Biochemical and Cellular Characterization of the Function of Fluorophosphonate-Binding Hydrolase H (FphH) in Staphylococcus aureus Support a Role in Bacterial Stress Response.

The development of new treatment options for bacterial infections requires access to new targets for antibiotics and antivirulence strategies. Chemoproteomic approaches are powerful tools for profiling and identifying novel druggable target candidates, but their functions often remain uncharacterized. Previously, we used activity-based protein profiling in the opportunistic pathogen Staphylococcus aureus to identify active serine hydrolases termed fluorophosphonate-binding hydrolases (Fph). Here, we provide the first characterization of S. aureus FphH, a conserved, putative carboxylesterase (referred to as yvaK in Bacillus subtilis) at the molecular and cellular level. First, phenotypic characterization of fphH-deficient transposon mutants revealed phenotypes during growth under nutrient deprivation, biofilm formation, and intracellular survival. Biochemical and structural investigations revealed that FphH acts as an esterase and lipase based on a fold well suited to act on a small to long hydrophobic unbranched lipid group within its substrate and can be inhibited by active site-targeting oxadiazoles. Prompted by a previous observation that fphH expression was upregulated in response to fusidic acid, we found that FphH can deacetylate this ribosome-targeting antibiotic, but the lack of FphH function did not infer major changes in antibiotic susceptibility. In conclusion, our results indicate a functional role of this hydrolase in S. aureus stress responses, and hypothetical functions connecting FphH with components of the ribosome rescue system that are conserved in the same gene cluster across Bacillales are discussed. Our atomic characterization of FphH will facilitate the development of specific FphH inhibitors and probes to elucidate its physiological role and validity as a drug target.

Occupancy of human brain GABA(A) receptors by the novel α5 subtype-selective benzodiazepine site inverse agonist α5IA as measured using [¹¹C]flumazenil PET imaging.

GABA(A) receptor α5-selective inverse agonists enhance cognitive performance in pre-clinical species. However, a key aspect of the clinical development of such compounds is the demonstration that in man such compounds are devoid of the anxiogenic-like activity associated with non-selective inverse agonists such as FG 7142. The triazolophthalazine α5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is an α5-selective inverse agonist which enhances cognitive performance in rodents and encouragingly in human Phase I Safety and Tolerability studies it was devoid of the anxiogenic-like activity associated with FG 7142. However, in order to appropriately interpret this latter observation, it was considered important to demonstrate that the absence of anxiogenic-like activity occurs at significant levels of receptor occupancy. Consequently, the occupancy of human brain GABA(A) receptors was measured using [¹¹C]flumazenil positron emission tomography in three healthy normal young male volunteers following a single oral dose of 2 mg α5IA. One hour after dosing, mean occupancy levels were 53% and this fell to 16% by 8 h post-dose, with the plasma α5IA concentration corresponding to 50% occupancy being 10 ng/mL. These data clearly show that an α5-selective inverse agonist is not associated with anxiogenic-like side effects at doses that give ~50% occupancy.

Sustained modulation of intestinal bacteria by exclusive enteral nutrition used to treat children with Crohn's disease.

BACKGROUND: The use of exclusive enteral nutrition to treat paediatric Crohn's disease (CD) is widely accepted, although the precise mechanism(s) of action remains speculative. AIM: To investigate the changes to key intestinal bacterial groups of Eubacteria, Bacteroides, Clostridium coccoides, Clostridium leptum and Bifidobacteria, during and after exclusive enteral nutrition treatment for CD in paediatric patients and correlate these changes to disease activity and intestinal inflammation. METHODS: Stool was collected from six children at diagnosis of CD, during exclusive enteral nutrition and 4 months post-therapy, and from seven healthy control children. The diversity of bacteria was assessed by polymerase chain reaction-denaturing gradient gel electrophoresis with changes to bacterial diversity measured by Bray-Curtis similarity, intestinal inflammation assessed by faecal S100A12 and the disease activity assessed by PCDAI. RESULTS: A significantly greater change in intestinal bacterial composition was seen with exclusive enteral nutrition treatment compared with controls. Further, the intestinal bacteria remained altered 4 months following exclusive enteral nutrition completion. Changes in the composition of Bacteroides were associated with reduced disease activity and inflammation. CONCLUSIONS: Exclusive enteral nutrition reduces bacterial diversity and initiates a sustained modulation of all predominant intestinal bacterial groups. Exclusive enteral nutrition may reduce inflammation through modulating intestinal Bacteroides species. The implications of these results for exclusive enteral nutrition therapy and CD pathogenesis should now be the subject of further investigation.

Attachment in individuals with social anxiety disorder: the relationship among adult attachment styles, social anxiety, and depression.

