The effect of inoculum on the performance of sulfate-reducing columns treating heavy metal contaminated water.

Sulfate-reducing permeable reactive zones (SR-PRZs) are a passive means of immobilizing metals and neutralizing the pH of mine drainage through microbially mediated reactions. In this bench-scale study, the influence of inoculum on the performance of columns simulating SR-PRZs was investigated using chemical and biomolecular analyses. Columns inoculated from two sources (bovine dairy manure (DM) and a previous sulfate-reducing column (SRC)) and uninoculated columns (U) were fed a simulated mine drainage and compared on the basis of pH neutralization and removal of cadmium, zinc, iron, and sulfate. Cadmium, zinc, and sulfate removal was significantly higher in SRC columns than in the DM and U columns, while there was no significant difference between the DM and U columns. Denaturing gradient gel electrophoresis (DGGE) analysis revealed differences in the microbial community composition among columns with different inocula, and indicated that the microbial community in the SRC columns was the first to reach a pseudo-steady state. In the SRC columns, a higher proportion of the DGGE band DNA sequences were related to microorganisms that carry out cellulose degradation, the rate-limiting step in SR-PRZ energy flow, than was the case in the other columns. The proportion of sulfate-reducing bacteria of the genus Desulfobacterium was monitored using real-time quantitative PCR and was observed to be consistently higher in the SRC columns. The results of this study suggest that the inoculum plays an important role in SR-PRZ performance. This is the first report providing a detailed analysis of the effect of different microbial inocula on the remediation of acid mine drainage.

Combining organic acid treatment with steam pasteurization to eliminate Listeria monocytogenes on fully cooked frankfurters.

An organic acid solution of 2% acetic, 1% lactic, 0.1% propionic, and 0.1% benzoic acids was combined with steam surface pasteurization to treat frankfurters during vacuum packaging to eliminate potential postcook contamination with Listeria monocytogenes. The thermal lethality of L. monocytogenes from steam was evaluated at an inoculation concentration of 1 to 6 log CFU/cm2. About 3-log reductions of L. monocytogenes were achieved when frankfurters were treated by steam for 1.5 s. Combining organic acid treatment with steam pasteurization further inhibited the growth of surviving L. monocytogenes cells for 19 and 14 weeks when the packaged frankfurters were stored at 4 and 7 degrees C, respectively. The results from this study provide meat processors with useful information for controlling L. monocytogenes on ready-to-eat meats.

Eradicating Listeria monocytogenes from fully cooked franks by using an integrated pasteurization-packaging system.

Surface pasteurization by applying steam or hot water before or after packaging of processed foods may be used to eliminate pathogens such as Listeria monocytogenes from ready-to-eat meat and poultry products. Surface pasteurization treatment with a mixture of pressurized steam and hot water was integrated into a continuous vacuum-packaging system to reduce L. monocytogenes from fully cooked franks. The franks (2.54 cm diameter by 15.24 cm length) were surface inoculated to contain up to 6 log CFU/cm2 L. monocytogenes. The inoculated franks were treated at 121 degrees C for 1.5 s in an arrangement of six franks per packaging chamber followed by immediate vacuum sealing of the top films of food packages in the same unit. A 3-log CFU/cm2 reduction of L. monocytogenes on fully cooked franks was obtained using the integrated pasteurization-packaging system. The pasteurization depth was 1.27 mm below the surfaces of the franks. This process provides a commercially applicable means of ensuring food safety by effectively eradicating L. monocytogenes from ready-to-eat meat and poultry products at the very last possible step of food packaging before reaching retail consumers.

Ty1-copia-retrotransposon behavior in a polyploid cotton.

Retrotransposons constitute a ubiquitous and dynamic component of plant genomes. Intragenomic and intergenomic comparisons of related genomes offer potential insights into retrotransposon behavior and genomic effects. Here, we have used fluorescent in-situ hybridization to determine the chromosomal distributions of a Ty1-copia-like retrotransposon in the cotton AD-genome tetraploid Gossypium hirsutum and closely related putative A- and D-genome diploid ancestors. Retrotransposon clone A108 hybridized to all G. hirsutum chromosomes, approximately equal in intensity in the A- and D-subgenomes. Similar results were obtained by hybridization of A108 to the A-genome diploid G. arboreum, whereas no signal was detected on chromosomes of the D-genome diploid G. raimondii. The significance and potential causes of these observations are discussed.

An E-mail assessment of undergraduates' attitudes toward smoking.

