Genotypic and phenotypic diversity of Lactobacillus rossiae isolated from beer.

AIMS: Over the past few years, the lactic acid bacteria (LAB) species Lactobacillus rossiae has appeared on occasion as a beer spoiler, in addition to its role as an inhabitant of sourdough and other foods. Many authors have described the L. rossiae sourdough isolates as phenotypically and genotypically extremely versatile. This characterization was confirmed in a comprehensive genotypic and phenotypic study based on 11 beer-related L. rossiae isolates. MATERIALS AND METHODS: The beer-related isolates and the L. rossiae type strain were classified in a polyphasic approach applying 16S rRNA, rpoA and pheS housekeeping gene sequence comparisons, DNA-DNA hybridization and rep-PCR technique. Additionally, carbohydrate fermentation and amino-acid metabolism were examined. In terms of the beer-spoilage ability, the growth in two different beer types was examined and the presence of three prominent hop resistance genes (horA, horC and hitA) and of one gene presumably responsible for the production of exopolysaccharides (gtf) was checked. CONCLUSION: The carbohydrate fermentation pattern (GTG)5 rep-PCR and the pheS gene sequence comparison showed deviations between sourdough and beer-related isolates. DNA-DNA hybridization values and the pheS gene sequence comparison between beer-related isolates point towards the need for expansion of the limits for species description. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus rossiae shows great phenotypic and genotypic variability stretching the limits of species description. The correlation between pheS gene sequence and the presence of the horC gene is important for brewing microbiologists and the search for beer-spoilage prediction methods.

Differentiation of probiotic and environmental Saccharomyces cerevisiae strains in animal feed.

AIMS: To establish an identification system for probiotic Saccharomyces cerevisiae strains based on artificial neural network (ANN)-assisted Fourier-transform infrared (FTIR) spectroscopy to improve quality control of animal feed. METHODS AND RESULTS: The ANN-based system for differentiating environmental from probiotic S. cerevisiae strains comprises five authorized feed additive strains plus environmental strains isolated from different habitats. A total of 108 isolates were used as reference strains to create the ANN. DHPLC analysis and δ-PCR were used as reference methods to type probiotic yeast isolates. The performance of the FTIR-ANN was tested in an internal validation using unknown spectra of each reference strain. This validation step yielded a classification rate of 99·1 %. For an external validation, a test data set comprising 965 spectra of 63 probiotic and environmental S. cerevisiae isolates unknown to the ANN was used, resulting in a classification rate of 98·2 %. CONCLUSIONS: Our results demonstrate that probiotic S. cerevisiae strains in feed can be differentiated successfully from environmental isolates using both genotypic approaches and ANN-based FTIR spectroscopy. SIGNIFICANCE AND IMPACT OF THE STUDY: FTIR-based artificial neural network analysis provides a rapid and inexpensive technique for yeast identification both at the species and at the strain level in routine diagnostic laboratories, using a single sample preparation.

High-resolution multitransistor array recording of electrical field potentials in cultured brain slices.

We report on the recording of electrical activity in cultured hippocampal slices by a multitransistor array (MTA) with 16,384 elements. Time-resolved imaging is achieved with a resolution of 7.8 microm on an area of 1 mm2 at 2 kHz. A read-out of fewer elements allows an enhanced time resolution. Individual transistor signals are caused by local evoked field potentials. They agree with micropipette measurements in amplitude and shape. The spatial continuity of the records provides time-resolved images of evoked field potentials and allows the detection of functional correlations over large distances. As examples, fast propagating waves of presynaptic action potentials are recorded as well as patterns of excitatory postsynaptic potentials across and along cornu ammonis.

Expression of eukaryotic translation initiation factors 4E and 2alpha is increased frequently in bronchioloalveolar but not in squamous cell carcinomas of the lung.

BACKGROUND: When resting cells are stimulated by growth factors, an increase in protein synthesis follows that depends in part on two key eukaryotic translation initiation factors, 4E and 2alpha (eIF-4E and eIF-2alpha, respectively). In the normal cell, expression and activity of both factors are increased transiently, whereas they become elevated constitutively in oncogene-transformed cultured cells, and overexpression of either initiation factor in rodent cells makes them tumorigenic. In this study, the authors investigated an association between the expression of these translation initiation factors and lung carcinogenesis. METHODS: The authors analyzed the expression of the protein synthesis initiation factors eIF-4E and eIF-2alpha by immunohistochemistry in bronchioloalveolar (BA) and squamous cell (SC) carcinomas of the lung. Western blot analysis was performed to validate the specificity of antibodies in detecting their cognate proteins. RESULTS: Both eIF-4E and eIF-2alpha were increased frequently in BA carcinomas, whereas only rarely did SC carcinomas demonstrate elevation of these translation initiation factors. An analysis of cyclin D1 expression did not show a strict correlation with the expression of eIF-4E and eIF-2alpha. CONCLUSIONS: Increased expression of either one or both translation initiation factors may facilitate accelerated growth and division of neoplastic cells in BA carcinoma of the lung. However, the current findings suggest a possibility that increased cell growth and proliferation in SC carcinoma may be achieved through a mechanism independent of increases in eIF-4E and eIF-2alpha expression.

Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS): the new thinking.

TTP and HUS are two disorders with many similarities. Though their first descriptions appeared at different time in history, there has been a trend among physicians to consider them as the same clinical entity. However, in recent years new research findings on the pathophysiology of TTP and HUS have revealed some differences between the two disorders. In this paper, we will review the current approaches to the clinical and laboratory diagnosis of TTP and HUS, as well as therapeutic strategies. We will also summarize the recent advances in three areas in the study of the pathophysiology of TTP and HUS, namely the newly discovered von Willebrand factor multimer-cleaving protease, endothelial cell apoptosis induced by serum from patients with TTP and atypical HUS and the activation of complement system. Since distinguishing and differentiating between TTP and HUS may help to develop more effective therapies targeted at key steps of the disease development, we will discuss possible ways of reclassifying the TTP-HUS disorders. In the end, we also present our views on possible future development.

Expression of eukaryotic translation initiation factors 4E and 2alpha correlates with the progression of thyroid carcinoma.

Cell growth and proliferation depend on protein synthesis that is regulated, in part, by two eukaryotic translation initiation factors, eIF-4E and eIF-2alpha. These factors are transiently increased as normal cells respond to growth factors and are constitutively elevated in transformed cells. In cultured cells, eIF-4E facilitates cell cycle progression by increasing the expression of cell cycle promoting proteins including cyclin D1. Our previous study revealed elevated cyclin D1 expression in histologically more aggressive thyroid carcinomas as compared to conventional papillary carcinoma. We hypothesized that the increased cyclin D1 expression might correlate with increased eIF-4E expression. We, therefore studied the expression of eIF-4E by immunohistochemistry in 25 cases of conventional papillary carcinoma (CPC) and 28 cases of aggressive thyroid carcinomas (ATC), the latter included 11 tall cell/columnar cell variant of papillary carcinoma, 5 insular carcinomas, and 12 anaplastic carcinomas. We also analyzed the expression of eIF-2a in the same samples as this factor is usually regulated similarly to eIF-4E in cell culture models. Of the 25 CPC, 13 were eIF-4E positive (11 weakly and 2 strongly), and 19 were eIF-2a positive (14 weakly and 5 strongly). Conversely, of the 28 ATC, 25 were eIF-4E positive (4 weakly and 21 strongly), and 23 were eIF-2alpha positive (4 weakly and 19 strongly). There was a significantly increased expression of both eIF-4E (p < 0.001) and eIF-2alpha (p < 0.001) in ATC compared to CPC, suggesting that these translation initiation factors may play a role in the progression of thyroid cancer.

The role of cell cycle regulatory protein, cyclin D1, in the progression of thyroid cancer.

Cell cycle progression is facilitated by cyclin-dependent kinases that are activated by cyclins including cyclin D1 and inactivated by cyclin-dependent kinase inhibitors (CDKIs) such as p27. Our previous studies have demonstrated decreased p27 expression in both papillary and more aggressive carcinomas of the thyroid compared to thyroid adenoma and almost similar level of cyclin D1 expression between thyroid adenoma and papillary carcinoma. These results indicate that CDKIs may have an important role in the carcinogenesis of the thyroid and that they probably have a limited role in malignant progression of the thyroid cancer. The role of cyclin D1 in malignant progression of thyroid carcinoma has yet to be established. We studied the expression of cyclin D1 by immunohistochemistry in 34 cases of conventional papillary carcinoma (CPC), 10 cases of minimally invasive follicular carcinoma (MIFC), and 32 cases of more aggressive thyroid carcinoma (ATC), which included 11 tall cell variants, one columnar cell variant of papillary carcinoma, seven insular carcinomas, and 13 anaplastic carcinomas. Cyclin D1 staining was classified by staining score as 0, negative; 1+, less than 25%; 2+, 25 to 50%; and 3+, more than 50% tumor cells staining positive. Kruskal-Wallis one-way ANOVA and Wilcoxon Rank Sum/Mann-Whitney U Test was used to assess the difference in the expression of cyclin D1 between the study groups. Twenty-eight out of the 34 CPCs were cyclin D1 positive, 24 (70%) were 1+, 3 (9%) were 2+, and one (3%) were 3+ positive. Seven of 10 MIFCs were cyclin D1 positive, five (71%) were 1+, and the remaining two (29%) were 2+ positive. On the other hand, 28 of 32 ATCs showed cyclin D1 immunostaining. Of these, three (9%) were 1+, five (13%) were 2+, and 20 (63%) were 3+ positive. This study demonstrates a significant overexpression of cyclin D1 in ATC compared CPC (P < .001) and MIFC (P < .005), suggesting that the cyclin D1 expression may play a role in tumor progression and may have prognostic significance in thyroid cancer.

