Anaerobic protoporphyrin biosynthesis does not require incorporation of methyl groups from methionine.

It was recently reported (H. Akutsu, J.-S. Park, and S. Sano, J. Am. Chem. Soc. 115:12185-12186, 1993) that in the strict anaerobe Desulfovibrio vulgaris methyl groups from exogenous L-methionine are incorporated specifically into the 1 and 3 positions (Fischer numbering system) on the heme groups of cytochrome c3. It was suggested that under anaerobic conditions, protoporphyrin IX biosynthesis proceeds via a novel pathway that does not involve coproporphyrinogen III as a precursor but instead may use precorrin-2 (1,3-dimethyluroporphyrinogen III), a siroheme and vitamin B12 precursor which is known to be derived from uroporphyrinogen III via methyl transfer from S-adenosyl-L-methionine. We have critically tested this hypothesis by examining the production of protoporphyrin IX-based tetrapyrroles in the presence of exogenous [14C]methyl-L-methionine under anaerobic conditions in a strict anaerobe (Chlorobium vibrioforme) and a facultative anaerobe (Rhodobacter capsulatus). In both organisms, 14C was incorporated into the bacteriochlorophyll precursor, Mg-protoporphyrin IX monomethyl ester. However, most of the label was lost upon base hydrolysis of this compound to yield Mg-protoporphyrin IX. These results indicate that although the administered [14C]methyl-L-methionine was taken up, converted into S-adenosyl-L-methionine, and used for methyl transfer reactions, including methylation of the 6-propionate of Mg-protoporphyrin IX, methyl groups were not transferred to the porphyrin nucleus of Mg-protoporphyrin IX. In other experiments, a cysG strain of Salmonella typhimurium, which cannot synthesize precorrin-2 because the gene encoding the enzyme that catalyzes methylation of uroporphyrinogen III at positions 1 and 3 is disrupted, was capable of heme-dependent anaerobic nitrate respiration and growth on the nonfermentable substrate glycerol, indicating that anaerobic biosynthesis of protoporphyrin IX-based hemes does not require the ability to methylate uroporphyrinogen III. Together, these results indicate that incorporation of L-methionine-deprived methyl groups into porphyrins or their precursors is not generally necessary for the anaerobic biosynthesis of protoporphyrin IX-based tetrapyrroles.     

Occurrence of bifurcated three-center hydrogen bonds in proteins

Analysis of 13 high-resolution protein X-ray crystal structures shows that 1204 (24%) of all the 4974 hydrogen bonds are of the bifurcated three-center type with the donor X-H opposing two acceptors A1, A2. They occur systematically in alpha-helices where 90% of the hydrogen bonds are of this type; the major component is (n + 4)N-H ... O = C(n) as expected for a 3.6(13) alpha-helix, and the minor component is (n + 4)N-H ... O = C(n + 1), as observed in 3(10) helices; distortions at the C-termini of alpha-helices are stabilized by three-center bonds. In beta-sheets 40% of the hydrogen bonds are three-centered. The frequent occurrence of three-center hydrogen bonds suggests that they should not be neglected in protein structural studies.     

Geometric criteria of hydrogen bonds in proteins and identification of "bifurcated" hydrogen bonds

Empirical criteria for identification of hydrogen bonds were analyzed to produce a set of geometrically consistent criteria. For a data set of 30 structures, application of a set of purely geometrical criteria, along with exclusion of abnormal backbone conformations, also excluded a common interaction of Ser/Thr side chains with Asp/Glu side chains ([ST]/[DE] pairs). These interactions were termed "bifurcated hydrogen bonds", which implies delocalization of a positively charged hydrogen of hydroxyl between the two acceptor atoms of the carboxylic group. These "bifurcated" interactions are among the most common packing patterns for [ST]/[DE] pairs of side chains. Therefore, the identification of hydrogen bonds cannot be based on geometrical criteria only and requires introduction of some physico-chemical criteria.     

