Ca2+ transport studied with arsenazo III in Tetrahymena microsomes. Effects of calcium ionophore A23187 and trifluoperazine

Transport of Ca2+ in microsomal membrane vesicles of the Tetrahymena has been investigated using arsenazo III as a Ca2+ indicator. The microsomes previously shown to carry a Mg2+-dependent, Ca2+-stimulated ATPase (Muto, Y. and Nozawa, Y. (1984) Biochim. Biophys. Acta 777, 67-74) accumulated calcium upon addition of ATP and Ca2+ sequestered into microsomal vesicles was rapidly discharged by the Ca2+ ionophore A23187. Kinetic studies indicated that the apparent Km for free Ca2+ and ATP are 0.4 and 59 microM, respectively. The Vmax was about 40 nmol/mg protein per min at 37 degrees C. The calcium accumulated during ATP-dependent uptake was released after depletion of ATP in the incubation medium. Furthermore, addition of trifluoperazine which inhibited both (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ uptake rapidly released the calcium accumulated in the microsomal vesicles. These observations suggest that Tetrahymena microsome contains both abilities to take up and to release calcium and may act as a Ca2+-regulating site in this organism.     

Three-dimensional and surface structures of rat filiform papillae

The three-dimensional and surface structures of the simple conical papillae of the rat tongue have been demonstrated with scanning electron microscopy. The papillary projection was organized into the anterior, posterior and central core cell populations, whereas the basal region of the papilla which consisted of circularly arranged cells showed no differentiation into three autonomic cell populations. It is considered that the anterior and posterior cell populations around the central core tend to be mutually attached at the bilateral sides, and that the posterior and core cell contacts are rather close than the anterior one. The anterior papillary cells showed relatively smooth surface with little micropits and without microridges. The reticular microridges on the basal cell surface of the posterior papillary cells appear to later develop the micropits and linear microridges on the tip cell surface. These suggest that the anterior cell surface is more highly keratinized than the posterior one. The microridges or micropits on the outer cell surface and the microprojections on the inner cell surface organizing filiform papilla are considered to be the structures for the purpose of cell adhesion.     

Cellular events during radiation-induced thymic leukemogenesis in mice: abnormal T cell differentiation in the thymus and defect of thymocyte precursors in the bone marrow after split-dose irradiation

Cellular events during the development of thymic lymphomas in young B10.BR mice given leukemogenic split-dose irradiation were studied by examining the differentiation of functional T lymphocyte precursors in the regenerating thymus. It was found that leukemogenic radiation treatment resulted in a sustained depression of the level of thymic cytotoxic T lymphocyte precursors (CTLp) and of mixed lymphocyte reactivity of thymus cells when assessed between 1 and 4 mo after irradiation, in spite of the fact that the total number of thymocytes was restored to the normal level within 2 mo and continued to increase thereafter. In vitro mixing studies of normal thymocytes with thymus cells from split-dose irradiated mice provided no evidence for active suppression as a mechanism for this depressed activity. The ability of bone marrow cells from split-dose irradiated mice to regenerate the thymus and to differentiate into functional CTLp was examined by use of supralethally irradiated Thy-1 congenic recipients. Reconstitution of supralethally irradiated B10.BR Thy-1.2 mice with normal bone marrow from B10.BR Thy-1.1 mice resulted in the complete repopulation of host-thymus with donor-derived cells when assessed at 4 wk after reconstitution. Lymphocytes from the regenerating thymus of these animals were shown to contain high levels of CTLp which were donor-derived. On the other hand, when the recipient mice were reconstituted with bone marrow cells from donor mice which had been split-dose irradiated 1 mo earlier, regeneration of the recipient thymus was severely depressed when assessed at 4 wk to 3 mo after reconstitution. Although variable but small numbers of donor-derived Thy-1+ cells were detected, CTL activity for alloantigen could not be induced in these donor-derived cells. The results suggest that T cell precursors derived from split-dose irradiated donor mice were unable to undergo active proliferation and differentiation into functional CTLp. The significance of these findings on radiation-induced thymic leukemogenesis is discussed.     

Transformation by extracellular DNA produced by Pseudomonas aeruginosa

Most Pseudomonas aeruginosa strains are capable of producing extracellular DNA. Very closely linked chromosomal markers (leu+ and trp+) were co-transferred to P. aeruginosa PAO1819 (leu9001, trp9008) by the extracellular DNA produced by P. aeruginosa strains IFO3445 and PAO1 at a frequency of 10(-7) to 10(-8). Treatment of the extracellular DNA with DNase, heating at 95 C or sonication completely destroyed its transforming ability. The R plasmid in the extracellular DNA produced by P. aeruginosa IFO3445 (RP4) or PAO2142 (RLb679) could be transferred to Escherichia coli ML4901 or P. aeruginosa PAO1819. The resultant transformants showed identical resistance patterns in the respective donors, and the sizes of the DNAs of RLb679 and RP4 isolated from the transformants were the same as those in the respective donors. These results demonstrate that the extracellular DNA contains both chromosomal DNA and plasmid DNA, and that it exhibits transforming ability. This implies that transformation by the extracellular DNA produced by P. aeruginosa may occur in nature and this seems to be of clinical importance in view of the spread of R plasmids among pathogens.     

