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PubMedDate not supplied

Synthesis and alkylation activity of a nitrogen mustard agent to penetrate the blood-brain barrier.

Nitrogen mustard agents are widely used for the clinical treatment of cancers. A nitrogen mustard (N-mustard) agent was synthesized utilizing nicotinic acid as the carrier of the alkylating substituent (-OCH2CH2N(CH2CH2Cl)2) that forms an ester group (R-C(O)-OR) on a heterocyclic ring. The N-mustard agent is a solid at room temperature and is stable for more than 6 weeks when stored at -10 degrees C. To determine the kinetics of alkylation activity a nucleophilic primary amine compound (4-chloroaniline) was placed in aqueous solution with the mustard agent at physiological pH 7.4 (pH of blood) and 37 degrees C. The alkylation reaction was found to be second-order with rate equation: rate = k2[N-mustard][Nu], where Nu = nucleophile and k2 = 0.0415 L/(mol x min). Pharmacological descriptors calculated showed values indicating a strong potential of penetrating the blood-brain barrier. The partition coefficient (Log P) of the mustard agent is 1.95 compared with 0.58 for nicotinic acid. Values of descriptors such as dipole, polar surface area, Log BB, molar refractivity, parachor, and violations of Rule of 5 were found to be 5.057 Debye, 42.44 A2, 0.662, 72.7 cm3, 607.7 cm3, and 0.0 for the N-mustard agent. Value of polar surface area for the mustard agent (42.44 A2) predicts that >90% of any amount present in the intestinal tract will be absorbed.

Algorithms↗
PubMed1982

The characteristics of peptide uptake in Streptococcus faecalis: studies on the transport of natural peptides and antibacterial phosphonopeptides.

Transport of natural peptides and antibacterial phosphonopeptide analogues was studied in Streptococcus faecalis ATCC 9790. Competition studies, and the isolation of peptide-transport deficient mutants, indicate the presence of two peptide permeases. One is a high-rate system used by dipeptides, and to a lesser extent tripeptides; the other is a low-rate oligopeptide system. Following uptake, peptides are cleaved and their amino acid residues may undergo rapid exodus. Different strains of S. faecalis differ in their rates of peptide transport.

Alanine↗
PubMed1977

On the competition between protamines and histones: studies directed towards the understanding of spermiogenesis.

Specific contacts between the crossinteracting histones effectively prevent their protamine-mediated displacement from nucleoproteins revealing a complex competition pattern not expected from their relative affinities to DNA. Histone H1 is the only species freed under a variety of conditions if native chromatin is used; the results found with chromatins obtained by conventional methods reflect the extent to which histone redistribution has occurred. It is concluded that the events occurring during spermiogenesis must be apt to disrupt histone interactions found in the nucleosomes. The possible role of a protamine phosphorylation/dephosphorylation mechanism is discussed on the basis of comparative experiments with protamines phosphorylated to different extents.

Animals↗
PubMed1980

The unmasking of proteolytic activity during the early development of Artemia salina. Identification of a precursor after hatching.

The proteolytic activities of Artemia salina immediately after hatching are found masked in a precursor of high molecular weight (approximately 100000). The molecular weight of this precursor decreases slightly as development proceeds. However, its kinetic and chromatographic properties vary greatly. Shortly after hatching, the activities are highly inhibited, can be activated severalfold by the chaotropic agent potassium iodide and are not proportional to the amount of enzyme-containing extract added. Later after hatching, its kinetics become normal. From these observations we have concluded that (a) the majority, if not all, of the proteinase activities found later in development are already present immediately after hatching, although in an inhibited state that is gradually activated; (b) the proteinases are not found free in the cytoplasm but in a complex which may allow regulation of their activity.

Animals↗
PubMed1985

Relative rates of transport of peptidyl drugs by Candida albicans.

A variety of peptide drugs are known to be active against Candida albicans; however, little is known about the transport of such agents into the target organism. To provide further information concerning transport of this type, we studied the uptake of two classes of small linear peptides: polyoxins which act intact within the cell and the m-fluorophenylalanyl (m-F-Phe) peptides which require peptidase cleavage to release m-F-Phe. Competition studies with a specific dipeptide detector (alanyl-alpha-thiophenylglycine) enabled us to determine Ki values of 2.6 microM for nikkomycin Z and 350 microM for polyoxin D. Rates of uptake of the peptidyl-nucleosides are approximately 30 times lower than those of the m-F-Phe peptides (apparent maximal velocities: nikkomycin Z, 62 pmol min-1 mg (dry weight) of cells-1; M-F-Phe alanine 1.3 nmol min-1 mg (dry weight) of cells-1). For both the m-F-Phe peptides and the peptidyl-nucleosides, the affinity of the drug for the transport system is an important determinant of its whole-cell activity.

Antifungal Agents↗
PubMedDate not supplied

Elastin-derived peptide binding to a hydrophobic domain on neutrophil elastase.

