Age and growth of round goby Neogobius melanostomus associated with depth and habitat in the western basin of Lake Erie.

Round goby Neogobius melanostomus were examined from the Bass Islands area in the western basin of Lake Erie, U.S.A., to determine age and growth correlations. A total of 188 specimens were collected and examined during summer 2011 with 90 aged using scale analysis. Fish were grouped by sex, depth of habitat and habitat type (anthropogenically modified shallows, natural shallows and open lake deep water). Fish ranged from 17 to 117 mm total length (LT ) and 0+ to 3+ years. Males dominated the population (1·94:1) and backcalculated age showed that both sexes grew exponentially, with male growth rate increasing faster than female. Males were significantly larger than females in LT and mass (both P < 0·001). The relative mass index (Wr ) was low for the sampled population (mean ± s.d. = 32·00 ± 26·87 g), implying that the health of the Bass Island area population is very poor when compared with the species throughout its range. This could be due to a lack of food resources related to population size or that the fish is not optimally utilizing the available food resources. In contradiction to these findings, regression slope coefficient (b), calculated using Fulton's condition factor (K) (mean ± s.d. =1·50 ± 0·20), was very low for each habitat, implying a healthy population throughout. This seemingly opposite effect may be due to more individuals per unit area in shallow waters, which would cause increased competition for resources. Poor condition may indicate that the Lake Erie population has reached saturation or may reflect indirect fitness costs associated with increasing anoxic or hypoxic hypolimnion conditions.

A growth study of Coxiella burnetii Nine Mile Phase I and Phase II in fibroblasts.

Coxiella burnetii, a slow-growing, gram-negative, obligate intracellular bacterium, is the causative agent of Q fever in humans. The avirulent Phase II C. burnetii Nine Mile strain can invade and establish persistent infections in a wide variety of laboratory cell lines, and is generally considered to be easier to grow in culture than the wild-type Phase I organism. Efforts to improve Phase I organism yield in the BHK-21 cell line demonstrated that high CO2 conditions and the use of Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose supplementation resulted in higher organism yields. Phase II organisms grown in the same cell line and conditions showed lower growth rates. Analysis revealed that increased average numbers of C. burnetii Phase I organisms within fibroblasts was due to higher growth rates within the hosts rather than to increased uptake or to increased cell-to-cell spreading. Addition of the nucleoside cytidine to the growth medium stimulated growth of Phase II but not Phase I organisms.

Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (crazy), of the Coxiella burnetii nine mile strain.

After repeated passages through embyronated eggs, the Nine Mile strain of Coxiella burnetii exhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol. 132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G+C contents were found within the deletions.

Expression of beta-lactamase in Coxiella burnetii transformants.

Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding beta-lactamase. However, non-plasmid emergence of resistance to ampicillin also develops. To validate the usefulness of the bla gene marker for selection and detection, transformed C. burnetii were examined for beta-lactamase expression by use of immunoblotting after SDS-PAGE. The 29-kDa mature form of the beta-lactamase protein was detected in C. burnetii lysates. Quantitation of these immunoblot signals showed that C. burnetii surprisingly expressed low levels of beta-lactamase. The results validate the use of plasmid-encoded ampicillin resistance as a means for selecting C. burnetii transformants; they also suggest that levels of ampicillin used for selection pressure should be empirically determined and that detection of beta-lactamase by antibody blotting done to confirm transformants.

Genetics of Coxiella burnetii.

Those organisms considered to be obligate intracellular bacteria are interesting objects for genetic studies. Little is known about their mechanisms for natural genetic exchange. Many genes from the bacterium Coxiella burnetii, an obligate intraphagolysosomal pathogen, have therefore been cloned and characterized using the heterologous host Escherichia coli. Recently, use of electroporation methodology followed by long-term selection periods have provided initial data on genetic transformation in C. burnetii.

Transformation of Coxiella burnetii to ampicillin resistance.

A 5.8-kb chromosomal fragment isolated from Coxiella burnetii initiates plasmid replication in Escherichia coli and was characterized as an autonomous replication sequence, ars (M. Suhan, S.-Y. Chen, H.A. Thompson, T.A. Hoover, A. Hill, and J.C. Williams, J. Bacteriol. 176:5233-5243, 1994). In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(+)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding beta-lactamase. pSKO(+)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing beta-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (beta-lactamase) gene.

Tissue-specific expression of novel messenger ribonucleic acids cloned from a renin-expressing kidney tumor cell line (As4.1).

