Photonic wire biosensor microarray chip and instrumentation with application to serotyping of Escherichia coli isolates.

A complete photonic wire molecular biosensor microarray chip architecture and supporting instrumentation is described. Chip layouts with 16 and 128 independent sensors have been fabricated and tested, where each sensor can provide an independent molecular binding curve. Each sensor is 50 µm in diameter, and consists of a millimeter long silicon photonic wire waveguide folded into a spiral ring resonator. An array of 128 sensors occupies a 2 × 2 mm2 area on a 6 × 9 mm2 chip. Microfluidic sample delivery channels are fabricated monolithically on the chip. The size and layout of the sensor array is fully compatible with commercial spotting tools designed to independently functionalize fluorescence based biochips. The sensor chips are interrogated using an instrument that delivers sample fluid to the chip and is capable of acquiring up to 128 optical sensor outputs simultaneously and in real time. Coupling light from the sensor chip is accomplished through arrays of sub-wavelength surface grating couplers, and the signals are collected by a fixed two-dimensional detector array. The chip and instrument are designed so that connection of the fluid delivery system and optical alignment are automated, and can be completed in a few seconds with no active user input. This microarray system is used to demonstrate a multiplexed assay for serotyping E. coli bacteria using serospecific polyclonal antibody probe molecules.

Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88-893: structural and serological similarities with S. flexneri provisional serotype Y394 (1c).

The structure of the repeating unit of the O-antigen polysaccharide from Shigella flexneri provisional serotype 88-893 has been determined. (1)H and (13)C NMR spectroscopy as well as 2D NMR experiments were employed to elucidate the structure. The carbohydrate part of the hexasaccharide repeating unit is identical to the previously elucidated structure of the O-polysaccharide from S. flexneri prov. serotype Y394. The O-antigen of S. flexneri prov. serotype 88-893 carries 0.7 mol O-acetyl group per repeating unit located at O-2 of the 3-substituted rhamnosyl residue, as identified by H2BC and BS-CT-HMBC NMR experiments. The O-antigen polysaccharide is composed of hexasaccharide repeating units with the following structure: →2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap2Ac-(1→3)[α-D-Glcp-(1→2)-α-D-Glcp-(1→4)]-ß-D-GlcpNAc-(1→. Serological studies showed that type antigens for the two provisional serotypes are identical; in addition 88-893 expresses S. flexneri group factor 6 antigen. We propose that provisional serotypes Y394 and 88-893 be designated as two new serotypes 7a and 7b, respectively, in the S. flexneri typing scheme.

Occurrence and characterization of Salmonella from chicken nuggets, strips, and pelleted broiler feed.

Raw, frozen chicken nuggets and strips have been identified as a significant risk factor in contracting foodborne salmonellosis. Cases of salmonellosis as a result of consuming partly cooked chicken nuggets may be due in part to Salmonella strains originating in broiler feed. This study was undertaken to determine the occurrence and characterize the strains of Salmonella contaminating chicken nuggets, strips, and pelleted feeds, in an attempt to demonstrate whether the same Salmonella strains present in broiler feed could be isolated from raw, frozen chicken nuggets and strips available for human consumption. Salmonellae were recovered using the Health Canada MFHPB-20 method for the isolation and identification of Salmonella from foods. Strains were characterized by serotyping, phage typing, antimicrobial resistance typing (R-typing), and by pulsed-field gel electrophoresis (PFGE). Salmonellae were isolated from 25-g samples in 27% (n=92) of nugget and strip samples, 95% (n=20) of chicken nugget meat samples, and from 9% (n=111) of pelleted feed samples. Salmonella Heidelberg, Salmonella Enteritidis, and Salmonella Orion were the most commonly isolated serovars from chicken nuggets and strips, nugget and strip meat, and pelleted broiler feeds, respectively. Salmonella Enteritidis phage type (PT) 13a with PFGE pattern SENXAI.0006 and R-type sensitive as well as Salmonella Enteritidis PT13a with PFGE pattern SENXAI.0068 and R-type sensitive were isolated from pelleted feed, and chicken nugget and strip meat in two separate instances. Data showed that Salmonella strains isolated from broiler feed were indistinguishable from strains isolated from packaged raw, frozen chicken nuggets and strips. However, results did not rule out the possibility that breeding stock or contamination during processing may have contributed to chicken meat contamination by Salmonella.

