True absorption and endogenous fecal excretion of manganese in relation to its dietary supply in growing rats

A conventional balance study with 48 male weanling rats was conducted to determine true absorption and endogenous fecal excretion of manganese (Mn) in relation to dietary Mn supply, following the procedures of a previously adapted isotope dilution technique. After 10 d on a diet with 1.5 ppm Mn, eight animals each were assigned to diets containing 1.5, 4.5, 11.2, 35, 65, or 100 ppm Mn on a dry-matter basis. Three days later, each rat was given an intramuscular⁵⁴Mn injection and kept on treatment for a balance period of 16 d. Apparent Mn absorption assessed for the final 8 d, averaged 8.6 μg/d without significant treatment effects, although Mn intake ranged from 18.6 to 1200 μg/d, in direct relation to dietary Mn concentrations. Mean fecal excretion of endogenous Mn for the six treatments was 0.9, 2.7, 7.4, 11.0, 16.3, and 17.7 μg/d, respectively. These values delineate the rates to which true absorption exceeded apparent rates. True absorption, as percent of Mn intake, averaged 28.7, 15.9, 11.7, 6.1, 3.4, and 2.0, respectively, as compared with mean values of 23.9, 10.9, 6.2, 3.4, 1.2, and 0.5 for percent apparent absorption. It was concluded that both true absorption and endogenous fecal excretion markedly responded to Mn nutrition and that the reduction in the efficiency of true absorption was quantitatively the most significant homeostatic response for maintaining stable Mn concentrations in body tissues.     

Direct evidence for Ca++-induced lateral phase separation in black membranes of lipid mixtures by the analysis of gramicidin A single-channels

Single-channel conductance fluctuations are analysed for gramicidin A incorporated into binary-mixed black lipid membranes of charged phosphatidic acid and neutral lecithin in different molar ratios. At very low Ca⁺⁺ concentrations in the electrolyte (i.e. in the presence of EDTA) homogeneous lipid mixtures are identified through their conductance and life time probability distributions for integral gramicidin pores. As for the pure lipid components, the conductance histograms each show a single maximum with regular width and for all channels a single mean lifetime is found.For Ca⁺⁺-levels (10^-6–10^-5 M) that are close to the critical demixing concentration (10^-4 M) unusually broad conductance distributions and reduced lifetimes are found provided the PC content, x, of the membrane is close to the critical mixture (x _crit0.5). We interpret this as a first example of the coupling of a membrane function (the transport of ions) to a lipid matrix with locally fluctuating composition close to a critical demixing point.For the conductance histogram of gramicidin A in an equimolar mixture of PA and PC shows two well-separated maxima. A correlation analysis between conductance and lifetime of the single pores shows that the two channel populations also differ significantly in their mean channel lifetime, ^*. This finding is interpreted as being direct evidence for Ca⁺⁺-induced lateral phase separation in black lipid membranes, as has been postulated recently.Abbreviations used HEPES N-2-hydroxyethyl-piperazine-N-2-ethane-sulfonic acid - EDTA ethylenediaminetetraacetic acid     

Minimum inhibitory concentration of drugs againstMycobacterium leprae as determined by anin vitro assay

The observations that liveMycobacterium leprae after entry into cultured peritoneal macrophages from mice, reduced the EA rosetting macrophages, have been exploited to determine the minimum inhibitory concentration of diamino diphenyl sulphone and rifampicin. Diamino diphenyl sulphone showed a minimum inhibitory concentration of 0.028 μg/ml and rifampicin 0.11μg/ml when given externally. However, there was accumulation of diamino diphenyl sulphone inside the macrophages. At an external concentration of 0.028 μg/ml the concentration inside the macrophage was 0.5μg/ml. The minimum inhibitory concentration for diamino diphenyl sulphone in this assay system is higher by several folds and that for rifampicin is slightly lower, than what is reported earlier with mice foot pad experiments. The minimum inhibitory concentration reported in this assay system is quite close to what is observed forin vitro inhibition ofMycobacterium lufu with both the drugs     

Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord

Receptors for thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) on thaw-mounted sections of rabbit spinal cord have been identified biochemically and visualized by light microscopic autoradiography. Binding of [3H] [3-Me-His2]TRH to 20 microns sections exhibited high apparent affinity and a pharmacological specificity almost identical to that previously demonstrated for spinal TRH receptors in membranes. In autoradiograms, the highest density of TRH receptors appeared in the substantia gelatinosa of the dorsal gray and around the central canal, with intermediate levels in the ventral gray.     

