Chronobiological Aspects of Jejunum Function in Humans

This review is intended to present our knowledge on both chronomorphology and chronophysiology of jejunum mainly in humans, as well as on implications of chronobiologic principles in the practice of gastroenterology.     

Wolf predation and snow cover as mortality factors in the ungulate community of the Bialowieża National Park,Poland

Summary Wolf-ungulate interactions were studied in the pristine deciduous and mixed forests of the Bialowiea National Park in 1985–1989. The study period included two severe and two mild winters. The community of ungulates inhabiting Bialowiea National Park consisted of red deer Cervus elaphus, 55% of all ungulates; wild boar Sus scrofa, 42%; and roe deer Capreolus capreolus, moose Alces alces, and European bison Bison bonasus, about 1% each. The average size of red deer groups increased from 2.7 (SD 2.35) in spring and summer to 6.9 (SD 6.84) in autumn and winter. In winter the group size of red deer was positively correlated with the depth of snow cover and negatively correlated with the mean daily temperature. Average group size of wild boar did not change significantly between seasons; it was 6.8 (SD 5.16) in spring and summer and 5.7 (SD 4.67) in autumn and winter. Analysis of 144 wolf scats showed that wolves preyed selectively on red deer. In October–April, Cervidae (mostly red deer) constituted 91% of biomass consumed by wolves, while wild boar made up only 8%. In May–September deer formed 77% of prey biomass, and the share of wild boar increased to 22%. In all seasons of the year wolves selected juveniles from deer and boar populations: 61% of red deer and 94% of wild boar of determined age recovered from wolves' scats were young <1 year old. Analysis of 117 carcasses of ungulates found in Bialowiea National Park showed that predation was the predominant mortality factor for red deer (40 killed, 10 dead from causes other than predation) and roe deer (4 killed, none dead). Wild boar suffered most from severe winter conditions (8 killed, 56 dead). The percentage of ungulates that had died from undernutrition and starvation in the total mortality was proportional to the severity of winter.     

The role of the plasma-membrane Ca2+-ATPase in Ca2+ homeostasis in Sinapis alba root hairs

The regulation of cytosolic Ca²⁺ has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca²⁺-ATPase. For this purpose, erythrosin B was used to inhibit the Ca²⁺-ATPase, and the Ca²⁺ ionophore A₂₃₁₈₇ was applied to manipulate cytosolic free [Ca²⁺] which was then measured with Ca²⁺-selective microelectrodes. (i) At 0.01 M, A₂₃₁₈₇ had no effect on the membrane potential but enhanced the Ca²⁺ permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A₂₃₁₈₇ first caused a decrease in cytosolic Ca²⁺ by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca²⁺ spontaneously reversed and became an increase, a process which strongly depended on the external Ca²⁺ concentration, (iv) Upon removal of A₂₃₁₈₇, the cytosolic free [Ca²⁺] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca²⁺ is an activation of Ca²⁺ transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca²⁺ from the cytosol. The two observations that after the addition of A₂₃₁₈₇, (i) Ca²⁺ gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca²⁺-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH⁺/Ca²⁺-ATPase. Based on the finding that the Ca²⁺-ATPase inhibitor erythrosin B had no effect on cytosolic Ca²⁺, but caused a strong Ca²⁺ increase after the addion of A₂₃₁₈₇ we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca²⁺-pump. We further conclude that this ATPase is a major Ca²⁺ regulator in stress situations where the cytosolic Ca²⁺ has been shifted from its steady-state level, as may be the case during processes of signal transduction.Abbreviations and Symbols EB erythrosin B - E_m membrane potential - pCa negative logarithm of the Ca²⁺ concentration This work was supported by the Deutche Forschungsgemeinschaft (H.F.) and the Alexander-von-Humboldt-Foundation (A.T.).     

Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid

Transformation of veratric (3,4-dimethoxybenzoic) acid by the white rot fungus Phlebia radiata was studied to elucidate the role of ligninolytic, reductive, and demeth(ox)ylating enzymes. Under both air and a 100% O₂ atmosphere, with nitrogen limitation and glucose as a carbon source, reducing activity resulted in the accumulation of veratryl alcohol in the medium. When the fungus was cultivated under air, veratric acid caused a rapid increase in laccase (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) production, which indicated that veratric acid was first demethylated, thus providing phenolic compounds for laccase. After a rapid decline in laccase activity, elevated lignin peroxidase (ligninase) activity and manganese-dependent peroxidase production were detected simultaneously with extracellular release of methanol. This indicated apparent demethoxylation. When the fungus was cultivated under a continuous 100% O₂ flow and in the presence of veratric acid, laccase production was markedly repressed, whereas production of lignin peroxidase and degradation of veratryl compounds were clearly enhanced. In all cultures, the increases in lignin peroxidase titers were directly related to veratryl alcohol accumulation. Evolution of ¹⁴CO₂ from 3-O¹⁴CH₃-and 4-O¹⁴CH₃-labeled veratric acids showed that the position of the methoxyl substituent in the aromatic ring only slightly affected demeth(ox)ylation activity. In both cases, more than 60% of the total ¹⁴C was converted to ¹⁴CO₂ under air in 4 weeks, and oxygen flux increased the degradation rate of the ¹⁴C-labeled veratric acids just as it did with unlabeled cultures.     