The effect of alendronate on fracture-related healthcare utilization and costs: the fracture intervention trial.

The Vertebral Fracture Arm (VFA) of the Fracture Intervention Trial (FIT) study demonstrated that alendronate reduced the incidence of spine, forearm and hip fractures in women with low bone mass and existing vertebral fractures by about 50%. The objective of the present study was to determine the effects of alendronate therapy versus placebo on fracture-related healthcare utilization and costs. Participants were randomly assigned to double-masked treatment with alendronate (5 mg/day for 2 years and then 10 mg/day for 1 year) or placebo for 3 years. For each patient experiencing a clinical fracture, we determined whether treatment in an emergency room, hospital, nursing home and/or rehabilitation hospital was a consequence of the fracture. The VFA of the FIT Study enrolled 2027 women aged 55-81 years with low bone mass and pre-existing vertebral fractures from population-based listings in 11 metropolitan areas of the United States. We measured (1) the proportion of patients who had any fracture-related healthcare event and (2) the estimated cost of fracture-related healthcare services. Alendronate significantly reduced the proportion of patients utilizing fracture-related healthcare (emergency room, hospital, rehabilitation hospital or nursing home) by 25% (p = 0.038). Alendronate significantly reduced the costs associated with hip-fracture-related care by 58%, or $181 per patient randomized (p = 0.036). The reduction in fracture-related total costs was 35% ($190 per patient randomized) in the alendronate group relative to the placebo group (p = 0.114). Alendronate thus not only reduces the incidence of clinical fractures and associated morbidity, but reduces the proportion of patients utilizing the associated healthcare resources.

Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae.

As part of our studies on the regulation of polyamine biosynthesis in Saccharomyces cerevisiae, we have investigated the effect of spermidine on the degradation of ornithine decarboxylase in this organism. We have found that in S. cerevisiae, as in other eukaryotic cells, the rate of degradation of ornithine decarboxylase, measured either enzymatically or immunologically, is increased by the addition of spermidine to a yeast culture. It is noteworthy that this effect of added spermidine is found even when the experiments are conducted with strains in which the ornithine decarboxylase is overexpressed several hundred-fold more than the wild-type level. The effect of added spermidine in the overexpressed SPE1 strains is best seen in spe2 mutants in which the initial intracellular spermidine is very low or absent. Experiments with cycloheximide show that new protein synthesis is required to effect the breakdown of the ornithine decarboxylase. These results indicate that S. cerevisiae contains an antizyme-like mechanism for the control of the level of ornithine decarboxylase by spermidine, even though, as contrasted with other eukaryotic cells, no specific antizyme homologue has been detected either in in vitro experiments or in the S. cerevisiae genome.

In vivo mechanism-based inactivation of S-adenosylmethionine decarboxylases from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.

S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits. The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation. A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product. Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078-4081].

Older women with diabetes have an increased risk of fracture: a prospective study.

To determine whether type 2 diabetes is associated with fracture in older women, we analyzed data from 9654 women, age 65 yr or older, in the Study of Osteoporotic Fractures. Diabetes with age at onset 40 yr or older was reported by 657 women, of whom 106 used insulin. A total of 2624 women experienced at least one nonvertebral fracture during an average follow-up of 9.4 yr, and 388 had at least one vertebral fracture during an average interval of 3.7 yr. Although diabetes was associated with higher bone mineral density, it was also associated with a higher risk of specific fractures. Compared with nondiabetics, women with diabetes who were not using insulin had an increased risk of hip [relative risk (RR), 1.82; 95% confidence interval (CI), 1.24-2.69] and proximal humerus (RR, 1.94; 95% CI, 1.24-3.02) fractures in multivariate models controlling for age, body mass index, bone density, and other factors associated with fractures and diabetes. Insulin-treated diabetics had more than double the risk of foot (multivariate adjusted RR, 2.66; 95% CI, 1.18-6.02) fractures compared with nondiabetics. This study indicates that diabetes is a risk factor for hip, proximal humerus, and foot fractures among older women, suggesting that fracture prevention efforts should be a consideration in the treatment of diabetes.

