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PubMed1995

A rapeseed cold-inducible transcript encodes a phosphoenolpyruvate carboxykinase.

We have isolated a clone corresponding to a new cold-regulated gene from a cDNA library made from rapeseed (Brassica napus cv Samourai) cold-acclimated etiolated seedlings. Sequence analysis and homology searches showed that this clone encodes a protein highly homologous to the ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) from Saccharomyces cerevisiae, Trypanosoma, Rhizobium sp., and Escherichia coli; we refer to the B. napus clone as BnPEPCK. A potential ATP-binding site existing in all PEPCK proteins was also found in BnPEPCK. Although there was a basal expression of BnPEPCK in seedlings grown at control, room temperature, the steady-state level of the transcripts increased at 4 degrees C and decreased to normal levels when the seedlings were returned to control temperature (22 degrees C). Using antibodies made against a recombinant histidine-BnPEPCK fusion protein, we demonstrated that BnPEPCK protein level is correlated with the accumulation of the BnPEPCK transcript.

Acclimatization↗
PubMed2005

Studies of helix fraying and solvation using 13C' isotopomers.

Both NMR and IR studies of carbonyl (13C') isotopomers of designed helices can provide residue-level details regarding the fractional occurrence and melting behavior of helical phi/psi angles along the sequence of helical peptides, details that cannot be obtained from CD or 1H-NMR studies. We have studied a classic series of helical models, Ac-YGG-(KAXAA)3K-NH2 (X=A,V), in both aqueous and helix-favoring media containing fluoroalcohol cosolvents, including a solvent system allowing the observation of cold denaturation. These studies confirmed the strong N-capping associated with this sequence and revealed more extensive C-terminal fraying than that calculated using current helicity prediction algorithms. In the X=A series, the central residues are somewhat resistant to thermal melting; it instead occurs predominantly at the frayable C terminus. For the X=V series under cold-denaturing conditions, the temperature of maximal helicity is not uniform along the sequence and both solvated and nonsolvated helical alanine sites (13C=O stretches at 1592 cm(-1) and 1615 cm(-1), respectively) are apparent. Correlation between the two spectroscopies employed yielded the intriguing observation that the valine side chain is able to desolvate the i - 4 amide in short monomeric helices. In addition, we report further measurements of the temperature dependence of alanine statistical coil chemical shifts, the temperature dependence of the 13C chemical shift of urea (employed as chemical shift reference), and a useful formula for converting 13C' shifts into fractional helicities.

Amino Acid Sequence↗
PubMed1997

The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins.

The psychrotrophic bacterium Pseudomonas fragi was subjected to cold shocks from 30 or 20 to 5 degrees C. The downshifts were followed by a lag phase before growth resumed at a characteristic 5 degrees C growth rate. The analysis of protein patterns by two-dimentional gel electrophoresis revealed overexpression of 25 or 17 proteins and underexpression of 12 proteins following the 30- or 20-to-5 degrees C shift, respectively. The two downshifts shared similar variations of synthesis of 20 proteins. The kinetic analysis distinguished the induced proteins into cold shock proteins (Csps), which were rapidly but transiently overexpressed, and cold acclimation proteins (Caps), which were more or less rapidly induced but still overexpressed several hours after the downshifts. Among the cold-induced proteins, four low-molecular-mass proteins, two of them previously characterized as Caps (CapA and CapB), and heat acclimation proteins (Haps) as well as heat shock proteins (Hsps) for the two others (TapA and TapB) displayed higher levels of induction. Partial amino acid sequences, obtained by microsequencing, were used to design primers to amplify by PCR the four genes and then determine their nucleotide sequences. A BamHI-EcoRI restriction fragment of 1.9 kb, containing the complete coding sequence for capB, was cloned and sequenced. The four peptides belong to the family of small nucleic acid-binding proteins as CspA, the major Escherichia coli Csp. They are likely to play a major role in the adaptative response of P. fragi to environmental temperature changes.

Adaptation, Biological↗
PubMed2002

Expression of human beta defensin (HBD-1 and HBD-2) mRNA in nasal epithelia of adult cystic fibrosis patients, healthy individuals, and individuals with acute cold.