In vivo and in vitro dielectric properties of animal tissues at radio frequencies

An open-ended coaxial line and an improved measurement method employing a computer controlled network analyzer were used to measure the permittivity of cat tissues. Muscle, spleen, kidney cortex, liver,and brain cortex were measured in vivo and in vitro at frequencies between 100 MHz and 8 GHz. The differences between the permittivities of these cat tissues, in the aforementioned range of frequencies, when measured in vivo and a few (up to four) hours after death, were found to be within the experimental uncertainty.     

Local secondary structure in ribonuclease A denatured by guanidine · HCl near 1 °C

Recent work has shown that a short α-helix can be stable in water near 1 °C when stabilized by specific interactions between side-chains, while earlier “host-guest” results with random copolymers have shown that a short α-helix is unstable in water at all temperatures in the absence of stabilizing side-chain interactions. As regards the mechanism of protein folding, it is now reasonable on energetic grounds to consider isolated α-helices and β-sheets as the first intermediates on the pathway of protein folding. Proton nuclear magnetic resonance is used here to detect isolated secondary structures in ribonuclease A denatured by guanidine · HCl (GuHCl). Temperatures near 1 °C are used because the low-temperature stability of the C-peptide helix may be a general property of isolated secondary structures in water.Our procedure is to titrate with GuHCl the C2H resonance lines of the four histidine residues of denatured ribonuclease A. Studies of model peptides (C-peptide (lactone) and C-peptide carboxylate, residues 1 to 13 of ribonuclease A; S-peptide, residues 1 to 20) show linear titration curves for the C2H resonance of His12 above 0.5 M-GuHCl, once helix unfolding is complete. Deviations from this line are used to monitor helix formation. The GuHCl titration curves of the other three histidine residues are also linear, once unfolding is complete. The results show that the helix found in C-peptide and S-peptide is also found in denatured ribonuclease A, where it behaves as an isolated helix not stabilized significantly by interactions with other chain segments. Studies of denatured S-protein show that the remaining three His residues, His48, His105 and His119, are involved in structure only below 1 m-GuHCl at 9 °C, pH 1.9. The nature of this structure is not known. The main conclusion from this work is that the His12 helix can be observed as a stable, isolated helix in denatured ribonuclease A near 1 °C, and that none of the other three His residues is involved in a comparably stable local structure. In native ribonuclease A, His12 is within an α-helix and the other three His residues are involved in a 3-stranded β-sheet structure.The helix-coil transition of C-peptide has also been studied for other side-chain resonances by GuHCl titration. Typically, but not always, the titration curves are linear after helix unfolding takes place and resonance lines from different residues of the same amino acid type can be resolved in GuHCl solutions. This is true of the four histidine residues of ribonuclease A although their pK values in 5 m-GuHCl are nearly the same. In C-peptide, the βCH₃ resonance of Ala6 is affected strongly by GuHCl while the lines of Ala4 and Ala5 are shifted only weakly by GuHCl. Evidently the interactions between GuHCl and side-chains in an unfolded peptide depend upon neighboring groups.     

Effects of Septal Lesions on Enzymes of Acetyl-CoA Metabolism in the Cholinergic System of the Rat Hippocampus

Abstract: Electrolytic lesions made in the medial septum of the rat brain caused an 80% decrease in the activity of choline acetyltransferase and a 33% reduction in ATP-citrate lyase activity in the synaptosomal fraction from the hippocampus. Decreases in the activities of the two enzymes in the cytosol (S₃) fraction were 70 and 13%, respectively. The activities of pyruvate dehydrogenase, citrate synthase, acetyl-CoA synthase, and carnitine acetyltransferase in crude hippocampal homogenates and in subcellular fractions were not affected by septal lesions. The data indicate that ATP-citrate lyase is linked to the septal-hippocampal pathway and that the enzyme is preferentially located in cholinergic nerve endings that terminate within the hippocampus.     

MRE11–RAD50–NBS1 is a critical regulator of FANCD2 stability and function during DNA double‐strand break repair

Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross‐links. It was reported earlier that FANCD2 co‐localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid‐S to G2 phase within sites containing single‐stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring‐like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11‐processed DNA double‐strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.