Body size and risk for clinical fractures in older women. Study of Osteoporotic Fractures Research Group.

BACKGROUND: Small body size predicts hip fractures in older women. OBJECTIVE: To test the hypothesis that small body size predicts the risk for other clinical fractures. DESIGN: Prospective cohort study. SETTING: Population-based listings in four areas of the United States. PATIENTS: 8059 ambulatory nonblack women 65 years of age or older. MEASUREMENTS: Weight, weight change since 25 years of age, body mass index, lean body mass and percent body fat, and nonspine fractures during 6.4 years of follow-up. RESULTS: Compared with women in the highest quartile of weight, women in the lowest quartile had relative risks of 2.0 (95% CI, 1.5 to 2.8) for hip fractures, 2.3 (CI, 1.1 to 4.7) for pelvis fractures, and 2.4 (CI, 1.5 to 3.9) for rib fractures. Adjustment for total-hip bone mineral density eliminated the elevated risk. Results were similar for other body size measures. Smaller body size was not a risk factor for humerus, elbow, wrist ankle, or foot fractures. CONCLUSIONS: Total body weight is useful in the prediction of hip, pelvis, and rib fractures when bone mineral density has not been measured.

Sensitivity of spermidine-deficient Saccharomyces cerevisiae to paromomycin.

Spermidine-deficient Saccharomyces cerevisiae cells are much more sensitive to paromomycin than nondeficient cells, resulting in cessation of growth and cell death.

It all started on a streetcar in Boston.

We first met on a Boston streetcar in 1940, being introduced by a mutual friend. Celia was returning from research work at the Massachusetts General Hospital as part of her senior thesis at Radcliffe College, and Herb was returning from a concert by the Boston Symphony. We were married in 1946 after Celia had finished her medical training. We started working together in 1952, and we are still actively collaborating in our studies on various aspects of the biosynthesis and function of polyamines. We are honored to have been invited by the editors of the Annual Review of Biochemistry to summarize our activities in biochemical research over the past 60 years. During most of this time we have been at the National Institutes of Health in Bethesda, Md., and we have witnessed the enormous expansion of biomedical research that has occurred during this period. In addition to summarizing our research, Herb summarizes his association with the Journal of Biological Chemistry and the remarkable developments that have occurred recently in electronic publication and dissemination of scientific literature.

Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant.

Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [Hamasaki-Katagiri, N., Tabor, C. W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35-43].

Spermidine biosynthesis in Saccharomyces cerevisae: polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase).

The Saccharomyces cerevisiae SPE3 gene, coding for spermidine synthase, was cloned, sequenced, and localized on the right arm of chromosome XVI. The deduced amino acid sequence has a high similarity to mammalian spermidine synthases, and has putative S-adenosylmethionine binding motifs. To investigate the effect of total loss of the SPE3 gene, we constructed a null mutant of this gene, spe3delta, which has no spermidine synthase activity and has an absolute requirement for spermidine or spermine for the growth. This requirement is satisfied by a very low concentration of spermidine (10(-8) M) or a higher concentration of spermine (10(-6) M).

Sensitivity of polyamine-deficient Saccharomyces cerevisiae to elevated temperatures.

Saccharomyces cerevisiae cells that cannot synthesize spermidine or spermine because of a deletion in the gene coding for S-adenosylmethionine decarboxylase are very sensitive to elevated temperatures when incubated in a polyamine-deficient medium; i.e., growth is inhibited and the cells are killed. This sensitivity is very pronounced at 39 degrees C, but a moderate effect is noted even at 33 to 34 degrees C. These findings support findings from other studies from our laboratory on the importance of polyamines in protecting cell components against damage. The sensitivity of spermidine-deficient cells to the temperature 39 degrees C provides a useful method for screening for polyamine auxotrophs.

SPE1 and SPE2: two essential genes in the biosynthesis of polyamines that modulate +1 ribosomal frameshifting in Saccharomyces cerevisiae.

We previously showed that a mutant of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene (spe2 delta), exhibits a marked elevation in +1 ribosomal frameshifting efficiency in response to the Ty1 frameshift sequence, CUU AGG C. In the present study, we found that spermidine deprivation alone does not result in increased +1 ribosomal frameshifting efficiency. The high level of +1 ribosomal frameshifting efficiency in spe2 delta cells is the result of the combined effects of both spermidine deprivation and the large increase in the level of intracellular putrescine resulting from the derepression of the gene for ornithine decarboxylase (SPE1) in spermidine-deficient strains.

