Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa

Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known as A band and B band. B band is the serospecific LPS, while A band is the common LPS antigen composed of a D-rhamnose O-polysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been identified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterization of two genes within the cluster, designated wzm and wzt. The Wzm and Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively, and are homologous to a number of proteins that comprise ABC (ATP-binding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-binding protein containing a highly conserved ATP-binding motif. Chromosomal wzm and wzt mutants were generated by using a gene replacement strategy in P. aeruginosa PAO1 (serotype 05). Western blot analysis and immunoelectron microscopy using A-band- and B-band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synthesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band polysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rather, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. These results demonstrate that A-band and B-band O-antigen assembly processes follow two distinct pathways, with the former requiring an ABC transport system for cell surface expression.     

Age- and sex-related differences in nuclear lipid content and nucleoside triphosphatase activity in the JCR:LA-cp corpulent rat

The putative role of the nuclear nucleoside triphosphatase (NTPase) is to provide energy to the nuclear pore complex for poly A(+) mRNA export. Previous work has demonstrated that liver nuclear NTPase activity is greater in 6 month old corpulent (cp/cp) female JCR:LA rats, a hyperlipidemic rat model, compared to lean (+/?) animals. This increase appeared to be related to increases in nuclear membrane cholesterol content. The current study extended these initial data to compare NTPase activity as a function of age and sex in isolated JCR:LA-cp rat liver nuclei, to further test the hypothesis that nuclear membrane cholesterol may modulate NTPase activity. NTPase activity was increased in cp/cp female animals compared to +/? females at all ages studied, with Vmax values increased by 60-176%. Membrane integrity of cp/cp female nuclei was reduced compared to +/? female nuclei. Nuclear membrane cholesterol levels increased linearly with age by 50, 150 and 250% in 3, 6 and 9 month old cp/cp females over leans. In contrast, nuclei from cp/cp males exhibited only minor, isolated changes in NTPase activity. Furthermore, there were no significant changes in nuclear cholesterol content or membrane integrity in the less hyperlipidemic male animals at any age. These data suggest that altered lipid metabolism may lead to changes in nuclear membrane structure, which in turn may alter NTPase activity and functioning of the nuclear pore complex.     

Tamoxifen downregulates TGF-beta production in keloid fibroblasts

BACKGROUND: It has recently been suggested that primary lactase deficiency might have been selected for by malaria, as occurred for beta-thalassaemia and glucose 6-phosphate dehydrogenase deficiency. However, recently we have found that the prevalence of primary lactase deficiency in the area of Sassari (Northern Sardinia), where, in the past, there was intermediate malarial endemicity, is comparable to that observed in the adult population from other areas of Southern Italy where malaria was less endemic. AIMS: To address the problem further, we have determined the prevalence of primary lactase deficiency, glucose 6-phosphate dehydrogenase deficiency deficiency and beta-thalassaemia trait in the populations of three Sardinian villages which differ in altitude above sea-level, socioeconomic features, history of endemic malaria and prevalence of b-thalassaemia and glucose 6-phosphate dehydrogenase deficiency. SUBJECTS: We tested 138 adult males: 53 were from Fonni (a non-malarial mountain village, with a strong pastoral tradition), 38 from Lodé (a village with a similar pastoral tradition, but high malarial endemicity in the past) and 47 from Terralba (a lowland fishing village with an agricultural tradition and heavy malarial morbidity and mortality). METHODS: A blood sample was obtained in all subjects for determination of HbA2 and glucose 6-phosphate dehydrogenase activity. Lactase deficiency was assessed by measuring breath hydrogen production after oral administration of lactose (50 g), by gas-chromatography. RESULTS: The frequencies of glucose 6-phosphate dehydrogenase deficiency and of beta-thalassaemia trait in the non-malarial village of Fonni were strikingly low, compared to frequencies found in the two villages (Terralba and Lodé) with a very high past malarial morbidity. In contrast, there was no significant difference in the prevalence of lactase deficiency in the three groups of subjects from the three villages. CONCLUSIONS: These data obtained in Northern Sardinia do not support the hypothesis of a selection of primary lactase deficiency by malaria. For definitive conclusions, however, the malaria hypothesis should be tested in other parts of the world.     

Glucose content in human skin: relationship with blood glucose levels

In order to ascertain the dynamic relationship between the extracellular glucose in upper skin layers and blood glucose, skin suction blisters were raised in six Type 1 diabetic patients during a three-step glucose clamp. Blister glucose closely paralleled venous glucose (mean of r = 0.998). However, in three patients blister glucose was constantly lower than plasma glucose and this appeared to be related to their slower formation of skin blisters. A substantial difference in skin blister suction time was noted among patients and it was found that suction time was linearly correlated to glycosylated haemoglobin (HbA1C) (n = 6, r = 0.865, p = 0.026). It is concluded that a non-invasive blood glucose monitoring system could be successfully based on measurement of alterations in skin glucose contents.     

