroism spectra, combined with peptide mapping of the tryptic digests of the reduced PLP-protein complexes, it could be concluded that the primary binding site is localized within the NH2-terminal part of the albumin molecule. The COOH-terminal part contains one or more secondary sites. It appeared that in albumin and in the largest NH2-terminal fragment, the environment of the primary binding site is rather apolar in character. However, in the smallest NH2-terminal fragment this site is more exposed to the solvent. This suggests that the part of the peptide chain which is not common in both fragments has a stabilizing effect on the structure around the primary binding site.

Fluorescamine was shown to be an excellent terminating agent for blocking unreacted amino groups during solid phase peptide synthesis. A comparison of the termination efficiency of fluorescamine versus that of acetylation revealed that the former method gave superior products as assessed by peptide analysis, dansyl-amino end group determination and biological assay. In addition, fluorescamine terminated fragments were converted to non-fluorescent spirolactones during the deprotection stage. These spirolactones were stable to subsequent solid phase reaction conditions and were readily removed from the target peptide.

The solid-phase syntheses of [Sar2]-, [Ala2]-, [D-Leu2]-, [D-Lys2]-beta-endorphins and [Pro5]-, [Leu5]-, [D-Leu5]-, [D-Ala2, D-Leu5]-beta-endorphins are described. The synthetic peptides were purified by chromatography on carboxymethylcellulose and partition chromatography on Sephadex G-50. They were characterized by partition chromatography on agarose, thin-layer chromatography, paper electrophoresis, and amino acid analyses of acid and enzymic hydrolysates. Bioassay of the synthetic analogs for analgesic activity by the tail-flick method showed the D-Leu2 analog to be 48% as potent as betah-endorphin while the Ala2, D-Lys2, Leu5, and [D-Ala2, D-Leu5] analogs were 8 to 17% as active. The Sar2, D-Leu5, and Pro5 analogs were less than 1% as potent.

A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl glycine in the dry state. Fast atom-bombardment (FAB) mass spectrometry and Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) showed that the product is a cyclic imide. Cyclic imide formation also was shown to occur with the tripeptides Gly-Asp-Gly and Gly-Asn-Gly. The cyclization of aspartyl peptides depends upon protonation of the beta-carboxyl group and can be promoted by acidification or by heating the NH4+ salt in the dry state.

The neurohypophyseal hormones of three species of Australian marsupials belonging to the family Macropodidae (Macropus rufus, Macropus giganteus, Macropus robustus). have been investigated. In these species three active peptides have been disclosed, one with oxytocic activity and two with pressor activity. The three hormones are present in all the individual glands which have been examined. Purification has been undertaken by ion-exchange chromatography and paper chromatoelectrophoresis. The more abundant of the pressor hormones (about 80% of the total pressor activity) has been identified as lysine-vasopressin by its biological properties, its chromatographic behaviour and its amino acid composition. The second pressor principle, more basic, has not yet been chemically characterized but its pharmacological properties and its chromatographic behaviour suggest that it is closely related or identical to arginine-vasopressin. Dimorphism of vasopressins seems peculiar to Metatheria since it has not been observed in Protheria and very rarely in Eutheria.

A method is suggested for the synthesis of multicomponent peptide mixtures. The method is a solid phase synthesis modified in order to give a closely equimolar mixture of peptides with predetermined sequences. The main point of modification is that before every coupling cycle the resin is divided into equal parts and each portion is coupled with a different amino acid. Then the portions are mixed and before the next coupling cycle the resin is again distributed into equal portions. The method is illustrated by the synthesis of a mixture of 27 tetrapeptides and that of 180 pentapeptides.

A single insulin component has been isolated by ion-exchange chromatography on CM-cellulose from a commercially available mixture of tuna, bonito, and swordfish insulin. The biological activity of this component is in the range of 17 i.u./mg as determined with the rat adipose tissue assay. This fish insulin ha s been reduced and aminoethylated. The two chain derivatives were separated by column chromatography on IRC-50. The A and B chain derivatives contain 21 and 30 amino acids, respectively. The two chain derivatives were digested with trypsin and the resulting peptides separated by high voltage paper electrophoresis, paper chromatography, and, in some cases, by ion-exchange chromatography and gel filtration. Certain tryptic peptides were further digested with chymotrypsin. The amino-acid sequences of the resulting peptides were determined by a modified “dansyl”-Edman technique and by the use of aminopeptidase M, carboxypeptidase A and B, and citrus carboxypeptidase. The complete amino-acid sequence of chains A and B is given.

