Increased muricide and decreased avoidance and discrimination learning in thiamine deficient rats.

This study assessed the effects of diet-induced thiamine deficiency in rats on two aspects of behavior, aggression and learning. Evidence of enhanced aggression (increased mouse killing) was noted with severe thiamine deficiency, but before the onset of overt neurological signs of thiamine deprivation. This behavioral change was rapidly reversible with thiamine. A similar degree of thiamine deficiency failed to alter learning of two-way shuttle-box avoidance acquisition. Animals with a gross neurological deficit did exhibit a major impairment in shuttle-box performance, but this was probably due to ataxia. However, when such rats were administered thiamine with total reversal of the neurological signs, testing in a three chambered Y-maze avoidance-discrimination apparatus also revealed impaired learning of both responses. These data demonstrate the presence of enhanced aggression during thiamine deprivation and of a persistent learning impairment in rats following reversal of this vitamin deficiency.     

Quantitative determination of myeloperoxidase using tetramethylbenzidine as substrate

Tetramethylbenzidine, a noncarcinogenic, nonmutagenic derivative of benzidine, has been used as a substrate to assay myeloperoxidase. The assay is sensitive to 0.1 μg of enzyme and can be used to quantitate myeloperoxidase over a pH range of 4.4 to 7.4.     

Quantitative gas-liquid chromatographic determination of ftorafur and 5-fluorouracil in biological specimens

A method developed for measuring the antitumor agent ftorafur and its biotransformation product 5-fluorouracil was applied to biological specimens. After extraction with ethylacetate, ftorafur, 5-fluorouracil, and the internal standard 2-methyl-4-hydroxy-6-chloromethylpyrimidine are converted to their chloromethyldimethylsilyl derivatives and assayed by glc, using either an electron-capture or a flame ionization detector. The minimum detectable amount is 100 pg/injection for ftorafur and 50 pg/injection for 5-fluorouracil employing electron-capture detection. Linearity was found up to microgram amounts of both substances, without any interference from endogenous substrates. Preliminary data are reported on the comparative serum kinetics of ftorafur and 5-fluorouracil in mice.     

Quantitative Cu(I) determination using X-ray absorption edge spectroscopy: oxidation of the reduced binuclear copper site in type 2 depleted Rhus laccase

We report a procedure, through difference comparison of X-ray absorption edge spectra, for the quantitative determination of Cu(I) content in copper complexes of mixed oxidation state composition. This technique is tested on copper model systems and then used to quantitatively determine that untreated T2D Rhus laccase contains 70 +/- 15% Cu(I). Whereas excess ferricyanide is demonstrated not to alter the Cu(I) content of the untreated T2D, aqueous peroxide and nitrite at pH 6.0 are shown to oxidize the cuprous type 3 site and generate met T2D protein forms.     

Evidence suggesting direct oxidation of human erythrocyte membrane sulfhydryls by copper

Results are presented which suggest that cupric ion can directly oxidize the sulfhydryls of human erythrocyte membrane proteins leading to the formation of disulfide links. When packed ghosts were incubated in cupric sulfate (0.3 to 0.7 mM) at pH 8, and electrophoresed on sodium dodecyl sulfate polyacrylamide gels in the absence of dithiothreitol bands 1, 2 (spectrin); 4.2 and 5 (actin) diminished in intensity concomitant with the appearance of high molecular weight material. Band 3 moved to its dimeric position on the gel. Evidence that these crosslinks result from formation of new disulfide links due to direct copper binding includes: (a) reversal of crosslinking upon addition of dithiothreitol; (b) blockage of the effect by N-ethylmaleimide, EDTA and mercuric chloride. The effect of copper was observed under N₂, suggesting that it is not related to air oxidation. Furthermore, the crosslinking effect does not require high copper concentrations if the ghost concentration is low. The possible implication of these results with regard to copper induced hemolytic anemias is briefly discussed.     

An automatic method for determination of urinary 3-methylhistidine: normal values.