Responses from 513 of 1,000 randomly selected undergraduate students who were sent an e-mail questionnaire, about cigarette smoking were analyzed. Thirteen percent of the respondents identified themselves as smokers. No statistically significant differences were observed between smokers and nonsmokers and year in college, sex, age, race, or having attended public or private high schools. Ninety-eight percent of the respondents considered themselves knowledgeable about adverse health consequences of smoking, yet 39.1% of current smokers seriously considered stopping smoking, and 11.5% of current nonsmokers intended to start smoking. The preferred quitting method of smokers and ex-smokers was stopping all at once ("cold turkey"). Fifty-two percent of the smokers did not want professional assistance to stop smoking; 40% of the nonsmokers wanted information on second-hand smoke.

Dispersed repetitive DNA has spread to new genomes since polyploid formation in cotton.

Polyploid formation has played a major role in the evolution of many plant and animal genomes; however, surprisingly little is known regarding the subsequent evolution of DNA sequences that become newly united in a common nucleus. Of particular interest is the repetitive DNA fraction, which accounts for most nuclear DNA in higher plants and animals and which can be remarkably different, even in closely related taxa. In one recently formed polyploid, cotton (Gossypium barbadense L.; AD genome), 83 non-cross-hybridizing DNA clones contain dispersed repeats that are estimated to comprise about 24% of the nuclear DNA. Among these, 64 (77%) are largely restricted to diploid taxa containing the larger A genome and collectively account for about half of the difference in DNA content between Old World (A) and New World (D) diploid ancestors of cultivated AD tetraploid cotton. In tetraploid cotton, FISH analysis showed that some A-genome dispersed repeats appear to have spread to D-genome chromosomes. Such spread may also account for the finding that one, and only one, D-genome diploid cotton, Gossypium gossypioides, contains moderate levels of (otherwise) A-genome-specific repeats in addition to normal levels of D-genome repeats. The discovery of A-genome repeats in G. gossypioides adds genome-wide support to a suggestion previously based on evidence from only a single genetic locus that this species may be either the closest living descendant of the New World cotton ancestor, or an adulterated relic of polyploid formation. Spread of dispersed repeats in the early stages of polyploid formation may provide a tag to identify diploid progenitors of a polyploid. Although most repetitive clones do not correspond to known DNA sequences, 4 correspond to known transposons, most contain internal subrepeats, and at least 12 (including 2 of the possible transposons) hybridize to mRNAs expressed at readily discernible levels in cotton seedlings, implicating transposition as one possible mechanism of spread. Integration of molecular, phylogenetic, and cytogenetic analysis of dispersed repetitive DNA may shed new light on evolution of other polyploid genomes, as well as providing valuable landmarks for many aspects of genome analysis.

A rapid procedure for the isolation of C0t-1 DNA from plants.

In situ hybridization (ISH) for the detection of single- or low-copy sequences, particularly large DNA fragments cloned into YAC or BAC vectors, generally requires the suppression or "blocking" of highly-repetitive DNAs. C0t-1 DNA is enriched for repetitive DNA elements, high or moderate in copy number, and can therefore be used more effectively than total genomic DNA to prehybridize and competitively hybridize repetitive elements that would otherwise cause nonspecific hybridization. C0t-1 DNAs from several mammalian species are commercially available, however, none is currently available for plants to the best of our knowledge. We have developed a simple 1-day procedure to generate C0t-1 DNA without the use of specialized equipment.

Use of meiotic FISH for identification of a new monosome in Gossypium hirsutum L.

The extensive use of molecular cytogenetics in human genetics and clinical diagnostics indicates that analogous applications in plants are highly feasible. One sort of application would be the identification of new aneuploids, which traditionally involves either direct karyotypic identification, which is feasible in only a few plant species, or tests with markers (cytogenetic, genetic, or molecular), which require sexual hybridization and at least one subsequent seed or plant generation. We have used meiotic fluorescence in situ hybridization (FISH) to analyze a new monosome of cotton (Gossypium hirsutum L., 2n = 4x = 52, 2(AD)1) that had a phenotype which seemed to be distinct from monosomes in the Cotton Cytogenetic Collection. Painting with A2-genome DNA revealed the monosome's D-subgenome origin. DAPI-PI staining showed that the monosome carries a major NOR, delimiting it to the major NOR-bearing chromosomes of the D-subgenome, i.e., 16 or 23. Dual-color FISH with 5S and 18S-28S rDNAs indicated that the monosome contains separate major clusters of each of these two tandemly repeated rDNA elements, thus delimiting the monosome to chromosome 23, for which the Cotton Cytogenetic Collection has previously been devoid of any sort of deficiency. Of the 26 chromosomes in the cotton genome, the Collection now provides coverage for 16 (70%) in the form of monosomy, and 20 (77%) in the form of monosomy and (or) telosomy. Use of molecular cytogenetic methods to identify a new plant aneuploid in cotton exemplifies the fact that a physicochemical karyotypic chromosome identification system is not required a priori for application of new molecular cytogenetic methods, thus indicating their potential applicability to nearly all plant species.