Despite their apparent implications for social functioning, adult attachment styles have never been specifically explored among persons with social anxiety disorder. In the current study, a cluster analysis of the Revised Adult Attachment Scale (N. L. Collins, 1996) revealed that 118 patients with social anxiety were best represented by anxious and secure attachment style clusters. Members of the anxious attachment cluster exhibited more severe social anxiety and avoidance, greater depression, greater impairment, and lower life satisfaction than members of the secure attachment cluster. This pattern was replicated in a separate sample of 56 patients and compared with the pattern found in 36 control participants. Social anxiety mediated the association between attachment insecurity and depression. Findings are discussed in the context of their relevance to the etiology, maintenance, and cognitive-behavioral treatment of social anxiety disorder.

An empirical approach to subtype identification in individuals with social phobia.

BACKGROUND: The present study used cluster analysis procedures to identify empirically subgroups of patients with social phobia in a large clinical sample. METHOD: The Liebowitz Social Anxiety Scale (LSAS) was administered to 382 patients from several studies of the treatment of social phobia. LSAS fear ratings were summed into four subscale scores (social interaction, public speaking, observation by others, eating and drinking in public) based on a previous factor analytical study of the LSAS. In order to produce a stable and robust solution, these factor scores were submitted to a two-stage clustering procedure consisting of an agglomerative-hierarchical clustering method followed by an iterative non-hierarchical clustering method. RESULTS: Three patient subgroups were identified based on their pattern of feared social situations on the LSAS. These groups were labelled: (1) pervasive social anxiety; (2) moderate social interaction anxiety; and (3) dominant public speaking anxiety. Clusters differed significantly on age and age of social phobia onset, as well as on measures of social anxiety, general anxiety and depressive symptomatology. Clusters also differed in the percentage of assigned patients who met criteria for the generalized subtype of social phobia and avoidant personality disorder. CONCLUSIONS: The results provide empirical support for the existence of three subgroups in a clinical sample of individuals with social phobia and contribute to the growing evidence for the heterogeneity of social phobia. Further study of the conceptual, clinical and aetiological significance of these subgroups is needed.

Use of COMPARE analysis to discover new natural product drugs: isolation of camptothecin and 9-methoxycamptothecin from a new source.

Analysis of cytotoxicity data of extracts from the National Cancer Institute's Active Repository by the COMPARE protocol was carried out using camptothecin as a reference point. Extracts identified by this process were further characterized by a selective yeast bioassay for inhibitors of topoisomerase I and by a biochemical assay for compounds that stabilize the topoisomerase I-DNA covalent binary complex. Five of the extracts were positive in the yeast bioassay, and eight extracts showed activity on the assay that monitors stabilization of the topoisomerase I-DNA complex. Four of the latter extracts were inactive in the yeast bioassay, and thus would not have been identified as hits without the COMPARE preselection process. One of the extracts, from Pyrenacantha klaineana, was selected for detailed investigation, and fractionation of this extract yielded camptothecin and 9-methoxycamptothecin as the bioactive constituents.

Non-invasive radiotracer imaging as a tool for drug development.

Non-Invasive Radiotracer Imaging (NIRI) uses either short-lived positron-emitting isotopes, such as 11C and 18F, for Positron Emis ion Tomography (PET) or single photon emitting nuclides, e.g., 123I, which provide images using planar imaging or Single-Photon Emission Computed Tomography (SPECT). These high-resolution imaging modalities provide anatomical distribution and localization of radiolabeled drugs, which can be used to generate real time receptor occupancy and off-rate studies in humans. This can be accomplished by either isotopically labeling a potential new drug (usually with 11C), or indirectly by studying how the unlabelled drug inhibits specific radioligand binding in vivo. Competitive blockade studies can be accomplished using a radiolabeled analogue which binds to the site of interest, rather than a radiolabeled version of the potential drug. Imaging, particularly PET imaging, can be used to demonstrate the effect of a drug through a biochemical marker of processes such as glucose metabolism or blood flow. NIRI as a development tool in the pharmaceutical industry is gaining increased acceptance as its unique ability to provide such critical information in human subjects is recognized. This section will review recent examples that illustrate the utility of NIRI, principally PET, in drug development, and the potential of imaging advances in the development of cancer drugs and gene therapy. Finally, we provide a brief overview of the design of new radiotracers for novel targets.

A role for NF-kappaB-dependent gene transactivation in sunburn.

Exposure of skin to ultraviolet (UV) radiation is known to induce NF-kappaB activation, but the functional role for this pathway in UV-induced cutaneous inflammation remains uncertain. In this study, we examined whether experimentally induced sunburn reactions in mice could be prevented by blocking UV-induced, NF-kappaB-dependent gene transactivation with oligodeoxynucleotides (ODNs) containing the NF-kappaB cis element (NF-kappaB decoy ODNs). UV-induced secretion of IL-1, IL-6, TNF-alpha, and VEGF by skin-derived cell lines was inhibited by the decoy ODNs, but not by the scrambled control ODNs. Systemic or local injection of NF-kappaB decoy ODNs also inhibited cutaneous swelling responses to UV irradiation. Moreover, local UV-induced inflammatory changes (swelling, leukocyte infiltration, epidermal hyperplasia, and accumulation of proinflammatory cytokines) were all inhibited specifically by topically applied decoy ODNs. Importantly, these ODNs had no effect on alternative types of cutaneous inflammation caused by irritant or allergic chemicals. These results indicate that sunburn reactions culminate from inflammatory events that are triggered by UV-activated transcription of NF-kappaB target genes, rather than from nonspecific changes associated with tissue damage.

