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K R Jennings

Publications and source records attributed to K R Jennings.

At least 19 recordsLinked to original sources
PubMed2001

New antigenic regions of streptokinase are identified by affinity-directed mass spectrometry.

Streptokinase (SK) is a bacterial protein used for the treatment of myocardial infarction, which is immunogenic in humans. Here we report the use of an affinity-directed MS approach to determine the minimal epitopes involved in the binding between SK and patient antibodies. Using this method we have identified two novel epitopes and mapped these to the minimal recognition regions formed by the amino acids D96-E99 and F323-D328. We have also located three previously identified antigenic regions and have now mapped them and shown that they can be defined more precisely as residues P4-L8, P171-P177 and K334-N338.

Antigens↗
PubMed1998

Rapid analysis of epitope-paratope interactions between HIV-1 and a 17-amino-acid neutralizing microantibody by electrospray ionization mass spectrometry.

Progress in therapeutic or prophylactic immune intervention in HIV-1 infections may only come about with a detailed understanding at the molecular/atomic level of how antibodies neutralize (inactivate) virus infectivity. Currently information on the molecular aspects of antibody-virus interaction comes predominantly from X-ray crystallography, a process that is dependent on the production of suitable crystals. NMR can also be valuable but is complex and time consuming, while mass spectrometry has been limited to matrix-assisted laser-desorption ionization (MALDI) analysis of peptides eluted from the cognate antibody. Here, we have used electrospray ionization mass spectrometry (ESI-MS) to detect directly the interactions of a novel 17-amino-acid microantibody (MicroAb) that has HIV-1-inhibitory activity, and peptides representing the V3 regions of primary HIV-1 strains isolated from Brazil (clade B) and Africa (clade A). The MicroAb is based on the third complementarity-determining region of the heavy chain (CDR-H3) of a murine monoclonal IGGI (F58) specific for the V3 loop of the gp120 envelope glycoprotein of HIV-1. ESI-MS proved to be rapid (taking < 3 h for the entire analysis), sensitive (analytes at 2 mmol/ml), and accurate (RMM estimation to 0.01-0.1%). With it, we showed that the MicroAb forms complexes with the V3 peptides, implying that its antiviral activity is mediated by binding directly to the virus particle. In addition, through controlled protease digestion of the V3 peptides, we concluded that the CDR-H3 MicroAb bound to RKXXXIGPGR, a region similar to the epitope of the whole IgG as determined by ELISA. We believe that the approach exemplified here will be applicable generally to the identification of groups involved in receptor-ligand interactions.

Amino Acid Sequence↗
PubMed1998

The hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) exists in several forms as shown by electrospray-ionisation mass spectrometry.

The hydroxylase of the soluble methane monooxygenase from the bacterium Methylococcus capsulatus (Bath) has been investigated by means of electrospray-ionisation mass spectrometry (ESI-MS) and liquid chromatography ESI-MS (LC/ESI-MS). The hydroxylase is a non-heme diiron protein consisting of three pairs of non-identical subunits (alpha approximately 60 kDa, beta approximately 45 kDa and gamma approximately 20 kDa). Liquid chromatographic separation of the hydroxylase subunits was required before MS analysis in order to detect the alpha-subunit. The masses measured for the three subunits were found to disagree with those calculated from their gene sequences. Experiments involving the use of CNBr and trypsin cleavage followed by LC/ESI-MS and MS/MS analyses permitted the location and correction of errors in the sequences deduced from the use of cDNA. The ESI-MS results also showed that the alpha-subunit of the hydroxylase exists in multiple forms which result from cleavage of the protein. This observation explains a number of enigmatic features of the protein previously reported in the literature and illustrates the pivotal role of ESI-MS in complementing data obtained from molecular biology for the characterisation of the primary sequence of proteins.

Cyanogen Bromide↗
PubMed1996

Use of electrospray ionization mass spectrometry and tandem mass spectrometry to study binding of F0 inhibitors to ceroid lipofuscinosis protein, a model system for subunit c of mitochondrial ATP synthase.

