Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture

The biomass concentration extant in potassiumlimited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K⁺ requirement. Significantly, this effect of pH was not eviden with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH₄Cl were added to the glutamate-containing medium. This suggested a functional replacement of K⁺ by NH ₄ ⁺ , a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 Å) and the ammonium ion (1.43 Å). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH₄Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h^-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassiumlimited medium containing initially 0.5 mM K⁺. Qualitatively similar findings were made with cultures of K. pneumoniae; and whereas one may not conclude that NH ₄ ⁺ can totally replace K⁺ in the growth of these bacteria, it can clearly do so very extensively.     

Abundant alkali-sensitive sites in DNA of human and mouse sperm

The DNA of human and mouse sperm cells was analyzed by single-cell microgel electrophoresis, by agarose gel electrophoresis, and by alkaline elution--three techniques that can detect single-strand DNA breaks and/or labile sites. Under these conditions a surprisingly large number of single-strand DNA breaks, approximately 10(6) to 10(7) per genome, were detected in human and mouse sperm but not in human lymphocytes or in mouse bone marrow cells. These breaks were also present in chicken erythrocyte DNA, which is also highly condensed. These breaks were not observed under neutral pH conditions nor under denaturing conditions not involving alkali, suggesting that these sites are alkali-sensitive and do not represent preexisting single-strand breaks. The high frequency of such sites in sperm from healthy mouse and human donors suggests that they represent a functional characteristic of condensed chromatin rather than DNA damage.     

Inactivation of two-electron reduced medium chain acyl-CoA dehydrogenase by 2-octynoyl-CoA

The acetylenic thioester, 2-octynoyl-CoA, inactivates medium chain acyl-CoA dehydrogenase from pig kidney by two distinct pathways depending on the redox state of the FAD prosthetic group. Inactivation of the oxidized dehydrogenase occurs with labeling of an active site glutamate residue and elimination of CoASH. Incubation of the reduced dehydrogenase with 2-octynoyl-CoA rapidly forms a kinetically stable dihydroflavin species which is resistant to reoxidation using trans-2-octenoyl-CoA, molecular oxygen, or electron transferring flavoprotein. The reduced enzyme derivative shows extensive bleaching at 446 nm with shoulders at 320 and 380 nm. Denaturation of the reduced derivative in 80% methanol yields a mixture of products which was characterized by HPLC, by uv/vis, and by radiolabeling experiments. Approximately 20% of the flavin is recovered as oxidized FAD, about 40% is retained covalently attached to the protein, and the remainder is distributed between several species eluting after FAD on reverse-phase HPLC. The spectrum of one of these species ressembles that of a N(5)-C(4a) dihydroflavin adduct. These data suggest that a primary reduced flavin species undergoes various rearrangements during release from the protein. The possibility that the inactive modified enzyme represents a covalent adduct between 2-octynoyl-CoA and reduced flavin is discussed. Analogous experiments using enzyme substituted with 1,5-dihydro-5-deaza-FAD show rapid and quantitative reoxidation of the flavin by 0.5 eq of 2-octynoyl-CoA.     

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.     

Binding of actinomycin D to [d(ATCGAT)]2: NMR evidence of multiple complexes

Actinomycin D (actD) binds to the oligonucleotide [d(ATCGAT)]2 with a hypochromatic and red-shifted visible absorbance band compared to free drug and a CD spectrum with double negative bands at 460 and 385 nm. These spectral features are similar to those of the actD-[d(ATGCAT)]2 complex, while actD-[d(AT)5]2 gives spectra similar to those of free drug. Upon dilution or raising the temperature, the spectral characteristics accompanying complex formation disappear in the actD-[(ATCGAT)]2 sample but remain in the actD-[d(ATGCAT)]2 complex under the same experimental conditions. These results suggest that (a) sequence-specific binding of actD occurs with [d(ATCGAT)]2 but not with [d(AT)5]2, (b) the binding is not as strong as with [d(ATGCAT)]2, and (c) actD binds [d(ATCGAT)]2 with the same mechanism as it binds [d(ATGCAT)]2, i.e., by intercalation. From NMR spectra of the actD-[d(ATCGAT)]2 complex, three types of signals can be detected below 20 degrees C, one major and two minor ones. At higher temperatures, exchange between the two minor ones becomes fast enough that only one type of minor signal was seen. Partial resonance assignments were made by using 2D nuclear Overhauser effect (NOE) and 2D homonuclear Hartmann-Hahn (HOHAHA) experiments. Proton chemical shift changes of the major complex are consistent with actD chromophore ring intercalation between hexamer base pairs. Data from NOE-detected dipolar interactions between actD and [d(ATCGAT)]2 protons were interpreted in terms of a major complex with the actD chromophore ring system intercalated at the CG position and minor complexes with the drug intercalated off center at the GA positions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of DNA secondary structure from solvent-accessible surfaces: the B- to Z-DNA transition as a model