BACKGROUND: Lack or inactivation of defensins may facilitate chronic bacterial colonization in the cystic fibrosis (CF) lung. CF nasal epithelium exhibits typical biochemical abnormalities and can be used to study defensin expression in CF. OBJECTIVES: To evaluate the expression of beta defensin (HBD-1 and HBD-2) mRNA and the presence of inflammatory markers (percentage of neutrophils and IL-8 mRNA expression) in CF and non-CF nasal mucosa. METHODS: Case-control study. Nasal brushing samples were obtained from 22 stable adult CF patients and 32 non-CF controls (25 healthy individuals and 7 individuals with acute cold). Samples were subjected to analysis involving mRNA expression (semiquantitative RT-PCR) and differential cell counting. RESULTS: Defensins and inflammatory markers were expressed at low levels in healthy individuals and at high levels in subjects with acute cold. In non-CF controls, defensin expression correlated significantly with inflammatory parameters (p < 0.001). In CF, defensin mRNA expression was comparable to healthy individuals (p = 0.2). In contrast to non-CF controls, in CF patients high levels of inflammatory markers did not correlate with defensin mRNA levels. CONCLUSIONS: Defensin expression is not upregulated in CF epithelium in response to inflammatory stimuli. Further studies are necessary to elucidate whether this is a consequence of the CF gene mutation.

Acute Disease↗
PubMed1997

Transport of sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, where C6-NBD-sphingomyelin is translocated across the plasma membrane by a multidrug transporter activity.

Sphingomyelin is a major lipid of the mammalian cell surface. The view that sphingomyelin, after synthesis in the Golgi lumen, reaches the outer leaflet of the plasma membrane on the inside of carrier vesicles has been challenged by inconsistencies in the results of transport studies. To investigate whether an alternative pathway to the cell surface exists for sphingomyelin, brefeldin A and mitotic cells were used to block vesicular traffic between the Golgi complex and the plasma membrane. Exogenous sphingomyelinase was applied in the cold to assay for the presence of sphingomyelin on the surface of CHO cells. Newly synthesized radiolabeled sphingomyelin was found to equilibrate with cell surface sphingomyelin within 1.5 hours at 37 degrees C. Brefeldin A and mitosis inhibited this transport but, surprisingly, not the surface appearance of the short-chain sphingomyelin analog N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl(C6-NBD)-sphingo myelin as assayed by depletion of this lipid in the medium by the scavenger albumin. Transport of C6-NBD-sphingomyelin in the presence of brefeldin A was blocked by cyclosporin A and PSC 833, inhibitors of the multidrug resistance P-glycoprotein. The same was observed in HepG2 and HeLa cells, and for short-chain glucosylceramide, which demonstrates the general nature of the transporter-dependent sphingolipid translocation across the plasma membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Cost of transport is increased after cold exposure in Monodelphis domestica: training for inefficiency.

Monodelphis domestica (Didelphidae: Marsupialia) lacks brown adipose tissue and thus relies on skeletal muscle as its primary thermogenic organ. Following cold exposure, the aerobic capacity of skeletal muscle in these animals is greatly increased. We investigated the effects of this plastic response to thermogenesis on locomotion and muscle mechanics. In cold-exposed animals, cost of transport was 15% higher than in controls but was unaffected by exercise training. Twitch kinetics in isolated semitendinosus muscles of cold-exposed animals were characteristic of slow-oxidative fiber types. Both time-to-peak tension and half-relaxation time were longer and maximal shortening velocity was slower following cold exposure compared to either thermoneutral controls or exercise-trained animals. Further, muscles from the cold-exposed animals had greater fatigue resistance than either control or exercise-trained animals, indicating greater oxidative capacity. Finally, we identified an uncoupling protein 3 homologue, whose gene expression was upregulated in skeletal muscle of cold-exposed Monodelphis domestica. Cold exposure provided a potent stimulus for muscle plasticity, driving a fast-to-slow transition more effectively than exercise training. However, linked to the dramatic shift in muscle properties is an equally dramatic increase in whole animal muscle energetics during locomotion, suggesting an uncoupled state, or 'training for inefficiency'.

Adaptation, Physiological↗
PubMed2004

Valproic acid uptake by bovine brain microvessel endothelial cells: role of active efflux transport.

