DISC ELECTROPHORETIC SEPARATION OF PROTEINS IN NEURONAL, GLIAL AND SUBCELLULAR FRACTIONS FROM CEREBRAL CORTEX

Abstract— Soluble proteins were studied in preparations from rabbit brain cortex enriched in neuronal or glial cells and in subcellular cortical fractions. Analytical polyacrylamide gels were used for acidic (pH 9-5) and basic (pH 4-3) proteins and qualitative and quantitative differences are described. The isozymes of lactic dehydrogenase, brain specific proteins and radioactive labelling patterns were used to characterize some soluble proteins.     

Protein kinase C modulates actin conformation in human T lymphocytes

We studied the effect of activators and inhibitors of protein kinase C on actin conformation in human blood lymphocytes by flow cytometry and gel electrophoresis. PMA, 1-oleyl-2-acetyl-glycerol, and mezerein, activators of protein kinase C, caused an increase in lymphocyte F-actin within 2 to 5 min. After stimulation with PMA, lymphocytes formed pseudopods containing an increased concentration of F-actin and had an increase of actin in the Triton-insoluble cytoskeletal fraction. Sphingosine and H-7, inhibitors of protein kinase C activation, inhibited the increase in F-actin induced by PMA. The increase in F-actin in response to PMA was striking in Th and Ts lymphocytes (2- to 3-fold increase), but B lymphocytes had only a slight increase (1.15-fold). Thus, activation of protein kinase C modulates actin conformation specifically in T lymphocytes.     

Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes

Molecular-genetic and muropeptide analysis techniques have been applied to examine the function in vivo of the Bacillus megaterium QM B1551 SleB and SleL proteins. In common with Bacillus subtilis and Bacillus anthracis, the presence of anhydromuropeptides in B. megaterium germination exudates, which is indicative of lytic transglycosylase activity, is associated with an intact sleB structural gene. B. megaterium sleB cwlJ double mutant strains complemented with engineered SleB variants in which the predicted N- or C-terminal domain has been deleted (SleB-ΔN or SleB-ΔC) efficiently initiate and hydrolyze the cortex, generating anhydromuropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-ΔN or SleB-ΔC, in which glutamate and aspartate residues have individually been changed to alanine, all retain the ability to hydrolyze the cortex to various degrees during germination, with concomitant release of anhydromuropeptides to the surrounding medium. These data indicate that while the presence of either the N- or C-terminal domain of B. megaterium SleB is sufficient for initiation of cortex hydrolysis and the generation of anhydromuropeptides, the perceived lytic transglycosylase activity may be derived from an enzyme(s), perhaps exclusively or in addition to SleB, which has yet to be identified. B. megaterium SleL appears to be associated with the epimerase-type activity observed previously in B. subtilis, differing from the glucosaminidase function that is apparent in B. cereus/B. anthracis.Spores of the genera Bacillus and Clostridium emerge from dormancy via the process of germination. The germination process comprises a series of sequential biophysical and biochemical reactions that result irreversibly in the spore losing its properties of metabolic dormancy and extreme resistance to various chemical and physical treatments (24, 34). Germination is initiated by the presumed binding of small molecular germinants, commonly amino acids or sugars, to cognate receptors located within the spore inner membrane (25, 28). In a process that is poorly understood at the molecular level, this interaction leads to a change in the permeability of the inner membrane, resulting in the release of various solutes from the spore core, including metal ions, calcium dipicolinate (Ca-DPA), and some amino acids (32, 33, 35). A degree of rehydration of the core is evident at or around the same time, although this is insufficient to permit a significant degree of vegetative metabolism (9, 31). These events, which appear common to all Bacillus species where examined, comprise stage I of germination (31, 32, 34).The major event in stage II of the germination process from a biochemical perspective involves depolymerization of the spore cortex. The spore cortex is a thick layer of peptidoglycan, characterized by the spore-specific muramic acid lactam (MAL) moiety (37, 38), which, together with the thin inner layer of germ cell wall peptidoglycan (36), forms contiguous layers that entirely envelope the spore protoplast. While the germ cell wall forms the initial cell wall during vegetative outgrowth, the spore cortex serves primarily to maintain the relatively dehydrated status of the spore protoplast during dormancy (13). Dissolution of the cortex permits complete hydration of the spore core and resumption of vegetative metabolism, leading ultimately to shedding of the spore coat and the emergence of a new vegetative cell (34).A number of studies have indicated that spores of various Bacillus species employ two cortex-lytic enzymes (CLEs), SleB and CwlJ, to initiate hydrolysis of the cortex during stage II of the germination process (16, 19, 32). These enzymes are semiredundant; hence, strains bearing null mutations in either structural gene can still degrade the cortex sufficiently to complete germination, whereas double mutant strains do not appear capable of degrading the cortex at all, resulting typically in a decrease of several orders of magnitude in colony-forming ability (15, 19, 32). Other enzymes, including Bacillus cereus/Bacillus anthracis SleL, are also involved in stage II of germination, apparently hydrolyzing peptidoglycan products of SleB and/or CwlJ to smaller peptidoglycan fragments that can more easily permeate through the spore coats to the surrounding germination medium (21).Studies with SleB and SleL purified from dormant and germinating spores indicate that whereas the latter enzyme degrades only cortical fragments of peptidoglycan (7), SleB has a requirement for intact peptidoglycan that has adopted the precise architecture present within the spore (12, 22). These substrate requirements appear to be important in maintenance of the respective autolysins, which are present in the spore in a mature form, in an inactive state during dormancy. Additionally, whereas the molecular mechanism of activation of SleB remains unclear—a change in cortical stress/architecture induced by stage I events has been hypothesized (12)—the efflux of Ca-DPA from the spore core to the cortex/coat boundary where CwlJ is localized (5) appears to be the mechanism by which this CLE is activated. CwlJ can also be activated by high concentrations of exogenous Ca-DPA, presenting an alternative germination pathway that bypasses the germinant receptors (27).The hydrolytic bond specificity of various CLEs has been examined by both direct and indirect biochemical means. Direct assays are typically conducted by incubation of purified or recombinant enzymes with peptidoglycan fragments or suspensions of spores in which the cortex is rendered accessible by first chemically compromising the permeability of the spore coats (7, 12, 22). Subsequent assays for the generation of reducing groups and/or free amino groups can yield information on the probable hydrolytic bond specificity of the respective enzyme(s) being assayed.More recently, the high-performance liquid chromatography/mass spectrometry (HPLC/MS)-based muropeptide analysis technique has been applied to characterize CLE activity during germination of various spore-forming species (2, 4, 10). This methodology has the resolution to reveal fine structural changes that occur to the peptidoglycan in vivo during germination, and when used in combination with CLE null mutant strains, it can be used to indirectly correlate the generation of certain classes of muropeptides, and therefore the hydrolytic bond specificity, with defined CLEs. Muropeptide analysis has revealed, for example, that an intact copy of the sleB gene in B. subtilis and B. anthracis is required for the presence of anhydromuropeptides in the germination exudates of these respective species, indicating that SleB is a lytic transglycosylase or generates substrate for subsequent lytic transglycosylase activity (6, 16). Conversely, B. cereus SleB was characterized as a probable amidase after enzyme purified from germinating spores was found to liberate a large amount of free amino groups when incubated with coat-stripped spores as a substrate (22). The hydrolytic bond specificity of SleB therefore remains ambiguous and perhaps varies between different species.Contrary to these observations, the overall structural architecture of SleB appears to be well conserved between different Bacillus species. Alignment of the primary amino acid sequence from different species indicates that the mature protein comprises an N-terminal domain that is connected to the C-terminal domain by a linker region that is variable in length and amino acid composition (Fig. ​(Fig.1).1). The N-terminal domain is thought to comprise the peptidoglycan binding domain by virtue of two direct sequence repeats that are reminiscent of cell wall-binding motifs observed in other proteins (26). The C-terminal domain shows homology with that of the other major Bacillus CLE, CwlJ, which lacks a corresponding peptidoglycan binding domain and is therefore thought to comprise the catalytic domain (19), although there is as yet no experimental evidence to substantiate this idea.Open in a separate windowFIG. 1.ClustalW alignment of SleB from various Bacillus species. Residues predicted to comprise putative structural domains are denoted. Stars indicate charged residues that were subjected to amino acid substitution in this work. BM, B. megaterium QM B1551; BC, B. cereus W; BCl, B. clausii KSM-K16; BS, B. subtilis 168.In the current study, we have investigated the molecular function of SleB during germination of Bacillus megaterium QM B1551 spores, employing engineered SleB N- and C-terminal deletion strains, site-directed mutagenesis (SDM), and muropeptide analyses. In addition to revealing several cortex-modifying activities during germination of this species, the presented data indicate that while the presence of either the N- or C-terminal domain of SleB is sufficient for the generation of anhydromuropeptides during germination, this may be an indirect effect, and at least a degree of lytic transglycosylase activity may result from the activity of another as yet unidentified enzyme.     

Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented.     

Mitigating the Impact of Bats in Historic Churches: The Response of Natterer’s Bats Myotis nattereri to Artificial Roosts and Deterrence

Bats frequently roost in historic churches, and these colonies are of considerable conservation value. Inside churches, bat droppings and urine can cause damage to the historic fabric of the building and to items of cultural significance. In extreme cases, large quantities of droppings can restrict the use of a church for worship and/or other community functions. In the United Kingdom, bats and their roosts are protected by law, and striking a balance between conserving the natural and cultural heritage can be a significant challenge. We investigated mitigation strategies that could be employed in churches and other historic buildings to alleviate problems caused by bats without adversely affecting their welfare or conservation status. We used a combination of artificial roost provision and deterrence at churches in Norfolk, England, where significant maternity colonies of Natterer’s bats Myotis nattereri damage church features. Radio-tracking data and population modelling showed that excluding M. nattereri from churches is likely to have a negative impact on their welfare and conservation status, but that judicious use of deterrents, especially high intensity ultrasound, can mitigate problems caused by bats. We show that deterrence can be used to move bats humanely from specific roosting sites within a church and limit the spread of droppings and urine so that problems to congregations and damage to cultural heritage can be much reduced. In addition, construction of bespoke roost spaces within churches can allow bats to continue to roost within the fabric of the building without flying in the church interior. We highlight that deterrence has the potential to cause serious harm to M. nattereri populations if not used judiciously, and so the effects of deterrents will need careful monitoring, and their use needs strict regulation.     

Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the ¹H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin–spin relaxation times, T₂, with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the ¹H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by ¹H n.m.r. spectroscopy.