Copper artifacts from prehistoric archeological sites in the dakotas.

Thirteen archeological specimens were analyzed spectrographlically, and within defined limits they were determined to be native copper. Twelve of the specimens show close elemental homogeneity and are believed to be of Lake Superior ore; the origin of the other specimen is deviolus.

Thermal inactivation D- and z-values of multidrug-resistant and non-multidrug-resistant Salmonella serotypes and survival in ground beef exposed to consumer-style cooking.

There has been speculation that multidrug-resistant (MDR) strains are generated by subtherapeutic antibiotic use in food animals and that such strains result in increased resistance to lethality by food processes such as heat and irradiation. The objective of this study was to evaluate the heat resistance of 20 strains, namely an MDR and a non-multidrug-resistant (NMDR) strain of each of 10 Salmonella serotypes isolated from cattle or cattle environments. MDR and NMDR Salmonella serotypes studied included Montevideo, Typhimurium, Anatum, Muenster, Newport, Mbandaka, Dublin, Reading, Agona, and Give. For phase I, stationary-phase cultures of the strains were aliquoted into sterile capillary tubes and immersed in a temperature-controlled water bath at 55, 60, 65, and 70 degrees C for appropriate times. Survivor curves were plotted for each temperature, and a best-fit linear regression was derived for each temperature. D-values (decimal reduction times) and z-values (changes in temperature required to change the D-values) were calculated for each strain. Although there was no overall significant difference in the heat resistance of MDR and NMDR serotypes, NMDR serotypes generally appeared to have slightly higher heat resistance than NMDR serotypes, especially at 55 and 60 degrees C. The highest relative heat resistance (highest z-values) was exhibited by Salmonella Anatum. Notably, the relative heat resistance of NMDR Salmonella Agona was similar to that of NMDR Salmonella Anatum and had the highest D-values at all four temperatures. For phase II, three serotypes (regardless of resistance profile) with the highest relative heat resistance and their drug-resistant counterparts were selected for thermal inactivation in ground beef patties cooked to endpoint temperatures. Salmonella Agona was able to survive in ground beef cooked to an internal temperature of 71 degrees C. Results of these studies suggest drug resistance does not affect the heat resistance of Salmonella and that serotype or strain is an important consideration in risk assessment of the pathogen with regard to survival at cooking temperatures.

Validation of individual and multiple-sequential interventions for reduction of microbial populations during processing of poultry carcasses and parts.

Changes in aerobic plate counts (APC), total coliform counts (TCC), Escherichia coli counts (ECC), and Salmonella incidence on poultry carcasses and parts and in poultry processing water were evaluated. Bacterial counts were estimated before and after individual interventions and after poultry carcasses were exposed to multiple-sequential interventions at various stages during the slaughter process. Individual and multiple-sequential interventions were evaluated at three processing plants: (i) plant A (New York wash, postevisceration wash, inside-outside bird washes 1 and 2, chlorine dioxide wash, chlorine dioxide wash plus chlorine chiller, chiller exit spray, and postchiller wash), (ii) plant B (New York wash, inside-outside bird washes 1 and 2, trisodium phosphate wash, and chlorine chiller), and (iii) plant C (trisodium phosphate wash and chlorine chiller). The majority of individual interventions effectively or significantly (P < 0.05) reduced microbial populations on or in carcasses, carcass parts, and processing water. Reductions in APC, TCC, and ECC due to individual interventions ranged from 0 to 1.2, 0 to 1.2, and 0 to 0.8 log CFU/ml, respectively. Individual interventions reduced Salmonella incidence by 0 to 100% depending on the type of process and product. Multiple-sequential interventions resulted in significant reductions (P < 0.05) in APC, TCC, ECC, and Salmonella incidence of 2.4, 2.8, and 2.9 log CFU/ml and 79%, respectively, at plant A; 1.8, 1.7, and 1.6 log CFU/ml and 91%, respectively, at plant B; and 0.8, 1.1, and 0.9 log CFU/ml and 40%, respectively, at plant C. These results enabled validation of in-plant poultry processing interventions and provide a source of information to help the industry in its selection of antimicrobial strategies.

Interaction of tRNA with domain II of 23S rRNA.