The basis for low brain permeability of valproic acid (VPA) appears to be the result of efflux transport at the blood-brain barrier (BBB); however, the identity of the putative efflux transporter has not been investigated. The objective of our studies was to determine whether the multidrug resistance-associated protein (MRP) might be involved in efflux transport of VPA. Brain microvessel endothelial cells (BMEC) were isolated from cow brains and grown to confluence. MRP messenger RNA (mRNA) in BMEC were verified by reverse transcriptase-polymerase chain reaction (RT-PCR). Functional activity was demonstrated using the steady-state retention of calcein and MRP inhibitors, indomethacin (IND) and probenecid (PRB). Probenecid (0.50 mM) and indomethacin (10 microM) produced a 26 and 13% ( P<0.05 ) elevation in steady-state cellular VPA uptake following a 30-min-incubation with tracer 3H-VPA and 30 microM cold VPA. In contrast, at higher concentrations of probenecid (2 mM) and indomethacin (500 microM), an 11 and 31% reduction in VPA uptake was observed. The biphasic pattern of VPA uptake suggested concurrent inhibition of uptake and efflux transporters by the inhibitor with differing sensitivities, i.e. the efflux transporter being more susceptible to inhibition than the influx transporter. Similar results were obtained in the MRP overexpressing cell line A549. Overall, the results suggest that MRP(s) is(are) involved in the efflux transport of VPA, but do not preclude the possible contribution(s) of other organic anion transporters. The findings also adds to the growing evidence that up-regulation of active drug efflux transporters at the BBB may contribute to the development of drug resistance to antiepileptic drug therapy.

Adenocarcinoma↗
PubMed2003

Effects of rewarming on nuclear factor-kappaB and interleukin 8 expression in cold-preserved alveolar epithelial cells.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) and interleukin (IL)-8 play important roles in the pathophysiology of acute lung injury after lung transplantation. Because alveolar epithelium is one of the most important sites at which IL-8 production takes place after reperfusion of donor lungs, we examined the effects of cold/rewarming on NF-kappaB and IL-8 expression in alveolar epithelial cells. METHODS: A549 cells were preserved at 4 degrees C for 5 hr and then rewarmed for up to 20 hr. NF-kappaB was analyzed by electrophoretic mobility shift assay. IL-8 mRNA expression was examined by reverse transcription-polymerase chain reaction. IL-8 concentration in the cell culture medium after rewarming was measured by enzyme-linked immunosorbent assay. RESULTS: NF-kappaB was increased in the nuclear extracts as early as 30 min after rewarming. There was a marked increase in the IL-8 mRNA expression at 1 and 3 hr after rewarming. IL-8 concentration in the cell culture medium was progressively increased during 20 hr following rewarming. The cell culture medium inhibited apoptosis of neutrophils significantly. The cold/rewarming-induced IL-8 production was reduced to approximately 50% by introducing an antisense oligonucleotide for the p65 subunit of NF-kappaB and by treatment with N-acetyl-leucinyl-leucinyl-norleucinal and pyrrolidine dithiocarbamate. The effect of dexamethasone treatment was dose dependent (reduced to approximately 30% at 10-5 M dexamethasone). CONCLUSIONS: Our results indicate that rewarming of cold-preserved alveolar epithelial cells itself may be an important initiator of the inflammatory cascades, including NF-kappaB activation and IL-8 release. Inhibition of NF-kappaB would be worth trying to control unnecessary IL-8 production and the inflammatory response in the donor lungs.

Apoptosis↗
PubMed2006

Exploring time-dependent structural changes during the cold crystallization process of isotactic polystyrene by infrared spectroscopy and multivariate curve resolution.

The present study attempts an application of Fourier transform infrared (FT-IR) spectroscopy in conjunction with multivariate curve resolution (MCR) techniques to explore the structural evolution of isotactic polystyrene (iPS) during the cold crystallization process. The focus of the present study is placed on the performance of MCR techniques, e.g., orthogonal projection (OP), alternating least squares (ALS), and fixed-size moving window evolving factor analysis (FSMWEFA), and the interpretability of spectral changes in the investigated chemical process. As a result, valuable information and conclusions about the structural evolution of iPS during the crystallization process can be extracted: when the amorphous phase of iPS changes, the ordering of the phenyl rings takes place first, and then the polymer chains adjust their local conformations to form short 3(1) helix structures. Furthermore, according to intensity profiles of the spectral variations, the ordering of the phenyl rings proceeds more intensely than the formation of ordered local chains, and the structural evolution of iPS occurs even during the induction period. The spectral variations resulting from the conformational changes in the 3(1) helical structures depend on the sequence length of the helical chains: the longer the polymer chain is, the smaller the corresponding band variations are. It has been demonstrated that the combination of FTIR spectroscopy and chemometric MCR techniques is very promising for the analysis of the crystallization process of polymers. MCR is a powerful tool for analyzing and visualizing spectral data and integrating them with other information, making spectral intensity variations more amenable to interpretation in order to explore the molecular dynamics of polymers.