Distribution of 5S and 18S-28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors.

The most widely cultivated species of cotton, Gossypium hirsutum, is a disomic tetraploid (2n=4x=52). It has been proposed previously that extant A- and D-genome species are most closely related to the diploid progenitors of the tetraploid. We used fluorescent in situ hybridization (FISH) to determine the distribution of 5S and 18S-28S rDNA loci in the A-genome species G. herbaceum and G. arboreum, the D-genome species G. raimondii and G. thurberi, and the AD tetraploid G. hirsutum. High signal-to-noise, single-label FISH was used to enumerate rDNA loci, and simultaneous, dual-label FISH was used to determine the syntenic relationships of 5S rDNA loci relative to 18S-28S rDNA loci. These techniques provided greater sensitivity than our previous methods and permitted detection of six new G. hirsutum 18S-28S rDNA loci, bringing the total number of observed loci to 11. Differences in the intensity of the hybridization signal at these loci allowed us to designate them as major, intermediate, or minor 18S-28S loci. Using genomic painting with labeled A-genome DNA, five 18S-28S loci were localized to the G. hirsutum A-subgenome and six to the D-subgenome. Four of the 11 18S-28S rDNA loci in G. hirsutum could not be accounted for in its presumed diploid progenitors, as both A-genome species had three loci and both D-genome species had four. G. hirsutum has two 5S rDNA loci, both of which are syntenic to major 18S-28S rDNA loci. All four of the diploid genomes we examined contained a single 5S locus. In g. herbaceum (A1) and G. thurberi (D1), the 5S locus is syntenic to a major 18S-28S locus, but in G. arboreum (A2) and G. raimondii (D5), the proposed D-genome progenitor of G. hirsutum, the 5S loci are syntenic to minor and intermediate 18S-28S loci, respectively. The multiplicity, variation in size and site number, and lack of additivity between the tetraploid species and its putative diploid ancestors indicate that the behavior of rDNA loci in cotton is nondogmatic, and considerably more complex and dynamic than previously envisioned. The relative variability of 18S-28S rDNA loci versus 5S rDNA loci suggests that the behavior of tandem repeats can differ widely.

Fluorescent in situ hybridization of a bacterial artificial chromosome.

Fluorescent in situ hybridization (FISH) of a 130 kilobase cotton (Gossypium hirsuitum L.) bacterial artificial chromosome (BAC) clone containing a high proportion of single-copy DNA produced a large pair of FISH signals on the distal end of the long arm of a pair of chromosomes of the D-genome species G. raimondii Ulbr. and produced a fainter pair of signals on a small submetacentric pair of chromosomes of the A-genome species G. herbaceum L. The signals were synthetic with a nucleolar organizer region in G. raimondii and G. herbaceum. Signal pairs were easily recognized in interphase and metaphase cells either with or without suppression of repetitive sequences with unlabeled G. hirsutum C0t-1 DNA. High quality FISH results were consistently obtained and image analysis was not required for viewing or photography. Results indicate that FISH of BAC clones is an excellent tool for the establishment of new molecular cytogenetic markers in plants and will likely prove instrumental in the development of useful physical maps for many economically important crop species.

Computed tomography of retroperitoneal perforation after barium enema.

A case of retroperitoneal perforation of the rectum complicating a single contrast barium enema is presented. The appearances on computed tomography (CT) allowed accurate depiction of the extent of barium extravasation not appreciated on plain films. Although this is an unusual complication of the barium enema, CT may prove useful when the suspicion of perforation arises.

Distribution of lung vascular resistance after chronic systemic-to-pulmonary shunts.

Congenital cardiac shunts produce pathological lesions on the arterial side of the lung vasculature. We examined the effects of chronic shunts (14.2 +/- 1.2 mo) in 10 young dogs, between the left subclavian and the left lower lobe (LLL) artery, on pulmonary vascular pressure and flow (P-Q) relationships, segmental resistance with arterial and venous occlusion (AVO), and sensitivity to drugs. At final thoracotomy, mean LLL pulmonary arterial pressure (Ppa) was 23.2 +/- 4.3 mmHg compared with 11.9 +/- 0.9 in the right lung (P less than 0.05); two animals had LLL Ppa of 41 and 48 mmHg. The LLL artery and vein were cannulated, and pressure-flow (P-Q) and AVO measurements were made and compared with previous control LLL (n = 11) and contralateral right lower lobe (RLL, n = 5). Responses to serotonin, histamine, and vasodilators (diltiazem and isoproterenol) were evaluated. Comparisons of morphometric measurements were made between LLL and RLL. We found a significant increase in arterial resistance as measured with AVO and a hypersensitivity to serotonin in the shunt LLL, without changes in total pulmonary vascular resistance or P-Q measurements; vasodilators had a small effect only in the hypertensive lobes. Our data suggest that chronic shunts to the pulmonary circulation increase arterial resistance and sensitivity to serotonin, even in the absence of discernible morphometric changes, and that vasoconstriction may be an important precursor to the development of morphological lesions.

