Human gut neuroanatomy: methodology for a quantitative analysis of nerve elements and neurotransmitter diversity in the human "enteric nervous system"

After a few decad of neglect, the "enteric nervous system" has recently regained the attention of investigators. Indeed, various studies, such as those which led to the isolation from the gut of a number of neuropeptides, subsequently demonstrated throughout the nervous system, have prompted major advancements of modern neuroscience. In spite of a wealth of animal investigations and a number of human studies, however, available information concerning the human "enteric nervous system" is comparatively sparse. In the opinion that such lack of information was largely due to unavailability of appropriate techniques, we have initiated and developed a new comprehensive methodology. This way, a quantitative analysis was made possible of both nerve structure and transmitter status, point-to-point along the gut, as well as within the various, functionally heterogeneous components of the gastrointestinal wall itself. After a general introduction, the present review is intended to summarize such methodology, with the addition of a few illustrative examples of application and a practically-oriented guideline to its use, in the form of technical appendix.     

Reactivity of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli with pyridoxal 5'-phosphate

The allosteric fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli was modified with pyridoxal 5'-phosphate in the presence and in the absence of phosphoenolpyruvate, fructose 1,6-bisphosphate, MgADP and MgATP. In all cases a time-dependent inactivation was observed, but the rate and the extent of inactivation varied according to the conditions used. The kinetic properties of the partially inactivated enzyme were differently modified by addition of substrates and effectors to the modification mixture, the parameters mostly affected being those concerning fructose 1,6-bisphosphate. Tryptic peptides obtained from fully inactivated pyruvate kinase in the different conditions have been separated. In all conditions three main 6-pyridoxyllysine-containing peptides were present, the amounts of which showed significant differences in the presence of fructose 1,6-bisphosphate and MgADP. The function of the labelled peptides and the evidence supporting the physical existence of different conformational states are discussed. The main conclusion concerns the involvement of one of the above peptides in the binding of the allosteric effector fructose 1,6-bisphosphate.     

Relationships Between Cytoplasmic Filaments and Nuclear Envelope in Teleost (Pimelodus maculatus) Endothelial Cells

In the present report a characteristic pattern showed by cytoplasmic filaments (intermediate-sized and actin-like) in the perinuclear area of a freshwater teleost (Pimelodus maculatus) endothelial cells is described for the first time. Thus, many intermediate-sized filaments are directly inserted in the nuclear envelope, but others are connected to one another and to the nucleus through microfilaments. It is suggested that these particular relationships between the nucleus and cytoplasmic filaments are responsible not only for nuclear anchorage, but also for nuclear movements     

Opioid Receptors of Neuroblastoma Cells Are in Two Domains of the Plasma Membrane that Differ in Content of G Proteins

Opioid receptors of NG 108-15 cell membranes are distributed in two membrane fractions sedimenting at 20,000 g (P2) and 200,000 g(P3). The number of receptors is identical in P2 and P3, but in P2 all sites are present in one high-affinity state (2 nM), whereas in P3 60% of these receptors display lower affinity (150 nM). Upon addition of GTP or pretreatment with pertussis toxin, 80% of the sites exist in low affinity in both P2 and P3. Therefore, the effect of GTP and pertussis toxin on agonist binding appears to be smaller in P2 than in P3. In contrast, sodium inhibits agonist binding in P2 and P3 to the same extent and with identical potency. Opioid-mediated stimulation of GTPase is much greater in P2 than in P3, whereas inhibition of adenylate cyclase does not differ in the two fractions. Using site-specific antibodies and pertussis toxin-catalyzed ADP-ribosylation, we found that the amount of G proteins in P3 is only 30-50% of that in P2. Treatment of intact cells with the hydrophilic protein-modifying agent sulfosuccinimido-biotin results in biotinylation of proteins from both fractions and in a similar reduction of opioid binding in P2 and P3. Likewise, exposure of intact cells to the alkylating opioid antagonist, chlornaltrexamine, produces identical degrees of receptor inactivation in P2 and P3. The rate of in vivo pertussis toxin-mediated modification of G proteins is not different in the two fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of irradiance on in vitro growth and proliferation of Vaccinium corymbosum (highbush blueberry) and subsequent rooting in vivo