The interaction of tRNA with domain II of 23S rRNA in E. coli ribosomes has been probed using short, complementary DNA oligodeoxyribonucleotides. Specifically, cDNA oligomers to the region 801-811 of the 23S rRNA were used to ascertain the interaction of this region with tRNA. It was found that when tRNA was bound to the P site, considerable competition occurred between tRNA and the cDNA oligomers which base paired with the nucleotides 807-811. However, A-site bound tRNA neither displaced, nor was displaced, by cDNA oligomers to this region. Additionally, the binding of tRNA lacking the CACCA nucleotides on the 3' terminus was unaffected by the presence a cDNA oligomer complementary to nucleotides 803-811, indicating that the cDNA-tRNA competition was dependent on the 3' terminal nucleotides of tRNA.

Ribosomal proteins: their structure and spatial arrangement in prokaryotic ribosomes.

During the last 15 years of ribosomal protein study, enormous progress has been made. Each of the proteins from E. coli ribosomes has been isolated, sequenced, and immunologically and physically characterized. Ribosomal proteins from other sources (e.g., from some bacteria, yeast, and rat) have been isolated and studied as well. Several proteins have recently been crystallized, and from the X-ray studies it is expected that much important information on the three-dimensional structure will be forthcoming. Many other proteins can probably be crystallized if suitable preparative procedures and crystallization conditions are found. Tremendous progress has also been made in deciphering the architecture of the ribosome. A battery of different methods has been used to provide the nearest neighbor distances of the ribosomal proteins in situ. Definitive measurements are now emanating from neutron-scattering experiments which also promise to give reasonably accurate radii of gyration of the proteins in situ. In turn, refined immune electron microscopy results supplement the neutron-scattering data and also position the proteins on the subunits themselves. This cannot be done by the other methods. Determination of the three-dimensional RNA structure within the ribosome is still in its infancy. Nonetheless, it is expected that by combining the data from protein-RNA and from RNA-RNA cross-linking studies, the structure of the RNA in situ can be unraveled. Of great interest is the fact that ribosomal subunits and ribosomes themselves have now been crystallized, and low-resolution structural maps have already been obtained. However, to grow suitable crystals and to resolve the ribosomal structure at a sufficiently high resolution remains a great challenge and task to biochemists and crystallographers.

Regions of 23 S ribosomal RNA proximal to transfer RNA bound at the P and E sites.

tRNAPhe transcribed in a T7 RNA polymerase system has been modified in such a way that 4-thiouridines have randomly replaced unmodified uridines. These 4-thiouridines serve as sites for conjugation of the cleavage reagent 5-iodoacetamido-1,10-phenanthroline (IOP). 1,10-Phenantholine, when complexed with Cu2+ in a reducing environment, causes hydrolysis of nearby nucleic acids. We show here that tRNA-phenanthroline (tRNA-OP) conjugates, when bound in situ to the P- and E-sites of 70 S ribosomes, cause cleavage, mainly in domains I, III and V of 23 S ribosomal RNA (rRNA). The cleavage sites in domain V predominantly occur very close to or in the peptidyl-transferase region. The regions of domain I and III that are cleaved are apparently folded in the 50 S ribosomal subunit so as to be proximal to the peptidyl-transferase center. Most of the cleavage events occur whether the tRNA-OP conjugate is bound to ribosomes alone, or yeast tRNA is also present in the P/P hybrid state. Cleavages that occur only in the absence of yeast tRNA are limited to the 1100 region of domain II, and the 2800 region of domain VI. Cleavages that occur only in the presence of yeast occur in the 2170 region of domain V. The regions of 23 S rRNA in which tRNA-OP induced cleavage occur complement those sites shown by chemical protection and cross-liking to be in a close proximity to the tRNA. However, the cleavage approach allows a more versatile and expanded view of the near neighborhood of rRNA surrounding the tRNA. These results provide considerable information which will allow a more detailed modeling of the tertiary structure of the 50 S ribosomal subunit.

Nationwide microbiological baseline data collected by sponge sampling during 1997 and 1998 for cattle, swine, turkeys, and geese.

