Cephalosporinase and penicillinase activities of a β-lactamase from Pseudomonas pyocyanea

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.     

Small population instability and island settlement patterns

A model of extinction probability, based on the general theory of island biogeography [MacArthur and Wilson, 1967], is proposed for humans on oceanic islands; extinction probability is determined by island carrying capacity, frequency and amplitude of fluctuations in resources determining carrying capacity, and the net costs of contact and exchange between population units. The model predicts that extinction probability will determine island settlement patterns within an island group resulting in nonsettlement of islands with low carrying capacities and settlement of all islands with high carrying capacities. Data examined from the Marshall Islands tend to support the model. The model is extended to initial atoll colonization patterns. Possible requirements for initial settlement are suggested.Deceased.     

Glycosyl conformational and inductive effects on the acid catalysed hydrolysis of purine nucleosides.

The log kobs vs. pH profiles were determined in the intermediate acidity region for the glycosyl hydrolysis of guanosine and its 8-amino, 8-monomethylamino, 8-dimethylamino and 8-bromo derivatives. The decreased rate of the 8-amino and enhanced rate of the 8-bromo compound compared to guanosine support an A type mechanism: base protonation followed by glycosyl bond cleavage. All three 8-amino guanosines exhibited log kobs - pH profiles clearly showing that both mono and di-base protonated nucleosides undergo hydrolysis. The 700 fold rate acceleration of 8-N(CH3)-guanosine compared to 8-NHCH3-guanosine and the 110 fold rate acceleration of 8-N(CH3)2-adenosine compared to 8-NHCH3-adenosine could be unequivocally attributed to the fixed syn glycosyl conformation of both 8-dimethylamino compounds and relief of steric compression upon hydrolysis in these molecules. The lack of anomerization of all substrates during the course of the reaction supports an A rather than a Schiff-base mechanism.     

Mouse model systems of autism spectrum disorder: Replicability and informatics signature

Phenotyping mouse model systems of human disease has proven to be a difficult task, with frequent poor inter‐ and intra‐laboratory replicability, particularly in behavioral domains such as social and cognitive function. However, establishing robust animal model systems with strong construct validity is of fundamental importance as they are central tools for understanding disease pathophysiology and developing therapeutics. To complete our studies of mouse model systems relevant to autism spectrum disorder (ASD), we present a replication of the main findings from our two published studies of five genetic mouse model systems of ASD. To assess the intra‐laboratory robustness of previous results, we chose the two model systems that showed the greatest phenotypic differences, the Shank3/F and Cntnap2, and repeated assessments of general health, activity and social behavior. We additionally explored all five model systems in the same framework, comparing all results obtained in this three‐yearlong effort using informatics techniques to assess commonalities and differences. Our results showed high intra‐laboratory replicability of results, even for those with effect sizes that were not particularly large, suggesting that discrepancies in the literature may be dependent on subtle but pivotal differences in testing conditions, housing enrichment, or background strains and less so on the variability of the behavioral phenotypes. The overall informatics analysis suggests that in our behavioral assays we can separate the set of tested mouse model system into two main classes that in some aspects lie on opposite ends of the behavioral spectrum, supporting the view that autism is not a unitary concept.     

Use of a Novel Probiotic Formulation to Alleviate Lactose Intolerance Symptoms—a Pilot Study

Probiotics and Antimicrobial Proteins - Lactose intolerance is a common condition caused by lactase deficiency and may result in symptoms of lactose malabsorption (bloating, flatulence, abdominal...     

3′-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors

3′-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3′-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3′-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem–loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation—CPSF, CstF, and CF I_m—are present in Drosophila nuclear extracts in a stable supercomplex.     

Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum

A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes. As C. acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution. Biochemical evidence for the expression of a cellulosomal protein complex was investigated. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C. cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present. In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C. acetobutylicum.     

Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules

Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs.