CspA, CspB, and CspG, major cold shock proteins of Escherichia coli, are induced at low temperature under conditions that completely block protein synthesis.
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CspA, CspB, and CspG, the major cold shock proteins of Escherichia coli, are dramatically induced upon temperature downshift. In this report, we examined the effects of kanamycin and chloramphenicol, inhibitors of protein synthesis, on cold shock inducibility of these proteins. Cell growth was completely blocked at 37 degrees C in the presence of kanamycin (100 microgram/ml) or chloramphenicol (200 microgram/ml). After 10 min of incubation with the antibiotics at 37 degrees C, cells were cold shocked at 15 degrees C and labeled with [35S]methionine at 30 min after the cold shock. Surprisingly, the synthesis of all these cold shock proteins was induced at a significantly high level virtually in the absence of synthesis of any other protein, indicating that the cold shock proteins are able to bypass the inhibitory effect of the antibiotics. Possible bypass mechanisms are discussed. The levels of cspA and cspB mRNAs for the first hour at 15 degrees C were hardly affected in the absence of new protein synthesis caused either by antibiotics or by amino acid starvation. (+info)
Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions.
(18/6450)
The most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is > or = 50 microM during early exponential growth at 37 degrees C; therefore, its designation as a major cold-shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-to-late exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5' untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA). (+info)
An ordered metastable phase in hydrated phosphatidylethanolamine: the Y-transition.
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By using time-resolved X-ray diffraction, differential scanning calorimetry and scanning densitometry, we observed rapid formation at low temperature of a metastable ordered phase, termed LR1 phase, in fully hydrated dihexadecylphosphatidylethanolamine (DHPE). The LR1 phase has the same lamellar repeat period as the gel Lbeta phase but differs from the latter in its more ordered, orthorhombic hydrocarbon chain arrangement. It forms at about 12 degrees C upon cooling and manifests itself as splitting of the sharp, symmetric wide-angle X-ray peak of the DHPE gel phase into two reflections. This transition, designated the 'Y-transition', is readily reversible and proceeds with almost no hysteresis between cooling and heating scans. Calorimetrically, the LR1-->Lbeta transition is recorded as a low-enthalpy (0.2 kcal/mol) endothermic event. The formation of the LR1 phase from the gel phase is associated with a small, about 2 microl/g, decrease of the lipid partial specific volume recorded by scanning densitometry, in agreement with a volume calculation based on the X-ray data. The formation of the equilibrium Lc phase was found to take place from within the LR1 phase. This appears to be the only observable pathway for crystallisation of DHPE upon low-temperature incubation. Once formed, the Lc phase of this lipid converts directly into Lbeta phase at 50 degrees C, skipping the LR1 phase. Thus, the LR1 phase of DHPE can only be entered by cooling of the gel Lbeta phase. The data disclose certain similarities between the low-temperature polymorphism of DHPE and that of long-chain normal alkanes. (+info)
Investigation of the most effective provocation test for patients with coronary spastic angina: usefulness of accelerated exercise following hyperventilation.
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This study sought to compare the clinical usefulness of the hyperventilation plus cold stress test or the hyperventilation combined with accelerated exercise test with other single tests in patients with coronary spastic angina. The study examined 24 patients (23 men, mean age 66 years) with angiographically confirmed coronary spastic angina and less than 50% stenosis. Moreover, none had spontaneous ST segment elevation before the study. Under no medication for at least 24 h prior, 4 procedures were performed from 09.00 h to 11.00 h: (i) a hyperventilation test for 5 min (HV(5)); (ii) HV(5) combined with a cold stress test for the last 2 min (HV(5)+CS(2)); (iii) a treadmill exercise test based on Bruce's protocol (TM(3)); and (iv) a treadmill exercise test accelerated at 1 min intervals according to Bruce's protocol immediately after HV(5) (HV(5)+TM(1)). The rate of appearance of chest pain and ischemia-induced ECG changes due to HV(5)+TM(1) were significantly higher than the other 3 tests. HV(5)+CS(2) was not superior to HV(5) alone. The incidence of provoked ST segment elevation due to HV(5)+TM(1) was higher than with the other 3 procedures. Thus, in patients with coronary spastic angina, no spontaneous ST segment elevation and near normal coronary arteries, HV(5)+CS(2) was no more useful than HV(5) alone. It is recommended that the newly designed HV(5)+TM(1) combination test be used for documenting evidence of ischemia in patients with coronary spastic angina, low disease activity and near normal coronary arteries. (+info)
Cluster headache-like attack as an opening symptom of a unilateral infarction of the cervical cord: persistent anaesthesia and dysaesthesia to cold stimuli.