To understand the contributions of binding of elastin to domains removed from the active site of neutrophil elastase, we isolated an elastin-derived peptide (EDP) fraction, which we have previously shown was tightly linked to neutrophil elastase after prolonged digestion of elastin but which can be released from the enzyme with hydroxylamine. Elastin from human aorta was incubated with human neutrophil elastase under conditions favoring proteolysis. Low molecular weight species, including free EDP, were separated from the protein fraction by a small centrifuged gel filtration column. The high molecular weight protein fraction was subjected directly to 0.5 M hydroxylamine. The reaction mixture was then fractionated on a phosphocellulose column using an ionic gradient. A fraction was collected that exhibited fluorescence with a peak at approximately 410 nm when excited at 320 nm, indicating the presence of desmosine and (or) isodesmosine. A second peak with amidolytic activity towards methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline (MeOSucAAPVpNa), but no fluorescence at 410 nm was also detected at the same elution volume where free elastase appeared. After removal of low molecular weight digestion products but prior to treatment with hydroxylamine, the putative elastase-EDP complex possessed no amidolytic activity towards MeOSucAAPVpNa. When the liberated EDP was added to elastase in an amidolytic assay, the EDP behaved as only a partial noncompetitive inhibitor (Vmax/Vmax approximately 90%), but bound with high affinity to neutrophil elastase (Ki congruent to 29 nM), as detected by its ability to quench elastase endogenous fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Aorta↗
PubMed1980

Sleep-promoting material from human urine and its relation to factor S from brain.

A sleep-promoting factor was extracted from human urine. Intraventricular infusion of the purified material induced excess slow-wave sleep in rats and rabbits for 5--10 h after the infusion. Chemical properties of the urinary factor were similar to those of factor S derived from whole brains of sleep-deprived goats, sheep, and rabbits. The behavior of the urinary factor in two ion exchange chromatographic steps, high voltage electrophoresis, gel-filtration, and ultrafiltration was similar to that of factor S. Effects of the purified urinary factor on slow-wave sleep of rats and rabbits were similar in time-course and duration to those of factor S from brain. However, the factor obtained from human urine did not increase the amplitude of cortical slow waves to the same extent as did factor S from brains of sleep-deprived animals.

Animals↗
PubMedDate not supplied

The location of the calcium precipitating protein in the sarcoplasmic membrane.

Fluram, a fast reacting reagent for primary amino groups does not label proteins enclosed in reconstituted sarcoplasmic vesicles (SR vesicles) when applied at low reagent/protein ratios. Hence, Fluram does not penetrate through SR membranes under these conditions. Likewise, the membrane of erythrocytes prevent the reagent from reacting with hemoglobin. However, at high reagent/protein ratios the membranes of the SR vesicles disintegrate. In contrast, liposomal membranes prepared from SR lipids are not affected even at a high degree of labeling. Disintegration of SR membranes starts to occur when the phosphatidylethanolamine fraction is completely substituted by Fluram and the transport protein is labeled with 3-4 mol of Fluram per 100000 d. When closed SR vesicles are labeled at low Fluram/protein ratios, the calcium precipitating protein is four times more intensely labeled than the transport protein. The labeling of the calcium transport protein in closed vesicles excludes its location in the intravesicular space. In SR vesicles solubilized with deoxycholate the relative degree of labeling of the calcium precipitating protein remains unchanged while the transport ATPase is more intensely labeled at the expense of the labeling of the amino lipids. The relative degree of labeling of the protein components depends not only on the number of labable groups but also on the rates with which these groups react with Fluram. Therefore, the experimental data do not give quantitative information concerning the distribution of the protein components in the SR membranes.

Adenosine Triphosphatases↗
PubMed2003

[HPLC-fluorescent spectrometric determination of serum mexiletine concentration after derivatization with fluram].

AIM: To establish an HPLC-fluorescent spectrometric method for the determination of mexiletine hydrochloride in plasma after derivatization with fluram. METHODS: Fluram acetone solution was added to the deproteinized plasma with acetone to obtain the derivative of mexiletine. The HPLC method was performed on a column of Allitima C18 (150 mm x 4.6 mm, 5 microns) with the mobile phase of methanol-water-diethylamine-phosphoric acid buffer (2.4 mol.L-1, pH 4.0) (70:28:2), and the detective wavelength were set at Ex 392 nm and Em 480 nm. RESULTS: Mexiletine has a liner range over the concentration range from 0.100-6.400 mg.L-1. The lowest detectable concentration of this method was 5 micrograms.L-1 (S/N > or = 4). The intra-day and inter-day RSDs were 1.34%-5.31%, respectively. CONCLUSION: This method is simple, selective and can be used for therapeutic drug monitoring (TDM) and pharmacokinetic studies of mexiletine.

Anti-Arrhythmia Agents↗
PubMed2003

In vivo efficacy of recombinant methioninase is enhanced by the combination of polyethylene glycol conjugation and pyridoxal 5'-phosphate supplementation.