Solvent structure and its interactions have been suggested to play a critical role in defining the conformation of polynucleotides and other macromolecules. In this work, we attempt to quantitate solvent effects on the well-studied conformational transition between right-handed B- and left-handed Z-DNA. The solvent-accessible surfaces of the hexamer sequences d(m5CG)3, d(CG)3, d(CA)3, and d(TA)3 were calculated in their B- and Z-DNA conformations. The difference in hydration free energies between the Z and the B conformations (delta delta GH(Z-B] was determined from these surfaces to be -0.494 kcal/mol for C-5 methylated d(CG), 0.228 kcal/mol for unmethylated d(CG), 0.756 kcal/mol for d(CA)-d(TG), and 0.896 kcal/mol for d(TA) dinucleotides. These delta delta GH(Z-B) values were compared to the experimental B- to Z-DNA transition energies of -0.56 kcal/mol that we measured for C-5 methylated d(CG), 0.69-1.30 kcal/mol reported for unmethylated d(CG), 1.32-1.48 kcal/mol reported for d(CA)-d(TG), and 2.3-2.4 kcal/mol for d(TA) dinucleotides. From this comparison, we found that the calculated delta delta GH(Z-B) of these dinucleotides could account for the previous observation that the dinucleotides were ordered as d(m5CG) greater than d(CG) greater than d(CA)-d(TG) greater than d(TA) in stability as Z-DNA. Furthermore, we predicted that one of the primary reasons for the inability of d(TA) sequences to form Z-DNA results from a decrease in exposed hydrophilic surfaces of adjacent base pairs due to the C-5 methyl group of thymine; thus, d(UA) dinucleotides should be more stable as Z-DNA than the analogous d(TA) dinucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for presence of peptide alpha-amidating activity in pancreatic islets from newborn rats.

The peptide alpha-amidating activity of a homogenate of pancreatic islets from 5-7-day-old rats was investigated, using as substrate a glycine-extended tripeptide (D-Tyr-Val-Gly). The islet homogenates had a marked amidating activity, with a Km of 57 microM, a Vmax. of 185 pmol/h per mg and a pH optimum of 7.0. This activity was dependent on the presence of ascorbic acid (in the reduced form) and Cu2+, the optimum concentrations being 4 mM and 40 microM respectively. On fractionation of the homogenate, the highest specific activity was found in the soluble fraction. Exocrine pancreatic tissue showed very low levels of amidating activity.     

Different patterns of glycolipid antigens are expressed following differentiation of TERA-2 human embryonal carcinoma cells induced by retinoic acid, hexamethylene bisacetamide (HMBA) or bromodeoxyuridine (BUdR)

NTERA-2 cl.D1 human embryonal carcinoma (EC) cells were induced to differentiate by either bromodeoxyuridine (BUdR) or hexamethylene bisacetamide (HMBA), and also by retinoic acid. Following exposure to each of these inducers, the globoseries glycolipid antigens stage-specific embryonic antigens -3 and -4 (SSEA-3 and -4) and the glycoprotein antigen TRA-1-60, all characteristic of the human EC cell surface, underwent a marked reduction in expression within about 7 days. At the same time, the lactoseries glycolipid antigen SSEA-1, and ganglioseries antigens A2B5 (GT3) and ME311 (9-0-acetyl GD3) were induced in BUdR- and retinoic acid-treated cells. However, these antigens did not appear during the first 7-14 days of HMBA-induced differentiation. The observations of cell surface antigen expression were paralleled by analysis of glycolipids isolated from the cells by thin-layer chromatography. This analysis, in which the new monoclonal antibodies VINIS-56 and VIN-2PB-22 were included, also revealed expression of gangliosides GD3 and GD2 in all differentiated cultures, albeit at much lower levels following HMBA exposure than following retinoic acid or BUdR-exposure. Further, disialylparagloboside was detected in retinoic acid and BUdR-induced, but not HMBA-induced, cultures. Taken with morphological observations, the results suggest that HMBA induces differentiation of NTERA-2 cl.D1 EC cells along a pathway distinct from the pathway(s) induced by retinoic acid and BUdR.     

Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis

The phycobilisomes and phycobiliproteins of Synechococcus sp. PCC 7002 wild-type strain PR6000 have been isolated and characterized. The hemidiscoidal phycobilisomes of strain PR6000 are composed of eleven different polypeptides: phycocyanin and subunits; allophycocyanin and subunits; subunit of allophycocyanin B; the allophycocyanin -subunit-like polypeptide of M_r 18 000; the linker phycobiliprotein of M_r 99 000; and non-chromophore-carrying linker polypeptides of M_r 33 000, 29 000, 9000, and 8000. Several of these polypeptides were purified to homogeneity and their amino acid compositions and amino-terminal amino acid sequences were determined. Analyses of the phycobiliproteins of Synechococcus sp. PCC 7002 were greatly facilitated by comparative studies performed with a mutant strain, PR6008, constructed to be devoid of the phycocyanin and subunits by recombinant DNA techniques and transformation of strain PR6000. The absence of phycocyanin did not greatly affect the allophycocyanin content of the mutant strain but caused the doubling time to increase 2–7-fold depending upon the light intensity at which the cells were grown. Although intact phycobilisome cores could not be isolated from this mutant, it is probable that functionally intact cores do exist in vivo.Abbreviations used SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate - 2D-PAGE two-dimensional gel electrophoresis in which the first dimension consisted of isoelectric focusing in the presence of 8.0 M urea in the pH range 4–6 and the second dimension consisted of electrophoresis in the presence of sodium dodecylsulfate. The nomenclature employed for the phycobiliprotein subunits and linker polypeptides is that defined by Glazer (1985)     

Yeast regulatory protein LEU3: a structure-function analysis.

Eleven mutations resulting in partially deleted or truncated LEU3 protein were generated by linker insertion or other modifications at restriction sites, deletion of restriction fragments, or oligonucleotide-directed mutagenesis. Functional studies of these mutants showed the following: (i) A specific DNA binding region is contained within the 173 N-terminal residues, but other regions of the protein are required for optimal binding. (ii) Activation of LEU2 expression depends on the C-terminal 113 residues of the LEU3 protein. (iii) Deletion of part or all of a central section of LEU3 eliminates the ability of the LEU3 protein to respond to the co-activator alpha-isopropylmalate, i.e. creates an unmodulated activator. (iv) Overproduction of unmodulated activator slows down cell growth. (v) Specific deletion of two short acidic regions, including one with net charge - 19, has only minor effects on activation and modulation.