Studies on the complex formed between glucagon and dicaprylphosphatidylcholine

The interaction between glucagon and dicaprylphosphatidylcholine (DCPC) was studied by fluorescence, circular dichroism and calorimetry, as well as by ¹H- and ³¹P-nuclear magnetic resonance. The water-soluble lipid-protein complex was also characterized by gel filtration and ultracentrifugation. The complex appeared to be monodisperse by sedimentation equilibrium measurements, with a molecular weight of (4.55 ± 0.57)·10⁴. This complex contained approximately 7 molecules of glucagon and 35 molecules of phospholipid. Proton-decoupled ³¹P-NMR spectra of the phospholipid in the lipid-protein complex display narrower resonances than those of sonicated vesicles of DCPC, and ¹H-³¹P coupling could be detected in proton coupled spectra. These NMR results, together with gel-filtration results, suggest that glucagon ‘solubilizes’ phospholipid aggregates, forming a lipid-protein complex which is smaller than sonicated preparations of DCPC. ¹H-NMR resonance of both the methionine methyl group (met-27) and the aromatic envelope of glucagon are broadened by the phospolipid, indicating that the C-terminal region and the aromatic residues are involved in the interaction with the phospholipid. Nuclear magnetic resonance titrations of the imidazole ring C(2) and C(4) protons of the histidine residue of glucagon show that DCPC lowers the pK of the imidazole. The alterations caused by the phospholipid in the far and near ultraviolet CD spectra of glucagon reflect, respectively, the increased helix content of the hormone and the fact that the aromatic residues are located in a more structured environment. The phospholipid also alters the fluorescence properties of glucagon, shifting the fluorescence emission maximum of the hormone to shorter wavelength, and enhancing its relative intensity. This suggests that the fluorophore is experiencing a more hydrophobic environment in the presence of the lipid. Binding of glucagon to the phospholipid was analysed by Scatchard plots of the enhancement of fluorescence caused by the phospholipid and showed that the equilibrium binding constants of glucagon to DCPC are (4.4 ± 0.5)·10⁴M^?1 and (7.5±0.5)·10⁴M^?1, at 15°C and 25°C, respectively. The average number of moles of phospholipid bound per mole of glucagon is 4.4±0.6. The isothermal enthalpy of reaction of glucagon with DCPC is ?20.5 kcal/mol of glucagon at 25°C and ?32.5 kcal/mol of glucagon at 15°C. The observed enthalpies can arise from glucagon-induced cyrstallization of the phospholipid, from the non-covalent interactions between the peptide and lipid as well as from the lipid-induced conformational change in the protein. These results demonstrate that, unlike the complexes formed between glucagon and phospholipids which form more stable bilayers, the complex formed between glucagon and DCPC is stable over a wide range of temperatures, including temperatures well above the phase transition.     

A high recovery method for concentrating microgram quantities of protein from large volumes of solution

A method is presented for the recovery of 40-80% of the protein from a 1 microgram/ml solution. The final protein pellet is free of detergent and other ionic compounds and is thus compatible with any denaturing solution. The primary structure of the protein is unaffected by the procedure, making the final pellet an ideal sample for any analytical procedure to determine protein structure.     

The effect of soil water status on critical phosphorus concentrations inStylosanthes hamata cv. Verano

Summary The effect of soil water status on the critical phosphorus concentration (CPC) determined in apices and whole tops ofStylosanthes hamata cv. Verano was investigated in a glasshouse trial. The species was grown with six rates of P and three ranges of soil water potential and was harvested at 10 and 14 weeks after germination. The CPC of both whole tops and apices decliced between the two harvests. At the first harvest the CPC of both whole tops and apices increased as the soil water potential decreased but at the second harvest there was no effect of soil water potential on CPC. It is suggested that at the earlier harvest water stress was delaying physiological development, resulting in a CPC characteristic of chronologically younger tissue, but that by the second harvest the decline in CPC with age had ceased for all water treatments.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.     

Seasonal nitrogen and carbon concentrations in white,brown and woody fine roots of sugar maple (Acer saccharum Marsh)

Small diameter (<1.0-mm) Acer saccharum Marsh roots were separated into white, brown and woody development state classes and analyzed for total N and C concentrations in April, July and October of 1988. White roots had greater concentrations of N and C than either brown or woody roots at each sampling date, and the N concentration of brown roots was consistently greater than that of woody roots. There were no temporal changes in N concentrations in any of the roots. C was slightly elevated in mid-summer in all three classes of roots. The data suggest the possible existence of an N translocation mechanism in ageing and developing fine roots. More research should be undertaken to establish the mechanisms of N loss in developing fine roots.     

ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.