A system for automatic analysis of urinary 3-methylhistidine is described, applying ion-exchange chromatography and using an automatic sample injector, a motoric selector valve, and a diode programmer, which controls the analytical system. The method permits a sampling rate of 22 samples/day. 3-Methylhistidine was completely separated from histidine in 37 min whereas 1-methylhistidine was eluted together with ammonia. The 3-methylhistidine concentration was linear up to 150 nmol/ml and no appreciable sample interaction was found at automatic sequential runs. The error, in a single determination based on duplicate samples, was 4.61% and, in duplicated determinations, 3.26%. The mean urinary 3-methylhistidine output was 299.4 ± 23.8 μmol/day in 12 healthy females and 545.5 ± 35.2 μmol/day in 12 healthy males. The 3-methylhistidine excretion was significantly higher in males than in females, when expressed as the absolute daily output or as the estimated ratio to body weight, body surface area, or creatinine.     

Lowry protein determination on membrane preparations: need for standardization by amino acid analysis

The protein content of three membrane protein preparations has been determined by the Lowry method with bovine serum albumin as a standard and also by quantitative amino acid analysis as an absolute method. The results differ considerably, the Lowry method giving 29–42% higher values. This implies that many published data for such proteins, based on Lowry protein determinations with bovine serum albumin as the generally applied standard, are in error. Suggestions are made on how to standardize the Lowry method so that reliable values can be obtained for membrane protein.     

Measurement of matrix enzyme activity in isolated mitochondria made permeable with toluene.

Mitochondria isolated from rat liver and heart were made permeable to normally nonpentrating substrates and cofactors by treatment with toluene. The optimal conditions for preparing stable, permeable mitochondria were 2% toluene for 2 min at 4 °C in a buffered, isotonic medium containing 8.5% polyethylene glycol (Mr 6000–7500). Without polyethylene glycol, the toluene-treated mitochondria were unstable and released their matrix enzymes. The treated mitochondria were particularly unstable in dilute suspension under normal assay conditions of their enzyme activities. The levels of matrix enzyme activities unmasked by toluene treatment of mitochondria were very close to those of sonicated mitochondria under identical assay conditions. Mitochondria made permeable with toluene lost only small amounts of their protein and retained a major fraction of the nucleotides and coenzymes. Electron microscopic examination of toluenetreated mitochondria indicated that they were relatively intact with swollen and vesiculated cristae membranes. Such preparations will allow the study of mitochondrial enzymes at approximate in vivo concentrations.     

Beta-Galactosidase immunoassay for the measurement of cyclic AMP

The previously described method for phenotyping of alpha₁-antitrypsin (alpha₁-protease inhibitor, Pi) that utilizes separator isoelectric focusing on thin-layer agarose gel (A. R. Qureshi and H. H. Punnett, in Electrophoresis '81, 3rd International Conference on Electrophoresis, pp. 83–87 (1981)) has been improved to give a better resolution of Pi pattern. A shallow pH gradient in the region of the isoelectric point of Pi pattern was obtained by the use of N-(2-acetamido)-2 aminoethanesulfonic acid (1%) and serine (0.8%). The present technique can resolve the Pi alleles. The patterns of Pi phenotypes were found to be similar to those observed on acrylamide gels. The method is fast, reliable, and reproducible.     

Phosphorylation site specificities of glycogen synthase kinases: determination by peptide mapping using high-performance liquid chromatography

A method is described which separates the various phosphorylation sites in glycogen synthase based on reverse phase high-performance liquid chromatography (HPLC) of tryptic 32P-peptides. Using this method we studied the phosphorylation site specificities of the kinases which act on glycogen synthase. The cAMP-dependent protein kinase phosphorylated sites 1a, 1b, and 2, whereas casein kinase II phosphorylated only site 5. Two calcium, calmodulin-dependent kinases, phosphorylase kinase and liver calmodulin-dependent synthase kinase, both phosphorylated site 2, and the latter enzyme also phosphorylated site 1b. A cAMP-independent kinase (kinase 4) purified from liver also specifically phosphorylated site 2. Synthase kinase 3 catalyzed the phosphorylation of only site 3. This HPLC method was also used to establish that all of these sites were subject to phosphorylation in vivo.     

