Primary structure of a 15-kDa protein from rat intestinal epithelium. Sequence similarity to fatty-acid-binding proteins

An abundant and novel cytosolic protein was purified from the rat intestinal epithelium by gel filtration, ion-exchange and hydroxylapatite chromatography. The protein was eluted into two different positions (fractions 1 and 2) on DEAE-cellulose chromatography. We have completed the primary structure of the protein of fraction 1 by Edman degradation. The protein (144565 Da) contains 127 amino acid residues and has an acetylated alanine at its NH2-terminus. Comparison of the primary structure of the protein with porcine gastrotropin [Walz, A. D., Wider, M. D., Snow, J. W., Dass, C. & Desiderio, D. M. (1988) J. Biol. Chem. 263, 14189-14195] and rat hepatic fatty-acid-binding protein revealed that identical residues within these proteins are found in 90 and 54 out of a total of 127 positions, respectively. Bioactivity studies demonstrated that neither the protein nor liver and intestinal fatty-acid-binding proteins influence gastric acid secretory activity in rats with gastric fistulas compared to pentagastrin. The protein showed very low affinity for palmitic-acid-binding in vitro assay system and only trace amounts of endogenous fatty acids were detected from the protein. The protein, rat intestinal 15-kDa protein is considered to be a new member of the fatty-acid-binding protein family based on its structural features.     

Possible role of rat fatty acid-binding proteins in the intestine as carriers of phenol and phthalate derivatives

Fatty acid-binding proteins of hepatic and intestinal type and gastrotropin-like protein (GTLP) were purified from rat intestinal cytosol by Sephadex G-75 gel filtration and DEAE-cellulose, CM-cellulose, and hydroxylapatite chromatographies. In addition to fatty acids, butylated hydroxytoluene (BHT), phthalate dibutyl, and di(2-ethylhexyl) esters (DBP and DEHP) were identified by gas liquid chromatography and mass spectrometry as endogenous ligands from the extract of either fatty acid-binding protein superfamily. These protein families in the intestine may have an important role as carriers in the initial step of arresting these exogenous pollutants.     

Utilization of vitamin A in rats with inflammation

Reductions in the concentration of retinol (vitamin A) in serum, lung and kidney were observed in rats subjected to inflammation-inducing treatments (turpentine oil injection of thermal injury). At the same time, the liver showed an almost normal vitamin A content. Feeding of retinol to vitamin A-depleted rats with inflammation revealed that intestinal absorption of retinol was still active in the inflamed state, and the livers of these rats showed good incorporation of retinol. The livers of normal and vitamin A-depleted rats subjected to the inflammatory treatments showed a normal RBP content (retinol-binding protein) and hepatic release of holo-RBP into the serum was not impaired functionally. These results suggest the possibility that the decreases of vitamin A in the lung, serum and kidney may be due primarily to enhanced local consumption of vitamin A related to the inflammation, rather than to a reduced supply of vitamin A from the liver or to decreased intestinal absorption. In bovine serum albumin (BSA)-sensitized rats produced by direct intubation of BSA into the lungs, the level of vitamin A in the lung decreased prior to that in the liver or serum, supporting the hypothesis that the decrease in vitamin A in the inflamed lungs of these rats may be due mainly to the consumption of vitamin A in the lung in response to inflammation.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.     

Selective differential staining of sister chromatids of the facultative heterochromatic X chromosome in the female mouse

Selective differential staining of sister chromatids for the facultative heterochromatic X chromosome in the female mouse has been achieved by the combination of two differential staining techniques; one for the heterochromatic X chromosome and the other for sister chromatids. Thermal hypotonic treatment moderately destroyed the chromosome structure except for the heterochromatic X in BrdU labelled metaphase cells, resulting in the selective sister chromatid differentiation of this X with Giemsa stain. This technique enables us to know the exact frequency of the spontaneous sister chromatid exchanges in the heterochromatic X without using ³H-TdR labelling for detecting the late DNA replication. The results indicate that the sister chromatid exchange frequency of the heterochromatic X chromosome is not affected by its late DNA replication during S phase, or by the genetic inactivation and the resulting heterochromatinization.     