The effects of light on in vitro proliferation and subsequent in vivo rooting and acclimatisation of Vaccinium corymbosum were investigated. The shoots were exposed in vitro to different irradiances (total radiation ranging from 55 to 240 μmol m⁻² s⁻¹) for 7 to 60 days. In vitro growth and proliferation and the possible consequences on in vivo rooting were observed.
As compared to the control treatment (55 μmol m⁻² s⁻¹), higher irradiances improved proliferation and rooting ratios only with short applications (7 days). Short but high (210 μmol m⁻² s⁻¹) exposures applied at the end of the proliferation phase increased in vivo growth and rooting of the shoots. The shoots treated with strong light for longer times (14 and 28 days) showed both inhibition of growth and red colour of leaves and sprouts, and were less vigorous when transferred in vivo.     

H1° and H3.3B mRNA levels in developing rat brain

Two overlapping rat cDNAs, covering a continuous region of 1107 base pairs, have been isolated and sequenced. The clones contain identical open reading frames, encoding a 136 amino acid long polypeptide which exhibits 100% identity to other mammalian H3.3 histone variants. We show that the inserts derive, in particular, from the H3.3B gene. We used these inserts and an insert from an H1° encoding clone, previously described (6), as probes to study the accumulation of mRNAs encoding the corresponding histone replacement variants (namely, H1° and H3.3) during rat brain development. We found that the concentration of both H1° and H3.3B mRNAs decreases from the embryonal day 18 (E18) to the postnatal day 10 (P10), with inverse correlation to protein accumulation.This paper is dedicated to our friend Paolo Carbone who devoted his life to research and teaching in Genetics. We will always remember him for scientific honesty and for his unique qualities of humanity.     

Enhanced Growth and Activity of a Biocontrol Bacterium Genetically Engineered To Utilize Salicylate

Plasmid NAH7 was transferred from Pseudomonas putida PpG7 to P. putida R20 [R20(NAH7)], an antagonist of Pythium ultimum. The plasmid did not affect growth or survival of R20(NAH7) and was stably maintained under nonselective conditions in broth and soil and on sugar beet seeds. Plasmid NAH7 conferred to R20(NAH7) the ability to utilize salicylate in culture, agricultural field soil, and on sugar beet seeds. The metabolic activity of R20(NAH7), but not the wild-type R20, was greatly increased in soil by amendment with salicylate (250 μg/g) as measured by induced respiration. Population densities of R20(NAH7) were also enhanced in salicylate-amended soil, increasing from approximately 1 × 10⁵ CFU/g to approximately 3 × 10⁸ CFU/g after 35 h of incubation. In contrast, population densities of R20(NAH7) in nonamended soil were approximately 3 × 10⁶ CFU/g of soil after 35 h of incubation. The concentration of salicylate in soil affected the rate and extent of population increase by R20(NAH7). At 50 to 250 μg of salicylate per g of soil, population densities of R20(NAH7) increased to approximately 10⁸ CFU/g of soil by 48 h of incubation, with the fastest increase at 100 μg/g. A lag phase of approximately 24 h occurred before the population density increased in the presence of salicylate at 500 μg/g; at 1,000 μg/g, population densities of R20(NAH7) declined over the time period of the experiment. Population densities of R20(NAH7) on sugar beet seeds in soils amended with 100 μg of salicylate per g were not increased while ample carbon was present in the spermosphere. However, after carbon from the seed had been utilized, population densities of R20(NAH7) decreased significantly less (P = 0.005) on sugar beet seeds in soil amended with salicylate than in nonamended soil.     

Structural and functional properties of rat liver mitochondria in hexachlorobenzene induced experimental porphyria

A possible link between changes in iron and porphyrin content in liver mitochondria, from rats treated with either hexachlorobenzene, iron, or hexachlorobenzene plus iron, as a function of treatment time and their structural-functional properties, has been investigated. Normal oxidative phosphorylation in mitochondria from rats treated with iron has been shown. By contrast a significant and constant uncoupling of the phosphorylative process, fully reversed by albumin, in mitochondria from rats treated with hexachlorobenzene and hexachlorobenzene plus iron has been presented. A possible involvement of pentachlorophenol in causing these abnormalities has been proposed.