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PubMed1972

Ribosome crystallization in chicken embryos. II. Conditions for the formation of ribosome tetramers in vitro.

Slow cooling of fertilized chicken eggs permits the elongation and termination of nascen(t) polypeptides in the polysomes but prevents the initiation of new protein chains. This leads to polysome disaggregation during the first 30 min of cooling, and to the formation, of a pool of inactive ribosomes prone to crystallization. After 2 hr these ribosomes began to form tetramers, which do not contain any labeled proteins synthesized during cooling. If protein synthesis is inhibited by cycloheximide, added to eggs before cooling, tetramer formation in the embryos is prevented. Puromycin, on the other hand, leads to polysome disassembly and does not prevent tetramer formation. Rapid cooling of explanted embryos after short incubation at 37 degrees C, with or without cycloheximide, largely prevents polysome disaggregation and the formation of tetramers. On the other hand, the addition of puromycin to explanted embryos promotes tetramer formation after rapid cooling. When cooled eggs are rewarmed, tetramers are disassembled into monomers, even if protein synthesis is inhibited. When those embryos were rapidly recooled tetramers reformed spontaneously from tetramer-derived monomers, even in the presence of cycloheximide. We conclude that the formation of tetramers at low temperature is an inherent property of the normal ribosomes.

Animals↗
PubMed2001

Glistening formation in an AcrySof lens initiated by spinodal decomposition of the polymer network by temperature change.

PURPOSE: To examine the effect of temperature change on the structural alteration and glistening formation in an AcrySof intraocular lens (IOL). SETTING: Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, Kobe, Japan. METHODS: The equilibrium water content of a Wagon-Wheel-packaged AcrySof IOL was determined at various temperatures by the gravimetrical method. Alterations in the microscopic structure of the lens were analyzed in situ with a reflecting microscope without staining, while the temperature of the lens was changed at different rates. RESULTS: Microvacuoles of 1.0 to 20.0 microm formed in the bulk of a swollen AcrySof IOL as the temperature decreased. The morphology of the vacuoles was similar to that of glistening particles observed in vivo. The size and density of the glistening-like vacuoles varied depending on the magnitude and rate of the temperature decrease. The glistening-like vacuoles were imprinted in the bulk of the IOL during the preceding cooling process in aqueous humor. CONCLUSIONS: Glistenings formed in the bulk of an AcrySof IOL as the medium temperature decreased. The mechanism may involve spinodal decomposition of the swollen polymer network, which initiates the formation of microvacuoles consisting of water and loosely packed network chains.

Acrylic Resins↗
PubMed1984

Isolation of small molecular forms of Factor VIII/von Willebrand factor from plasma.

Cryoprecipitated factor VIII/von Willebrand factor (FVIII/vWF), freed of fibrinogen by clotting with calcium and Defibrase, was chromatographed on Sepharose CL-2B. Fractions containing lower-molecular-weight forms of FVIII/vWF comprised coprecipitated plasma proteins of similar molecular weights. The major contaminants, fibronectin and IgM, were removed by affinity chromatography on gelatin- and anti-IgM-agarose, respectively. Finally, pure low-molecular-weight FVIII/vWF protein was harvested in the void volume fraction of a Sepharose CL-6B column. The smallest multimers had the size of the tetramer of the basic subunit chain of FVIII/vWF.

Blood Coagulation Factors↗
PubMed1984

Conformation and packing of unsaturated chains in crystals of cholesteryl nervonate at 123 K.

At 123 K, cholesterol cis-15-tetracosenoate (cholesteryl nervonate, C51H90O2) is monoclinic, space group P2(1) with a = 12.948(5), b = 8.805(5), c = 42.98(5) A, beta = 105.93(3) degrees, [lambda(CuK alpha) = 1.5418 A], having two independent molecules (A) and (B) in the unit cell. The crystal structure at 123 K has been determined from 6329 reflections with sin theta/lambda less than 0.54 A-1, of which 1990 gave I greater than 2 sigma(I). Structure refinement by Fourier methods and block diagonal least squares gave R = 0.232 for all reflections, R = 0.135 for those with I greater than 2 sigma(I). The crystal structure consists of layers in which there is close packing of cholesteryl groups and the proximal segments of the ester chains. This layer structure occurs also in cholesteryl palmitoleate and the alkanoate esters C9 through C12. Because the nervonate chains are longer, they become aligned in the interface region between layers, but without a regular subcell structure being established. The nervonate (A)-chain is almost extended, while the (B)-chain has two bends, one in the saturated region. Both chains have complex dislocations at the cis-double bond.