Algorithms↗
PubMed1996

Prediction and evaluation of side-chain conformations for protein backbone structures.

A common approach to protein modeling is to propose a backbone structure based on homology or threading and then to attempt to build side chains onto this backbone. A fast algorithm using the simple criteria of atomic overlap and overall rotamer probability is proposed for this purpose. The method was first tested in the context of exhaustive searches of side chain configuration space in protein cores and was then applied to all side chains in 49 proteins of known structure, using simulated annealing to sample space. The latter procedure obtains the correct rotamer for 57% and the correct chi 1 value for 74% of the 6751 residues in the sample. When low-temperature Monte-Carlo simulations are initiated from the results of the simulated-annealing processes, consensus configurations are obtained which exhibit slightly more accurate predictions. The Monte-Carlo procedure also allows converged side chain entropies to be calculated for all residues. These prove to be accurate indicators of prediction reliability. For example, the correct rotamer is obtained for 79% and the correct chi 1 value is obtained for 84% of the half of the sample residues exhibiting the lowest entropies. Side chain entropy and predictability are nearly completely uncorrelated with solvent-accessible area. Some precedents for and implications of this observation are discussed.

Algorithms↗
PubMed2004

Elucidation of polysaccharide origin in Ramalina peruviana symbiosis.

A structural elucidation of polysaccharides extracted from the aposymbiotically cultured mycobiont of the lichen Ramalina peruviana was carried out in order to determine whether the polysaccharides found previously in the symbiotic thalli are produced by the mycobiont or photobiont or both. The mycobiont isolate was cultivated on a solid malt-yeast extract-medium and the freeze-dried colonies were defatted and the polysaccharides extracted successively with hot water and aq. 2% KOH, each at 100 degrees C. The alkaline extract was obtained in much higher yield (31.5%) and submitted to a freeze-thawing treatment, giving rise to a precipitate (PK2) of a mixture of (1-->3),(1-->4)-alpha-glucan (1.2:1 ratio, nigeran) and a (1-->3)-beta-glucan (laminaran). The mother liquor was treated with Fehling solution to give a precipitate (galactomannan). This had a (1-->6)-linked alpha-d-mannopyranosyl main chain, substituted at O-4 and in small proportion at O-2,4 by beta-Galp units. All three polysaccharides have previously been found in the symbiotic thalli of R. peruviana, showing that these are produced by the fungus, without the participation of the Trebouxia photobiont. Surprisingly, isolichenan, a cold-water soluble (1-->3),(1-->4)-alpha-linked-glucan (3:1 ratio) was not found in the isolated mycobiont, despite being the main polysaccharide found in the thalli.

Ascomycota↗
PubMed2005

Telomerase activity as a potential diagnostic marker for triage of abnormal Pap smears.

OBJECTIVES: To determine whether there is an association between high levels of telomerase and premalignant cervical disease and to provide a preliminary analysis of telomerase activity as a potential triage strategy. MATERIALS AND METHODS: Premenopausal women were invited to participate in the study during routine gynecologic visits as well as visits where colposcopy was performed. Samples were taken from the cervix using a broom device and placed in cold phosphate-buffered saline. A total of 92 samples were evaluated. Cells were counted and lysed, and a semiquantitative measure of telomerase activity was determined using a commercially available telomerase enzyme-linked immunosorbent assay kit. The presence of human papillomavirus (HPV) types 16 and 18 was assessed by polymerase chain reaction analysis. One-way analysis of variance was used to test for the association of telomerase activity with cytology, HPV type 16 or 18 status, and colposcopy and/or biopsy findings. RESULTS: When telomerase levels were analyzed according to Pap smear results, there were no differences among four groups of cytology findings (normal, atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesion, and high-grade squamous intraepithelial lesion). When colposcopy and/or biopsy results were considered, significantly higher levels of telomerase were detected in cervical intraepithelial neoplasia (CIN) 2,3 samples than in normal Pap smear samples and CIN 1 samples (p = .035). There was no significant difference in telomerase levels between samples that tested positive for HPV type 16 or 18 and those that did not (p = .111). CONCLUSIONS: Telomerase levels were significantly higher in cytologic samples from women with biopsy-proven CIN 2,3 than in samples from women with normal cytology results or CIN 1. These results warrant larger studies to determine whether telomerase activity may be a useful triage tool for abnormal cytologic findings.