Inhibition of biogenic amines uptake by imipramine,desipramine, 2 OH-imipramine and 2 OH-desipramine in rat brain

The tricyclic antidepressant imipramine and its metabolites desipramine, 2-hydroxyimipramine and 2-hydroxydesipramine are all pharmacologically active in the central nervous system as determined by $\text{in vitro}$ inhibition of biogenic amine uptake by rat brain synaptosomes and their $\text{in vivo}$ effect on spontaneous and forced motor activity. Since $\text{in vivo}$ hydroxylation of both imipramine and desipramine produced compounds of similar pharmacological activity as the parent compounds, these results suggest that clinical studies relating plasma levels of tricyclic antidepressants to efficacy should also take into consideration the levels of hydroxylated metabolites.     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons: I. Sympathetic neurons

Quantitative studies on the nerve growth factor (NGF) requirement of chick embryo sympathetic neurons in dissociated cell culture revealed the following. (i) The minimum concentration of 2.5 S NGF required for survival of maximal numbers of neurons is about 0.5 ng/ml (～2 × 10^?11M). In culture, this concentration of NGF appears not to be stable for more than 24 hr. Long-term neuronal maintenance with medium changes twice weekly requires a minimum of 5 ng/ml of NGF. (ii) At 24 hr after plating in medium containing 10% fetal bovine serum, neuronal survival is less than optimal at NGF concentrations above 5 ng/ml; in medium with 5% horse serum, survival is constant with up to 5000 ng/ml of NGF. (iii) Survival of neurons after 1 week in culture was less than optimal at NGF concentrations greater than 50 ng/ml, even in medium containing horse serum. (iv) No correlation was observed between the level of NGF (0.5–500 ng/ml) and the estimated neuronal somatic volumes up to 1 month in vitro. (v) Withdrawal of NGF, even after 4 weeks of culture, resulted in degeneration of nerve cell bodies and processes.     

A sensitive method for the determination of cytolytic activity

A new method is described for the determination of the cytolytic activity of extremely low levels of stable as well as very labile cytotoxins. The method involves the application of the cytotoxin to a column of immunobilized erythrocytes or other suitable cells and a continuous monitoring of the column eluate for the presence of hemoglobin or other cell constituents. The cytotoxic activity of horseradish peroxidase at concentrations as low as 10^?12, m can be measured with this technique. The column hemolytic assay is compared with a static (batch) hemolytic assay with respect to sensitivity and reproducibility. Furthermore, a method is described to determine the true rates of lysis, i.e., the number of cells lysed per minute.     

Colorimetric determination of arginine residues in proteins by p-nitrophenylglyoxal

A new method is presented for the colorimetric determination of arginine residues in proteins. Under mildly alkaline conditions, p-nitrophenylglyoxal reacted with arginine to produce a stable colored solution in the presence of 0.15 m sodium ascorbate. Complete color development was obtained after 30 min at pH 9.0 and 30°C. The color produced at 475 nm obeyed Beer's law in the range 0.03–0.33 mm arginine. This color reaction was used to determine the number of arginine residues in several proteins of known arginine content. Best results were obtained when the protein samples were digested with a mixture of trypsin and subtilisin prior to assaying. The arginine contents obtained by this method agreed well with either the published values or with the results of amino acid analysis.     