As4.1 cells are derived from a renin-expressing kidney tumor induced by tissue-specific oncogene-mediated tumorigenesis in transgenic mice. These cells express high levels of renin messenger RNA (mRNA) and synthesize prorenin and renin; they were therefore used as a model to further investigate the molecular biology of renin-producing kidney cells by cloning and characterizing novel mRNAs expressed in these cells. One clone, designated 1.5, was randomly selected from an As4.1 complementary DNA (cDNA) library, and two other cDNA clones, designated 4.9 and 6.9, were obtained by screening the cDNA library using a strategy to identify As4.1 cell-specific mRNAs. Each clone exhibited a highly restricted tissue-specific expression profile, including high level expression in As4.1 cells and low level expression in kidney. No homology was found between the sequence of the partial 1.5 and 4.9 cDNAs and sequences in Genbank. Southern blot analysis revealed that clone 4.9 is encoded by a single copy gene containing at least two separate exons. A homology search of the sequence of clone 6.9 revealed it to encode a cDNA to serum amyloid A protein; consistent with this identification, expression of 6.9 mRNA was highly induced in both kidney and liver after treatment of mice with Escherichia coli lipopolysaccharide.

Secretion of proteins by Coxiella burnetii.

Viable Coxiella burnetii organisms were isolated from the culture medium of persistently infected Baby Hamster Kidney (BHK-21) fibroblasts. When these organisms were incubated in host-cell-free medium at low pH, some of the de novo-synthesized protein made by the bacteria was translocated to the exterior of the cell. The exported protein was detectable after 2-7 h incubation at 37 degrees C. No evidence was found to suggest that protein accumulation in the medium was due to leakiness caused by cell damage. Both DCCD (dicyclohexylcarbodiimide) and CCCP (carbonyl cyanide m-chlorophenylhydrazone) inhibited the process to some extent. Exported protein was represented largely by three polypeptides with molecular masses of 34, 24 and 12 kDa. De novo-synthesized proteins corresponding to these molecular masses were not detected in cytoplasmic fractions, but a membrane fraction might possess a similar form. It was concluded that a physiological process of protein translocation occurred in C. burnetii during acid activation in a defined medium. Organisms that were extracted directly from the cytoplasm of infected fibroblasts by a mechanical disruption procedure were also active in de novo protein synthesis; however they exported much less of the protein.

Cloning and characterization of an autonomous replication sequence from Coxiella burnetii.

A Coxiella burnetii chromosomal fragment capable of functioning as an origin for the replication of a kanamycin resistance (Kanr) plasmid was isolated by use of origin search methods utilizing an Escherichia coli host. The 5.8-kb fragment was subcloned into phagemid vectors and was deleted progressively by an exonuclease III-S1 technique. Plasmids containing progressively shorter DNA fragments were then tested for their capability to support replication by transformation of an E. coli polA strain. A minimal autonomous replication sequence (ARS) was delimited to 403 bp. Sequencing of the entire 5.8-kb region revealed that the minimal ARS contained two consensus DnaA boxes, three A + T-rich 21-mers, a transcriptional promoter leading rightwards, and potential integration host factor and factor of inversion stimulation binding sites. Database comparisons of deduced amino acid sequences revealed that open reading frames located around the ARS were homologous to genes often, but not always, found near bacterial chromosomal origins; these included identities with rpmH and rnpA in E. coli and identities with the 9K protein and 60K membrane protein in E. coli and Pseudomonas species. These and direct hybridization data suggested that the ARS was chromosomal and not associated with the resident plasmid QpH1. Two-dimensional agarose gel electrophoresis did not reveal the presence of initiating intermediates, indicating that the ARS did not initiate chromosome replication during laboratory growth of C. burnetii.

Electron microscopy of Coxiella burnetii in tissue culture. Induction of cell types as products of developmental cycle.

An in vitro ultrastructural study was carried out on tissue cultures (J774, murine macrophage-like tumour cell line, and BHK-21, baby hamster kidney cell line) persistently infected with C. burnetii to investigate whether the events of cellular differentiation could be visualized. At a given stage of the developmental cycle, a proportion of the cells within the affected phagolysosomes clearly underwent cellular differentiation. The cells initially showed asymmetrical septation, the primary stage of cellular differentiation, and ended with the formation of the differentiated product, a precursor to the small cell. The results verified our initial observation that the events occurring during growth in a phagolysosome represent stages of a complex developmental cycle consisting not only of i) vegetative growth by typical transverse binary fission, but also ii) cellular differentiation.

Directors' and officers' liability.

Charities or business associations frequently ask physicians--due to their status in the community--to serve as directors or officers. Just as frequently, physicians agree to serve in those positions without fully understanding the responsibilities and possible liabilities they are assuming. This article describes the duties of directors and officers and the liabilities associated with the job.

Employment law primer.

All physicians regardless of their practice size will find themselves one day in the uncomfortable situation of dismissing an employee who may threaten legal action because of termination. Knowledge of and adherence to the basics of employment law as outlined in this article may not stop an angry dismissed employee from pursuing legal action. However, it should allow the physician to feel somewhat secure that such action likely will not be successful.

From informed consent to informed refusal.

This article traces the development of the informed consent concept, focusing on Texas case law and statutory provisions. We describe the proper use of the Texas Medical Disclosure Panel forms in establishing informed consent and illustrate common problems in proving that informed consent has been obtained. The need for effective physician-patient dialogue and communication is discussed.