Adolescent↗
PubMed1999

Loss of bactericidal capacity of long-chain quaternary ammonium compounds with protein at lowered temperature.

Amphiphilic betaine esters are quaternary ammonium compounds (QACs) with rapid microbicidal effect, which spontaneously hydrolyze into nontoxic products. thus being referred to as soft antimicrobial agents. The bactericidal effect of 1-decyl (B10), 1-dodecyl (B12), and 1-tetradecyl (B14) betaine esters on Salmonella typhimurium was strongly influenced by temperature, pH and length of hydrocarbon chain. At pH 6.0, presence of 1.5 mM (10% w/v) BSA raised the concentration of B14 for 99% killing (BC2) from 0.006 mM to 1.8 mM. There was a stoichiometric relationship between concentration of BSA and BC2 of B14, indicating that one molecule of B14 was bound per BSA molecule when 99% killing was achieved. When the temperature was lowered to 0 degrees C only minor killing was seen in 1.5 mM BSA at the highest concentration of B14 tested, 57 mM. With B10 at 30 degrees C and pH 6.0, the presence of 1.5 mM BSA raised the bactericidal concentration (BC2) from 0.69 mM to 4.1 mM, and at 0 degrees C and 1.5 mM BSA the BC2 was 11 mM. Thus, the impairment caused of the bactericidal effect of B10 by BSA and lower temperature was less than for B14, since B14 is much more active than B10 at 30 degrees C in the absence of BSA, somewhat more active than B10 at 30 degrees C in the presence of 1.5 mM BSA, and much less active than B10 at 0 degrees C in the presence of BSA. B12 showed properties intermediate between B10 and B14. Lowered pH reduced the bactericidal effect particularly when reduced from pH 5.0 to 4.0 with B10. In the presence of 1.5 mM BSA, the bactericidal effect of 1-dodecyl (DTAB) and 1-hexadecyl (CTAB) trimethylammonium bromide decreased in the same manner as for B10 and B14, respectively. Increasing the time of incubation at 0 degrees C to 50 min, a 99% killing effect was seen with 17 mM CTAB, whereas the same killing effect was reached in 8 min with 17 mM DTAB. Binding of [3H]CTAB to S. typhimurium was also reduced at 0 degrees C in the presence of BSA. Thus, in the presence of 1.5 mM BSA, QACs with the longer hydrocarbon chain were most efficient at 30 degrees C, whereas at 0 degrees C those with the shorter hydrocarbon chain were most active. Consequently, QACs with shorter tails should be used for disinfection in the presence of proteins at lower temperatures.

Cetrimonium↗
PubMed2005

Cold preservation of islets in UW solution--with special reference to apoptosis.

BACKGROUND: Apoptosis progresses in cultured islets. Little is known with regard to apoptosis under cold preservation. We examined viability and function of islets in University of Wisconsin (UW) solution. MATERIALS AND METHODS: Isolated rat islets were cultured overnight (overnight group) and further treated with 7-day culture in RPMI 1640 medium at 37 degrees C (culture group) or 7-day preservation in UW solution at 4 degrees C (preservation group). They were evaluated by glucose-stimulated insulin secretion test. Apoptosis was examined by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Expression of caspase mRNA and the ratio of Bax to Bcl-2 were evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: Islet recovery after 7 days was significantly lower in culture group than in preservation group (44.0 +/- 3.7% versus 75.0 +/- 4.9%, P < 0.05). The stimulation index in the culture group was significantly lower than in the overnight group (2.1 +/- 0.2 versus 4.1 +/- 0.4, P < 0.05). The apoptotic index in the culture group was significantly higher than both in the overnight group and in the preservation group (38.0 +/- 3.0% versus 10.8 +/- 2.0 and 27.0 +/- 4.0%, P < 0.05). Caspase 3, 8, and 9 mRNA in the culture group expressed more than in the other groups. Bax/Bcl-2 in the culture group was significantly lower than in the overnight group (3.2 +/- 0.66 versus 8.1 +/- 0.95, P < 0.05), suggesting that apoptosis had been already destined early after isolation. CONCLUSIONS: The preservation group showed better recovery and function than the culture group. Apoptosis contributed to islet loss under culture and it was significantly suppressed under cold preservation.

