The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter.

Legionella pneumophila requires the dot/icm genes to create an organelle inside eukaryotic host cells that will support bacterial replication. The dot/icm genes are predicted to encode a type IV-related secretion apparatus. However, no proteins have been identified that require the dot/icm genes for secretion. In this study we show that the DotA protein, which was previously found to be a polytopic membrane protein, is secreted by the Dot/Icm transporter into culture supernatants. Secreted DotA protein was purified and N-terminal sequencing of the purified protein revealed that a 19 amino acid leader peptide is removed from DotA prior to secretion. Extracellular DotA protein did not fractionate in membrane vesicles. Structures containing secreted DotA protein were visualized by electron microscopy and were shaped like hollow rings. These data indicate that the large poly topic membrane protein DotA is secreted from L.pneumophila by a unique process. This represents the first target secreted by the dot/icm-encoded apparatus and demonstrates that this transporter is competent for protein secretion.

How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane.

Within five minutes of macrophage infection by Legionella pneumophila, the bacterium responsible for Legionnaires' disease, elements of the rough endoplasmic reticulum (RER) and mitochondria attach to the surface of the bacteria-enclosed phagosome. Connecting these abutting membranes are tiny hairs, which are frequently periodic like the rungs of a ladder. These connections are stable and of high affinity - phagosomes from infected macrophages remain connected to the ER and mitochondria (as they were in situ) even after infected macrophages are homogenized. Thin sections through the plasma and phagosomal membranes show that the phagosomal membrane is thicker (72+/-2 A) than the ER and mitochondrial membranes (60+/-2 A), presumably owing to the lack of cholesterol, sphingolipids and glycolipids in the ER. Interestingly, within 15 minutes of infection, the phagosomal membrane changes thickness to resemble that of the attached ER vesicles. Only later (e.g. after six hours) does the ER-phagosome association become less frequent. Instead ribosomes stud the former phagosomal membrane and L. pneumophila reside directly in the rough ER. Examination of phagosomes of various L. pneumophila mutants suggests that this membrane conversion is a four-stage process used by L. pneumophila to establish itself in the RER and to survive intracellularly. But what is particularly interesting is that L. pneumophila is exploiting a poorly characterized naturally occurring cellular process.

Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth.

Legionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV-related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein-protein interactions. Specific interactions between different Icm proteins were detected by yeast two-hybrid and gel overlay analysis. These data support a model in which the IcmQ-IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS-IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.

Identification and subcellular localization of the Legionella pneumophila IcmX protein: a factor essential for establishment of a replicative organelle in eukaryotic host cells.

The gram-negative respiratory pathogen Legionella pneumophila infects and grows within mammalian macrophages and protozoan host cells. Upon uptake into macrophages, L. pneumophila establishes a replicative organelle that avoids fusion with endocytic vesicles. There are 24 dot/icm genes on the L. pneumophila chromosome required for biogenesis of this vacuole. Many of the Dot/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems. We have been investigating the function of L. pneumophila dot/icm gene products that do not have obvious orthologs in other type IV transfer systems, since these determinants could govern processes unique to phagosome biogenesis. The icmX gene product falls into this category. To understand the role of the IcmX protein in pathogenesis, we have detailed interactions between an L. pneumophila icmX deletion mutant and murine bone marrow-derived macrophages. These data demonstrate that icmX is required for biogenesis of the L. pneumophila replicative organelle. Immunoblot analysis indicates that the icmX gene product is a polypeptide with an estimated molecular mass of 50 kDa. The IcmX protein was localized to the bacterial periplasm, and periplasmic translocation was mediated by an N-terminal sec-dependent leader peptide. A truncated IcmX product was secreted into culture supernatants by wild-type L. pneumophila growing extracellularly in liquid media; however, transport of the IcmX protein into eukaryotic host cells was not detected. Proteins similar in molecular weight to IcmX were identified in other Legionella species by immunoblot analysis using a monoclonal antibody specific for L. pneumophila IcmX protein. From these data, we conclude that the IcmX protein is an essential component of the dot/icm secretion apparatus, and that a conserved mechanism of host cell parasitism exists for members of the Legionellaceae family.

A profile of cardiovascular disease in northern Ontario: public health planning implications.