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.     

In vivo sister chromatid exchange in cells of various organs of the mouse

In vivo sister chromatid exchange (SCE) in mouse cells derived from various organs was studied by infusing BrdU from the tail vein. It was found that at BrdU concentrations ranging from 2.2–13.5 g/g/h, the SCE frequency in bone marrow cells seemed to stay at a constant level (1.5–2/cell/two cell cycles) whereas it started to rise as the BrdU dose exceeded this dose range. When BrdU within this dose range was infused continuously from the tail vein for appropriate hours to label chromosomes in various organs, the average SCE frequencies per cell were found to be 1.64 in bone marrow cells, 1.82 in spermatogonia, 1.99 in splenic cells, 2.89 in intestinal cells and 3.69 in cells from adjuvant stimulated lymph nodes. It is suggested that the spontaneous level of the in vivo SCE frequency might be about 1.5–2/cell/two cell cycles in the mouse. In cells derived from intestine and adjuvant stimulated lymph node, some unknown factors might work as a inducer of SCEs resulting in a significant increase in the SCE frequency in these organs.     

Corelike structures in a stable L-form ofStreptococcus pyogenes

Corelike structures, which were interpreted as straight, large cylinders containing ribosome-like particles surrounded by an amorphous substance of low electron density, were found in a stable L-form ofStreptococcus pyogenes grown in the absence of antibiotics.     

Crossed inhibition as revealed by cross-correlogram between bilateral homonymous motor unit spikes in man.

Neuromuscular unit (NMU) spikes activated during the tonic vibration reflex (TVR) in man were studied by means of the cross-correlogram test. Those NMUs which preferredly fired at some definite interval with good correlation with vibration were called "locked" spikes. The amount above zero correlation level seen in the cross-correlogram was expressed as ipsilateral "excitatory frequency", Ef. TVR contraction of the ipsilateral quadriceps femoris muscle was stimulated by voluntary contraction of the contralateral quadriceps femoris. I. Cross-correlogram test of the contralateral voluntary NMU spikes with ipsilateral vibration revealed "contralateral inhibitory modulation". Such inhibitory modulation was observed in 24% of the contralateral NMUs during voluntary tracking contraction with visual feed-back and in 22% of NMUs during blind simulated contraction without visual feed-back. The "subtracted" amount, i.e., contralateral inhibitory modulation, was also expressed as the contralateral "inhibitory frequency", If, which was calculated from the indentations of the cross-correlogram. 2. When varied vibratory frequency was applied to the muscle both Ef and If attained their maximum value around an "optimal frequency" of vibration, i.e., 80-95 HZ. 3. The ratio Ef/If was proposed as the reciprocal co-activation ratio which was fairly independent of the change of vibratory frequency. An average value of Ef/If ratio was calculated at 2.2 +/- 0.9 from 19 experiments.     

Dielectric relaxation of DNA in aqueous solutions.

Using a four-electrode cell and a new electronic system for direct detection of the frequency differences specturm of solution impedance, the complex dielectric constant of calf thymus DNA (M_r = 4 × 10⁶) in aqueous NaCl at 10°C is measured at frequencies ranging from 0.2 Hz to 30 kHz. The DNA concentrations are C_p = 0.01% and 0.05%, and the NaCl concentrations are varied from C_s = 10^?4 M to 10^?3 M. A single relaxation regions is found in this frequency range, the relaxation frequency being 10 Hz at C_p = 0.01% and C_s = 10^?3 M. At C_p = 0.05% it is evidenced that the DNA chains have appreciable intermolecular interactions. The dielectric relaxaton time τ_d at C_p = 0.01% agrees well with the rotational relaxation time estimated from the reduced visocisty on the assumption that the DNA is not representable as a rigid rod but a coiled chain. It is concluded that the dielectric relaxiatioinis ascribed to the rotation of the molecule. Observed values of dielectric increment and other experimental findings are reasonably explained by assuming that the dipole moment of DNA results from the slow counterion fluctuation which has a longer relaxation time than τ_d.