The targets for normal human serum antibodies that react with proteins c(alpha) and R4 isolated from group B streptococci (GBS; Streptococcus agalactiae) have been studied and compared with the targets for murine monoclonal and rabbit polyclonal antibodies raised against these proteins. The proteins were extracted by trypsin digestion and purified by precipitations and gel filtration and testing was based on enzyme immunoassays. The immune antibodies showed specificity for the corresponding protein, targeted that protein in Western blotting and recognized their targets after heat treatment (100 degrees C) of the proteins. Human antibodies in a commercial gammaglobulin preparation targeted a site(s) common to c(alpha) and R4. This target failed to bind the antibodies in Western blotting and was destroyed by heating. c(alpha)- and R4-reactive antibodies in sera from healthy pregnant women recognized the common, heat-labile determinant(s), but contained little or no antibodies against the heat-stable c(alpha)- or R4-specific determinants. These results are consistent with the notions that (i) the normal human antibodies and the immunization-induced animal antibodies targeted different sites on the c(alpha) and R4 proteins and that (ii) the natural human antibodies targeted conformational epitopes and the immune antibodies targeted linear epitopes. These findings are important for further clarification of GBS immunology and immunoprotection in humans.
Cold adaptation of enzymes: structural, kinetic and microcalorimetric characterizations of an aminopeptidase from the Arctic psychrophile Colwellia psychrerythraea and of human leukotriene A(4) hydrolase.
The relationships between structure, activity, stability and flexibility of a cold-adapted aminopeptidase produced by a psychrophilic marine bacterium have been investigated in comparison with a mesophilic structural and functional human homolog. Differential scanning calorimetry, fluorescence monitoring of thermal- and guanidine hydrochloride-induced unfolding and fluorescence quenching were used to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a low structural stability versus thermal and chemical denaturants and a greater structural permeability to a quenching agent relative to the mesophilic homolog. These findings support the hypothesis that cold-adapted enzymes maintain their activity at low temperatures as a result of increased global or local structural flexibility, which results in low stability. Analysis of the thermodynamic parameters of irreversible thermal unfolding suggests that entropy-driven factors are responsible for the fast unfolding rate of the cold-adapted aminopeptidase. A reduced number of proline residues, a lower degree of hydrophobic residue burial and a decreased surface accessibility of charged residues may be responsible for this effect. On the other hand, the reduction in enthalpy-driven interactions is the primary determinant of the weak conformational stability.
A comparative evaluation on the effect of carbon dioxide (CO2 ) on quality and shelf life of Atlantic salmon loins pasteurized with microwave and conventional technology was conducted. The experimental design allowed CO2 to enter the salmon muscle before (soluble gas stabilization [SGS] + vacuum) or after pasteurization (CO2 emitter + vacuum), whereas the control samples (vacuum only) were not presented for CO2 . This setup resulted in six different groups; three heated with microwaves and three with conventional pasteurization. The core temperature of microwave samples was 58.8 ± 2.2 °C, whereas the surface temperature was equal to the oven temperature (62 °C) during conventional pasteurization and close to the core temperature during microwave pasteurization (57.6 ± 1.4 °C). Microwave-heated samples showed higher microbial growth; decreased shelf life; and darker (lower L* -value), more reddish (higher a* -value), and yellowish (higher b* -value) colors compared to conventional-heated salmon. Lowest liquid loss (LL) was observed in salmon packaged with the CO2 emitter, whereas a SGS step prior to pasteurization did not affect the LL negatively as compared to samples packaged in vacuum only. Treatment with CO2 , independent of the prestep using SGS or an emitter, resulted in increased shelf life. Protein denaturation, microbial growth, product color, product shelf life, and sensory properties of the salmon loin were significantly affected by the applied pasteurization method (microwave- or conventional pasteurization). However, the heat load was probably too high to detect differences resulting from the pretreatment using SGS or packaging with CO2 emitter. PRACTICAL APPLICATION: Recent developments with increased time pressure from both work and past time activities have led to a tremendous increase in the demand for convenient, tasty ready-to-use food options. Furthermore, contemporary trends for consumption of fresh or lightly processed seafood stress the need to develop processing methods that allow a fulfillment of these demands, while still offering a reasonable shelf life. Carbon dioxide in combination with either microwave or conventional pasteurization is innovative processing technology that can meet consumer's demand of such products.
Denaturation of alpha-amylase from Aspergillus oryzae was studied under the effect of heating urea and some other denaturating agents. Inhibition in the enzyme denaturation, deviation from the first order equation and, consequently, establishment of the false equilibrium in the system are shown. The values are calculated for the reaction rate constants of alpha-amylase denaturation under the effect to heat (40 degrees C) and urea. A method is developed for isolating native amylase stabilized by heating at 40 degrees C during the period of inactivation slowing down and preservation to the 50-70% activity in the system. It is shown that in the presence of calcium ions the stability of the isolated native enzyme is 13.0 +/- 2.5% hihger on the average to heating up to 40 degrees C, 28.4 %/- 7.2% higher - to the effect of 5.5 M urea and 18.4 +/- 3.6% higher - to 18% alcohol.