Cardiovascular disease (CVD) is a leading cause of death in Northern Ontario and therefore considered an important issue. To this end, this paper examines CVD trends in Northern Ontario and the prevalence of known risk factors that give an insight into these trends. Ontario Health Survey 1990, Ontario Health Survey 1996, Canadian Institute for Health Information (1990-95) and Vital Statistics (1990-95) were examined. It was determined that CVD rates in Northern Ontario significantly exceeded those of the province. Further, high prevalence of modifiable risk factors, such as smoking, fat intake, physical inactivity and obesity are all experienced in Northern Ontario when compared to the province. Planning implications, as they relate to collaboration, delivery of services, determinants of health, multiple risk factors and monitoring and evaluation are also discussed.

Pore-forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth.

Bacterial pathogens often subvert eukaryotic cellular processes in order to establish a replicative niche and evade host immunity. Inhibition of phagosome lysosome fusion is a strategy used by several intracellular bacteria that grow within mammalian cells. It was shown recently that Legionella pneumophila possesses a cytolytic activity that results from the insertion of pores in the macrophage membrane upon contact, and that this activity requires the dot/icm gene products, which are necessary for intracellular growth and phagosome trafficking. Other bacteria that inhibit phagosome lysosome fusion, such as Mycobacterium tuberculosis, demonstrate similar cytolytic activities, which suggests that formation of pores in the phagosome membrane may account for the defects observed in phagosome trafficking. In this study, we identify a new class of L. pneumophila mutant that retains the pore-forming activity found in virulent bacteria, but is defective in phagosome lysosome fusion inhibition and intracellular growth. These data indicate that cytolytic activity is not sufficient for L. pneumophila-induced alterations in phagosome trafficking. Rather, the pore may be a vehicle that facilitates delivery of bacterial-derived effector molecules to the host cell cytoplasm.

Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake.

Numerous intracellular bacterial pathogens modulate the nature of the membrane-bound compartment in which they reside, although little is known about the molecular basis for this control. Legionella pneumophila is a bacterial pathogen able to grow within human alveolar macrophages and residing in a phagosome that does not fuse with lysosomes. This study demonstrates that the dotA product is required to regulate trafficking of the L. pneumophila phagosome. Phagosomes containing L. pneumophila dotA+ bacteria exhibited differential trafficking profiles when compared with isogenic dotA mutants. Phagosomes containing dotA mutants showed rapid accumulation of the lysosomal glycoprotein LAMP-1 as early as 5 min after uptake, whereas the majority of wild-type L. pneumophila phagosomes did not acquire LAMP-1. The association of LAMP-1 with phagosomes containing dotA mutant bacteria was concomitant with the appearance of the small GTP-binding protein Rab7 on the vacuolar membrane. These data demonstrate that phagosomes containing replication-competent L. pneumophila evade early endocytic fusion events. In contrast, the kinetics of LAMP-1 and Rab7 association indicate that the dotA mutants are routed along a well-characterized endocytic pathway leading to fusion with lysosomes. Genetic studies show that L. pneumophila requires DotA expression before macrophage uptake in order to establish an intracellular site for replication. However, the bacteria do not appear to require continuous expression of the DotA protein to maintain a replicative phagosome. These data indicate that DotA is one factor that plays a fundamental role in regulating initial phagosome trafficking decisions either upon or immediately after macrophage uptake.

The measurement of solar ultraviolet radiation.

High skin cancer rates, stratospheric ozone depletion and increased public interest and concern have resulted in a strong demand for solar ultraviolet radiation measurements and information. The Australian Radiation Laboratory (ARL) has been involved since the mid-1980s in the measurement of solar ultraviolet radiation (UVR) using spectroradiometers (SRM) and a network of broadband detectors at 18 sites in Australia and Antarctica and in Singapore through a collaborative agreement with the Singapore Institute of Science and Forensic Medicine. Measurement locations range from equatorial (Singapore, 1.3 degrees N) through tropical (Darwin, 12.4 degrees S) to polar (Mawson, 67.6 degrees S) and as a result there are many difficulties associated with maintenance and calibration of the network detectors, and transfer of data to ensure an accurate and reliable data collection. Calibration procedures for the various detectors involve the comparison with simultaneous spectral measurements using a portable SRM incorporating a double monochromator, calibrated against traceable standard lamps. Laboratory measurements of cosine response and responsivity are also made. Detectors are intercompared at the Yallambie site for a number of months before installation at another location. As an additional check on the calibrations, computer models of solar UVR at the earth's surface for days with clear sky and known ozone are compared with the UV radiometer measurements.