Comparison of S-100 versus hematoxylin and eosin staining for evaluating dermal invasion and peripheral margins by desmoplastic malignant melanoma.

Desmoplastic malignant melanoma (DMM) is a rare and locally aggressive variant of malignant melanoma, which is difficult to diagnose clinically and microscopically. A retrospective study of 21 DMM were collected during 7 years, representing 1.7% of melanomas. By comparing S-100 and hematoxylin and eosin (H & E) staining in DMM, we sought to determine whether S-100 staining offered a more accurate means of assessing dermal and neural invasion, tumor thickness, and peripheral margins. Eleven cases were excluded because the tumor extended past the deep margin. Six cases that were stained with S-100 showed a greater tumor thickness than by H & E (difference of 0. 13 to 2.79 mm). In two cases, the tumor thickness was greater by using H & E than S-100, and there was no difference in the remaining two cases. Of the eight cases in which the peripheral margins were positive by S-100, neoplastic cells were only apparent at those margins in four cases when examined with H & E. The remaining two cases showed negative margins by both stains. Clinical follow-up was obtained from nine cases, and none had recurrence of melanoma. Particularly for hypocellular and amelanotic tumors, S-100 staining proved to be a valuable adjunct in determining the extent of the tumor at the peripheral margins.

Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway.

In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.

Positron emission tomography neuroreceptor imaging as a tool in drug discovery, research and development.

Improved communication and cooperation between research-driven drug companies and academic positron emission tomography (PET) centers, coupled with improvements in PET camera resolution, the availability of small animal PET cameras and a growing list of neuroreceptor-specific PET tracers, have all contributed to a substantial increase in the use and value of PET as a tool in central nervous system drug discovery and development.

Nevus with fat: clinical characteristics of 100 nevi containing mature adipose cells.

BACKGROUND: The presence of mature adipose cells among the dermal melanocytes of nevi (nevi with fat) has previously been reported. However, its clinical features are not well characterized. OBJECTIVE: Our goal was to develop a better clinical understanding and insight into the histogenesis of nevi with fat by characterizing the patients' age, sex, height, weight, and location of lesion. METHODS: This was a prospective study of 100 nevi with fat from 89 patients over an 18-month period. RESULTS: Nevi with fat occur 4 times more commonly in women than men. They were diagnosed most often in patients between the ages of 40 to 49 years. The most frequent location is the head and neck. Most nevi with fat are intradermal. CONCLUSION: The appearance of fat within nevi is probably a multifactorial process. Age, weight, and sun exposure may be factors associated with the occurrence of nevi with fat.

Tasks and stages of professional growth.

This paper combines data from two surveys, one in 1986 and one in 1992, which used the same instrument. Practice problems, and responses to the problems, were found to differ according to the number of years in practice, validating a stage explanation of professional growth.

Practice profile: the first twelve years.

In a mailed questionnaire study of a 1/3 sample of recent graduates of the UOP School of Dentistry (57 percent response rate), most respondents felt competent in the majority of procedures they perform. Most procedures not performed were not performed because they are easily referred. Sources of learning about procedures differed according to frequency of performance (frequently performed: 95 percent-dental school; infrequently performed: 82 percent-continuing education). Differences also were noted according to years of practice.

Tasks and stages of professional growth.

This paper combines data from two surveys, one in 1986 and one in 1992, which used the same instrument. Practice problems, and responses to the problems, were found to differ according to the number of years in practice, validating a stage explanation of professional growth.

The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth.

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.

Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu.

The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity.

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

[Pyrimidine 5'-nucleotidase deficiency as the congenital cause of nonspherocytic hemolytic anemia]. / Pyrimidin-5'-Nucleotidase-Mangel als kongenitale Ursache einer nicht-sphärozytären hämolytischen Anämie.

An 11-year-old girl had marked haemolytic anaemia since the first year of life. Physical examination revealed scleral and cutaneous icterus and slight splenomegaly. Haemoglobin concentration was reduced to 9.5 g/dl, while platelet count and bilirubin concentration were increased (350,000/microliter and 2.2 mg/dl, respectively). The erythrocytes showed marked basophilic stippling, its extent typical of pyrimidine-5'-nucleotidase deficiency. The enzyme activity in the erythrocytes was 15% of normal. Deficiency of this enzyme, inherited as an autosomal recessive, is probably one of the most common erythrocyte enzyme deficiencies causing haemolytic anaemia. It brings about the intracellular accumulation of pyrimidine nucleotides which via secondary metabolic changes causes an accelerated destruction of erythrocytes. There is no known causative treatment: splenectomy is ineffective against the anaemia.