Superior vena cava syndrome secondary to an angiotropic large cell lymphoma.

BACKGROUND: Angiotropic large cell lymphoma (ALCL) is characterized by the intravascular proliferation of malignant lymphoid cells in small and medium-sized blood vessels. In the current study, the authors report an unusual case in which the initial presentation of the ALCL was that of superior vena cava (SVC) syndrome. METHODS: The case is presented, followed by a general review of the literature regarding ALCL. RESULTS: Surgical intervention was required for diagnosis in this case. Successful treatment with chemotherapy followed by involved field radiation ensued with a maintained disease remission at 48 months of follow-up. CONCLUSIONS: Although usually presenting in small blood vessels, ALCL can present initially with large blood vessel involvement and should be considered in the differential diagnosis of this condition, even in the absence of extravascular lymph node involvement. Aggressive treatment with antineoplastic therapy is warranted and may result in long term recurrence free survival.

Expression of the eukaryotic translation initiation factors 4E and 2alpha in non-Hodgkin's lymphomas.

Transition of cells from quiescence to proliferation requires an increase in the rate of protein synthesis, which is regulated in part by two key translation initiation factors, 4E and 2alpha. The expression and activity of both factors are increased transiently when normal resting cells are stimulated to proliferate. They are constitutively elevated in oncogene transformed cultured cells, and overexpression of either initiation factor in rodent cells makes them tumorigenic. In this study we investigate an association between the expression of translation initiation factors and lymphomagenesis. We have analyzed the expression of the protein synthesis initiation factors 4E and 2alpha by immunohistochemistry in reactive lymph nodes and several types of non-Hodgkin's lymphoma representing a wide range of clinical behaviors based on the Revised European-American Lymphoma behavioral classification. The study included 7 benign lymph nodes with follicular hyperplasia, 26 indolent lymphomas (6 marginal zone lymphomas, 7 small lymphocytic lymphomas, and 13 follicular lymphomas, grades 1 and 2), 16 moderately aggressive lymphomas (8 mantle cell lymphomas and 8 follicular lymphomas, grade 3), 24 aggressive lymphomas (14 large-B-cell lymphomas and 10 anaplastic large-cell lymphomas), and 15 highly aggressive lymphomas (7 lymphoblastic lymphomas and 8 Burkitt's lymphomas). Strong expression of initiation factors 4E and 2alpha was demonstrated in the germinal centers of reactive follicles. Minimal or no expression was seen in the mantle zones and surrounding paracortices, indicating that high expression of initiation factors 4E and 2alpha is associated with the active proliferation of lymphocytes. Most cases of aggressive and highly aggressive lymphomas showed strong expression of initiation factors 4E and 2alpha, in contrast to the cases of indolent and moderately aggressive lymphoma, in which their expression was intermediate between the germinal centers and the mantles of reactive follicles. A positive correlation was found between the expression of both initiation factors 4E and 2alpha and the Revised European-American Lymphoma behavior classification (P < 0.05). Thus, constitutively increased expression of initiation factors 4E and 2alpha may play an important role in the development of lymphomas and is correlated with their biological aggressiveness.

Electrical resonance of isolated hair cells does not account for acoustic tuning in the free-standing region of the alligator lizard's cochlea.

The cochlea of the alligator lizard is divided into two morphologically and physiologically distinct regions. In the "tectorial region," hair bundles of hair cells are draped by a tectorial membrane, whereas in the "free-standing region," hair bundles are said to be free-standing because there are no overlying tectorial structures. The acoustic tuning of the free-standing region depends at least in part on mechanical resonances of the hair bundles. In the turtle cochlea, in contrast, acoustic tuning depends in large part upon the electrical properties of the hair cells. We have investigated the electrical properties of hair cells isolated from the free-standing region of the alligator lizard's cochlea. When injected with steps of depolarizing current, these "free-standing hair cells" exhibited electrical resonances that were comparable in frequency and quality to electrical resonances in cochlear hair cells from turtles, chicks, and alligators, and in saccular hair cells from frogs and fish. In the lizard's free-standing hair cells, however, the electrical resonance frequencies (< 300 Hz) were a decade below the cells' acoustic characteristic frequencies (between 1 and 4 kHz), showing that the electrical resonance is not likely to contribute to acoustic tuning. The electrical resonances were not apparent at rest. The cells' resting potentials were significantly more negative than the activation voltage (approximately -40 mV) of the Ca(2+)-dependent K+ current upon which the electrical resonance has been shown to depend in other hair cells. At potentials more negative than -50 mV, an inwardly rectifying K+ conductance dominated. Because we observed no electrical tuning above 300 Hz, our results indirectly support a mechanical origin for acoustic tuning in the free-standing region of the alligator lizard cochlea. These results further show that acoustic tuning cannot be inferred solely from the electrical resonances of isolated hair cells.

Marijuana and spontaneous abortion of known karyotype.

We tested whether marijuana use in the 2 months before the last menstrual period and during pregnancy affects the risk of spontaneous abortions of known karyotype. Spontaneous abortions (cases) were defined as chromosomally normal (n = 567) or chromosomally aberrant (n = 393) and, within the latter, by type of aberration (212 trisomies, 71 monosomies X, 49 triploidies, 61 others). Controls were women with prenatal care before 22 weeks gestation and delivering at 28 weeks or later (n = 2042). In comparison with controls, adjusted odds (OR) of reported marijuana use in chromosomally normal cases were 1.1 (95% confidence interval (CI) 0.7, 1.5) and in chromosomally aberrant cases combined 1.2 (95% CI 0.7, 1.9). With respect to specific aberrations, use in the perifertilisation period did not differ significantly from that in controls for trisomies (adjusted OR = 0.8, 95% CI 0.4, 1.8), monosomies X (adjusted OR = 1.8, 95% CI 0.7, 4.3), and triploidies (adjusted OR = 1.3, 95% CI 0.4, 4.5). Comparison of karyotype groups with each other yielded similar results. Our data do not support causal associations of marijuana use, at the levels represented in our sample, with either chromosomally normal or trisomic spontaneous abortion. With monosomy X and triploidy, no statistically significant associations were detected although numbers were insufficient to rule out moderate effects.

Paternal age and trisomy among spontaneous abortions.

The relationship of paternal age to specific types of trisomy and to chromosomally normal loss was investigated in data drawn from a case-control study of spontaneous abortions. Differences in paternal age between karyotype groups and controls delivering after 28 weeks gestation were tested using an urn model analysis which adjusted, by regression, for maternal age and, by stratification, for the effects of design variables (payment status, phase of study) and demographic factors (language, ethnicity). The magnitude of paternal age differences was estimated using least squares regression analysis. For chromosomally normal cases there was no association with paternal age. Among the fourteen trisomy categories examined, four (7, 9, 18, 21) showed increased paternal age (greater than or equal to 1 year above expectation), three (13, 20, 22) showed decreased paternal age and the rest, including the most common, trisomy 16, showed negligible differences. Only the association with trisomy 22 was statistically significant (P = 0.012), with a predicted reduction in paternal age of 2.1 years (95% CI -4.9, -0.5 years). This association did not vary with maternal age, payment status, phase of study, language or ethnicity. Because previous observations are extensive, the relation of paternal age to trisomy 21 was examined further. The overall association was not significant (beta = 0.8 years; 95% CI -0.8, 2.4 years). Moreover, there was evidence that the magnitude and direction of paternal age associations vary significantly within the sample, although not between subgroups defined on the basis of payment, phase of study, language or ethnicity. With respect to maternal age, the trend is towards a greater paternal age difference for trisomy 21 losses in younger women (P = 0.058). Given the number of tests performed, the finding for trisomy 22 and reduced paternal age could be due to chance. Among trisomy types, the direction of paternal age associations was not consistent for chromosomes grouped according to characteristics that might relate to the probability of nondisjunction, such as size, arm ratio, or nucleolar organizer region content, or to the potential viability of the trisomy. Thus, neither on statistical nor biological grounds do the data provide compelling evidence of paternal age effects on the trisomies found among spontaneous abortions, or on chromosomally normal losses.

Does the karyotype of a spontaneous abortion predict the karyotype of a subsequent abortion? Evidence from 273 women with two karyotyped spontaneous abortions.

