Development of monoclonal antibodies to 3-hydroxy-3-methylglutaryl-coenzyme A reductase

This report describes the development of a series of monoclonal antibodies to rat liver 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR). Sera from hybridoma tumor-bearing mice were used to remove and characterize HMGR activity from a mixture of rat liver proteins. Two IgG₂ monoclonal antibodies removed separately greater than 80% HMGR activity while non-immune mouse or negative hybridoma-derived sera were ineffective. Radiolabeled immunoprecipitates of enzyme preparations resolved in one- and two-dimensional SDS-PAGE showed two predominant subunits at M_r 52,000 and 54,000. Our results indicate that in these preparations of rat liver proteins HMGR exists as a heteropolymer with at least two distinct subunits of different molecular weights.     

Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase

Purified prostaglandin H synthase contains cyclooxygenase activity that forms the hydroperoxide, prostaglandin G, and peroxidase activity which removes hydroperoxides. Since hydroperoxides are necessary activators of cyclooxygenase activity, the paradoxical presence of two apparently opposing activities requires careful interpretation. Kinetic studies indicate that the concentration of hydroperoxide needed for full cyclooxygenase activity is much less than that which gives 50 percent effectiveness with the peroxidase. Thus, the peroxidase activity of the synthase is very ineffective in decreasing the hydroperoxide concentration below levels that still permit rapid cyclooxygenase action.     

Uptake of horseradish peroxydase by the testis of Locusta migratoria during the last larval instar; relation with variations of ecdysteroid levels in haemolymph

Summary By using horseradish peroxydase (HRP) as a tracer, it is shown that the gonial region of the locust testis is an open compartment which is almost always freely penetrated by the tracer. During the last larval instar, however, the penetration of HRP decreases and ceases at the time when high levels of ecdysteroids are detected in the haemolymph by radioimmunoassay. A cause and effect relationship between tracer uptake and hormonal level could not be demonstrated by the experiments carried out up to now. From ultrastructural observations of the testis, it is concluded that the temporary isolation of the gonial compartment is not based upon any morphological structure which could act as a barrier. Penetration of the macromolecule is considered as the expression of an active uptake by the testis and the short period of nonpenetrability as a state of inertia whose significance remains to be discovered.     

Peripartum Sex Steroid Profiles and Endocrine Correlates of Postpartum Maternal Behavior in Captive Gorillas (Gorilla gorilla gorilla)

An association between pregnancy levels of estrogen and progesterone and maternal behavior has been demonstrated in several taxonomic orders of nonprimate and primate mammals, but has not so far been investigated in the gorilla. In this study we investigated whether prepartum titers of urinary estrone conjugates (E1C) or pregnanediol-3alpha-glucuronide (PdG) were related to postpartum maternal behavior in eight multiparous Western lowland gorilla females (Gorilla gorilla gorilla) housed in four zoological gardens. Urine samples were collected from each study animal for 14 days prepartum and 14 days postpartum, and measures of maternal responsiveness were scored during the first 15 days postpartum. Urine samples were assayed with radioimmunoassay for E1C and PdG. Results for the peripartum profiles of urinary E1C, as well as postpartum profiles of PdG, agree with previous findings for the gorilla, while results for late-pregnancy profiles of urinary PdG were inconclusive in confirming a prepartum increase or decrease. Neither prepartum levels of E1C or PdG, nor the E1C/PdG ratio were found to be related to measures of postpartum maternal behavior. This lack of association between late-pregnancy E1C titers per se and postpartum maternal behavior is contrary to findings in nonprimate and other primate species.     

On ecdysone-binding proteins and ecdysone-like material in nematodes.

Homogenates of Panagrellus redivivus and extracts of P. redivivus, Aphelenchus avenae and Haemonchus contortus were examined for ecdysone-binding proteins and ecdysone-like material, respectively. Sucrose density-gradient centrifugation, polyacrylamide gel electrophoresis and charcoal-dextran-binding assay failed to detect ecdysone-binding protein(s) in homogenates of P. redivivus. Ecdysone-like material was detected in extracts of the three nematodes by radioimmunoassay (RIA). The titres detected in A. avenae and P. redivivus were correlated with their population profiles. The extraction efficiency for endogenous ecdysone-like material was estimated to be 92%. The RIA did not cross-react with cholesterol at the concentrations added.     

Pyrroloquinoline quinone ameliorates l‐thyroxine‐induced hyperthyroidism and associated problems in rats

Pyrroloquinoline quinone (PQQ) is believed to be a strong antioxidant. In this study, we have evaluated its hitherto unknown role in l ‐thyroxin (L‐T₄)‐induced hyperthyroidism considering laboratory rat as a model. Alterations in the serum concentration of thyroxin (T₄) and triiodothyronine (T₃); lipid peroxidation (LPO) of liver, kidney, heart, muscles and brain; in the endogenous antioxidants such as superoxide dismutase, catalase and glutathione and in serum total cholesterol, high‐density lipoprotien, triglycerides, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and urea were evaluated. Administration of l ‐T₄ (500‐µg kg^?1 body weight) enhanced not only the serum T₃ and T₄ levels but also the tissue LPO, serum SGOT, SGPT and urea with a parallel decrease in the levels of antioxidants and serum lipids. However, on simultaneous administration of PQQ (5 mg kg^?1 for 6 days), all these adverse effects were ameliorated, indicating the potential of PQQ in the amelioration of hyperthyroidism and associated problems. Possibly, the curative effects were mediated through inhibition of oxidative stress. We suggest that PQQ may be considered for therapeutic use for hyperthyroidism after dose standardization. Copyright © 2014 John Wiley & Sons, Ltd.     

Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.     

Improved liquid chromatographic determination of serum cortisol with double internal standardization compared to radioimmunoassay and fluorometry, and evaluated by isotope dilution/mass spectrometry

A sensitive and specific high-performance liquid chromatographic (HPLC) method for the determination of cortisol in only 200 microliters of serum is described. Cortisol and two internal standards, 19-nortestosterone (IS1) and 6 alpha-methylprednisolone (IS2) are extracted with dichloromethane and analyzed on a C18 reversed-phase column eluted with a mobile phase of methanol:water at a flow rate of 0.75 ml/min. Ultraviolet absorption at 254 nm is used for detection and quantitation is performed by peak height ratio measurement. Using 200 microliters of serum, the lower limit of detection for cortisol is 10 ng/ml, the analytical recovery is 104 +/- 3.6% (n = 8), and the day-to-day precision was 1.69% at a level of 90 ng/ml (n = 16). Cortisol values obtained by this method were generally lower than those obtained by radioimmunoassay or by fluorometry. A serum pool was analyzed both by HPLC and by isotope dilution/mass spectrometry (ID/MS). A mean value of 90.1 ng/ml was obtained by HPLC (n = 16, CV = 1.7%), whereas ID/MS yielded a mean of 90.8 ng/ml (n = 28, CV = 0.4%). These results clearly demonstrate the high specificity and the accuracy of the HPLC procedure. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of medication by either of these two products.     

Central and peripheral forms of C-type natriuretic peptide (CNP): evidence for differential regulation in plasma and cerebrospinal fluid

Aminoterminal proCNP (NTproCNP), a stable product of CNP gene expression and readily measured in human plasma, provides a new approach to studies of CNP which is rapidly degraded at source. CNP is detectable in human CSF but the presence and proportions of NTproCNP in CSF are unknown. Since CNP is widely expressed throughout the CNS, we hypothesized that the ratio of NTproCNP to CNP in CSF is greatly increased when compared to plasma and that CSF CNP peptides may contribute to their concentrations in the systemic circulation. Concurrent plasma and CSF concentrations of CNP forms were measured in 51 subjects undergoing spinal anesthesia for arranged orthopedic procedures. Elevated concentrations of NTproCNP (1045 ± 359 pmol/L), characterized by HPLC-RIA, were found in CSF and greatly exceeded those of CNP (7.9 ± 3.2 pmol/L). The ratio of NTproCNP to CNP in CSF (145 ± 55) was much higher than in plasma (31 ± 27). A significant inverse relation was found between plasma and CSF CNP concentrations (r = −0.29, p < 0.05). cGMP and neprilysin were unrelated to CNP levels in CSF. We conclude that CNP is differentially regulated across the brain in normal health. Despite markedly elevated levels of NTproCNP in CSF, it is unlikely that these contribute to systemic levels in healthy adults. Identifying NTproCNP as the dominant CNP form in CSF opens up the possibility of its use in future studies exploring CNP regulation within the CNS and possible applications in the diagnosis and monitoring of subjects with central neural disorders.     

Isoprostanes and asthma

Isoprostanes are prostaglandin (PG)-like compounds generated in vivo following oxidative stress by non-enzymatic peroxidation of polyunsaturated fatty acids, including arachidonic acid. They are named based on their prostane ring structure and by the localization of hydroxyl groups on the carbon side chain; these structural differences result in a broad array of isoprostane molecules with varying biological properties. Generation of specific isoprostanes is also regulated by host cell redox conditions; reducing conditions favor F₂-isoprostane production while under conditions with deficient antioxidant capacity, D₂- and E₂-isoprostanes are formed. F₂-isoprostanes (F₂-isoP) are considered reliable markers of oxidative stress in pulmonary diseases including asthma. Importantly, F₂-isoP and other isoprostanes function as ligands for PG receptors, and potentially other receptors that have not yet been identified. They have been reported to have important biological properties in many organs. In the lung, isoprostanes regulate cellular processes affecting airway smooth muscle tone, neural secretion, epithelial ion flux, endothelial cell adhesion and permeability, and macrophage adhesion and function. In this review, we will summarize the evidence that F₂-isoP functions as a marker of oxidative stress in asthma, and that F₂-isoP and other isoprostanes exert biological effects that contribute to the pathogenesis of asthma. This article is part of a Special Issue entitled Biochemistry of Asthma.