Protection against solar ultraviolet radiation.

Interest in protection against solar ultraviolet radiation (UVR) among the general public in Australia has been increasing steadily as a result of the 'SunSmart' campaigns run by the various state cancer councils. This increasing awareness is due in part to the requirements for occupational protection of outdoor workers and to provision of UVR protection for the recreational market. Behaviour outdoors can significantly affect exposure to solar UVR and use of items of personal protection can provide a substantial reduction in the UVR dose received. The protective properties of sunscreens, sunglasses, hats and clothing against UVR have been the subject of considerable research for some time, and over the last few years interest has extended to the provision of shade structures and the UVR protection provided by various commonly used materials. These materials include shadecloth, plastics, glass, windscreens and applicable tints. Australia has rigorous standards covering protection and UVR, in particular for sunscreens [Standards Australia/Standards New Zealand, Sunscreen products-evaluation and classification, Report No. AS 2604, Sydney/Wellington, 1993.], sunglasses [Standards Australia, Sunglasses and fashion spectacles-nonprescription types, Report No. AS 1067.1, Sydney, 1990.], protective eyewear [Standards Australia/Standards New Zealand, Eye protectors for industrial applications, Report No. AS/NZS 1337, Sydney/Wellington, 1992.] and shadecloth [Standards Australia, Synthetic shadecloth, Report No. AS 4174, Sydney, 1994.]. Compliance with the sunglass standard became mandatory in 1988 and UVR protection provided by sunglasses has increased substantially since then. In July 1996 a standard on 'sun protective textiles' [Standards Australia/Standards New Zealand, Sun protective clothing-evaluation and classification, Report No. AS/NZS 4399, Sydney, 1996.] incorporating ultraviolet protection factors (UPFs) and a rating scheme with protection categories, was introduced; this was the first of its kind in the world. Australian Radiation Laboratory (ARL) UPF swing tags with UVR protection advice from the Australian Cancer Society on the reverse side are used to denote the amount of protection against solar UVR provided by clothing. To date in excess of 5 million ARL swing tags have been issued. Work on the various standards is continuing. The maximum allowed 'sun protection factor' (SPF) limit for sunscreens may be increased to SPF 30 + in the near future, and additions to the sun protective textiles standard are also planned. This paper discusses measurement methods, results, the rationale used in formulating the Australian Standards and the current state of UVR protection in Australia.

UV protection by clothing: an intercomparison of measurements and methods.

In an attempt to reduce the incidence of skin cancer, cancer foundations have run educational campaigns which encourage the general population to limit their solar UVR exposures. An important part of these campaigns, in particular in Australia, but also more recently in Europe and the U.S., has been the adoption of protective measures such as sunscreens, hats, sunglasses and clothing. The protective properties of fabrics and clothing against ultraviolet radiation (UVR) have been known for some time, but recently there has been considerable interest in quantifying the degree of protection. This has been generated, in part, by the requirements for occupational protection for outdoor workers as well as the provision of UVR protection for the recreational market. The quantification of UVR protection has been laboratory based using in vitro test methods. Development of a standard test method has become an important part of the testing process, and this paper presents results from an intercomparison involving five independent testing laboratories. Agreement is good, in particular for samples with protection factors below 50. Technical difficulties and sources of errors associated with the measurements are discussed.

Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages.

The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 and 73 amino acids and a carboxyl-terminal cytoplasmic domain of 122 amino acids. Protein fractionation studies were consistent with DotA residing in the inner membrane. An alkaline phosphatase fusion located 9 amino acids upstream from the C terminus of DotA still retained function and was able to restore intracellular growth when harbored by two L. pneumophila dotA mutants. A hybrid protein from which the carboxyl-terminal 48 amino acids of DotA were deleted was unable to complement the intracellular growth defect in the dotA mutants, indicating that this cytoplasmic region is required for function.

Effect of seasonal ultraviolet radiation fluctuations on vitamin D homeostasis during an Antarctic expedition.