Cholesterol Esters↗
PubMed1987

Dexamethasone impairs growth and collagen synthesis in condylar cartilage in vitro.

The in vitro effects of dexamethasone on condylar cartilage from normal newborn mice were tested by measuring protein and DNA content, collagen synthesis, prolyl hydroxylase activity, collagen chains and by immunofluorescence the localization of type I and II collagen and fibronectin. The biochemical assays were complemented by structural studies of hormone-treated and control cultured specimens. It became apparent that both the protein and DNA content of the tissue decreased immediately following the addition of dexamethasone of the incubation system. Protein synthesis was significantly decreased by the hormone by 24 h. The degree of collagen hydroxylation was decreased by 24 h. Dexamethasone-treated condyles did not reveal a significant increase in the percentage of cold hydroxyproline of the total protein. Using the indirect immunofluorescence method, hormone-treated condyles revealed an enhancement of positive reactivity for type I collagen and fibronectin. SDS-gel electrophoresis of 3H-labeled collagen chains isolated by CM-cellulose chromatography indicated that dexamethasone did not significantly affect the ratios of the collagen chains. For further characterization, each chain was subjected to cyanogen bromide cleavage showing that the peptide maps of alpha 1(I) and alpha 1(II) chains were not different in dexamethasone-treated tissues in comparison to controls.

Animals↗
PubMed1970

Controlled proteolysis of nascent polypeptides in rat liver cell fractions. I. Location of the polypeptides within ribosomes.

Free ribosomes containing nascent polypeptide chains labeled in vitro were submitted to proteolysis at 0 degrees by a mixture of trypsin and chymotrypsin. Sucrose gradient analysis showed that polysome patterns are retained even after 24 hr of proteolysis in the cold, while messenger RNA-free ribosomes (generated progressively during in vitro incorporation) are, within 2 hr, completely dissociated into subunits by trypsin. Although ribosomes and subunits are not extensively degraded into smaller fragments during low temperature proteolysis, changes in the acrylamide gel electrophoresis pattern showed that most ribosomal proteins are accessible to and are partially degraded by the proteases. Ribosome-bound nascent polypeptides are partially resistant to proteolysis at 0 degrees , although they are totally digested at 37 degrees or when the ribosomal subunit structure is disrupted by other means. Radioactivity incorporated into nascent chains during incubation times shorter than 3 min was mostly resistant to digestion at 0 degrees . A larger fraction of the initial radioactivity became degraded in ribosomes which incorporated for longer times. In these ribosomes, the amount of radioactivity which was resistant to proteolysis was constant and independent of the initial value, which reflects the labeled length of the nascent chains. These results suggest that the growing end of the nascent polypeptide is resistant to digestion and is protected from proteolytic attack by the ribosomal structure. A pulse and chase experiment confirmed this suggestion, showing that the protected segment is located at the carboxy-terminal end of the nascent chain. The protected segment was contained in the large ribosomal subunit and had a length of approximately 39 amino acid residues, as estimated by chromatography on Sephadex G-50.

Animals↗
PubMed1988

Vasoconstrictor response to cold in forestry workers: a prospective study.