My graduate student Regina Vidaver and I published the first article in the second issue of RNA in April 1995. Our paper reported a surprising property of human U6 RNA that provides insight into mechanisms for modulating the stability of RNA–RNA complexes. That article represents the mid-point in my career in RNA science so far. Below I describe what led up to that article, and very briefly consider how things have changed since it was published.

We present an origami paper-based electrophoretic device (oPAD-Ep) that achieves rapid (∼5 min) separation of fluorescent molecules and proteins. Due to the innovative design, the required driving voltage is just ∼10 V, which is more than 10 times lower than that used for conventional electrophoresis. The oPAD-Ep uses multiple, thin (180 μm/layer) folded paper layers as the supporting medium for electrophoresis. This approach significantly shortens the distance between the anode and cathode, and this, in turn, accounts for the high electric field (>1 kV/m) that can be achieved even with a low applied voltage. The multilayer design of the oPAD-Ep enables convenient sample introduction by use of a slip layer as well as easy product analysis and reclamation after electrophoresis by unfolding the origami paper and cutting out desired layers. We demonstrate the use of oPAD-Ep for simple separation of proteins in bovine serum, which illustrates its potential applications for point-of-care diagnostic testing.

Exorphin C is a peptide with five amino acids [(Tyr-Pro-Ile-Ser-Leu) Trifluoroacetate salt] (Sigma) that has an affinity to opioid receptor-expressing tissues and tumors. Exorphin-C was labeled with 99m Tc using glucoheptonate (GH) as bifunctional chelating agent. Then, we investigated its radiopharmaceutical potential as opioid receptor-expressing tissue on rabbits. Quality controls were performed by ITLC, paper electrophoresis and HPLC. Labeling efficiency was higher than 98%. The compound was stable for at least 5 h at room temperature. Scintigraphic imaging with 99m Tc–GH–exorphin C ( 99m Tc–GE) was performed on male Albino rabbits. Static images were obtained from anterior projection using a Camstar XR/T gamma camera at several time intervals. Although a significant amount of activity was seen in the brain, less activity was seen on receptor saturation studies at 30 min. Slight hepatobiliary excretion was seen, though the main excretion route was renal. After saturating, the receptor hepatobiliary excretion was not seen; the only excretion route was renal.

A facile, two-step synthesis of the bis(hydroxamamide)-based tetradentate ligands for 99m Tc-radiopharmaceuticals is described. Firstly, the hydroxamamide was converted to hydroximic acid chloride by reaction with sodium nitrite in hydrochloric acid at 0 °C. Secondly, treating the halide with the ethylenediamine or 1,3-propylenediamine in absolute ethanol formed the desired products, N , N ′-ethylene bis(1-(4-nitroimidazole-1-yl)–propan-hydroxyiminoamide) ( I ) and N , N ′-propylene bis(1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide) ( II ). The corresponding 99m Tc complexes showed high yields and were found by paper electrophoresis to be electrically neutral under physiological conditions. The partition coefficients indicated a distinct difference between the two complexes.

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.

Exposure of the skin to sunlight results in an increase of the content of epidermal trans-urocanic acid, a key metabolite of L-histidine, and also in occurrence of the isomerization of trans-urocanic acid to the cis isomer. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of urocanic acid and glutathione, is a presumed origin of a urinary compound S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). The formation of GS(CIE) is stimulated by exposing the skin to sunlight irradiation. In this study we investigated an enzymatic formation of GS(CIE) from glutathione and cis-urocanic acid by incubation with rat liver extract that contained glutathione S-transferase (GST) at high activity. The formation of GS(CIE) was suppressed significantly when a liver extract depleted of GST activity was used. Enzymatic degradation of GS(CIE) with gamma -glutamyl transpeptidase resulted in the formation of N-[S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteinyl]glycine, a metabolic intermediate between the glutathione adduct and Cys(CIE). A hydrolyzed product of GS(CIE) by HCl was identical with the urinary Cys(CIE). Compounds were analyzed by high-voltage paper electrophoresis, capillary electrophoresis, and fast atom bombardment mass spectrometry. From these results, we suggest that GS(CIE) formed from cis-urocanic acid and glutathione is an origin of the urinary compound Cys(CIE) and that the formation reaction is catalyzed mostly by the action of GST.

