Identification and sequence of a Na+-linked gene from the marine bacterium Alteromonas haloplanktis which functionally complements the dagA gene of Escherichia coli

A 4.0 kb fragment from a plasmid genomic DNA library of the marine bacterium Alteromonas haloplanktis ATCC 19855 was found in the presence of Na+ to complement the dagA gene of Escherichia coli. We have completely sequenced this fragment and the position of the Na(+)-linked D-alanine glycine permease gene (dagA) on the fragment has been determined by complementation. The predicted carrier protein consists of 542 amino acid residues (M(r) 58,955). Its hydropathy profile suggests it is composed of eight transmembrane segments with a long hydrophilic region between segments six and seven. Significant similarity has been found between this Na(+)-linked permease and the Na+/proline permeases of E. coli and Salmonella typhimurium and the human and rabbit intestinal Na+/glucose cotransporters.     

Sensitivity of some marine bacteria, a moderate halophile, and Escherichia coli to uncouplers at alkaline pH.

The inhibitory effects of uncouplers on amino acid transport into three marine bacteria, Vibrio alginolyticus 118, Vibrio parahaemolyticus 113, and Alteromonas haloplanktis 214, into a moderate halophile, Vibrio costicola NRC 37001, and into Escherichia coli K-12 were found to vary depending upon the uncoupler tested, its concentration, and the pH. Higher concentrations of all of the uncouplers were required to inhibit transport at pH 8.5 than at pH 7.0. The protonophore carbonyl cyanide m-chlorophenylhydrazone showed the greatest reduction in inhibitory capacity as the pH was increased, carbonyl cyanide p-trifluoromethoxyphenylhydrazone showed less reduction, and 3,3',4',5-tetrachlorosalicylanilide was almost as effective as an inhibitor of amino acid transport at pH 8.5 as at pH 7.0 for all of the organisms except A. haloplanktis 214. Differences between the protonophores in their relative activities at pHs 7.0 and 8.5 were attributed to differences in their pK values. 3,3',4',5-Tetrachlorosalicylanilide, carbonyl cyanide m-chlorophenylhydrazone, 2-heptyl-4-hydroxyquinoline-N-oxide, and NaCN all inhibited Na+ extrusion from Na+-loaded cells of V. alginolyticus 118 at pH 8.5. The results support the conclusion that Na+ extrusion from this organism at pH 8.5 occurs as a result of Na+/H+ antiport activity. Data are presented indicating the presence in V. alginolyticus 118 of an NADH oxidase which is stimulated by Na+ at pH 8.5.     

Mass spectrometry and chromatography of t-butyldimethylsilyl derivatives of cytokinin bases

Di-(t-butyldimethylsilyl) derivatives of the cytokinin bases zeatin, cis-zeatin, and dihydrozeatin may be prepared quantitatively in the presence of dimethylaminopyridine. These derivatives have good gas chromatographic properties and are very suitable for gas chromatography-mass spectrometry analysis of cytokinin bases. The t-butyldimethylsilyl (tBuDMS) group at N-9 may be selectively hydrolyzed and the resulting mono-O-silyl derivatives are sufficiently stable to be subjected to thin-layer chromatography and high-performance liquid chromatography. The mass spectral fragmentation of the mono- and di-tBuDMS derivatives of adenine, zeatin, cis-zeatin, and dihydrozeatin and also of the mono-tBuDMS derivatives of N6-isopentenyladenine and 6-benzylaminopurine have been rationalized. The 9-tBuDMS moiety was characterized by an elimination of isobutene (M-56) and of isobutene plus a methyl radical (M-56-15).     

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system

Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.     

