Detection of antineoplastic agent induced cardiotoxicity by 31P NMR of perfused rat hearts

Development of dose dependent chronic irreversible cardiotoxicity is a key problem encountered in chemotherapy with adriamycin. Here it has been demonstrated that infusion of this agent produced distinct and largely irreversible changes in levels of phosphate metabolites and substantial acidosis that are detected by 31P NMR of the Langendorf perfused heart. Administration of the antioxidant, butylated hydroxytoluene minimizes these spectral changes but does not substantially diminish the antineoplastic activity of adriamycin. Bisantrene (CL 216,942), a noncardiotoxic anthracene with antineoplastic activity, produces only minor perturbations of the 31P spectrum of the perfused rat heart. These studies demonstrate the potential utility of employing 31P NMR to monitor acute or chronic cardiotoxicity in the perfused rat heart and for developing noninvasive in vivo NMR techniques for monitoring cardiotoxicity in experimental animals and humans.     

A solid-phase immunoassay for human immunoglobulin E: use of 125I-labeled protein A as the tracer

A solid-phase immunoassay has been developed for human immunoglobulin (Ig) E. The specific binding of ¹²⁵I-labeled protein A (¹²⁵I-PA) to the Fc region of rabbit IgG anti-IgE served as a quantitative measure of specific anti-IgE antibody bound to the IgE beads under optimal assay conditions. Inhibition of antibody binding by known amounts of standard IgE was reflected in a decreased binding of ¹²⁵I-PA. The degree of inhibition of ¹²⁵I-PA binding was related to the amount of fluid-phase IgE present and gave a standard curve which was used to determine the concentration of IgE in test samples. The sensitivity of this method and a double antibody radioimmunoassay (RIA), which was developed using the same IgE preparation and anti-IgE antibody, was approximately the same. These assays gave similar results when used to determine levels of IgE in normal human sera that had been absorbed with protein A—Sepharose to remove components responsible for specific and nonspecific interference in the assays.     

A rapid specific radioimmunoassay for unconjugated estriol in plasma.

A rapid, non-chromatographic radioimmunossaay for unconjugated estriol in pregnancy plasma has been developed which utilizes a commonly available antiestrogen antisera. Estradiol-17beta and estrone demonstrate 135% relative cross-reactivity with our antiserum, as compared with 100% for estriol. Specificity is achieved by purification of estriol with solvent partitioning using benzene: petroleum ether (1:1). The results obtained using this method are similar to a radioimmunoassay utilizing a highly specific, but commercially unavailable, antiestriol antiserum. The method is precise, with coefficients of variation ranging from 3.0 to 8.2%.     

Adenosine 3',5'-cyclic monophosphate dependent and independent protein kinase activities in 1,2-dimethylhydrazine induced rat colon cancer

The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent and cAMP-independent kinase activities were measured in the 1,2-dimethylhydrazine (DMH) induced rat colon cancer and in untreated colon. Previous studies had shown that intestinal tumors induced by chronic exposure to DMH contained 2-fold less intracellular cAMP. The present findings indicate that reduction in cAMP-dependent protein kinase activities also occur in colon cancer cells. Similar hydrogen ion dependence (pH 6-7) and approximate association constants (Ka approximately 0.1 microM) were observed for the enzymes existing in both normal and tumor tissues, while the cAMP-dependent tumor protein kinase was found to phosphorylate phosvitin and casein to a greater degree. These recent findings are consistent with the concept that the concentrations of cAMP and activities of its associated enzyme system are inversely related to the cell proliferation state.     

Generation of free radicals and initiation of radical reactions in nitrones - Fe2+ - phosphate buffer systems

Three antigenic fractions were isolated from collagenase-solubilised human glomerular basement membrane utilising affinity columns containing purified human anti-glomerular basement membrane autoantibodies. The three fractions were able to inhibit the binding of sera containing anti-glomerular basement membrane autoantibodies when measured by solid phase radio-immunoassay. Amino acid and carbohydrate analyses revealed differences in the composition of the three fractions and were consistent with them being derived from the non-collagenous components of glomerular basement membrane.     

Immunospecificity of albumin detected in 1.6 million-year-old fossils from Venta Micena in Orce,Granada, Spain

The Orce skull fragment from southern Spain, dated at 1.6 Myr, has been a subject of heated controversy since it was first discovered in 1982. If it is hominid, as its discoverers contend, it is by far the oldest fossil hominid yet found in western Europe and implies that human populations settled this region much earlier than was previously realized. Numerous stone artifacts found at the Orce sites provide evidence that hominids were indeed present there in the Lower Pleistocene. Some paleontologists maintain that the 8 cm diameter occipital fragment is from a horse, not a hominid. Two independent investigations of the residual proteins in the skull were undertaken, one at the University of Granada in Spain, the other at the University of California, San Francisco. Two immunological methods of comparable sensitivity were employed for detection and species attribution of protein extracted from fossil bone: the Granada team used an enzyme-linked-immunosorbent assay (ELISA), and the UCSF team used a radioimmunoassay (RIA). Both teams obtained reactions characteristic of human albumin in the Orce skull and horse albumin in some of the horse fossils. These results support the lithic evidence that hominids were living in Andalusia 1.6 million years ago. Am J Phys Anthropol 103:433–441, 1997. © 1997 Wiley-Liss, Inc.     

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.     

beta-Endorphin: deletion of a single amino acid residue abolishes immunoreactivity but retains opiate potency.

Two synthetic analogs of camel β-endorphin, one with omission of Leu-14 and the other with omission of Asn-20, have been assayed for immunoreactivity by radioimmunoassay, opiate activity in the guinea pig ileum preparation and analgesic potency in mice. It was found that the omission analogs had no immunoreactivity, but retained significant biological activities. As far as we are aware, this is the first instance in which deletion of a single amino acid residue in a biologically active peptide abolished immunoreactivity.     

A designed ankyrin repeat protein evolved to picomolar affinity to Her2

Designed ankyrin repeat proteins (DARPins) are a novel class of binding molecules, which can be selected to recognize specifically a wide variety of target proteins. DARPins were previously selected against human epidermal growth factor receptor 2 (Her2) with low nanomolar affinities. We describe here their affinity maturation by error-prone PCR and ribosome display yielding clones with zero to seven (average 2.5) amino acid substitutions in framework positions. The DARPin with highest affinity (90 pM) carried four mutations at framework positions, leading to a 3000-fold affinity increase compared to the consensus framework variant, mainly coming from a 500-fold increase of the on-rate. This DARPin was found to be highly sensitive in detecting Her2 in human carcinoma extracts. We have determined the crystal structure of this DARPin at 1.7 A, and found that a His to Tyr mutation at the framework position 52 alters the inter-repeat H-bonding pattern and causes a significant conformational change in the relative disposition of the repeat subdomains. These changes are thought to be the reason for the enhanced on-rate of the mutated DARPin. The DARPin not bearing the residue 52 mutation has an unusually slow on-rate, suggesting that binding occurred via conformational selection of a relatively rare state, which was stabilized by this His52Tyr mutation, increasing the on-rate again to typical values. An analysis of the structural location of the framework mutations suggests that randomization of some framework residues either by error-prone PCR or by design in a future library could increase affinities and the target binding spectrum.     

Biotechnological significance of toxic marine dinoflagellates

Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review.