In a five year prospective study of the vasoconstrictor response to cold 37 forestry workers were investigated in 1978 and again in 1983. The subjects were classified into three groups: group A (n = 13): no subjective finger symptoms in 1978 and continued sawing until 1983; group B (n = 12): no symptoms in 1978 and stopped sawing before 1983; group C (n = 12): vibration induced white finger (VWF) in 1978. A cold provocation test measuring the finger systolic blood pressure with a cuff and strain gauge technique during combined body cooling and finger cooling to 30 degrees, 15 degrees, and 6 degrees C was applied to all subjects at both investigations. In 1978 all groups had an increased cold response when compared with that of 20 non-exposed controls (p less than 0.05), and the response was more exaggerated in group C than in groups A and B (p less than 0.01). From 1978 to 1983 the vasoconstrictor response increased in group A (p less than 0.05), was in group B (p greater than 0.10), and improved in group C (p less than 0.05). The changes in cold response in groups A and C were significantly larger than the within day variation of the method (p less than 0.01). The aggravation of group A indicates that a further five years use of anti-vibration chain saws induced an increased hyperreactivity in the vasoconstrictor mechanisms but led to subjective symptoms of VWF in only one subject. The improvement in group C was in accordance with the improvements of subjective finger symptoms and was associated with a previous shift from saws without vibration damping devices to anti-vibration saws and with cessation of work. The results of group C indicate that VWF may improve or subjectively disappear if exposure to vibration is reduced or stopped. The cold provocation test was more sensitive than the Taylor-Pelmear stage assessments (p<0.05). The test may be of guidance in evaluation preventive measures.

Adolescent↗
PubMed2000

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers.

Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells. day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system.

Amino Acids, Branched-Chain↗
PubMed1999

An in vitro system using HeLa cytoplasmic extracts that reproduces regulated mRNA stability.

The pathways and machinery involved in the regulated turnover of mRNAs in mammalian cells are largely unknown. We have developed an in vitro system using HeLa cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that faithfully reproduces in vivo aspects of regulated mRNA turnover. RNA substrates for use in the system that contain a poly(A) tail precisely at their 3' end can be readily prepared using a ligation-polymerase chain reaction approach. The system also uses standard cytoplasmic S100 extracts that are activated through the sequestration of poly(A)-binding proteins by the addition of cold poly(A) RNA. On incubation in the system, the poly(A) tail is removed from RNA substrates by a sequence-specific deadenylase activity and the body of the transcript is ultimately degraded in the system with no apparent intermediates by an ATP-dependent ribonulceolytic activity. AU-rich destability elements can regulate the rates of both deadenylation and degradation in the system. This in vitro system, therefore, should allow the elucidation of pathways of mRNA turnover, identification of the cellular factors involved, and insights into the mechanisms that regulate the half-life of a mRNA.

Cell-Free System↗
PubMed2006

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as -26 degrees C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.

Acclimatization↗
PubMed2006

Two new Tetramitus species (Heterolobosea, Vahlkampfiidae) from cold aquatic environments.

Characterisation of the protists of cold environments provides important background for assessing the effects of climate change on microbial communities. Tetramitus angularis n. sp., from aquatic environments in Iceland and Switzerland, is the first vahlkampfiid recognised to have a characteristic Tetramitus flagellate stage combined with pre-formed excystment pores, which are not typical of this genus. T. angularis amoebae have a typical vahlkampfiid locomotive form and contain prominent lipid inclusions. Flagellates have a collar and cytostome, and can be mono- to multi-nucleate with corresponding change in cell shape from cylindrical to ellipsoidal and variable number of flagella. Cysts are round to semi-angular and have 2-5 pores closed by protruding, translucent plugs. A second organism, T. parangularis n. sp. from Alaska, has similar cysts but a flagellate stage has not been recognised; ITS sequence divergence is consistent with species criteria in the Vahlkampfiidae. Phylogenetic analysis of sequence data for the 5.8S rDNA region clusters the new spp. with T. rostratus, T. entericus and T. waccamawensis.

Alaska↗
PubMed2004

Listeria monocytogenes isolated from cold-smoked fish products in Osaka City, Japan.

Listeria monocytogenes contamination of ready-to-eat seafood products commercially available in Osaka was examined between 1999 and 2000. L. monocytogenes was isolated from 12 (13%) of the 95 products tested. All positive samples were from cold-smoked fish with 9 being obtained during the summer. Thirteen isolates of L. monocytogenes were typed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR)-based typing methods. Isolates of the same serotype originating from the same manufacturer gave similar DNA profiles, irrespective of the type of sample or date of isolation. The finding suggest that persistent strains in each manufacturing facility proliferate during the summer and contaminate products during manufacturing processes.