Recombinant methioninase (rMETase) is an enzyme active in preclinical mouse models of human cancer. The efficacy of rMETase is due to depletion of plasma methionine, an amino acid for which tumors generally have an abnormally high methionine requirement. Furthermore, transient methionine depletion results in a markedly increased sensitivity of the tumors to several chemotherapeutic agents. This study characterized methods to prolong the half-life of rMETase to extend the in vivo period of depletion of plasma and tumor methionine. In the present study, rMETase was coupled to methoxypolyethylene glycol succinimidyl glutarate-5000 in order to prolong the half-life of rMETase and thus extend the in vivo period of depletion of plasma and tumor methionine. Matrix-assisted laser desorption ionization mass spectrometry indicated that one sub-unit of rMETase was modified by approximately 4, 6 and 8 PEG molecules when rMETase was PEGylated at molar ratios of PEG/rMETase of 30/1, 60/1, and 120/1, respectively. PEG-rMETase (120/1) had a serum half-life increase of 20-fold, and methionine depletion time increased 12-fold compared to unmodified rMETase. The increase in in vivo half-life depended on the extent of PEGylation of rMETase. In addition, a remarkable prolongation of in vivo activity and effective methionine depletion by the PEG-rMETase was achieved by the simultaneous administration of pyridoxal 5'-phosphate. PEGylation also reduced the immunogenicity of rMETase. The extent of reduction in immunogenicity depended on the number of residues PEGylated. PEG-rMETase 30/1 had a 10-fold decrease in IgG titer while PEG-rMETase 120/1 had a 10(4)-fold decreased titer compared to naked rMETase. Thus, the molecular modification of PEGylation confers critical new properties to rMETase for development as a cancer therapeutic.

Animals↗
PubMed2004

Identification of a new peptide deformylase gene from enterococcus faecium and establishment of a new screening model targeted on PDF for novel antibiotics.

OBJECTIVE: To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF. METHODS: A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E. coli B121(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics. RESULT: A new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples. CONCLUSION: A new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.

Amidohydrolases↗
PubMed1988

Substructure and higher structure of chicken smooth muscle alpha-actinin molecule.

Substructure of chicken gizzard smooth muscle alpha-actinin molecule was deduced by domainal mapping of the proteolytic fragments with alpha-chymotrypsin. There were three chymotryptic cleavage sites (Sites I, II, and III, from the amino terminus). Cleavage at Site I generated two fragments, i.e. an NH2-terminal 36-kDa fragment and a COOH-terminal 70-kDa fragment. The 70-kDa fragment generated either a 55-kDa fragment by cleavage at Site II or a 65-kDa fragment by cleavage at Site III. Purified NH2-terminal 36-kDa fragment bound to F-actin, whereas the 55-kDa fragment formed a dimeric molecule. Circular dichroism and electron microscopic experiments demonstrated that the alpha-helical content of the 55-kDa fragment was 14% higher than that of native gizzard alpha-actinin, coinciding with the apparently rod-shaped configuration of this fragment. A 110-kDa product was generated from two 55-kDa fragments in a cross-linking study with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Two cross-linkable sites in the 55-kDa, A- and B-site, were shown to be involved in this reaction. Further, it was demonstrated by using N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide labeling and immunoblotting analyses that the A-site on one 55-kDa fragment was cross-linked to the B-site on the other. These results suggest that smooth muscle alpha-actinin formed an antiparallel dimeric molecule in which the 55-kDa fragments connected the two actin-binding domains composed of the 36-kDa fragments.

Actinin↗
PubMed1986

Fusion of negatively charged phospholipid vesicles by insulin. Relationship with lipid fluidity.

We have studied, by fluorescence methods, the association of insulin to liposomes, the modification of lipid fluidity, and the fusion of vesicles induced by insulin. All parameters showed a similar dependence on pH and ionic strength of the medium and on negative charges in liposomes. The influence of temperature indicated that the association of insulin to liposomes per se was not sufficient to produce a decrease in lipid fluidity and fusion of liposomes. The modification of lipid fluidity induced by insulin in biological membranes is discussed as a possible general event in the action of the hormone.

Fluorescamine↗
PubMed1978

The influence of enzymatic cleavage and chemical modification of human and rabbit IgG on their reactivity with staphylococcal protein A.

Proteolytic cleavage fragments from rabbit IgG have been isolated and characterized in an attempt to locate the sites involved in the reactivity with Staphylococcal protein A. The plasmin cleavage product Facb together with the pepsin cleavage products F(ab')2 and pFc' failed to react in contrast to the papain Fc fragment. These data, together with data from unfractionated plasmin digests, in which the Facb fragment remains associated with the plasmin pFc' fragment, indicate that inter-domain interactions are important in the maintenance of this activity. beta2-microglobulin was also shown to be unreactive with protein A. Chemical modification studies employing flurescamine, tetranitromethane and potassium cyanate indicate that lysine and tyrosine residues are not involved in the reactivity of human and rabbit IgG with protein A.

Animals↗