The effect of muscle repair on postoperative facial skeletal growth in children with bilateral cleft lip and palate

Systems for testing genetic toxicology are components of carcinogenic and genetic risk assessment. Present routine genotoxicity-testing is based on at least 20 years of development during which many different test systems have been introduced and used. Today, it is clear that no single test is capable of detecting all genotoxic agents. Therefore, the usual approach is to perform a standard battery of in-vitro and in-vivo tests for genotoxicity. Work-groups of the European Union (EU), the Organization for Economic Co-operation and Development (OECD), and, very recently, the work-group of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) have defined such standard battery tests. These and some currently used supplementary or confirmatory tests are briefly discussed here. Additional test systems for the assessment of genotoxic and carcinogenic hazard and risk are seriously needed. These tests must be more relevant to man than are current assays and less demanding in respect of cost, time and number of animals. Another aspect for reassessment derives from the actual situation in the pharmaceutical industry. Companies have to prepare for the world economy of the 21st century. Therefore, pharmaceutical research is speeding up tremendously by use of tools such as genomics, combinatorial chemistry, high throughput screening and proteomics. Toxicology and genotoxicology need to re-evaluate their changing environment and must find ways to respond to these needs. In conclusion, genetic toxicology needs to answer questions coming from two major directions: hazard and risk identification and high throughput testing.     

Genetic polymorphisms of cytochrome P4502E1 related to the development of alcoholic liver disease

BACKGROUND/AIMS: Because heavy drinkers do not always develop alcoholic liver disease (ALD), genetic factors may be involved. Cytochrome P4502E1 is the main enzyme that oxidizes ethanol in the non-alcohol dehydrogenase pathway. Recently, the presence of genetic polymorphisms of this enzyme was confirmed. In the present study, the genotypes of P4502E1 were analyzed in patients with or without ALD. METHODS: After extraction of DNA from white blood cells, genotypes of P4502E1 were determined by restriction fragment length polymorphisms using two endonucleases. The genotypes were separated into three types: type A, type C (homozygous for the c1 or c2 gene), and type B (heterozygous for both genes). RESULTS: In 50 patients with ALD, the prevalence of type A was 16% and that of the c2 gene was 84%. The genotypes in 10 heavy drinkers without ALD were all type A. In 34 patients with non-alcoholic liver disease and in 88 patients without hepatobiliary disease, the prevalence of type A was 65% and 71%, respectively, indicating a significantly higher prevalence of the c2 gene in ALD. In healthy nonalcoholics, the prevalence of type A was 62%-68%. CONCLUSIONS: These results suggest that polymorphisms of P4502E1 may be related to the development of ALD.     

Isolated lung perfusion with tumor necrosis factor for pulmonary metastases

BACKGROUND: A phase I trial was initiated to define the feasibility and safety of single-lung isolation perfusion with tumor necrosis factor-alpha, interferon-gamma, and moderate hyperthermia for patients with unresectable pulmonary metastases. METHODS: Twenty patients with lung metastases (Ewing's, 2; sarcoma, 8; melanoma, 6; other, 4) were considered for single-lung isolation perfusion with 0.3 to 6.0 mg of tumor necrosis factor-alpha and 0.2 mg interferon-gamma delivered through an oxygenated pump circuit. Sixteen perfusions were performed in 15 patients (bilateral in 1). Metastases were completely resected (no single-lung isolation perfusion) in 3 patients, 1 patient had extrapulmonary disease, and one single-lung isolation perfusion was aborted for mechanical reasons. RESULTS: There were no significant changes in systemic arterial blood pressure or cardiac output during perfusion. Systolic pulmonary artery pressure increased with isolation, but returned to pre-single-lung isolation perfusion levels after clamp release. The maximum systemic tumor necrosis factor-alpha level was 8 ng/mL, whereas pump-circuit levels ranged from 200 to 10,976 ng/mL. There were no deaths, and the mean hospitalization period was 9 days (range, 5 to 34 days). A short-term (6 to 9 month) unilateral decrease in perfused nodules was noted in 3 patients (melanoma in 1, adenoid cystic carcinoma in 1, renal cell carcinoma in 1). CONCLUSIONS: Future studies using a combination of biologic modifiers, chemotherapy, and hyperthermia should be pursued to define active cytotoxic agents that will preserve underlying pulmonary function.