Replicative aging of the yeast does not require DNA replication

Glucose addition to a stationary culture of wild-type Saccharomyces cerevisiae BY4742 cells with zero activity of MDR pumps resuspended in a fresh medium causes pump resynthesis (measured as pump-effected diS-C3(3) efflux). In a stationary culture in its original growth medium, this glucose-induced pump resynthesis fails to occur due to depletion of essential nutrients or to extracellular metabolites produced by cells during growth. Direct pump inactivation by metabolites is excluded since exponential cells with high MDR pump activity cultured in a medium with high concentration of extracellular metabolites retain this activity for at least 2 h. The metabolites also do not affect pump synthesis on the level of gene expression as addition of concentrated growth medium or an amino acid mixture to stationary cells in spent growth medium restores glucose-induced pump synthesis. The block of MDR pump synthesis is therefore due to the lack of essential nutrients in spent medium.     

Yeast thermotolerance does not require protein synthesis.

Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance. Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C. This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.     

Highly efficient multiplex PCR of noninvasive DNA does not require pre-amplification

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.     

m-Calpain activation in vitro does not require autolysis or subunit dissociation

Calpains are Ca(2+)-dependent, intracellular cysteine proteases involved in many physiological functions. How calpains are activated in the cell is unknown because the average intracellular concentration of Ca(2+) is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Two of the proposed mechanisms by which calpains can overcome this Ca(2+) concentration differential are autoproteolysis (autolysis) and subunit dissociation, both of which could release constraints on the core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, we show that while the anchor helix is one of the first targets to be cut, this occurs in the same time-frame as several potentially inactivating cleavages in Domain III. Thus autolytic activation would overlap with inactivation. We also show that the small subunit does not dissociate from the large subunit, but is proteolyzed to a 40-45k heterodimer of Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit homodimer produced by subunit dissociation. We propose a model for m-calpain activation that does not involve either autolysis or subunit dissociation.     

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate.

The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.     

Formation of primary constriction and heterochromatin in mouse does not require minor satellite DNA.

Whereas the major satellite fraction in mouse extends its domain from the centromere to the distal end of the pericentric heterochromatin, the minor satellite DNA is present specifically in the centromere or primary constriction. We hybridized the biotinylated minor satellite sequence to L929 cells of mouse origin. The sequence hybridized to all chromosomes. Whereas hybridization was detected on all active centromeres, the inactive centromeres in certain dicentrics did not show any signal. This satellite, however, was detected in all inactive centromeres in a heptacentric chromosome. The intensity of fluorescence on the inactive centromeres of the heptacentric was similar to that present on the active centromeres. Several heterochromatin blocks, which were not associated with any centromere, were also found to lack hybridization with the minor satellite. The inactive centromeres, whether carrying the minor satellite DNA fraction or not, generally do not react with the antikinetochore antibodies present in the scleroderma serum. These studies are interpreted to show that (1) the primary constriction in mouse can be formed without the participation of minor satellite, (2) heterochromatin in mouse may constitute without this fraction, (3) the major and minor satellite may not be interspersed but are joined at some defined boundary, and (4) the binding of CENP-B does not depend upon the quantity of minor satellite or the number of CENP boxes present in the inactive centromeres.     

Induction of myogenic differentiation in serum-free medium does not require DNA synthesis

Cells of the myogenic rat cell line L6 can be obtained as a confluent, quiescent population of undifferentiated myoblasts after growth in F12 medium supplemented with fetal calf serum. Myogenic differentiation can be induced in these cells by changing to Dulbecco's modified Eagle's (DME) medium containing insulin as the only protein component. Labeling of the cells with [3H]thymidine demonstrates that this induction of fusion occurs in the absence of DNA synthesis in about 85% of the cells. This result was confirmed using cytosine arabinoside: fusion of quiescent L6 cells was induced in the presence of this inhibitor of DNA synthesis. The myotubes formed in DME + insulin medium, with or without cytosine arabinoside, synthesize or accumulate proteins characteristic of differentiated muscle cells including myosin heavy and light chains, alpha-actin, alpha- and beta-tropomyosins, and the acetylcholine receptor. These experiments represent a direct demonstration that DNA synthesis is not required for the induction of myogenic differentiation in undifferentiated quiescent cells.     