Adenosine↗
PubMed2006

Protective role of 3-nitro-N-methyl-salicylamide on isolated rat heart during 4 hours of cold storage and reperfusion.

OBJECTIVE: Cardiac ischemia/reperfusion (I/R) injury, a necessary consequence of transplantation, is probably related to the formation of reactive oxygen species (ROS). The ROS burst within the first moments of reperfusion is associated with injury, continuously generate O2- at about 3% to 5% of total O2 consumption owing to electron leak by mitochondrial oxidoreductases, especially complexes I and III. 3-nitro-N-methyl-salicylamide (NNMS) displays inhibitory effects on succinate-cytochrome C reductase, but also reduces effects on creation of O2- radical and H2O2 by isolated rat mitochondria. Presumably NNMS inhibits electron leakage from the mitochondrial respiratory chain. We investigated effect of NNMS on heart protection after hypothermic ischemia. METHODS: A Langendorff-prepared rat heart model was employed after the heart had been preserved for 4 hours under hypothermic conditions of ischemia with subsequent reperfusion/rewarming for 60 minutes. RESULTS: The group of hearts treated with NNMS showed increased recovery of heart function compared with a group of mEC. The lactate dehydrogenase (LDH) activity in coronary flow (CF) by hearts treated with NNMS was lower than that with mECs, as was the content of malonedialdehyde (MDA) and conjugated diene (CD). CONCLUSIONS: NNMS improved heart physiology after reperfusion following 4 hours of hypothermic ischemia.

Animals↗
PubMed2002

Deficiency of a cytosolic ascorbate peroxidase associated with chilling tolerance in soybean.

We investigated the isozyme profiles of antioxidant enzymes in cultivars and lines with different seed productivity in cool climate conditions as a step towards understanding the physiological and genetical mechanisms underlying chilling tolerance in soybean. While no difference in superoxide dismutase, or catalase isozyme profiles was observed among the cultivars and lines tested, we found polymorphism in the ascorbate peroxidase isozyme profile; there were two types, with or without a cytosolic isoform (APX1). The cultivars and lines lacking APX1 proved more tolerant to chilling temperatures, as evaluated by yielding ability. The genotype-dependent deficiency of APX1 was consistent in plants and tissues under various oxidative stress conditions including the exposure to low-temperatures. In addition, the genetic analysis of progeny derived from crossing between cultivars differing in the isozyme profile indicated that the APX1 deficiency is controlled by a single recessive gene (apx1), and is inherited independently of the genes that have previously been identified for their association with chilling tolerance. Molecular and linkage analyses suggested that the variant gene of the APX1-absent genotype coding for a cytosolic APX, which contained a single nucleotide substitution and a single nucleotide deletion in the coding region, is responsible for the genotype-dependent deficiency of APX1. The association of APX1 deficiency with chilling tolerance is discussed in detail.

Amino Acid Sequence↗
PubMed1999

PCR-based 16S ribosomal DNA detection technique for Clostridium estertheticum causing spoilage in vacuum-packed chill-stored beef.

Chill stored vacuum-packaged meat is sometimes spoiled by psychrotrophic or psychrophilic clostridia. Clostridium estertheticum was first isolated from vacuum-packed beef from southern Africa, but has recently been found in beef originating from northern Europe. This organism is difficult to isolate using conventional methods, and two PCR-based methods have been devised for use in measures to control the bacterium in the abattoir and to study its ecology. In the first method, primers were designed having a high annealing temperature of 65 degrees C to increase specificity, producing a PCR product of 567 bp from the 16S rDNA. Two species of Enterobacteriaceae found in meat cross-reacted in this test, and so it was necessary to use a second step, digesting the PCR product with two restriction enzymes. Subsequently a further set of primers was designed, producing a PCR product of 641 bp, and using an annealing temperature of 60 degrees C. The second procedure was more specific and did not require subsequent restriction analysis of the PCR product. The two sets of primers appeared to have similar sensitivity, detecting 10-100 cells of C. estertheticum in broth, meat or meat purge (drip). A semiquantitative method is described for estimating numbers of the target bacterium.

Animals↗