A new method has been developed for the isolation and rapid identification of anthocyanins from two floricultural crops based on the use of high-voltage paper electrophoresis with bisulphite buffer. Using this method, anthocyanin pigments were successfully purified as their negatively charged bisulphite-addition compounds from crude extracts of plant tissue. In conjunction with liquid chromatography–electrospray mass spectrometry, the method enabled the anthocyanins from the flowers of two Banksia species and the leaves of two Acacia species to be identified. The Banksia flowers contained both cyanidin and peonidin-based pigments, while the Acacia leaves contained cyanidin and delphinidin derivatives. Copyright © 2003 John Wiley & Sons, Ltd.

Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. `Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on `Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [3H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeat-units of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins.

A few years ago (99m)Tc-ethylenedicysteine ((99m)Tc-L,L-EC) had been proposed as an interesting substitute for technetium-99m labeled mercaptoacetyltriglycine (MAG3) as renal function tracer agent. It possesses in its structure two carboxylate functions and is in this respect different from other renal tracers such as (99m)Tc-N, N'-bis-(mercaptoacetyl)-2,3-diaminopropionate ((99m)Tc-CO(2)DADS), (99m)Tc-MAG3, and Hippuran, which have only one carboxylic group. To study whether both carboxylic acid groups of (99m)Tc-L,L-EC contribute to the efficient renal handling of this compound we synthesized and biologically evaluated the technetium-99m labeled isomers of L- and D-ethylenecysteamine cysteine (ECC), the mono-acid derivative of (99m)Tc-L,L-EC. Labeling of L-ECC or D-ECC with (99m)Tc using a direct or exchange labeling method yields for each of them two diastereomeric (99m)Tc complexes (A and B, in the order of elution during reversed phase high performance liquid chromatography) in relative amounts depending on the pH during labeling. In mice, all four isomers of (99m)Tc-ECC (LA, LB, DA, and DB) are cleared rapidly from the blood, mainly by the renal system. The isomers LB and DB show the most efficient renal handling, but none of the mono-acid derivatives has a urinary excretion rate as high as that of (99m)Tc-L,L-EC. The renal handling of the isomers of (99m)Tc-ECC is partly due to tubular secretion because the urinary excretion of these compounds is significantly lower in mice pretreated with probenecid. In the baboon, isomers DA and DB show a plasma clearance comparable to that of (99m)Tc-L,L-EC. The plasma clearance of isomers LA and LB is lower but still comparable to or higher than that of (99m)Tc-MAG3. In a human volunteer, isomer DB shows a plasma clearance rate only slightly lower than that of (99m)Tc-L,L-EC. Thus, it appears that the presence of one carboxylate in (99m)Tc-EC-like compounds can be sufficient for efficient renal handling. However, it is also evident that the configuration at the chiral carbon atom and the orientation of the oxotechnetium core determine in a significant way the biological characteristics.

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.

A 13-kDa phosphoamidase was isolated as a single band on SDS-PAGE from bovine liver. Its Stokes' radius, sedimentation coefficient, molecular mass, and optimal pH were estimated to be 1.6 nm, 1.8 s, 13 kDa, and 6.5, respectively. The enzyme released P(i) from 3-phosphohistidine, 6-phospholysine, and amidophosphate at rates of 0.9, 0.6, and 2.6 micromol/min/mg protein, respectively. However, it did not dephosphorylate phosphocreatine, N(omega)-phosphoarginine, imidodiphosphate, or O-phosphorylated compounds including inorganic pyrophosphate. It also dephosphorylated succinic thiokinase and nucleoside diphosphate kinase autophosphorylated at His residues, indicating that it works as a protein histidine phosphatase. A thiol reagent, 30 microM N-ethylmaleimide, depressed the activity by half, while a thiol compound, 2-mercaptoethanol, protected the enzyme from heat-inactivation. Five millimolar divalent cations, such as Mg2+ and Mn2+, and 5 mM EDTA, had no effect on the activity.

ABSTRACT. Novel polydentate amine-pyrrole and amine-thiophene ligands were synthesized and characterized by 1 H and 13 C NMR spectroscopy. Radiochemical studies with 99m Tc were carried out at 0.1–100 μM of technetium. Complexation yields were estimated from thin layer chromatography (TLC), paper electrophoresis, and solvent extraction studies. The 99m Tc complexes formed were found to be neutral and lipophilic. Complexes with the corresponding imine-ligands were formed in lower yields. Biodistribution studies of the 99m Tc complexes of these ligands showed no significant uptake in brain or heart, and the clearance was mainly through the hepatobiliary system.

