Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or?) [α and β]. Inactivation or deletion of or?β results in SegA^? plasmids. Better than random segregation requires an active segB region. The segB region contains two or?s (or?? and or?ζ). Inactivation of either of the orfs does not lead to an increase in cell death, but or?ζ^? plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.     

Re-localization of Actsk-1 to mouse Chromosome 8, a new region of homology with human Chromosome 1

We present here the genetic mapping of the -skeletal actin locus (Actsk-1) on mouse Chromosome (Chr) 8, on the basis of the PCR analysis of a microsatellite in an interspecific backcross. Linkage and genetic distances were established for four loci by analysis of 192 (or 222) meiotic events and indicated the following gene order: (centromere)-Es-1-11.7 cM-Tat-8.3 cM-Actsk-1-0.5 cM-Aprt. Mapping of ACTSK to human Chr 1 and of TAT and APRT to human Chr 16 demonstrates the existence of a new short region of homology between mouse Chr 8 and human Chr 1. Intermingling on this scale between human and mouse chromosomal homologies that occurred during evolution creates disorders in comparative linkage studies.     

Streamlined approach for assessing embedded consumption of lithium and cobalt in the United States

In today's complex global supply chains, time and data intensive analyses are required to understand global flows of mineral commodities from mine to consumer, particularly for mineral commodities in products (electronics, automobiles, etc.) that contain multiple parts with many mineral commodities. National and regional analyses require additional time and data to incorporate international trade flows. However, data limitations and time constraints often prohibit global and national material flow analyses for minor metals. Here we present a methodological approach to circumvent these constraints by utilizing readily available industry-level global data from the United Nations Statistics Division and national industrial data to estimate total requirements for a mineral commodity. We apply this approach to lithium and cobalt use in the United States for the year 2018 and distinguish between apparent raw material consumption versus inferred embedded consumption of lithium and cobalt materials in all forms. The results show that more than half of the United States’ total requirements for both lithium and cobalt is in parts and products that were manufactured outside the United States. In large part, this is due to limited US manufacturing capability for lithium-ion battery materials and cells and the United States’ high import reliance for electronics that use those batteries.     

Interleukin-2 Modulates Evoked Release of [3H]Dopamine in Rat Cultured Mesencephalic Cells

Abstract: Mesencephalic cell cultures were used as a model to investigate the effects of interleukin-2 (IL-2) on evoked release of [³H]dopamine ([³H]DA) and γ-[³H]-aminobutyric acid ([³H]GABA). At low concentrations (10^?13-10^?12M), IL-2 potentiated [³H]DA release evoked by the excitatory amino acids N-methyl-D-aspartate (NMDA) and kainate, whereas higher IL-2 concentrations (10^?9-10^?8M) had no effect. IL-2 (10^?14-10^?8M) modulated K⁺-evoked [³H]DA release in a biphasic manner, with low concentrations (10^?12-10^?11M) of IL-2 potentiating and higher concentrations (10^?9-10^?8M) inhibiting K⁺-induced [³H]DA release. IL-2 (10^?14-10^?8M) by itself failed to alter spontaneous [³H]DA release. The inhibition by IL-2 of K⁺-evoked [³H]DA release was reversible and not due to neurotoxicity, as preexposure to IL-2 (10^?8M) had no significant effect on the subsequent ability of dopaminergic cells to take up and to release [³H]DA. Under our experimental conditions, IL-2 (10^?8 M) did not alter Ca²⁺-independent [³H]GABA release evoked by either K⁺ or NMDA. The results of this study indicate that IL-2 is able to potentiate [³H]DA release evoked by a number of different stimuli, including K⁺ depolarization and activation of both NMDA and non-NMDA receptor subtypes in mesencephalic cell cultures. IL-2 is active at very low concentrations, a finding that indicates a potent effect of IL-2 on dopaminergic neurons and implicates a physiological role for this cytokine in the modulation of DA release.     

Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span

Comparing the properties of young and senescent (aged) O⁺ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a -galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows:

Functional analysis of Gln-237 mutants of HhaI methyltransferase.