Mutations at position 187 in secreted gelsolin enable aberrant proteolysis at the 172-173 and 243-244 amide bonds, affording the 71-residue amyloidogenic peptide deposited in Familial Amyloidosis of Finnish Type (FAF). Thermodynamic comparisons of two different domain 2 constructs were carried out to study possible effects of the mutations on proteolytic susceptibility. In the construct we consider to be most representative of domain 2 in the context of the full-length protein (134-266), the D187N FAF variant is slightly destabilized relative to wild type (WT) under the conditions of urea denaturation, but exhibits a T(m) identical to WT. The D187Y variant is less stable to intermediate urea concentrations and exhibits a T(m) that is estimated to be approximately 5 degrees C lower than WT (pH 7.4, Ca(2+)-free). Although the thermodynamic data indicate that the FAF mutations may slightly destabilize domain 2, these changes are probably not sufficient to shift the native to denatured state equilibrium enough to enable the proteolysis leading to FAF. Biophysical data indicate that these two FAF variants may have different native state structures and possibly different pathways of amyloidosis.
Dietary caloric restriction (CR) is the only experimental intervention that can reliably retard the development of cataract in a normal animal model. Here we have studied the possible mechanisms by which CR retards the age-related degeneration of the lens of Brown Norway rats. We have found that CR slowed protein insolubilization and blunted declines of the total soluble thiols, protein thiols, reduced glutathione and ascorbic acid levels in the lenses of old BN rats. From the lens protein point of view, the development of cataract in rat lenses has 3 stages: (1) the precipitation of gamma-crystallin, (2) the insolubilization of beta-crystallin, and (3) the final precipitation of alpha-crystallin which was saturated with other denatured lens proteins. A similar sequence is also observed when the lens proteins are subjected to oxidative stress in vitro. These data are the first to suggest that CR may retard the age-related degeneration of the lens by attenuating the oxidative stress in the lens. Since oxidative stress is likely a main cause of human cataract, CR intervention may be relevant to humans as well.
We studied the effect of changes in erythrocyte volume and irreversible thermal denaturation of cytoskeleton proteins and lipid matrix on activity of Ca(2+)-activated K+ channels in erythrocytes of alcoholic and patients with II type diabetes mellitus. Changes in Ca(2+)-dependent potassium permeability of erythrocyte membrane in alcoholic patients and patients with II type diabetes mellitus are related to modification of cytoskeleton, rather than to changes in lipid matrix.
Human phosphofructokinase (PFK) is a tetrameric enzyme, encoded by muscle, liver, and platelet genes. Deficiency of muscle PFK (PFK-M), glycogenosis type VII (Tarui disease), is an autosomal recessive disorder characterized by an exertional myopathy and hemolytic syndrome. Several disease-causing mutations have been identified in the PFK-M gene in Japanese, Ashkenazi Jewish, and Italian patients. We describe the genetic defects in French Canadian and Swiss patients with the disease, and we use a genetically well-defined yeast system devoid of endogenous PFK for structure-function studies of the mutant PFKs. A G-to-A transition at codon 209-in exon 8 of the PFK-M gene, changing an encoded Gly to Asp, is responsible for the disease in a homozygous French Canadian patient. Gly-209-mutated protein is completely inactive in the yeast system. The Swiss patient is a genetic compound, carrying a G-to-A transition at codon 100 in exon 6 (Arg to Gln) and a G-to-A transition at codon 696 in exon 22 (Arg to His). The mutants expressed in yeast generate functional enzyme with modest changes in thermal stability. The advantages and limitations of the yeast system for expression of human mutant PFKs are discussed.
Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer-susceptibility condition characterized by early onset colorectal cancer. Germ-line mutations in one of four DNA mismatch repair (MMR) genes, hMSH2, hMLH1, hPMS1, or hPMS2, are known to cause HNPCC. Although many mutations in these genes have been found in HNPCC kindreds complying with the so-called Amsterdam criteria, little is known about the involvement of these genes in families not satisfying these criteria but showing clear-cut familial clustering of colorectal cancer and other cancers. Here, we applied denaturing gradient-gel electrophoresis to screen for hMSH2 and hMLH1 mutations in two sets of HNPCC families, one set comprising families strictly complying with the Amsterdam criteria and another set in which at least one of the criteria was not satisfied. Interestingly, hMSH2 and hMLH1 mutations were found in 49% of the kindreds fully complying with the Amsterdam criteria, whereas a disease-causing mutation could be identified in only 8% of the families in which the criteria were not satisfied fully. In correspondence with these findings, 4 of 6 colorectal tumors from patients belonging to kindreds meeting the criteria showed microsatellite instability, whereas only 3 of 11 tumors from the other set of families demonstrated this instability. Although the number of tumors included in the study admittedly is small, the frequencies of mutations in the MMR genes show obvious differences between the two clinical sets of families. These results also emphasize the practical importance of the Amsterdam criteria, which provide a valid clinical subdivision between families, on the basis of their chance of carrying an hMSH2 or an hMLH1 mutation, and which bear important consequences for genetic testing and counseling and for the management of colorectal cancer families.