Antarctica is a unique and challenging environment where members of expeditions face a range of conditions not normally experienced. Ultraviolet (uv) radiation levels show marked variation during the year. The 25-hydroxy metabolite of vitamin D [25(OH)D] is largely produced by sunlight and shows a yearly variation in concentration that corresponds to uv radiation levels. The active metabolite 1,25-dihydroxyvitamin D [1,25(OH)2D] does not generally show any such variation provided 25(OH)D concentrations are sufficient. Previous studies have shown a seasonal variation in 25(OH)D with a significant winter drop. No other study of 1,25(OH)2D has been reported on members of Antarctic expeditions. A group of 19 men wintering at Davis Station (68 degrees 34' S) had four blood samples taken at 3-monthly intervals beginning in the Antarctic summer. Analysis for 25(OH)D showed a drop in concentration for each of the latter three sampling periods (P < 0.005). This correlated with uv radiation levels and would suggest that endogenous production of 25(OH)D ceases for at least the duration of the Antarctic winter. There were no significant alterations in 1,25(OH)2D or calcium concentrations over the same period. Providing that individuals with pre-existing vitamin D deficiencies are detected before departure for Antarctica and missions are limited in duration, clinical deficiency is unlikely to occur.

Personal dosimetry of solar UV radiation for different outdoor activities.

Quantifying individual exposure to ultraviolet radiation (UVR) is critical to understanding the etiology of a number of diseases including nonmelanotic and melanotic skin cancers. Measurements of personal exposure to solar UVR were made in Hobart, Tasmania in February (summer) 1991 for six different outdoor activities using UVR-sensitive polysulfone (PS) film attached at seven anatomical sites. Concurrent behavioral and environmental observations were also made. To date many studies have relied on subject recall to quantify past solar UVR exposures. To gain insight into the accuracy of subject recall the measured UVR exposures received by different subjects using the PS film were compared to those calculated from personal diaries and ambient solar UVB levels from a monitoring station. In general, when UVR exposure activities took place under close supervision, good correlations were obtained between the PS badges and the ambient measurements/diaries approach. Ultraviolet radiation exposures for the field study involving 94 subjects engaged in a number of outdoor activities are presented.

Ultraviolet radiation protection factors for clothing.

There has been considerable interest and activity in Australia during recent years in providing ultraviolet radiation protection for the public and the work force. Many requests from the public and manufacturers have been received by the Australian Radiation Laboratory for the quantification of the protection provided by different articles of clothing. The laboratory has been making spectral measurements of the ultraviolet radiation transmission of materials and fabrics for a number of years and, to this date, have tested and rated > 2,000 samples. The laboratory test methods for making spectral transmission measurements of both dry and wet fabric samples are described along with the radiometric test methods to determine ultraviolet radiation transmission. A scheme to designate the amount of ultraviolet radiation protection of materials using ultraviolet protection factors is outlined and used to compare the results obtained by different test methods. Some typical measurement results are also summarized and presented.

Positive hydrocele cytology accompanying testis seminoma.

Accurate palpation of the testis may be obscured by the presence of a hydrocele. Analysis of hydrocele fluid is seldom reported. We describe the cytologic demonstration of malignant cells within a hydrocele accompanying testicular seminoma. Although this connection is not unexpected, it is only rarely documented.

Identification of Bordetella pertussis regulatory sequences required for transcriptional activation of the fhaB gene and autoregulation of the bvgAS operon.

Transcription of numerous virulence genes in Bordetella pertussis is positively regulated by the products of the bvgAS genes. In this study a series of lacZYA fusions containing deletions in either the fhaB or bvgA promoter regions was used to identify cis-acting regulatory regions required for bvg activation of these two genes. Gel retardation and DNase I protection analyses have shown that specific protein-DNA interactions occur at these regulatory regions and that these interactions require the transcriptional activator protein BvgA. The regulatory regions found upstream of fhaB and bvgA which are involved in protein binding both contain the sequence TTTCCTA. This sequence is part of an inverted repeat upstream of fhaB and a direct repeat upstream of bvgA. Homologous repeats are not apparent upstream of other bvg-activated genes, such as ptx and cyaA. These data suggest that the mechanism for transcriptional regulation of the other bvg-activated genes is complex and may require regulatory factors in addition to the bvgAS gene products.

Incidental carcinoma of prostate, Long-term follow-up.

Stage A1 carcinoma of the prostate because of its small volume and low grade has been regarded as clinically insignificant and requiring no treatment. Recent long-term studies of the untreated natural history of this disease suggests otherwise. Review of our long-term follow-up of untreated incidental carcinoma of the prostate diagnosed between 1966 and 1975 has demonstrated a 16 percent progression rate requiring therapy. This finding suggests Stage A1 prostate carcinoma is not a dismissible diagnosis but demands accurate staging, closer follow-up to uncover progression, and consideration of definitive therapy following diagnosis.