Blood–Brain Transfer of Glucose and Glucose Analogs in Newborn Rats

Little is known of the selectivity of the blood-brain barrier at birth. Hexoses are transported through the barrier by a facilitating mechanism. To study the capacity of this mechanism to distinguish between analogs of D-glucose, we compared the transport of fluorodeoxyglucose, deoxyglucose, glucose, methylglucose, mannose, galactose, mannitol, and iodoantipyrine across the cerebral capillary endothelium in newborn Wistar rats. Cerebral blood flow, glucose consumption, and the blood-brain permeabilities of the hexoses were 25-50% of the adult values but the ratios between the permeabilities of the individual hexoses were similar to the ratios observed in adult rats. The mannitol clearance into brain was considerably higher than in adult rats (about 10-fold), indicating a higher endothelial permeability to small polar nonelectrolytes. The brain water content was higher in newborn than in adult rats and was associated with a higher steady-state distribution of labeled methylglucose between brain and blood. Hexose concentrations were determined relative to whole blood because the apparent erythrocyte membrane permeability to glucose was as high as in humans and thus considerably higher than in adult rats. The half-saturation concentration of glucose transport across the blood-brain barrier was considerably higher than in adult rats, about three-fold, suggesting that net blood-brain glucose transfer is less sensitive to blood glucose fluctuation in newborn than in adult rats.     

Cell cycles in the male accessory glands of mealworm pupae

During the pupal stage of Tenebrio molitor, the accessory reproductive glands of males grow by cell division. Within the secretory epithelium of the bean-shaped accessory glands (BAGs), cell numbers triple. In the tubular accessory glands (TAGs), the increase is 14-fold. There are two mitotic maxima in each gland. The first maximum occurs at 1-2 days while the second is at 4-5 days. The second maximum coincides with the major ecdysteroid peak described by Delbecque et al. [Dev. Biol. 64, 11-30 (1978)]. Nuclei were isolated from TAGs during the pupal mitotic bouts and during mitotic inactivity in the adult. After Feulgen or propidium iodide staining, the DNA content of these nuclear populations was measured by absorption cytophotometry or by fluorescence flow cytometry, respectively. The proportion of cells in each phase of the cycle was calculated using an iterative model. After mitoses have ended in the late pupa, the cells were arrested in G2. [3H]Thymidine was injected into 1- and 4-day pupae to pulse-label cells of the TAGs. After allowing various periods from 4 to 60 hr for cells to progress through G2 to reach mitosis, fractions of labelled mitoses were determined by autoradiography. From the combined cytometric and autoradiographic data, the duration of each phase of the cell cycle was calculated assuming the population was in exponential growth. Cell cycles in 4-day pupal TAGs take 48 hr. G1, S, G2, and, M lasted 13, 14, 17, and 4 hr, respectively.     

Cytokinin biosynthesis in plant tumour tissues

A range of endogenous cytokinins have been identified inDatura crown-gall tissue by GC-MS. Incorporation of [³H]adenine into zeatin riboside, zeatin and its nucleotide(s) is also shown. Metabolism studies usingcis- andtrans-isomers of zeatin riboside indicate that interconversion of the two isomers does not occur in this tissue. Data on the identity of major endogenous cytokinins in a genetic tumour line of tobacco is also provided.     

Separation of histones by reverse-phase high-performance liquid chromatography: analysis of the binding of carcinogens to histones

Reverse-phase high-performance liquid chromatography (RP-HPLC) has been examined as an approach to the rapid analysis of carcinogen-modified histones. H1 and core histone fractions were prepared by differential acid extraction of 0.35 M NaCl-extracted rat liver nuclei previously exposed to [3H]-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [( 3H]BPDE-I). Using a sodium perchlorate-phosphate (PCP)/acetonitrile solvent system, the H1 histone fraction was eluted from an Aquapore RP-300 column in five peaks (P1-P5). The core histone fraction was resolved into eight peaks (C1-C8) using a PCP/acetonitrile-methanol solvent system. The histones of each peak were identified by sodium dodecyl sulfate and Triton/acid/urea gel electrophoresis or amino acid analysis as follows: P1, H1 degrees; P2-P5, four different H1 variant fractions; C1, H4 + A24; C2, H2B; C3, H2A X 2 + to one H2A variant; C4, H2A.1; C5, H2A.1 + two H2A variants; C6, H3.2; C7, H3.3; C8, H3.1. The bulk of radioactivity was covalently bound to histone H2A, which had higher specific activities of BPDE-I than other histones. Significant amounts of radioactivity were observed in histones H3 and H1, but not in histones H2B and H4. These RP-HPLC systems have the advantages of an analysis time within 60 min, the identification of H1, H2A, and H3 variants, and the quantitative analysis of radioactive histones. These results indicate that these RP-HPLC systems are very useful to analyze the binding of carcinogens to histones.     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.