The presence of an active S-adenosylmethionine decarboxylase gene increases the growth defect observed in Saccharomyces cerevisiae mutants unable to synthesize putrescine, spermidine, and spermine.

Saccharomyces cerevisiae spe1 delta SPE2 mutants (lacking ornithine decarboxylase) and spe1 delta spe2 delta mutants (lacking both ornithine decarboxylase and S-adenosylmethionine decarboxylase) are equally unable to synthesize putrescine, spermidine, and spermine and require spermidine or spermine for growth in amine-free media. The cessation of growth, however, occurs more rapidly in spe1 delta SPE2 cells than in SPE1 spe2 delta or spe1 delta spe2 delta cells. Since spe1 delta SPE2 cells can synthesize decarboxylated adenosylmethionine (dcAdoMet), these data indicate that dcAdoMet may be toxic to amine-deficient cells.

Campylobacter fetus diarrhea in a Hutterite colony: epidemiological observations and typing of the causative organism.

Following a case of Campylobacter fetus sepsis and meningitis in a 4-month-old female member of a Hutterite colony, an epidemiological investigation revealed at least 18 cases of diarrhea in other members of the colony. C. fetus was isolated from 7 of 15 fecal samples submitted from affected persons. A case control study suggested that persons who worked in the abattoir were 2.03 times more likely to have had diarrhea, but none of the risk factors studied were significant. The epicurve of the outbreak was inconclusive as to the likely mode of spread of C. fetus. All of the C. fetus strains isolated from the blood of the infant and from the fecal samples were the same by biochemical and antibiotic susceptibility tests. Pulsed-field gel electrophoresis showed that all isolates produced identical restriction endonuclease patterns and differed from other nonepidemiologically related strains of C. fetus.

Spermidine deficiency increases +1 ribosomal frameshifting efficiency and inhibits Ty1 retrotransposition in Saccharomyces cerevisiae.

Polyamines have been implicated in nucleic acid-related functions and in protein biosynthesis. RNA sequences that specifically direct ribosomes to shift reading frame in the -1 and +1 directions may be used to probe the mechanisms controlling translational fidelity. We examined the effects of spermidine on translational fidelity by an in vivo assay in which changes in beta-galactosidase activity are dependent on yeast retrovirus Ty +1 and yeast double-stranded RNA virus L-A -1 ribosomal frameshifting signals. In spe2 delta mutants of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene, there is a marked elevation in +1 but no change in -1 ribosomal frameshifting. The increase in +1 ribosomal frameshifting efficiency is accompanied by a striking decrease in Ty1 retrotransposition.

Oxygen toxicity in a polyamine-depleted spe2 delta mutant of Saccharomyces cerevisiae.

When a mutant of Saccharomyces cerevisiae (spe2 delta) that cannot make spermidine or spermine was incubated in a polyamine-deficient medium in oxygen, there was a rapid cessation of cell growth and associated cell death. In contrast, when the mutant cells were incubated in the polyamine-deficient medium in air or anaerobically, the culture stopped growing more gradually, and there was no significant loss of cell viability. We also found that the polyamine-deficient cells grown in air, but not those grown anaerobically, showed a permanent loss of functional mitochondria ("respiratory competency"), as evidenced by their inability to grow on glycerol as the sole carbon source. These data support the postulation that polyamines act, in part, by protecting cell components from damage resulting from oxidation. However, since the mutant cells still required spermidine or spermine for growth when incubated under strictly anaerobic conditions, polyamines must also have other essential functions.

Deletion mutations in the speED operon: spermidine is not essential for the growth of Escherichia coli.

Null mutants of Escherichia coli were constructed that cannot synthesize spermidine, because of deletions in the gene encoding S-adenosylmethionine decarboxylase. These mutants are still able to grow at near normal rates in purified media deficient in polyamines. These results in E. coli differ from recent findings that null mutants of Saccharomyces cerevisiae and of Neurospora crassa have an absolute growth requirement for spermidine.