Comprehensive transposon mutant library of Pseudomonas aeruginosa

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.     

Interleukin- 1 gene polymorphism disease activity and bone mineral metabolism in rheumatoid arthritis

Objective To determine whether interleukin- 1α and 1β gene polymorphism is associated w ith rheumatoid arthritis disease activity and bone mineral metabolism, and whet her there is any relationship between IL- 1β and rheumatoid arthritis (RA) moti f gene. Methods IL- 1 gene polymorphisms were analyzed in 65 RA patients who met American College of Radiology (ACR) criteria and 60 controls. From genomic DNA, 2 polymo rphisms in each gene for IL1α- 889 and IL- 1β+3953 were typed by PCR- RFLP and HLA- DRB1 allele typing was also undertaken by PCR- SSOP. Some clinical and l aboratory parameters were collected. The allelic frequencies and carriage rate s were compared between RA patients and controls and between patients with acti ve and quiescent disease. Comparison was also made between IL- 1 polymorphism a nd parameters of bone mineral metabolism and between patients with the HLA- DRB1 RA motif plus IL- 1β2 and patients without the two alleles. Fisher test an d the analysis of variance was used to analyze the data. Results There was no significant difference in the frequency and carriage rate of IL- 1 α polymorphisms between RA patients and the controls. The β2/2 genotype of IL - 1β was more common in female RA patients compared with controls (P=0. 001 ). A lower carriage rate of IL- 1β2 occurred in male RA patients (P=0. 0 01). A higher carriage rate of IL- 1α2 is associated with a higher ESR (P =0. 008), HAQ score (P=0. 03), and vit- D(3) (P<0. 001), but conversely a lower SJC (p=0. 002), a lower RF (P=0. 002) and a lower BMD at the l um bar spine (P=0. 001). A higher frequency of IL- 1α1 is associated with a l ow er CRP value (P=0. 009). An increased IL- 1β2 carriage is associated with active rheumatoid disease as indicated by a higher CRP (P<0. 001), ESR ( P<0. 001) and pain score (P=0. 001) and a higher BMD at the lumbar spin e (P=0. 007), lower vit- D(3) and. Udpd/Crea level The presence of the HLA DR B1 RA motif and IL- 1β allele 2 at same time did not contribute to disease acti vity. Conclution Polymorphisms of the IL- β gene may affect the RA occurrence. Carriage of IL- 1β2 polymorphisms is associated with more active disease in RA and the presenc e of both the IL- 1α2 and the IL- 1β1 allele in RA influences bone resorption .     

Comparison of the Aachen Aphasia Test,clinical study and Aachen Aphasia Beside Test in brain tumor patients

In the clinical routine examination of patients with brain tumors, aphasic symptoms are often not recognized. In order to document the incidence of such symptoms, three diagnostic methods of testing for aphasia were compared: the Aachen aphasia test (AAT), which is the German standard aphasia test, clinical examination, and the Aachen aphasia bedside test (AABT), which was designed to test patients in the acute phases of illness. In the AAT, 50% of patients with left-sided tumors and 36% of those with right-sided tumors showed aphasic disturbances. The AAT results were defined as the gold standard. Clinical examination showed only low sensitivity; less than half of the aphasic patients were diagnosed as such. The AABT also detected only about half of the patients with aphasic disturbances. The low sensitivity is caused mainly by the results of the patients with right-hemisphere tumors, in which the mental set of the examiner during clinical examination (aphasic symptoms are not expected in patients with right-hemisphere lesions) and the pattern of disturbances in the AABT (deficits may be less severe and different in nature) may prevent detection of aphasic symptoms. Both clinical examination and AABT are thus not suitable for aphasia diagnostics in brain tumor patients. As the AAT is very time-consuming in everyday clinical routine, however, the development of an aphasia screening test seems desirable.     