Determination of protein-bound phosphate by continuous flow analysis: Automated procedure for the determination of alkali-labile phosphate

A method is described for the specific, quantitative determination of protein-bound phosphorus by a continuous flow procedure using a Technicon AutoAnalyzer. It is based on the exceptional alkali lability of serine phosphate linkages to β-elimination when the serine residues are present in a polypeptide chain. The results are reproducible within about 3, 5, or 10%, respectively, when the analytical sample contains about 100, 10, or 3 nmol of protein-bound P. The presence of less than 1 nmol protein-bound P can be detected. The method tolerates wide variations of the pH and ionic composition of the sample, making it suitable for the automatic, serial analysis of chromatographic effluent fractions. Low-molecular-weight phosphomonoesters, ribonucleic acid (phosphodiester), and nucleotide phosphates (pyrophosphate) do not react measurably. Carboxyphenyl phosphate is partially hydrolyzed (10–15%). In contrast, the release of P from various phosphoproteins is quantitative.     

Proteolytic dissection of rat brain hexokinase: determination of the cleavage pattern during limited digestion with trypsin

Limited treatment of rat brain hexokinase (ATP: D-hexose-6-phosphotransferase; EC 2.7.1.1) with trypsin causes cleavage of the Mr 98K enzyme into three major fragments having molecular weights of 10K, 40K, and 50K, with intermediates of Mr 60K and 90K being detected. This information, in conjunction with N- and C-terminal analysis of the intact enzyme and tryptic cleavage products, has established the tryptic cleavage pattern as where T1 and T2 indicate tryptic cleavage sites; cleavage at only T1 or T2 gives rise to the 90K or 60K intermediate, respectively. Confirmation of this cleavage pattern has been provided by two-dimensional peptide mapping using Staphylococcus aureus V8 protease, and epitope mapping with two monoclonal antibodies directed against rat brain hexokinase. The epitopes recognized by one of the monoclonal antibodies is located within the 40K C-terminal fragment while the epitope for the other monoclonal antibody lies within the 50K fragment. A two-dimensional peptide mapping-immunoblotting technique has permitted a more defined localization of these epitopes to specific regions within these major tryptic cleavage fragments. Complete tryptic cleavage of the enzyme occurs with only modest (approximately 20%) loss of catalytic activity, and the cleaved enzyme retains many of the properties of intact hexokinase. Specifically, there was no effect of cleavage on the Km for Glc or the Ki for Glc-6-P, though a slight decrease in Km for ATP was consistently noted to result from cleavage. Furthermore, like the intact enzyme, cleaved hexokinase retained the ability to bind to outer mitochondrial membranes in a Glc-6-P-sensitive manner. Under nondenaturing conditions, the cleaved fragments remain associated by noncovalent forces. Thus, the cleaved enzyme sedimented at a rate comparable to intact enzyme during centrifugation on sucrose density gradients, and migrated only slightly faster when electrophoresed on gradient acrylamide gels under nondenaturing conditions.     

Linear one-step assay for the determination of orthophosphate

A rapid one-step spectrophotometric assay for orthophosphate that requires a single stable reagent solution is presented. The reagent solution, an aqueous mixture of ammonium molybdate and zinc acetate at pH 5.0, produces a stable complex with orthophosphate that absorbs strongly in the near-visible region of the light spectrum. Response to concentration of phosphate was linear up to 300 microM phosphate with a molar absorptivity of 7200 M-1 cm-1 at 350 nm. The mild conditions for phosphate determination employed in this method are unique, making it particularly suitable for the assay of orthophosphate in the presence of labile organophosphates.     

Automated fluorometric determination of free and total tryptophan in plasma

We have developed a sensitive automated fluorometric method based upon the manual procedure of Denckla and Dewey [(1967) J. Lab. Clin. Med.69, 160–169] for determining both free and total tryptophan in plasma. Free tryptophan is measured in a series of increasingly diluted aliquots of a sample of plasma after tryptophan bound to albumin is removed by continuous-flow dialysis. Free and total tryptophan are then derived from Scatchard plots of the data. The method can be used for nutritional assessments, clinical investigation of behavioral disorders in which serotonin is implicated in the pathogenesis, and studies on tryptophan transport and metabolism.