During 1997 and 1998, the U.S. Food Safety and Inspection Service completed nationwide microbiological baseline studies on four separate categories of livestock and poultry. Data were collected by sponge-sampling techniques. These studies were designed to provide nationwide estimates of the prevalence of Salmonella and prevalence and concentrations of Escherichia coli in cattle (n = 1,881), swine (n = 2,127), turkeys (n = 1,396), and geese (n = 102) in establishments under federal inspection. Salmonella prevalence ranged from 1.2% in cattle to 6.9% in swine, 13.7% in geese, and 19.6% in turkeys. The prevalence of E. coli was 16.6% in cattle (geometric mean = 0.26 CFU/cm2), 44.1% in swine (mean = 0.78 CFU/cm2), 92.7% in turkey (mean = 2.46 CFU/cm2), and 96.5% in geese (mean = 1.97 CFU/cm2). These values are similar to or somewhat lower than previous baseline values obtained for steers and heifers, cows and bulls, market hogs, and young turkeys. This study is the first in which nationwide microbiological baseline data have been compiled for geese. These data will be useful to individuals working with hazard analysis critical control point plans and risk assessment and to the research and academic communities.

Probing ribosome structure using short oligodeoxyribonucleotides: the question of resolution.

The structure of ribosomal RNA in situ can be probed using short, complementary DNA oligomers. As these oligomers bind to exposed, single-stranded regions of rRNA, the stability of the hybridized complex can be assayed. Differences in binding stability between cDNA probes of similar length and composition may be indicative of the presence of competing structure, such as base-paired rRNA regions, tRNA interactions or protein interactions. In this study the degree to which such interactions can be distinguished is studied. It is found that by using suitable controls, interactions between rRNA and tRNA or rRNA can be discriminated to a resolution of one or two bases. This resolution promises to be important in delineating the higher-order structure of the rRNA.

Probing the initiation complex formation on E coli ribosomes using short complementary DNA oligomers.

Interactions between Escherichia coli 16S rRNA sequences (as components of 30S ribosomal subunits or tight-couple 70S ribosomes) with the ligands poly(U), poly(AGU), tRNAPhe, tRNAfMet, and the initiation factors have been studied. The ligands were employed as competitors for selected sites on 16S rRNA known to be accessible for hybridization to cDNA oligomers, regions 517-528, 1397-1404, and 1534-1542. The binding of cDNAs 1534-1541 and 1398-1403 decreased in the presence of the ligand pair poly(U)/tRNAPhe. Only the binding of cDNA 1534-1541 was affected by poly(AGU), while none of the complementary DNA oligomer binding was affected by tRNAPhe or tRNAfMet alone. The poly(AGU)/tRNAfMet ligand pair caused an additional decline in the binding of cDNA 1534-1541, relative to that caused by poly(AGU) alone, but the ligand pair did not affect the binding of the cDNA oligomers 517-528 or 1398-1403. The inclusion of the initiation factors did not significantly alter the binding level decreases observed for cDNA 1534-1541 in the presence of mRNAs or tRNA. At the 517-528 and 1398-1403 regions, the inclusion of the initiation factors, in either the presence or absence of the other ligands, caused a large decrease in the binding of the cDNA oligomers. The oligomers complementary to 16S bases 517-528 and 1398-1403 did not bind to tight-couple or reassociated 70S ribosomes. The data are discussed in terms of the decoding site hypothesis, and in terms of the mRNA alignment mechanism proposed by Trifonov [1].

Comparative sensitivity of 13 species of pathogenic bacteria to seven chemical germicides.

BACKGROUND: The relative resistance of diverse human bacterial pathogens to commonly used germicidal agents has not been established. METHODS: We measured by titration the survival of thirteen different bacteria after exposure to glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate, sodium hypochlorite, or phenol. RESULTS: Our comparative experiments allowed classification of the organisms' survival into four groups: (a) Pseudomonas aeruginosa and Staphylococcus aureus showed the most resistance, (b) Clostridium perfringens, Salmonella typhimurium, Staphylococcus epidermidis, and Escherichia coli O157:H7 showed intermediate resistance, (c) Listeria monocytogenes, Shigella sonnei, and Vibrio parahaemolyticus survived some treatments with chemical agents only in the presence of protecting protein (serum albumin), and (d) Vibrio cholerae, Vibrio vulnificus, Bacillus cereus, and Yersinia enterocolitica did not survive any of the treatments applied. CONCLUSION: We found species that more frequently survived exposure to germicidal agents were also those most commonly reported in association with hospital infections. Our findings suggest that resistance to disinfectants may be more important than pathogenicity in determining the relative prominence of an organism as an agent responsible for nosocomial infections.