Proteins with R–CO–CO–NH– at the N-terminus, rather than the usual R–CH(– NH 3 + )–CO–NH–, are produced by non-enzymic transamination and also occur in the pyruvoyl enzymes. The oxoacyl group may be specifically removed from a model peptide, in yields of 70–80%, by treating them in 0.1 M phosphate buffer at 37°C for 24 h with 25 mM of the N -phosphonomethyl derivative of phenylene-1,2-diamine. This provides mild conditions for the stepwise removal of N-terminal residues without denaturation.

15th International Symposium on Radiopharmaceutical Chemistry -- AUG 10-14, 2003 -- Sydney, AUSTRALIA, WOS: 000234790900001, PubMed ID: 16394539, The aim of this study was to label ascorbic acid with Te-99m and to investigate its radiopharmaceutical potential in rats. Ascorbic acid was labeled with Tc-99m using the stannous chloride method. The radiochemical purity of [Tc-99m]ascorbic acid (Tc-99m-AA) was determined by RTLC, paper electrophoresis, and RHPLC methods. The labeling yield was found to be 93 +/- 5.0%. The maximum labeling yield of Tc-99m-AA was determined at pH 5 and 25 degrees C. The biodistribution studies related to Tc-99m-AA were done in male albino Wistar rats. Tc-99m-AA, which has a specific activity of 13.02 GBq/mmol, was administered into the tail vein of the rats. The rats were sacrificed at 15, 30, 60, and 120 min after the injection by heart puncture under ether anaesthesia. The organs were weighed after removal. Their activities were counted using a Cd(Te) detector equipped with a RAD 501 count system. The %ID/g (% of injected dose per gram of tissue weight) in each organ and in blood was calculated. Maximum uptake of Tc-99m-AA was observed in prostate and kidneys at the 60th min. Tc-99m-AA may be a promising radio-pharmaceutical for the imaging of prostate and kidneys.

In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentration is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bisphosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allow us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phosphoglycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM.

Extracellular polysaccharide produced by suspension-cultured Mentha cells consisted of 50% neutral sugars, 32% uronic acid and 10% of protein. The ammonium oxalate-soluble fraction of this polysaccharide contained 30% hemicellulose and 70% pectic substances. The purified hemicellulose contained xylose, glucose, arabinose, galactose, mannose and fucose residues in a molar ratio of 41.6: 31.3: 13.1: 11.1: 1.3: 1.6. It was identified as a xyloglucan from its neutral sugar composition and by methylation analysis and cellulase treatment: the principal neutral sugar was arabinose. The presence of O -acetyl residues was confirmed with 1 H NMR, 13 C NMR and GC-mass spectrometry. The total acetyl content in the polysaccharide was 4%. The point of attachment of the O -acetyl residue was shown to be at position 6 of the galactosyl residue.

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.

The major oligosaccharide chains of bovine, frog and human rhodopsins are abridged, hybrid, asparagine-linked structures that contain only mannose and N-acetylglucosamine as the constituent sugars. Isomers that also contain galactose have been detected in varying amounts in rhodopsins from different species. As part of studies to examine the mechanisms used by the human retina to control the glycosylation of rhodopsin, the kinetics of the retinal galactosyltransferase was examined using Golgi-enriched fractions of human retina as the enzyme source and rhodopsin, opsin, and the oligosaccharide isolated from rhodopsin as acceptors. These reactions were compared to those using bovine retina and rat liver Golgi. A comparison of the Vmax/Km ratios revealed that the efficiency of the human retinal galactosyltransferase was some 20-fold lower than that of the rat liver. In addition, consistent with the higher state of galactosylation of human as compared to bovine rhodopsin, greater efficiency was observed with the human preparation. While the conformation of the visual pigment exerted an effect, its low galactosylation was not due to a major directing influence on glycosylation by the polypeptide matrix as indicated by the even lower activity toward the isolated oligosaccharide. The galactosylated oligosaccharides obtained by these procedures were identified by chromatographic methods. The relatively low activity of the retinal galactosyltransferases observed in this study may help explain the limited glycosylation that is typical of rhodopsin.