Involvement of upper-airway muscles in extrapyramidal disorders. A cause of airflow limitation.

To identify the site and cause of airflow limitation in patients with parkinsonism, we tested pulmonary function in 27 patients with extrapyramidal disorders. In 24 patients, an abnormal flow-volume loop contour, showing either regular (18 patients) or irregular (6 patients) flow oscillations, was found. On direct fiberoptic visualization of the upper airway, these oscillations corresponded to either rhythmic (4 to 8 Hz) or irregular involuntary movements of glottic and supraglottic structures. Ten patients had physiologic evidence of upper-airway obstruction, which was symptomatic in four. We conclude that the upper-airway musculature is frequently involved in extrapyramidal disorders. This causes upper-airway dysfunction that can be severe enough to limit airflow.

Radiology of the diaphragm as two muscles.

In order to investigate the postulate that the diaphragm consists of two muscles, cineradiographic studies and plain roentgenograms were taken of the diaphragmatic movement in eight dogs. A characteristic pattern of contraction was seen following direct electric stimulation of each of the sections of the diaphragm, namely the costal muscle, mainly anterior to the central tendon, and the smaller, posterior crural muscle. Stimulation of the roots of the phrenic nerves in the neck showed that, with C5 root stimulation, contraction of the costal area occurred, but that with C6 and C7 root stimulation, contraction of the crural area occurred, although the movement was larger with C6 than with C7 root stimulation. Downward displacement of the entire diaphragm followed stimulation of the intrathoracic phrenic nerves. These results support the concept that the canine diaphragm actually consists of two muscles with a different segmental innervation of each part.

Metastatic carcinoma. The lung as the site for the clinically undiagnosed primary.

Of 387 patients who died with lung cancer, 28 cases were reviewed (7.2%) which were clinically undiagnosed. The male:female ratio was 3.6 and mean age was 64 years in the males, 47 years in the females. The most frequent presenting symptoms were neurologic. Prior to death, 21 patients had known or suspected metastatic disease (biopsy-proven in 12), while a malignant diagnosis was not considered in seven patients. Mean survival was 3.5 months. Despite a mean tumor size of 2.8 cm, most of the chest x-rays were not diagnostic even in retrospect. At autopsy, 65% of the tumors were adenocarcinomas (compared to 32% in the other 359 patients); 53% of these showed vascular and lymphatic invasion around the primary tumor, explaining their wide dissemination. In patients with small cell carcinomas (25% of the cases reviewed) or with solitary metastases (14% of the cases reviewed) therapeutic intervention could possibly have been beneficial.

Primate retroviruses: envelope glycoproteins of endogenous type C and type D viruses possess common interspecies antigenic determinants.

The major 70,000- to 80,000-molecular-weight envelope glycoproteins of the squirrel monkey retrovirus, Mason-Pfizer monkey virus, and M7 baboon virus and the related endogenous feline virus, RD114, were isolated and immunologically characterized. Immunoprecipitation and competition immunoassay analysis revealed these viral envelope glycoproteins to possess several distinct classes of immunological determinants. These include species-specific determinants, group-specific antigenic determinants unique to endogenous primate type C viruses, and group-specific determinants for type D viruses such as Mason-Pfizer monkey virus and squirrel monkey retrovirus. In addition, a class of broadly reactive antigenic determinants shared by envelope glycoproteins of both type C viruses of the baboon/RD114 group and type D viruses of the Mason-Pfizer monkey virus/squirrel monkey virus group are described. Other mammalian oncornaviruses tested, including isolates of nonprimate origin and representative type B viruses, lacked these determinants. The demonstration of antigenic determinants specific to envelope glycoproteins of type C and type D primate viruses indicates either that these viruses are evolutionarily related or that genetic recombination occurred between their progenitors. Alternatively, endogenous type D oncornaviruses may be replication defective, and acquisition of endogenous type C viral genetic sequences coding for envelope glycoprotein determinants may be necessary for their isolation as infectious virus.