On the role of NMDA receptors in blood pressure regulation in spontaneously hypertensive rats (SHR)

Summary In the present study the binding of [³H]MK-801 to glutamatergic receptors of the NMDA type was compared in spontaneously hypertensive (SHR) and normotensive (WKY) rats in various brain structures (including nucleus tractus solitarii) by quantitative receptor autoradiography. Additionally, blood pressure changes after treatment with the NMDA antagonist MK-801 were studied in both strains. There were no differences between SHR and WKY rats either in the level of [³H]MK-801 binding or in the hypertensive reaction to MK-801.     

Neural and smooth muscle development in the chicken gizzard

The development of the autonomic ganglia of Auerbach's plexus and gizzard smooth muscle was studied in chicken embryos. Nervous system and smooth-muscle-specific antibodies were employed in immunofluorescence stainings on tissue sections to investigate the temporal and spatial frame of neural and muscular differentiation in relation to each other. Subserosal clusters of neural cells were clearly demonstrable at embryonic day 5 (ED5), the earliest stage analysed, with the monoclonal antibody El (SGIII-1). Fine nerve fibres (ED6) and, later, large axon bundles projecting from subserosal neuron clusters towards the lumen were followed and found to reach the luminal border by ED11. Already in early development the area of the future laminar tendons on the ventral and dorsal surface of the gizzard was devoid of neuroblasts, and nerve fibres were not extending to the muscle-tendon borderline until ED16. Double stainings with antibodies to smooth muscle myosin (SMM) and El revealed that SMM expression, taken as an indicator for muscle differentiation, followed neural growth. It was first detectable in close apposition to the differentiating neuroblasts in the caudal and cranial portion of the gizzard at ED6. With further development, myosin expression proceeded inward towards the lumen in a wave which followed the ingrowth of E1-positive nerve fibres from the prospective Auerbach plexus. Neuromuscular differentiation deviated from this pattern in the lateral tendon area where nerve growth was delayed and myosin expression preceeded the arrival of E1-positive nerve fibres. The findings suggest that the gizzard could serve as a model system for the analysis of potential early nervous system imprints on smooth premuscle mesenchyme differentiation.     

Photodynamic herbicides: 1. Concept and phenomenology

A new approach to the design of conceptually and phenomenologically new herbicides is described. It involves the joint utilization of tetrapyrrole precursors, such as δ-aminolaevulinic acid (a biodegradable amino acid) and activators of the chlorophyll biosynthetic pathway, such as 2,2′-dipyridyl, in order to induce treated plants to biosynthesize and accumulate massive amounts of tetrapyrrole intermediates of the chlorophyll biosynthetic pathway in the dark (i.e. at night). During the subsequent light period (daylight) the accumulated tetrapyrroles act as potent photodynamic sensitiziers, which in turn result in the death of susceptible plants in a matter of hours. We have therefore proposed to name herbicides that act via this mechanism as photodynamic herbicides, or more pictorially as laser herbicides. From a limited survey of agricultural plant and weed species it appears that photodynamic herbicides exhibit a very pronounced organ, age and species-dependent selectivity. For example, dicotyledonous weeds such as mustard, red-root pigweed, common purslane and lambsquarter are very susceptible while monocotyledonous plants such as corn, wheat, barley and oats are not. The biochemical basis of this selectivity seems to lie, among other things, in the rates of tetrapyrrole turnover and in a differential enhancement by the applied chemicals of the monovinyl and divinyl tetrapyrrole biosynthetic pathways in the various species. A survey of various groups of chemicals (herbicides and other selected biochemicals) that are likely to exhibit photodynamic herbicidal properties is currently under investigation.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Discrimination of flash-induced fast and slow electric potential components

This work aimed at the resolution of the multi-component electric potential changes induced by single-turnover flash illumination of Photosystem-I-enriched subchloroplast vesicles. If supplemented with ferredoxin and under carefully adjusted redox poising, these vesicles show a pronounced slow-rising and -decaying electric potential component, as monitored by endogenous and exogenous field-sensitive probes, carotenoids and oxonol VI, respectively. The fast and slow potential components can be easily discriminated without the need for computer-assisted deconvolution after selective presaturation of the slow component by preillumination or a transmembrane ΔpH, after selective suppression of the slow component by low valinomycin or uncoupler concentrations or in the absence of ferredoxin. The slow electric potential component, as compared to the fast one, is relatively sensitive to low concentrations of ionophores and uncouplers, detergent, ageing and lower temperatures (4–12°C), is associated with electrogenic proton displacements and is interpreted to respond to a field that is more located on the membrane-bulk interface. Temperature effects show transition temperatures around 20°C for both the rise and decay of the slow potential component. The results provide further evidence that the carotenoids and oxonol VI sense the same (slow) electric field, but may be differently located in the thylakoid membrane.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