Hospital Topics: Examination of the Whole Colon with the Fibreoptic Colonoscope

During examination of the intact colon with the Olympus CF LB 185-cm colonoscope it has proved possible to reach the caecum or terminal ileum in 47 out of 50 cases (94%). Careful bowel preparation, moderately heavy sedation, and some x-ray screening are necessary for the procedure, but it was well tolerated by all patients and there was no morbidity. The average time taken to the caecum was 40 minutes and the average time to completion of examination 90 minutes. The long colonoscope was as convenient to use as the fibresigmoidoscope in examinations confined to the sigmoid colon or in patients with a colostomy, ileostomy, or ileorectal anastomosis. Of the two, the long colonoscope is the instrument of choice for clinical use.Because of the expense, time, and equipment involved colonoscopy appears to be best offered as a specialist service after x-ray studies. There is alo a limited place for colonoscopy during abdominal surgery, when it is technically an easier procedure. In this series 10 patients were saved exploratory laparotomy by examination with the colonocope, and we also diagnosed four resectable carcinomas not seen on double-contrast barium-enema studies. The colonoscope provides an effective new means of diagnosis of lesions throughout the colon.     

Lipid composition of human serum lipoproteins

1. The lipid compositions of the low-density lipoproteins, the high-density lipoproteins and the ultracentrifugal residue of human serum are presented, with emphasis on certain lipoprotein classes and lipid components not previously described. 2. Except for the lipoproteins with the lowest and highest densities, there is a trend for stepwise successive increase or, respectively, decrease in the relative amounts of the main constituents of lipoproteins. 3. High-density lipoprotein-2 and high-density lipoprotein-3 have different amounts of certain lipids; high-density lipoprotein-2 has relatively more free cholesterol and sphingomyelin; high-density lipoprotein-3 has more free fatty acids, diglycerides and ceramide monohexosides. 4. All the lipoproteins contain hydrocarbons of the alkane series. The greatest amount, which averages 4.4% of total lipid extracted, is in the ultracentrifugal residue; n-alkanes comprise 18-50% of the hydrocarbons. 5. All the lipoproteins contain ceramide monohexosides. The highest relative contents of these glycolipids are in high-density lipoprotein-3 and in the ultracentrifugal residue. 6. The ultracentrifugal residue contains 55% of the total quantity of free fatty acids present in serum. The remaining free fatty acids are distributed among the other lipoprotein classes. 7. The choline-containing phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) comprise about 90% of the phospholipids in all the lipoprotein classes except the low-density lipoprotein-2, which contains about 80% of these phospholipids. 8. The presence of a large amount of lysophosphatidylcholine in the ultracentrifugal residue and the successive decrease of sphingomyelin from the low-density lipoprotein-1 to the ultracentrifugal residue was confirmed. 9. The low-density lipoprotein-2 and the ultracentrifugal residue are characterized by relatively high contents of the lower glycerides.     

Ca(2+)-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: partial purification and Ca(2+)-dependent association of the enzyme to the microsomes.

Ca(2+)-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE-Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The Km values for ATP and casein were 11 microM and 300 micrograms/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48/80, but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca2+ and was eluted by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca(2+)-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.     

Purification of Chlamydomonas 28-kDa ubiquitinated protein and its identification as ubiquitinated histone H2B.

One of the most predominantly ubiquitinated protein species in Chlamydomonas, of which the apparent molecular mass in SDS-PAGE was 28 kDa, was found to exist abundantly in nuclei. The 28-kDa ubiquitinated protein was purified to homogeneity from the isolated nuclei of Chlamydomonas, and its partial amino acid sequence was determined. The N-terminal peptide sequence was identical with that of ubiquitin. Sequences homologous to those Chlamydomonas ubiquitin [corrected] and wheat histone H2B, and paired sequences of both of them were found in arginylendopeptidase-digested or protease V8-digested polypeptide fragments of the 28-kDa ubiquitinated protein. Based on these results, it was concluded that Chlamydomonas 28-kDa ubiquitinated protein is monoubiquitinated histone H2B.     

The secondary structure of the colicin E3 immunity protein as studied by 1H-1H and 1H-15N two-dimensional NMR spectroscopy.

By performing 1H-1H and 1H-15N two-dimensional (2D) nuclear magnetic resonance (NMR) experiments, the complete sequence-specific resonance assignment was determined for the colicin E3 immunity protein (84 residues; ImmE3), which binds to colicin E3 and inhibits its RNase activity. First, the fingerprint region of the spectrum was analyzed by homonuclear 1H-1H HOHAHA and NOESY methods. For the identification of overlapping resonances, heteronuclear 1H-15N (HMQC-HOHAHA, HMQC-NOESY) experiments were performed, so that the complete 1H and 15N resonance assignments were provided. Then the secondary structure of ImmE3 was determined by examination of characteristic patterns of sequential backbone proton NOEs in combination with measurement of exchange rates of amide protons and 3JHN alpha coupling constants. From these results, it was concluded that ImmE3 contains a four-stranded antiparallel beta-sheet (residues 2-10, 19-22, 47-49, and 71-79) and a short alpha-helix (residues 31-36).     

A site-directed mutagenesis study on the role of isoleucine-23 of human epidermal growth factor in the receptor binding.

The isoleucine-23 residue of human epidermal growth factor (hEGF) was substituted by a variety of amino acid residues and the receptor-binding activities of variant hEGFs were determined by the use of human KB cell. Tight receptor binding was found of variants with hydrophobic amino acid residues in position 23. The size of the isoleucine residue was nearly optimum for the receptor binding as compared with other hydrophobic residues. The structure analysis by two-dimensional nuclear magnetic resonance spectroscopy showed that the substitution at position 23 only slightly affected the tertiary structure of hEGF. These indicate that the side chain of isoleucine residue in position 23, which is exposed on the protein surface, directly binds to a hydrophobic pocket of the receptor.