At least two spontaneous abortions were karyotyped in 273 women during cytogenetic surveys in New York City and Honolulu. These pairs were analyzed using maximum-likelihood logistic-regression analysis to adjust for maternal age and location. There was a significantly increased risk for a chromosomally normal spontaneous abortion after a previous abortion with a normal karyotype. There was no increased risk for trisomy in a second spontaneous abortion following either a previous trisomic abortion or an abortion with another abnormal karyotype. This is unexpected, given the increased risk for trisomy found among live births and at prenatal diagnosis in young women with a previous trisomic birth. The most likely explanation is that the increased recurrence risk for trisomy is restricted to trisomy for only one or a few chromosomes, for reasons such as parental trisomy mosaicism. These data predict no increased risk of chromosome abnormality in future pregnancies after either (1) spontaneous abortions with trisomies of a kind that are always lethal in utero or (2) multiple early abortions in the presence of normal parental karyotypes.

Cigarettes, alcohol and marijuana: varying associations with birthweight.

The relations of cigarette smoking, alcohol drinking and marijuana use during pregnancy to birthweight were examined in two prospectively studied pregnancy cohorts (Phases I and II). After analytic adjustment in ordinary least squares regressions for other factors that influence birthweight, cigarette smoking during at least half the pregnancy was associated with a significant decrease in mean birthweight (159 grams in Phase I, 202 grams in Phase II). In Phase II only, beer drinking was associated with a significant decrement of 8.4 grams in estimated birthweight per ounce of absolute alcohol per month. Neither wine nor liquor drinking in the Phase II data nor any of the three beverages in the Phase I data was associated with significant decrements in predicted birthweight. Furthermore, with one exception (drinking once a week in Phase II only), alcohol drinking, defined as the number of occasions per month on which any alcoholic beverage was consumed, was not associated with a change in birthweight. Regarding marijuana use, the data are not consistent between the two phases. In the Phase I data, no coherent trend in association with birthweight was observed. In the Phase II data, marijuana use 2-3 times per week, 4-6 times per week and daily was associated with increasing decrements in estimated birthweight: 127 g, 143 g and 230 g respectively. The inconsistent findings for alcohol drinking and marijuana use between phases stand in marked contrast to the consistent findings for cigarette smoking.

Environmental modulation of lipopolysaccharide chain length alters the sensitivity of Escherichia coli to the neutrophil bactericidal/permeability-increasing protein.

We have shown previously that the sensitivity of Escherichia coli to the neutrophil bactericidal/permeability-increasing protein (BPI) depends mainly on the polysaccharide chain length of outer membrane lipopolysaccharides (LPS) (J. Weiss, S. Beckerdite-Quagliata, and P. Elsbach, J. Clin. Invest. 65:619-628, 1980). Thus, rough strains of E. coli producing only short-chain LPS are more sensitive to BPI than smooth strains that produce LPS with varied chain lengths. We now show that changes in the bacterial growth environment can modify BPI sensitivity of smooth E. coli as much as 30-fold depending on the bacterial strain and the growth conditions examined. Changes in BPI sensitivity paralleled differences in binding affinity of E. coli for BPI and closely correlated with changes in the chain length of LPS produced under different growth conditions, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No concomitent changes in either the number of LPS molecules per cell or the bacterial protein profile were detected. Rough strains showed little or no growth-dependent variation in BPI sensitivity, further indicating that subtle alterations in bacterial constituents other than LPS do not significantly affect bacterial sensitivity to BPI. Thus, the BPI sensitivity of E. coli can be modulated not only by the genotypic conversion of the LPS phenotype, but also by environmental effects on LPS-polysaccharide formation in wild-type strains.

Maternal employment and the chromosomal characteristics of spontaneously aborted conceptions.

The employment histories of 1,252 women experiencing a spontaneous abortion and 2,126 controls were compared to examine the relation between maternal employment before and during pregnancy and karyotype of the spontaneously aborted conceptus. Among private patients, there was no evidence of a positive association of work only before pregnancy, only during pregnancy, or both before and during pregnancy (v no work) with either chromosomally normal or abnormal abortions. In contrast, among public patients the odds of working only during pregnancy or both before and during pregnancy were raised for chromosomally abnormal abortions (adjusted odds ratios = 3.11 and 1.86, respectively) and slightly raised for chromosomally normal abortions (adjusted odds ratios = 1.35 and 1.31, respectively). Among workers, the frequencies of work before pregnancy in specific employment circumstances were compared among karyotyped cases and controls. For two work locations--factory and hospital/nursing facility--the data were of sufficient size to suggest that associations of twofold or greater with most types of abortion are unlikely.