Unfolded 30 S ribosomal subunits.

The 30 S ribosomal subunit from Escherichia coli was unfolded into discrete particles upon which physical studies were carried out. These particles were found to be homogeneous and were characterized using sedimentation velocity, diffusion, density and viscosity measurements. The results of these studies clearly certify two distinct stages of unfolding, neither involving a significant loss of protein. However, the results also clearly show that the measurement of only one characteristic (e.g., the sedimentation coefficient) is not sufficient to suggest a structural change. The significance and importance of the apparent specific volume are stressed.

Synthesis, characterization and anti-tumour testing of some platinum(II) amine complexes containing 1,1- and 1,2-cyclobutanedicarboxylate ligands.

A series of new platinum(II) amine complexes containing 1,1- and 1,2-cyclobutanedicarboxylate ligands, cis-[PtA2(1,1-CBDCA)] (A = RNH2, where R = C2H5, n-C3H7, n-C4H9, n-C5H11, n-C6H13, c-C3H5, c-C5H9, c-C6H11; A2 = ethylenediamine, 1,3-diaminopropane), cis-[PtA2(1,2-CBDCA)] (A = NH3, RNH2 where R = CH3, C2H5, n-C3H7, n-C4H9, c-C3H5) and trans-[Pt(NH3)2(1,1-CBDCAH)2] (CBDCA, CBDCAH = dianion and monoanion of the dicarboxylic acid, respectively) have been synthesized by an improved route. These complexes are stable in aqueous solution and show good aqueous solubility. The [Pt(c-C3H5NH2)2(1,1-CBDCA)] can be isolated in white, grey and blue forms. The grey and blue forms exhibit ESR signals analogous to the so-called platinum blues. The existence of the blue form in aqueous solution is time and temperature dependent. Several of the complexes have been tested against leukaemia L1210 in male BDF mice and activity appears to decrease with the increase in length of the aliphatic chain (or increase in size of the alicyclic ring) of the primary amine. The Yoshida lymphoscarcoma screen, usually insensitive to platinum drugs, was found to respond well to [Pt(n-C4H9NH2)2(1,1-CBDCA)] in 5-day subcutaneously implanted tumours in female Wistar rats.

Identification of foodborne pathogens by nucleic acid hybridization.

Nucleic acid hybridization methods have been developed and used to identify microorganisms in foods. Tests performed on mixed cultures save the time required to establish pure cultures. Enterotoxigenic or invasive strains of foodborne bacterial pathogens are detected with probes that identify genes responsible for virulence. Hybridization tests signal the presence or absence of a particular strain or an entire genus and are especially well suited for screening foods for specific pathogens. With the colony hybridization assay format, foodborne bacteria harboring a specific gene can be enumerated. However, hybridization tests require the presence of 10(5) to 10(6) cells to yield a positive result, thereby limiting sensitivity and necessitating a time-consuming growth step. In vitro DNA amplification techniques increase the amount of DNA segments 10(5)-10(6)-fold in 2 to 3 h, thus enhancing test sensitivity.

In vitro breakage of plasmid DNA by mutagens and pesticides.

Covalently closed circular molecules of the colicinogenic plasmid E1 can serve as sensitive indicators for detecting in vitro breakage of DNA. After these molecules are radioactively labeled and purified by cesium chloride density-gradient centrifugation, they are incubated with the compounds to be tested. Samples are analyzed on alkaline sucrose gradients to determine the fraction of unbroken molecules and a breakage rate is calculated. Positive results were obtained for all three mutagenic alkylating agents (MMS, EMS, and MNNG) and of the 11 pesticides tested, dexon, dichlorvos, malathion, and methyl parathion induced breaks in molecules at a rate significantly greater than the controls.

Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of vibrio parahaemolyticus.

