Solution conformations of DNAs containing binding sites of the catabolite gene activator protein and lac repressor protein: characterization by Raman spectroscopy

Raman spectra from three subfragments of the Escherichia coli lactose promoter region were obtained in 0.1 M NaCl. The three DNAs are 21, 40, and 62 bp in length. The 21 and 62 bp DNAs contain the binding site for the catabolite gene activator protein (CAP). The 40 bp DNA contains the binding site for the lac repressor. A quantitative analysis of Raman band characteristics indicates an overall B-type conformation for these gene regulatory sites. Bands which correspond to A-family (807 cm-1) and B-family (834 cm-1) deoxyribose phosphate vibrations have the same intensities as bands found in heterogeneous DNAs. The spectra of the 21 bp CAP site have, however, a small band at 867 cm-1 and several other small differences similar to some characteristics observed in C-DNA spectra. Several dG nucleosides in the CAP site appear to be altered from the conventional C2'-endo/anti conformation. At 45 degrees C, well below the melting region of these DNAs, small changes occur in the spectra of the 40 bp lac repressor site which are not observed in the other DNAs. A weak band occurs at 705 cm-1, and intensity changes are observed at 497, 682, and 792 cm-1. The changes suggest that the conformations of several dG nucleosides are altered and that a small region may exist with characteristics of an A-family backbone. This conformational change at 45 degrees C coincides with previous NMR observations indicating an enhanced imino proton exchange rate at a GTG sequence within the lac operator site.     

Cl-/HCO3- exchange modulates intracellular pH in rat type II alveolar epithelial cells

The role of an anion exchange pathway in modulating intracellular pH (pHi) under steady-state and alkaline load conditions was investigated in confluent monolayers of rat type II alveolar epithelial cells using the pH-sensitive fluorescent probe 2'-7'-biscarboxy-ethyl-5,6-carboxylfluorescein. Under steady-state conditions in the presence of 25 mM HCO3-, 5% CO2 at pHo 7.4, pHi was 7.32 in a Na+-replete medium and 7.33 in the absence of Na+. Steady-state pHi was 7.19 in a nominally HCO3(-)-free medium at pHo 7.4, and 7.52 in a Cl(-)-free medium, with both values significantly different from that obtained in the presence of both HCO3- and Cl-. Monolayers in which pHi was rapidly elevated by removal of HCO3-/CO2 from the bathing medium demonstrated an absolute requirement for Cl- to recover toward base-line pHi. The Km of Cl- for the external site of the exchange pathway was 11 +/- 1 mM. Recovery of pHi from the alkaline load in the presence of Cl- was inhibited 60% by the stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Removal of Cl- from the medium of cells bathed in HCO3-/CO2 resulted in a rapid increment in pHi which returned to base line when Cl- was reintroduced into the bathing medium. In contrast, pHi was not perturbed by removal or addition of Cl- to monolayers bathed in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered medium, indicating that HCO3- was the preferred species for transport. Recovery of pHi from an alkaline load was not affected by the presence or absence of Na+. These findings define the transport pathway as Na+-independent Cl-/HCO3- exchange. This pathway contributes importantly to determining resting pHi of pneumocytes and enables the cell to recover from an alkaline load.     

Evidence for heterogeneity of low-density lipoprotein metabolism in the cynomolgus monkey

