NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis

Heme d₁ plays an important role in denitrification as the essential cofactor of the cytochrome cd₁ nitrite reductase NirS. At present, the biosynthesis of heme d₁ is only partially understood. The last step of heme d₁ biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d₁. Instead, the NirS purified from this strain contains the heme d₁ precursor dihydro-heme d₁ lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d₁ was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d₁ and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d₁.     

Circadian pancreatic enzyme pattern and relationship between secretory and motor activity in fasting humans.

It is unknown whether nonparallel pancreatic enzyme output occurs under basal conditions in humans. We aimed to determine whether the circadian or wake-sleep cycle influences the relationship among pancreatic enzymes or between pancreatic secretory and jejunal motor activity. Using orojejunal multilumen intubation, we measured enzyme outputs and proximal jejunal motility index during consecutive daytime and nighttime periods in each of seven fasting, healthy volunteers. Enzyme outputs were correlated tightly during daytime phases of wakefulness and nighttime phases of sleep (r > 0.72, P < 0.001). During nocturnal phases of wakefulness, output of proteases (r = 0.84, P < 0.001), but not of amylase and trypsin (r = 0.12), remained associated. Nocturnally, particularly during sleep, pancreatic secretory activity was directly correlated with jejunal motility index (r > 0.50, P < 0.001). In conclusion, parallel secretion of pancreatic enzymes dominates throughout the circadian cycle. Nonparallel secretion during nocturnal phases of wakefulness may be due to merely circadian effects or to the coupling of the wake-sleep and the circadian cycle. The association between fluctuations of secretory and motor activity appears to be particularly tight during the night.     

Müller glia endfeet,a basal lamina and the polarity of retinal layers form properly in vitro only in the presence of marginal pigmented epithelium

Summary Dissociated embryonic chicken retinal cells regenerate in rotary culture into cellular spheres that consist of subareas expressing all three nuclear layers in an inside-out sequence (rosetted vitroretinae). However, when pigmented cells from the eye margin (peripheral retinal pigment epithelium) are added to the system, the sequence of layers is identical with that of an in-situ retina (laminar vitroretinae). In order to elucidate further the lamina-stabilizing effect exerted by the retinal pigment epithelium, we have compared both systems, laying particular emphasis on the ultrastructure of the basal lamina and of Müller glia processes. Ultrastructurally, in both systems, an outer limiting membrane, inner segments of photoreceptors and the segregation of cell bodies into three cell layers develop properly. Synapses are detectable in a premature state, although only in the inner plexiform layer of laminar vitroretinae. Although present in both systems, radial processes of juvenile Müller glia cells are properly fixed at their endfeet only in laminar vitroretinae, since a basal lamina is only expressed here. Large amounts of laminin are detected immunohistochemically within the retinal pigment epithelium and along a basal stalk that reaches inside the laminar vitroretinae. We conclude that the peripheral retinal pigment epithelium is essential for the expression of a basal lamina in vitro. Moreover, the basal lamina may be responsible both for stabilizing the correct polarity of retinal layers and for the final differentiation of the Müller cells.     

A new molecular form of PYY: structural characterization of human PYY(3-36) and PYY(1-36)

A radioimmunoassay was developed using an antibody raised in rabbits against synthetic porcine PYY. This radioimmunoassay was used to detect PYY immunoreactivity in human intestinal extracts. Human colonic mucosa was extracted with acid, centrifuged and the supernatant concentrated by low pressure preparative reverse phase chromatography. A subsequent C-18 reverse phase HPLC step separated two peaks of PYY immunoreactivity. Each peak was purified by sequential steps of ion-exchange FPLC and reverse phase HPLC. In the final purification step single absorbance peaks were associated with PYY immunoreactivity. Microsequence, amino acid, and mass spectral analysis of the intact and tryptic fragments of the two peptides were consistent with the structures: YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(1-36)] and--IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(3-36)]. Human PYY(1-36) differs from porcine PYY only at position 3, with Ile instead of Ala, and position 18, with Asn instead of Ser. PYY(3-36) may differ in its biological activity from the intact peptide. Its high proportions in the colon suggest that it is released into the circulation where it could act as a partial antagonist of PYY(1-36).     

Independent spatial waves of biochemical differentiation along the surface of chicken brain as revealed by the sequential expression of acetylcholinesterase

Summary AChE-positive cells suddenly amass in a superficial layer of the neuroepithelium; this layer finally covers, in a sheat-like manner, the entire surface of the embryonic chicken brain. This feature is functionally not understood; however, it appears shortly after the neurons become post-mitotic, and the lateral extensions of this layer can easily be traced using histochemistry on serial brain sections. The layer can therefore be exploited to delineate spatially the waves of onset of biochemical tissue differentiation. We have studied whole brains between stages 11 and 30 and provide the first complete spatial schemes of brain differentiation based on computer-reconstructed, two- and three-dimensional maps. The brain does not differentiate in one smooth coherent wave, but instead five separate primary AChE-activation zones are detected: the first originating at stage 11 (rhombencephalic wave), the second at the same time (midbrain wave), the third at stage 15 ("tectal wave). A fourth zone develops later, at stage 18, from the bottom part of the telencephalon to the top. Retinal development also starts at stage 18. In a given area, it appears that AChE-development shortly precedes that of the formation of major fiber tracts. AChE might therefore represent a prerequisite for fiber growth and pathfinding.Abbreviations AChE acetylcholinesterase - BChE butyrylcholinesterase - ATC acetylthiocholine - OS ora serrata - ON optic stalk - OP otic placode