Production of the thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus is associated with pathogenicity of the organism and is encoded by the tdh gene. The timely resolution of seafood-associated outbreaks requires rapid and accurate detection of pathogenic V. parahaemolyticus. The specificity of alkaline phosphatase- and digoxigenin-labeled tdh gene probes was evaluated against 61 strains of V. parahaemolyticus (including isolates from recent outbreaks involving oysters from the Pacific Northwest, Texas, and New York), 85 strains of other vibrios, and 7 strains of non-vibrio species from clinical and environmental sources. The probes were specific for detection of the V. parahaemolyticus tdh gene.

Development of digoxigenin-labeled PCR amplicon probes for use in the detection and identification of enteropathogenic Yersinia and Shiga toxin-producing Escherichia coli from foods.

By including digoxigenin-11-dUTP in a polymerase chain reaction (PCR), amplification products were produced that contained nonisotopic markers for use as DNA hybridization probes. Because these labeled amplicons encode pathogenic traits for specific foodborne bacteria, they can be used to detect the presence of potentially virulent organisms that may be present in foods. This technology allows the synthesis of a variety of shelf-stable probe reagents for detecting a number of foodborne microbes of public health concern. We used this technology to detect four genes in two potential pathogens: virF and yadA in enteropathogenic Yersinia and stx1 and stx2 in Shiga-like toxin-producing Escherichia coli. Results of DNA hybridizations of dot blots of 68 Yersinia strains and 24 of 25 E. coli strains were consistent with results of equivalent PCR analyses. DNA colony hybridization with nonisotopic virF probes of colonies arising on spread plates from artificially contaminated food homogenates was able to detect potentially pathogenic Y. enterocolitica. When compared with oligonucleotide probes, amplicon probes are much less sensitive to changes in hybridization and wash temperatures, allowing greater reproducibility. Labeled probe preparations were reused more than five times and have been stored at -20 degrees C for more than 8 months. This method conveniently generates probes that are safe, stable, inexpensive, reusable, and reliable.

Template preparation for PCR and RFLP of amplification products for the detection and identification of Cyclospora sp. and Eimeria spp. Oocysts directly from raspberries.

Raspberries were epidemiologically associated with cyclosporiasis outbreaks during 1996 and 1997. The 18S rRNA genes of Cyclospora cayetanensis and several species of a closely related genus, Eimeria, were sequenced and primers for a nested PCR developed in a previous study. The ability to distinguish amplified products of Cyclospora sp. from those of Eimeria spp. is important for testing food and environmental samples. Therefore, an RFLP analysis of amplified products was used to differentiate Cyclospora cayetanensis from Eimeria spp. PCR inhibitors and the low levels of Cyclospora oocysts present in raspberries make template preparation for PCR challenging. Several approaches for PCR template preparation from raspberry samples were evaluated. Template preparation methods using various washing and concentration steps, oocyst disruption protocols, resin matrix treatment, DNA precipitation, and/or the addition of nonfat dried milk solution to a PCR using modified primers were evaluated first with oocysts of Eimeria tenella then refined with oocysts of C. cayetanensis. Approximately 10 E. tenella oocysts per PCR or approximately 19 C. cayetanensis oocysts per PCR were detected with the optimized template preparation method. The addition of 20 microliters of raspberry wash sediment extract and nonfat dried milk solution did not inhibit the amplification of DNA from as few as 10 E. tenella and 25 C. cayetanensis oocysts in a 100-microliter PCR. The nucleotide sequences of C. cayetanensis and the Eimeria spp. are 94 to 96% similar in the amplified region, but the amplification products from the two genera were distinguished using an RFLP analysis with the restriction enzyme MnlI.

An oligonucleotide-ligation assay for the differentiation between Cyclospora and Eimeria spp. polymerase chain reaction amplification products.

An oligonucleotide-ligation assay (OLA) was developed and compared to a restriction fragment length polymorphism (RFLP) test for distinguishing between 294-bp polymerase chain reaction (PCR) amplification products of the 18S rRNA gene from Cyclospora and Eimeria spp. The PCR/OLA correctly distinguished between three Cyclospora, three E. tenella, and one E. mitis strains and the ratio of positive to negative spectrophotometric absorbance (A490) values for each strain ranged from 4.086 to 15.280 (median 9.5). PCR/OLA provides a rapid, reliable, spectrophotometric alternative to PCR/RFLP.