Isolation from urine of two Serratia marcescens strains excreting a diffusible yellow pigment

Two bacterial strains excreting a yellow pigment were isolated from human urine and identified as Serratia marcescens. The pigment was produced in the late exponential and early stationary phases of growth. Minimal media supplemented with tyrosine, phenylalanine, 3,4-dihydroxyphenylacetate or tryptophan, as well as complex media, induced pigment production. UV-visible spectra of the extracted pigment had peaks characteristic of 2-hydroxy-5-carboxymethylmuconate semialdehyde, produced from meta-cleavage of 3,4-dihydroxyphenylacetate by the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15). This enzyme was active when the bacteria were grown under conditions promoting pigment production. The kinetics and factors affecting pigment production are also reported.     

XY gonadal dysgenesis with female phenotype (author's transpl)]

In this paper, we are dealing with the study of a case of multiple somatic malformations, with external female genitals and 46 XY caryotype. The anatomical and histological study of the genital organs, allows us to verify the existence of internal genital organs; consisting essentially in tubes, bicornous uterus, a gonadal ligament in a normotopical position, Wolffian remains and the absence of a vagina. The external female genitals are completely normal. When we interpreted these findings, we paid special attention to the relation existing between the abnormal presence of the Wolffian remains, male genotype, and typical female genital structures. Taking account of the latest scientific advances concerning genital development, we considered the possibility of the existence of secretions of a "masculinizing" substance from the gonad, before its morphological differentiation, which was interrupted by an etiological undetermined noxa. When this evolution was arrested, together with the secretions of the masculinizing substance, the genital development continued normally for a female. The terminal teratogenic period for this malformation is situated from the 5th to the 6th week of gestation (human embryos from 11 to 14 mm., Streeter Horizon XVII).     

A fluorescence-based homogeneous assay for measuring activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is one of the key enzymes of bacterial lipid A biosynthesis, catalyzing the removal of the N-acetyl group of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. The lpxC gene is essential in Gram-negative bacteria but absent from mammalian genomes, making it an attractive target for antibacterial drug discovery. Current assay methods for LpxC are not suitable for high throughput screening, since they require multiple product separation steps and the use of radioactively labeled material that is difficult to prepare. A homogeneous fluorescence-based assay was developed that uses UDP-3-O-(N-hexyl-propionamide)-N-acetylglucosamine as a surrogate substrate. This surrogate can be prepared from commercially available UDP-GlcNAc by enzymatic conversion to UDP-MurNAc, which is then chemically coupled to n-hexylamine. Following the LpxC reaction, the free amine of the deacetylation product can be derivatized by fluorescamine, thus generating a fluorescent signal. This surrogate substrate has a K(m) of 367 microM and k(cat) of 0.36 s(-1), compared to 2 microM and 1.5 s(-1) for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC(50)s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.     

Analysis and interpretation of dynamic FDG PET oncological studies using data reduction techniques

Background  

Dynamic positron emission tomography studies produce a large amount of image data, from which clinically useful parametric information can be extracted using tracer kinetic methods. Data reduction methods can facilitate the initial interpretation and visual analysis of these large image sequences and at the same time can preserve important information and allow for basic feature characterization.     

Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1/GRO1-dependent manner

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.     

The Activity of Hyaluronan Synthase 2 Is Regulated by Dimerization and Ubiquitination

Hyaluronan is a component of the extracellular matrix, which affects tissue homeostasis. In this study, we investigated the regulatory mechanisms of one of the hyaluronan-synthesizing enzymes, HAS2. Ectopic expression of Flag- and 6myc-HAS2 in COS-1 cells followed by immunoprecipitation and immunoblotting revealed homodimers; after co-transfection with Flag-HAS3, also heterodimers were seen. Furthermore, the expressed HAS2 was ubiquitinated. We identified one acceptor site for ubiquitin on lysine residue 190. Mutation of this residue led to inactivation of the enzymatic activity of HAS2. Interestingly, K190R-mutated HAS2 formed dimers with wt HAS2 and quenched the activity of wt HAS2, thus demonstrating a functional role of the dimeric configuration.     

The role of combichem in antibiotic discovery

Combinatorial chemistry (combichem) has had a significant impact on the discovery of new antibiotics. Most of the successes have come from the use of small libraries to explore a specific pharmacophore. However, large diverse libraries are more appropriate when identifying hits by screening specific bacterial or fungal targets. Combichem has been used to optimize new azole and oxazolidinone leads. An entirely new class of antibiotics, inhibitors of bacterial peptidyl-deformylase, has been discovered by combining mechanism-based drug design and combichem. These compounds are active in vivo. The impact of combichem on discovery projects that aim to develop new antibiotics for the treatment of infectious diseases is discussed.     

Hyaluronidase activity is modulated by complexing with various polyelectrolytes including hyaluronan.

Hyaluronidase (HAase) plays an important role in the control of the size and concentration of hyaluronan (HA) chains, whose biological properties strongly depend on their length. Our previous studies of HA hydrolysis catalyzed by testicular HAase demonstrated that, whilst the substrate-dependence curve has a Michaelis-Menten shape with a 0.15 mol L(-1) ionic strength, at low ionic strength (5 mmol L(-1)), a strong decrease in the initial hydrolysis rate is observed at high substrate concentrations; the HA concentration for which the initial rate is maximum increases when the HAase concentration is increased. After examination of various hypotheses, we suggested that this could be explained by the ability of HA to form non-specific complexes with HAase, which thus becomes unable to catalyze HA hydrolysis. In order to verify this hypothesis, we first showed from turbidimetric measurements that HAase, like albumin, is able to form electrostatic complexes with HA. Albumin then was used as a non-catalytic protein able to compete with HAase for the formation of non-specific complexes with HA, allowing HAase to be free and catalytically active. The kinetic results showed that the HA-HAase non-specific complex inhibits HAase catalytic activity towards HA. Depending on the albumin concentration with respect to the HAase and HA concentrations, albumin can either remove this inhibition or induce another type of inhibition. Finally, the extent of such non-specific interactions between polyelectrolytes and proteins in HAase inhibition or activation, in particular under in vivo conditions, is discussed.     

New antibacterial tetrahydro-4(2H)-thiopyran and thiomorpholine S-oxide and S,S-dioxide phenyloxazolidinones

Combinatorial libraries of N-acylated 5-(S)-aminomethyloxazolidinone derivatives of S-oxide and S,S-dioxide tetrahydro-4(2H)-thiopyranyl and thiomorpholine phenyloxazolidinone series have been synthesized on a solid phase and evaluated for antimicrobial activity. Several novel potent leads have been identified, including orally active oxazolidinones with enhanced activity against respiratory tract infection pathogens Haemophilus influenzae and Moraxella catarrhalis.