Preliminary studies were performed to establish whether there was kinetic heterogeneity in the metabolism of subclasses of low-density lipoproteins (LDL) in the cynomolgus monkey. Previous studies of the effects of inhibition of hepatic triglyceride lipase in this species had shown an increase in the mass of lighter LDL (Sf greater than 9) and a decrease in the mass of denser LDL. LDL (1.019 less than d less than 1.063) were subdivided into two subfractions LDL1 (1.019 less than d less than 1.035) and LDL2 (1.035 less than d less than 1.063) by ultracentrifugation. The lipoproteins in these two fractions could be shown to have different flotation by analytic and isopycnic ultracentrifugation. When tracer amounts of homologous 125I-labeled very-low-density lipoproteins (VLDL) were injected into chow-fed cynomolgus monkeys, apoB radioactivity appeared in LDL1 prior to its appearance in LDL2. [125I]LDL1 injected into the monkey was removed from the LDL1 density subclass with a half-life of 5.5-10.3 h. Much of the radioactivity injected as LDL1 was converted to denser LDL (LDL2). Labeled LDL2 injected into the monkey was not converted to LDL1. Thus, at least two kinetically distinct subpopulations of LDL circulate in the plasma of this species. The lighter LDL is to a large extent a metabolic precursor of the more dense LDL (LDL2).     

A comparison of the inhibitory effects of somatostatin-14, -25, and -28 on motility of the guinea pig ileum

A number of studies have suggested that somatostatin-14 (SS-14) and somatostatin-28 (SS-28) exhibit a similar spectrum of biological activities but have different potencies. In the present study the effects of SS-14, SS-28, and somatostatin-25 on electrically induced contractions of the guinea pig ileum have been compared. All three peptides exhibited equipotent inhibitory effects. Inhibition was obtained at a threshold concentration less than 10(-10) M, with maximal inhibition at 10(-7) M and IC50 values of 6.0-6.5 X 10(-10) M. The N-terminal 14 amino acid fragment of SS-28 had no effect either on motility, when added alone, or on the actions of SS-28, suggesting that this region of the molecule is not critical for biological activity.     

Regulation of B-lymphocyte activation, proliferation, and immunoglobulin secretion

Lymphocyte growth and differentiation are controlled by signals resulting from the interaction of antigen and cellular products, such as lymphokines, with specific cell membrane receptors. Resting B lymphocytes can be activated by low concentrations (1-5 micrograms/ml) of antibodies to membrane IgM, which is the B-lymphocyte receptor for antigen. The binding of anti-IgM to B cells causes a rapid increase in intracellular free calcium concentration ([Ca2+]i), in inositol phosphate concentration, and in protein kinase activity. Moreover, the effects of anti-IgM on B cells are mimicked by the combined use of calcium ionophores and phorbol esters. Since phorbol esters activate protein kinase c, this suggests that the increase in [Ca2+]i and in phosphatidylinositol metabolism stimulated by anti-IgM are critical events in B-cell activation. The entry into S phase of B cells stimulated with anti-IgM depends on the action of a T-cell-derived factor designated B-cell stimulatory factor (BSF)-1. This is a 20,000-Da protein which is a powerful inducer of class II major histocompatibility complex molecules. Although an important cofactor for B-cell proliferative responses to anti-IgM, its major locus of action is on resting B cells. B cells stimulated with anti-IgM and BSF-1 do not synthesize secretory IgM. However, if two additional T-cell-derived factors, B151-TRF and interleukin-2, are added to cultures, a substantial proportion of stimulated B cells produce secretory IgM. BSF-1 has also been shown to participate in the "switch" in Ig class expression. Resting B cells cultured with lipopolysaccharide will switch to IgG1 secretion in the presence of purified BSF-1.     

A deletion map of the human Y chromosome based on DNA hybridization.

The genomes of 27 individuals (19 XX males, two XX hermaphrodites, and six persons with microscopically detectable anomalies of the Y chromosome) were analyzed by hybridization for the presence or absence of 23 Y-specific DNA restriction fragments. Y-specific DNA was detected in 12 of the XX males and in all six individuals with microscopic anomalies. The results are consistent with each of these individuals carrying a single contiguous portion of the Y chromosome; that is, the results suggest a deletion map of the Y chromosome, in which each of the 23 Y-specific restriction fragments tested can be assigned to one of seven intervals. We have established the polarity of this map with respect to the long and short arms of the Y chromosome. On the short arm, there is a large cluster of sequences homologous to the X chromosome. The testis determinant(s) map to one of the intervals on the short arm.     

Method for determining the temporal response of microbial phosphate transport affinity.

Nutrient transport affinities of nutrient-starved microbial populations were measured as initial slopes of plots of limiting-nutrient transport rates versus extracellular limiting-nutrient concentrations. A method was devised for the determination of soluble reactive phosphate (Pi) affinity in Pi-limited continuous culture (aT), which was then used as an indicator of the effects of light/dark cycle (LD) perturbations on the temporal Pi transport abilities of three species of freshwater algae. Cell division was asynchronous for the green alga Selenastrum capricornutum grown in continuous cultures exposed to LD cycles. An apparent rhythm in aT for Pi was greatly affected by the population size parameter. Cell division was phased for the green alga Scenedesmus quadricauda grown in LD continuous culture. A rhythm in aT for Pi was not greatly affected by the biomass parameter. Cell division was also phased in LD continuous culture for the blue-green alga (cyanobacterium) Synechococcus Nägeli, but rhythms in other parameters could not be detected. Synechococcus Nägeli was an extremely efficient Pi transporter at low Pi concentrations in LD continuous culture, and so aT could not be calculated. The results demonstrate that aT is well suited to describing the temporal response of Pi transport in LD-perturbed, Pi-limited continuous culture.     

Pentachlorophenol degradation: a pure bacterial culture and an epilithic microbial consortium.

The steady-state growth of a Flavobacterium strain known to utilize pentachlorophenol (PCP) was examined when cellobiose and PCP simultaneously limited its growth rate in continuous culture. A concentration of 600 mg of PCP per liter in influent medium could be continuously degraded without affecting steady-state growth. We measured specific rates of PCP carbon degradation as high as 0.15 +/- 0.01 g (dry weight) of C per h at a growth rate of 0.045 h-1. Comparable specific rates of PCP degradation were obtained and maintained by PCP-adapted, natural consortia of epilithic microorganisms. The consortium results suggest that a fixed-film bioreactor containing a PCP-adapted natural microbial population could be used to treat PCP-contaminated water.     

Rhizobium trifolii 0403 Is Capable of Growth in the Absence of Combined Nitrogen

Rhizobium trifolii 0403 was treated with 16.6 mM succinate and other nutrients and thereby induced to grow in nitrogen-free medium. The organism grew microaerophilically on either semisolid or liquid medium, fixing atmospheric nitrogen to meet metabolic needs. Nitrogen fixation was measured via ¹⁵N incorporation (18% ¹⁵N enrichment in 1.5 doublings) and acetylene reduction. Nitrogen-fixing cells had a K_m for acetylene of 0.07 atm (ca. 7.09 kPa), required about 3% oxygen for optimum growth in liquid medium, and showed a maximal specific activity of 5 nmol of acetylene reduced per min per mg of protein at 0.04 atm (ca. 4.05 kPa) of acetylene. The doubling time on N-free liquid medium ranged from 1 to 5 days, depending on oxygen tension, with an optimum temperature for growth of about 30°C. Nodulation of white clover by the cultures showing in vitro nitrogenase activity indicates that at least part of the population maintained identity with wild-type strain 0403.     

Cloning and expression of a cDNA encoding a catalytically active fragment of calf thymus DNA polymerase alpha

A calf thymus cDNA expression library was constructed in the EcoRI site of lambda gt11 and probed with an antibody raised against calf thymus DNA polymerase alpha. Three classes of antibody-reactive clones were isolated. The largest class carried a 1.9 kilobase calf cDNA insert and expressed a 165-175 kilodalton beta-galactosidase:calf fusion protein which displayed DNA polymerase activity. The characteristic responses of the polymerase activity to alpha-specific inhibitors and antibodies identified the 1.9 kilobase cDNA as a sequence specifically derived from the structural gene encoding the pol alpha catalytic core.