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A 54 year old man experienced excruciating left retro-orbital pain with lacrimation and redness of the eye representative of a cluster headache attack. This was followed by left hemiparesis with plegia of the lower limb and left Horner's syndrome. Five days later the hemiparesis recovered while the patient developed hypoanaesthesia to cold stimuli that evoked painful burning dysaesthesia on the right side below the C4 level. MRI disclosed a discrete infarct in the left lateral aspect of the cord at C2 level concomitant to a left vertebral artery thrombosis. This limited infarct and the clinical symptoms suggest a hypoperfusion in the peripheral arterial system of the left hemicord, supplied both by the anterior and posterior spinal arteries. Cluster headache-like attack and persistent dysaesthesia to cold stimuli are discussed respectively in view of the central sympathetic involvement and partial spinothalamic system dysfunction. (+info)
von Willebrand factor contained in a high purity FVIII concentrate (Fanhdi) binds to platelet glycoproteins and supports platelet adhesion to subendothelium under flow conditions.
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BACKGROUND AND OBJECTIVE: There is evidence suggesting that von Willebrand factor (VWF) from high purity factor VIII concentrates could be of clinical use in the management of patients suffering from VWD. We analyzed structural and functional characteristics of VWF present in a high purity factor VIII concentrate VWFHPC (Fanhdi). The multimeric structure, the ability to bind to platelet GP Ib/IX or GP IIb/IIIa, and the capacity of VWFHPC to promote platelet adhesion on injured vessels were investigated and compared with that present in standard plasma cryoprecipitates [VWFCRYO]. DESIGN AND METHODS: Binding studies were carried out by incubating radiolabeled VWF and washed platelets, which were activated with either ristocetin (1 mg/mL; for GP Ib/IX), or thrombin (2.5 U/mL; for GP IIb/IIIa). Platelet adhesion was assessed in a perfusion system (shear rate = 800 s-1, 10 min) in which the source of VWF was added (at 0.4 or 0.8 U/mL VWF:Ag) to washed platelets and red cells suspended in a human albumin solution. The deposition of platelets onto the perfused subendothelial surface was morphometrically evaluated and expressed as percentage of surface coverage (%SC). RESULTS: The VWFHPC (152 Units VWF:RCof/mg protein; VWF:RCof/VWF:Ag = 0.97), lacked only a small proportion of high-molecular-weight multimers present in VWFCRYO. Binding affinities (Kd values, nM) of VWFHPC were similar to those of VWFCRYO (5.3 +/- 0.86 vs 5.2 +/- 0.95, for GP Ib/IX; and 11.6 +/- 2.7 vs 15.4 +/- 1.7 for GPIIb-IIIa). A slightly, though not significantly, higher binding capacity for these receptors (Bmax values, molecules/pit) was obtained for VWFHPC. The %SC in perfusions in the presence of albumin was < 10%. Addition of VWFHPC or VWFCRYO significantly increased the %SC, with values of 27.1 +/- 4.9 and 17.5 +/- 2.8%, respectively with 0.4 U/mL (p < 0.004 and p < 0.02 vs albumin); and 30.8 +/- 4.9% and 20.03 +/- 4.1%, respectively, at 0.8 U/mL (p < 0.001 and p < 0.02 vs albumin). INTERPRETATION AND CONCLUSIONS: Our data show that VWF present in the high purity FVIII concentrate Fanhdi retains the functional capacity to bind to GPs Ib/IX and IIb/IIIa and to promote platelet adhesion onto exposed subendothelium. (+info)
Effect of cold shock on lipid A biosynthesis in Escherichia coli. Induction At 12 degrees C of an acyltransferase specific for palmitoleoyl-acyl carrier protein.
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Palmitoleate is not present in lipid A isolated from Escherichia coli grown at 30 degrees C or higher, but it comprises approximately 11% of the fatty acyl chains of lipid A in cells grown at 12 degrees C. The appearance of palmitoleate at 12 degrees C is accompanied by a decline in laurate from approximately 18% to approximately 5.5%. We now report that wild-type E. coli shifted from 30 degrees C to 12 degrees C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo2-lipid IVA. The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12 degrees C. The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear. Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo2-lipid IVA (Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase. Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase. Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid. The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA. The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12 degrees C. (+info)
Effects of temperature and preservation time on the pharmacological response of isolated vascular endothelial and smooth muscle function.
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In clinical transplantation and cardiovascular surgery, cold preservation is usually used because it is a simple method. However, the established temperature is by no means exact. The aim of this study was to find the optimum storage temperature for preservation of the vasculature by observing the pharmacological endothelium and smooth muscle response. The thoracic aorta of 36 male Wister rats were studied in organ baths: as fresh control after 24 hours, 48 hours and 72 hours of storage at 0.5 degree C, 4 degrees C and 8 degrees C in Krebs-Henseleit bicarbonate (KHB) solution. Acetylcholine (Ach) was used to elicit endothelium-dependent relaxation, and sodium nitroprusside (SNP) to elicit smooth muscle-dependent relaxation. The contractility caused by Phenylephrine (Ph) was influenced by time but before 48 hours it was not influenced by preservation temperature. Significant responsive deterioration by Ach and SNP was seen after 24 hours of storage at 0.5 degree C as compared with storage at 4 degrees C. The endothelium-dependent relaxing function and smooth muscle-dependent relaxing function were best preserved at 4 degrees C and 8 degrees C. These results indicate that precise temperature control is necessary for vessel preservation in clinical situations. (+info)