When the HhaI (cytosine-5) methyltransferase (M.HhaI) binds DNA it causes the target cytosine to be flipped 180 degrees out of the helix. The space becomes occupied by two amino acids, Ser-87 and Gln-237, which enter the helix from opposite sides and form a hydrogen bond to each other. Gln-237 may be involved in specific sequence recognition since it forms three hydrogen bonds to the orphan guanosine, which is the partner of the target cytosine. We have prepared all 19 mutants of Gln-237 and tested their biochemical properties. We find that mutations of this residue greatly affect the stability of the M.HhaI-DNA complex without affecting the enzyme's specificity for the target sequence. Surprisingly, all mutants retain detectable levels of enzymatic activity.     

Substrate specificities of recombinant murine Golgi alpha1, 2- mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1,2-mannosidases

The catalytic domains of murine Golgi alpha1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted proteins in P.pastoris . Recombinant mannosidases IA and IB both required divalent cations for activity, were inhibited by deoxymannojirimycin and kifunensine, and exhibited similar catalytic constants using Manalpha1,2Manalpha-O-CH3as substrate. Mannosidase IA was purified as a 50 kDa catalytically active soluble fragment and shown to be an inverting glycosidase. Recombinant mannosidases IA and IB were used to cleave Man9GlcNAc and the isomers produced were identified by high performance liquid chromatography and proton-nuclear magnetic resonance spectroscopy. Man9GlcNAc was rapidly cleaved by both enzymes to Man6GlcNAc, followed by a much slower conversion to Man5GlcNAc. The same isomers of Man7GlcNAc and Man6GlcNAc were produced by both enzymes but different isomers of Man8GlcNAc were formed. When Man8GlcNAc (Man8B isomer) was used as substrate, rapid conversion to Man5GlcNAc was observed, and the same oligosaccharide isomer intermediates were formed by both enzymes. These results combined with proton-nuclear magnetic resonance spectroscopy data demonstrate that it is the terminal alpha1, 2-mannose residue missing in the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate. When rat liver endoplasmic reticulum membrane extracts were incubated with Man9GlcNAc2, Man8GlcNAc2was the major product and Man8B was the major isomer. In contrast, rat liver Golgi membranes rapidly cleaved Man9GlcNAc2to Man6GlcNAc2and more slowly to Man5GlcNAc2. In this case all three isomers of Man8GlcNAc2were formed as intermediates, but a distinctive isomer, Man8A, was predominant. Antiserum to recombinant mannosidase IA immunoprecipitated an enzyme from Golgi extracts with the same specificity as recombinant mannosidase IA. These immunodepleted membranes were enriched in a Man9GlcNAc2to Man8GlcNAc2- cleaving activity forming predominantly the Man8B isomer. These results suggest that mannosidases IA and IB in Golgi membranes prefer the Man8B isomer generated by a complementary mannosidase that removes a single mannose from Man9GlcNAc2.      

Glucocorticoids Provoke a Shift from α2- to α1-Adrenoreceptor Activities in Cultured Hypothalamic Slices Leading to Opposite Noradrenaline Effect on Corticotropin-Releasing Hormone Release

Abstract: We have shown previously that noradrenaline (NA) stimulated or inhibited the release of corticotropin-releasing hormone (CRH) according to the availability of adrenal steroids. The aim of the present work was to examine whether the changes in the NA modulation of CRH release from hypothalamic neurons result from a steroid-induced plasticity of the adrenergic transduction pathways. From anterior hypothalamic slices cultured in standard medium (i.e., containing adrenal steroids at a final dilution of 61 ± 9 ng/ml), (a) the stimulatory effect of NA on CRH release was reversed in a dose-dependent manner by increasing concentrations of the α₁-adrenoreceptor antagonist prazosin, (b) activation of protein kinase C by acute treatment with phorbol 12-myristate 13-acetate (0.5 µ M , 1 h) mimicked NA stimulation of CRH secretion, and (c) the activation of L-type Ca²⁺ channels by Bay K 8644 also produce an increased CRH secretion. In contrast, the inhibitory effect of NA on CRH secretion from slices cultured in steroid-free medium was markedly reversed by the α₂-adrenoreceptor antagonist yohimbine, by pretreatment with pertussin toxin, or by the addition of 4-aminopyridine, a K⁺-channel blocker. Acute treatment with phorbol 12-myristate 13-acetate did not change the inhibitory NA effect. Moreover, all these effects were reversed by daily corticosterone supplementation, for as long as they were tested. These results are consistent with a steroid-dependent change in the nature of adrenergic receptors and its associated transduction pathways involved in the regulation of CRH secretion in the hypothalamus.