Detection of variant Creutzfeldt-Jakob disease infectivity in extraneural tissues

Abnormal accumulations of prion protein (PrP) can be detected in the spleen, lymph nodes, and tonsils of patients with variant Creutzfeldt-Jakob disease (vCJD). Therefore, it has been assumed, but not shown, that these tissues harbour infectivity, which in turn presents the potential for iatrogenic spread through surgery. Here, we show and measure levels of infectivity in spleen and tonsil from two patients with vCJD, by bioassay in intracerebrally inoculated RIII mice. Similar bioassays failed to detect infectivity in buffy coat and plasma.     

Inhibition of Ras p21 synthesis by antisense undecamers with uniform and specifically arranged phosphorothioate linkages

The design of chimeric oligodeoxynucleotides (ODNs) in which certain phosphodiester linkages are replaced by phosphorothioate (PS) aims to decrease non-sequence-specific effects of uniform PS ODNs and to preserve the PS-provided protection against exo- and endonucleases. This study has, for the fist time, directly compared the differences in nuclease resistance, cellular uptake, antisense potency and sequence specificity of PS and end-capped, pyrimidine-protected (PPS) undecamer ODNs, that are complementary to the initiation codon region of human Ha-ras mRNA. At concentrations above 5 microM, both PS and PPS undecamers were moderately and equally stable for over 48 h in complete medium with RS485 cells overexpressing Ha-ras. They were completely stable at 0.4 microM when complexed with Lipofectin reagent that enhanced cellular uptake up to 9-fold. Both the antisense PPS and PS undecamers produced well-defined inhibition of Ras p21 synthesis in both cell-free and cell-based assays. However, non-sequence-specific effects of the uniform phosphorothioates were still significant. In contrast, the antisense PPS undecamer, when delivered to RS485 cells with Lipofectin reagent, inhibits human Ras p21 synthesis by more than 90% at a concentration of 3.2 microM, while the effect of controls with inverted, mismatched or scrambled sequence was minimal (5% or less) on p21 synthesis and RS485 cell growth.     

The transmission of prions to humans

The identification of new-variant Creutzfeldt-Jakob disease (nvCJD) in 1996 led to the proposal that this new disease was caused by the transmission of bovine spongiform encephalopathy (BSE) to the human population. The ramifications of such a proposal have been extensive and profound, both politically and on the general public in the UK and other countries. Patients with nvCJD exhibit a consistent set of clinicopathological features, and cases of nvCJD continue to be reported almost exclusively in the UK, the country with by far the highest incidence of BSE. Laboratory studies, including transmission experiments in mice, provide strong support for the hypothesis that nvCJD is caused by BSE.     

Infantile osteopetrosis; bone marrow transplantation from a cousin donor.

The successful correction of infantile osteopetrosis in an Asian child by bone marrow transplantation (BMT) from an HLA-A,B matched cousin donor is reported. Retrospective HLA molecular analysis revealed that patient and donor were incompatible for HLA-DPB1. Donor type cells detected in the patient after transplantation indicate successful engraftment. The patient is currently alive and well.     

Effects of post-weaning environment and apparatus dimension on spontaneous alternation as a function of phenotype in “dwarf” mice

The spontaneous alternations of “dwarf” mice estimated in a Y maze proportioned to the size of control mice demonstrated the necessity of reducing the size of the apparatus for testing the “dwarf” mice. Tested in a maze of reduced dimension, the “dwarf” mice showed a significant deficit in this task but only in a T shaped maze, and not in a Y shaped maze. For one month after weaning, “dwarf” and control mice were reared in either an enriched or an impoverished environment. These rearing conditions did not modify the spontaneous alternation of control mice whether estimated in a T or a Y maze, but they significantly altered the behavior of “dwarf” mice, yet only when evaluated in a T maze: “isolated” “dwarf” mice alternating less than “enriched” “dwarf” mice. It is suggested that this behavioral profile displayed by “dwarf” mice in spontaneous alternation might be explained by a deficit in perceiving, integrating and/or utilizing complex spatial information.