Animals↗
PubMed1989

Thermal stabilities of globular proteins.

Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the dependence on temperature. Folding is assumed to be driven by solvophobic interactions and opposed by the conformational entropy. The temperature dependence of the solvophobic interaction is taken from the transfer experiments on amino acids by Tanford and Nozaki and on model solutes by Gill and Wadsö. One long-standing puzzle has been why proteins denature upon heating, since the solvophobic force to fold strengthens with increasing temperature. This is resolved by the theory, which predicts two first-order phase transitions. "Cold denaturation" is driven principally by the weakening of the solvophobic interaction, but normal denaturation is driven principally by the gain of conformational entropy of the chain. Predictions of the thermodynamic state functions are in reasonable agreement with the calorimetric experiments of Privalov and Khechinashvili. Comparison of the theory with experiments suggests that there may be an additional enthalpic driving force toward folding which is not due to the solvophobic interactions.

Drug Stability↗
PubMed1978

Immunoglobulin genes in DNA restriction fragments.

We have investigated the organization of immunoglobulin genes in mice. High molecular weight DNA from myelomas and Krebs ascites cells was cleaved with EcoRI restriction endonuclease and fractionated using preparative agarose gel electrophoresis. Each fraction was then hybridized to an immunoglobulin mRNA or a cDNA transcribed from the mRNA. In two series of experiments, one with a kappa chain probe (MOPC 41 mRNA), the other with a lambda chain probe (SAPC 178 mRNA), we analyzed a variety of myeloma DNAs and Krebs DNA. In contrast to previously reported findings (Tonegawa, S., et al. (1976) Cold Spring Harbor Symp. Quant. Biol. 41, 877), we did not observe any unique restriction map pattern in the DNA from cells which exress a given immunoglobulin gene. We also found that restriction fragments containing c region genes do not appear to transpose, while DNA sequences corresponding to other portions of the kappa and lambda mRNAs do in some cases.

Animals↗
PubMed2002

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.

Aldose-Ketose Isomerases↗
PubMed2002

PCR detection of psychrotolerant clostridia associated with deep tissue spoilage of vacuum-packed chilled meats.

AIMS: To develop a practical molecular procedure that directly (without isolation) and specifically detects the presence of clostridia, which cause the deep tissue spoilage condition. METHODS AND RESULTS: A primer set was designed and a PCR amplification procedure developed to detect the presence of Clostridium algidicarnis and Cl. putrefaciens 16S rDNA gene fragments in meat. The procedure yielded amplicons of the expected size with homologous DNA templates, but failed to give PCR products with DNAs from 47 food clostridia and common meat spoilage micro-organisms. The minimum level of detection was 10(4) cfu g-1 for nonenriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, the presence of Cl. algidicarnis and/or Cl. putrefaciens was confirmed in a swab sample taken from the cartilage of an ovine stifle joint, which on opening exhibited strong offensive odours. CONCLUSIONS: The developed method can be used for rapid detection of clostridia causing deep tissue spoilage in commercial vacuum packs. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of deep tissue clostridial spoilage in commercial vacuum-packed chilled meats.

Animals↗
PubMed2003

Cold storage affects survival and growth of Erwinia amylovora on the calyx of apple.

AIMS: To determine the effect of cold storage on the survival of Erwinia amylovora. METHODS AND RESULTS: The survival of E. amylovora was assessed during storage at 2 degrees C. Populations of E. amylovora inoculated into phosphate-buffered saline remained static, whereas in nutrient media populations increased at low temperatures. In contrast, populations of E. amylovora on tissue in the apple calyx decreased during cold storage. CONCLUSIONS: Erwinia amylovora has the ability, in nutrient media, to multiply at low temperatures. However, populations of E. amylovora on tissue in the apple calyx decrease with the time spent in cold storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Cold storage of apples will provide assurance that mature fruit from orchards, free of fire blight, or even with low levels of fire blight, may be exported with a negligible risk of introducing the disease into countries free of fire blight.

Cold Temperature↗