Ceroid lipofuscinosis protein (CLP), the major accumulating protein in several forms of ceroid lipofuscinosis, has an amino acid sequence that is identical to that of the F0 subunit c of normal bovine ATP synthase. Electrospray ionization mass spectrometry (ESI-MS) has shown that ovine CLP and normal bovine F0 subunit c are identical, including a 42 mass unit post-translational modification. Although the identity and the location of this modification have not been fully established in both species, CLP can be used as a convenient and a unique source of subunit c for studies of F0 inhibitor interactions by ESI-MS analysis. Analysis of mixtures of CLP incubated with several known F0 inhibitors showed that N, N'-dicyclohexylcarbodiimide and organotins bind covalently to CLP but interactions with oligomycin and venturicidin were not observed. The sulphydryl inhibitors, 2,3-dimethoxy-5-methyl-1,4,-benzoquinone (UQ0) and N-ethyl maleimide (NEM) were also shown to bind covalently to the protein. The binding stoichiometry and the relative rate of reaction were then determined for each inhibitor. Tandem mass spectrometry experiments performed on the [M+5H]5+ ion of the intact CLP and of the complexes UQ0-CLP and NEM-CLP allowed the identification of 80% of the CLP sequence and revealed that UQ0 and NEM are both bound to cysteine-64. This work shows the exceptional utility of ESI-MS in studies of the interaction of CLP with a range of inhibitors which are applicable to studies of the F0 component of ATP synthase.

Adenosine Triphosphatases↗
PubMed1994

High resolution 1H NMR spectroscopic studies of the metabolism and excretion of ampicillin in rats and amoxycillin in rats and man.

High resolution proton nuclear magnetic resonance (1H NMR) spectroscopy has been used to investigate the metabolism and urinary excretion of the aminopenicillins, ampicillin and amoxycillin, in rats and of amoxycillin in man. 1H NMR resonances of the aminopenicillins, together with those for their 5R, 6R and 5S, 6R penicilloic acids and diketopiperazine metabolites were detected, assigned and quantified in urine samples with the aid of spin-echo NMR techniques. The dimer of amoxycillin was detected in rat urine for the first time together with novel drug-related resonances assigned to amoxycillin carbamate. Quantitative 1H NMR spectroscopic results were consistent with HPLC and microbiological data considering that only single measurements were recorded. Due to the short analysis time and simple sample preparation, NMR was particularly useful for studying the metabolism of the aminopenicillins for which sample degradation poses analytical problems. The non-invasive character of 1H NMR spectroscopic analysis of urine also provided unique information on a reversible reaction between amoxycillin and bicarbonate, an endogenous urinary metabolite.

Amoxicillin↗
PubMed1994

Delphinium alkaloids as inhibitors of alpha-bungarotoxin binding to rat and insect neural membranes.

A series of C19-diterpenoid alkaloids purified from Delphinium were evaluated as inhibitors of alpha-bungarotoxin binding to rat and house fly neural membranes. In comparing these diterpenoid analogs, a wide range of inhibition potencies (IC50) were observed, with calculated IC50 values ranging six orders of magnitude. The most potent inhibitory alkaloids in this series possessed the succinimide aromatic ester moiety in the C18 position. Glaudelsine had an IC50 value of 42 pM at the insect nicotinic acetylcholine receptor.

Alkaloids↗
PubMed1991

Determination of the amino acid sequence of cystine-containing peptides by tandem mass spectrometry.

A method has been developed for the determination of the amino-acid sequence of a cyclic peptide containing cystine. It is based on the reduction of the peptide in a reductive matrix prior to ionization by fast-atom bombardment. The amino-acid sequence of the resulting linear peptide is then determined by tandem mass spectrometry from the spectrum produced by the collision-induced decomposition of the [M + H]+ ion of the peptide.

