Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.     

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure

AIMS: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). METHODS AND RESULTS: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. CONCLUSION: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. Significance and Impact of the Study: This information should help in an understanding of genetic exchanges in lactic acid bacteria.     

Novel non-isotopic method for the localization of receptors in tissue sections.

We describe a novel fluorescent method for the detection of receptors for chimeric proteins in tissue sections. The technique was developed using a recombinant human insulin-like growth factor (IGF-1) chimera, bearing six additional histidine residues at the carboxy-terminal end (IGF-1-His). We demonstrated that dehydration of the tissue sections was detrimental for binding and that its prevention dramatically increased sensitivity. The specificity of IGF-1-His interaction was shown by gradual abolition of the fluorescent signal in the presence of increasing concentrations of IGF-1. Combining immunofluorescence with in situ ligand binding, we showed that IGF-1-His binding corresponded to the IGF-1 receptor (IGFR-1) distribution in human fetal kidney. Moreover, incubation of the tissue sections with an anti-IGFR-1 blocking antibody abolished IGF-1-His binding, demonstrating that the interaction was mediated by the IGFR-1. The method was also used to localize the IGFR-1 in E18 rat embryo sagittal sections. The IGF-1-His binding pattern was observed in brain, cartilage, lung, skin, heart, diaphragm, and tongue, and paralleled the previously reported IGFR-1 distribution. We believe that this new non-isotopic in situ ligand binding method will facilitate rapid and accurate localization of receptors in tissue sections.     

Expansion of repetitive DNA into cytogenetically visible elements.

Two cases of amplified repetitive elements accidentally identified in cancer samples are reported. In both cases, repeated DNA that is normally not visible by traditional chromosome banding had increased in amount to become cytogenetically visible. In one case, an addition to the short arm of chromosome 1 was originally diagnosed. However, upon molecular analysis the diagnosis could be corrected to an amplification of the D1Z2 repeat. In the second case, a strongly DAPI-positive band was visible at the top of the short arm of chromosome 22, and the original diagnosis was add(22). Staining for telomeric repeats revealed their presence inside the DAPI-positive element, thus confirming that the element in question was truly added to the end of the chromosome. Curiously, no telomeric repeats could be detected distal to the DAPI-positive element. The identity of the DAPI-positive element could not be established, as it was not stained by any of the specific probes applied, nor in a scanning hybridization with labeled Cot-1 DNA. It thus seems to represent an expansion from some lowly repetitive AT-rich DNA translocated to the tip of chromosome 22.     

Mechanisms of long-term synaptic depression in the hippocampus]

Long-term depression (LTD) was studied in hippocampal slices obtained from neonatal rats at the synapses between CA3 and CA1 pyramidal neurons. The induction of the LTD required pairing of Ca2+ influx into the postsynaptic CA1 neuron through voltage-gated Ca2+ channels with activation of metabotropic glutamate receptors. The expression of this form of LTD is at least partly presynaptic, suggesting the need for a retrograde messenger. We present evidence that arachidonic acid might serve such a function. Thus applications of arachidonic acid simulate LTD whereas blockade of arachiidonic acid release inhibits LTD.     

Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane.

A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder.     

L-glutamate activated membrane conductivity in spinal cord neurons during early chick embryogenesis

Transient and steady-state components of L-glutamate-activated membrane currents were investigated using intracellular perfusion, voltage clamp, and concentration clamp techniques in spinal cord neurons of 6–11 day chick embryos. Hill's coefficient was found to equal 1 for transient and 2 for steady-state components. It was shown that the L-glutamate-activated receptors are present, which appear in the membrane of spinal neurons at the early stages of development.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 251–258, March–April, 1987.