Double and bifurcated hydrogen bonds in alpha-helices of globular proteins]

The statistical analysis of hydrogen bonds distribution in space structures of globular proteins has been done. The parameters of H-bonds in the different secondary structures of globular proteins were collected. In alpha-helices besides the canonical 1-5 H-bonds (the mean length 3 A), 1-4 H-bonds were observed (the mean length 3.2 A). The histograms of length and angular distributions of the bonds are presented. It was found on the basis of quantum chemistry calculations that most H-bonds in alpha-helices are double or bifurcated.     

Transformation of Kluyveromyces lactis cells by plasmid DNA does not require alkaline cations

Summary A procedure for the transformation ofKluyveromyces lactis based on the Li salt method for introducing plasmid DNA into intact yeast cells is described. Contrary toSaccharomyces cerevisiae, lithium salts are dispensable for inducing competence inK. lactis. 2-Mercaptoethanol, a compound that stimulates transformation inS. cerevisiae, showed an opposite effect. inK. lactis. On the other hand, the presence of PEG 4000 and a heat shock were absolutely required to obtain high transformation efficiency.     

Differential stain for mycobacteria that does not require heat.

A stain of 0.1% toluidine blue in a 1.0% aqueous solution of triethylene glycol followed by decolorization with acid-alcohol resulted in mycobacteria retaining the stain (violet), whereas non-acid-fast bacteria were decolorized.     

The antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action

Insulin antagonized the lipolytic actions of epinephrine in rat epididymal adipocytes when the phosphodiesterase inhibitor, Ro 20-1724, was present. Adipocytes were depleted of functional cAMP by inhibiting adenylate cyclase with N6-phenylisopropyladenosine in the presence of adenosine deaminase such that Ro 20-1724 no longer stimulated lipolysis. The cAMP analogs 8-thioisopropyl-cAMP or 8-thiomethyl-cAMP, which are resistant to phosphodiesterase hydrolysis, were subsequently added to bypass adenylate cyclase and phosphodiesterase action. Under these conditions, insulin antagonized the lipolytic effects of these analogs, even in the presence of Ro 20-1724.     

Autoimmune myocarditis does not require B cells for antigen presentation.

T cells constitute the pathogenic effector cell population in autoimmune myocarditis in BALB/c mice. Using mice rendered deficient for B cells by a targeted disruption to the IgM transmembrane domain or by treatment with anti-IgM Ab from birth, we asked whether B cells are a critical APC in the induction of autoimmune myocarditis. B cell-deficient mice immunized with cardiac myosin develop myocarditis comparable in incidence and severity to that in wild-type mice, suggesting that autoreactive T cells that cause myocarditis in BALB/c mice are activated by macrophages or dendritic cells. Since it does not appear that presentation of cryptic epitopes is critical for the breakdown of self tolerance, potentially pathogenic T cells recognizing dominant myosin epitopes must have escaped tolerization. Either anatomic sequestration of cardiac myosin peptide-MHC complexes or subthreshold presentation of cardiac myosin peptides by conventional APC can explain the survival of these autoreactive T cells.     

DNA methylation can enhance or induce DNA curvature.

Oligomers of different palindromic sequences (EcoRI, BamHI, and ClaI linkers) were ligated to form distributions of multimers. These long ligation ladders were methylated using corresponding methylases. The migration of the unmethylated as well as the methylated multimer distributions was analysed in 10% polyacrylamide gels. The migration anomaly of these sequences is interpreted in terms of the curvature of the DNA helix axis. The double-stranded oligomer dCGGAATTCCG is considerably curved in its unmodified form. Its curvature is strongly enhanced when the central dAs are methylated. This result is predicted by a model for DNA curvature. Multimers of the closely related sequence dCGGGATCCCG are straight. When methylated at the central dAs or at the most central dCs, a small curvature of the helix axis is induced. The double-stranded oligomer dCCATCGATGG is straight in its unmodified form as well as when it is methylated. Thus, DNA curvature can be induced or enhanced by methylation. However, DNA methylation at palindromic sequences seems not always to influence the linear path of the DNA helix axis.