Amino Acid Sequence↗
PubMed1986

N-formylpenicillamine and penicillamine as degradation products of penicillins in solution.

The degradation of several penicillins in unbuffered aqueous solution produces N-formylpenicillamine, in some cases in high yield. Very little or no penicillamine is formed under these conditions. N-Formylpenicillamine was produced from benzylpenicillin at pH values between 2.5 and 7, with a maximum yield of 30% at pH 5, whereas penicillamine was produced only at pH 5 or below in a yield of less than 1%. Benzylpenicillenic acid at pH 5 gave a 20% yield of N-formylpenicillamine and no penicillamine whereas benzylpenicilloic, penilloic and penillic acids gave no N-formylpenicillamine and a small amount of penicillamine.

Amoxicillin↗
PubMed1984

The effects of diltiazem on Periplaneta americana.

The effects of the calcium antagonist diltiazem on nerve and muscle in the cockroach Periplaneta americana were examined. Diltiazem was observed to inhibit myogenic and glutamate-induced contractions of the visceral muscle while having either a potentiating, an inhibiting or a biphasic effect against proctolin-induced contractions. Against the isolated nervous system, diltiazem induced an increase in spontaneous discharge activity, followed by nerve block. Injection of diltiazem into cockroaches produced behavioral and toxic effects.

Action Potentials↗
PubMed1983

Structure re-assignment of a metabolite of ampicillin and amoxycillin and epimerization of their penicilloic acids.

Products formed in-vitro from ampicillin and amoxycillin penicilloates have been examined by high-performance liquid chromatography, ultraviolet and nuclear magnetic resonance spectroscopy and thiol group determination and were found to be the 5S epimers of the penicilloic acids. This is in contrast to a published claim that the corresponding penamaldic acids were formed by the treatment used.

Amoxicillin↗
PubMed1982

An early sodium and a late calcium phase in the afterdischarge of peptide-secreting neurons of Aplysia.

Following brief electrical stimulation of a pleuroabdominal connective nerve, the clusters of peptidergic bag cell neurons of Aplysia generate a long-lasting (or approximately 30 min) synchronous afterdischarge. In a normal seawater medium, the afterdischarge comprises an early phase of rapid firing (2-6 Hz) lasting for less than one minute, followed by a second, prolonged phase of lowering firing rate (less than 0.5 Hz). We have found that the transition from the first, rapid-firing phase to the prolonged second phase of afterdischarge is associated with an increase both in the width and height of intracellularly recorded action potentials. In addition, we find that in the presence of the potassium channel-blocker, TEA, such biphasic afterdischarges may be triggered by depolarizing current that evokes action potentials in a single bag cell neuron in a cluster. Afterdischarges resembling either the first or the second phase of a normal afterdischarge may be produced by using media deficient in calcium or sodium, respectively, in combination with potassium channel-blockers. Brief stimulation in media deficient in calcium give rise to afterdischarges of high firing frequency (2-6 Hz) lasting for less than one minute (mean duration = 41 s) while, in a sodium-free medium, longer afterdischarges (mean duration = 18 min) of lower firing rate (less than 0.3 Hz) were generated. At the end of afterdischarge in a normal seawater medium, containing both sodium and calcium ions, the bag cell neurons become refractory to stimulation. Thus, further stimulation either fails to induce afterdischarge or results in afterdischarges that are attenuated both in frequency of firing and in duration compared with the first afterdischarge. In these experiments we found that the brief afterdischarges, observed in low-calcium media, which resemble the first phase of a normal afterdischarge, did not induce subsequent refractoriness while afterdischarges evoked in the normal calcium, sodium-free medium did result in refractoriness to further afterdischarge. Our data indicate that both phases of a bag cell afterdischarge are due to intrinsic bag cell mechanisms and suggest that the firing pattern in the first phase is largely sodium dependent and that of the second, slow phase is calcium dependent. Moreover, our data indicate that it is the second, calcium-dependent phase that induces the refractoriness that follows bag cell afterdischarge.

Animals↗