Serum indole-3-acetic acid in control subjects and newly abstinent alcoholics after an oral loading with L-tryptophan: a preliminary study using liquid chromatography with amperometric detection

Described in this paper is a rapid, isocratic assay for serum indole-3-acetic acid (IAA). The sample preparation involves only protein precipitation using sulfosalicylic acid, and the sensitivity of amperometric detection is in the picogram range. The chromatographic analysis time is approximately 4 min. The devised method was used for a longitudinal study of IAA levels in serum samples from control subjects and newly abstinent alcoholics. Dietary variations were eliminated by administering a 2.0-g loading dose of L-Trp to all subjects investigated. The results are presented in the form of cumulative frequency polygons. Preliminary data indicate no differences in IAA levels between newly abstinent alcoholics and control subjects.     

HMG17 protein facilitates the DNA catenation reaction catalyzed by DNA topoisomerases

HMG17 protein is shown to greatly facilitate the catention of double-stranded DNA rings catalyzed by DNA topoisomerases. Even at low DNA concentrations such that catenanes are not observable in the absence of HMG17, the addition of the protein promotes the catenation of greater than 95% of the input DNA into networks that do not enter the gel upon electrophoresis. Electron microscopy and restriction enzyme cleavage experiments indicate that these networks are large structures containing many catenated DNA rings. The HMG17-promoted DNA network formation has been observed with calf thymus type II DNA topoisomerase and the type I topoisomerases of Escherichia coli, Micrococcus luteus, and calf thymus.     

Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies

We have identified three germ cell surface carbohydrate antigens that exhibit a common, stage-specific pattern of expression during spermatogenesis in the mouse. IgM-class monoclonal antibodies designated "J1," "C6," and "A5" were absorbed by adult testis, but not by any adult somatic tissue tested. In indirect immunofluorescence assays using collagenase-dissociated prepuberal and adult testicular cells, these antibodies labeled the surfaces of early and late pachytene spermatocytes and round spermatids. Gonocytes from fetal and neonatal testes were not labeled. In paraffin sections of prepuberal and adult testes, sialidase treatment exposed antigens recognized by antibodies C6 and A5 on preleptotene, leptotene, and zygotene spermatocytes located near the perimeter of seminiferous tubules. The determinants recognized by antibodies J1, C6, and A5 were characterized partially using a sugar hapten inhibition assay. The binding of J1 to adult testicular cells was inhibited specifically by N-acetylglucosamine and the binding of both C6 and A5 was inhibited by N-acetyllactosamine. The glycoconjugates recognized by J1, C6, and A5 eluted from gel filtration columns with an apparent molecular weight greater than 1 X 10(6) and were sensitive to endo-beta-galactosidase (keratanase) treatment. The apparent high molecular weight of these glycoconjugates was confirmed by immunolabeling Western blots of testis extracts separated by SDS-polyacrylamide gel electrophoresis. The results suggest that polylactosamine (keratan) glycoconjugates of high molecular weight are associated with the plasma membranes of meiotic and haploid male germ cells. The effects of sialidase on antibody labeling patterns suggest that changes in cell surface sialylation accompany the transition of early meiotic germ cells to pachytene spermatocytes during spermatogenesis.     

A chemiluminescent probe specific for singlet oxygen

We have synthesized a methoxyvinylpyrene (MVP) in order to model the mechanism for the observed microsomal chemiluminescence of benzo[a]pyrene 7,8-dihydrodiol, the proximate carcinogenic metabolite of benzo[a]pyrene. This MVP analog has been found to be a highly efficient and specific chemiluminescent probe for picomole quantities of singlet oxygen and singlet oxygen equivalents, and it produces significant chemiluminescence when reacted with cytochrome P-450 enzymes.