Kinetics of the thermal degradation of patulin in the presence of ascorbic acid.

Degradation of the mycotoxin patulin between 25 and 85 °C without and with added ascorbic acid was studied, and the effectiveness of linear and nonlinear models for predicting reaction rates was compared. In agreement with previous reports, ascorbic acid significantly increased (P ≤ 0.05) the rate of patulin degradation at all temperatures studied. The data for patulin degradation in the absence of ascorbic acid were adequately modeled using a zero-order linear kinetic model. However, the predictive abilities of zero and higher-order linear models were not adequate to describe the more complex reactions that likely occurred when ascorbic acid was added. In contrast, the nonlinear Weibull model adequately described the patulin-ascorbic acid reaction throughout the temperature range studied. Zero-order rate constants and Weibull scale values for each of the respective reactions followed the Arrhenius law. Activation energies of 58.7 ± 3.9 and 29.6 ± 1.9 kJ mol⁻¹ for the reaction without and with ascorbic acid, respectively, confirmed decreased patulin stability in the presence of ascorbic acid and suggested that the mechanisms for the 2 degradation reactions were different.

Pilot study exploring lung allograft surfactant protein A (SP-A) expression in association with lung transplant outcome.

Primary graft failure and chronic lung allograft dysfunction (CLAD) limit lung transplant long-term outcomes. Various lung diseases have been correlated with surfactant protein (SP) expression and polymorphisms. We sought to investigate the role of SP expression in lung allografts prior to implantation, in relation to posttransplant outcomes. The expression of SP-(A, B, C, D) mRNA was assayed in 42 allografts. Posttransplant assessments include pulmonary function tests, bronchoscopy, broncho-alveolar lavage fluid (BALF) and biopsies to determine allograft rejection. BALF was assayed for SP-A, SP-D in addition to cytokines IL-8, IL-12 and IL-2. The diagnosis of CLAD was evaluated 6 months after transplantation. Lung allografts with low SP-A mRNA expression prior to implantation reduced survival (Log-rank p < 0.0001). No association was noted for the other SPs. Allografts with low SP-A mRNA had greater IL-2 (p = 0.03) and IL-12 (p < 0.0001) in the BALF and a greater incidence of rejection episodes (p = 0.003). Levels of SP-A mRNA expression were associated with the SP-A2 polymorphisms (p = 0.015). Specifically, genotype 1A1A(0) was associated with lower SP-A mRNA expression (p < 0.05). Lung allografts with low levels of SP-A mRNA expression are associated with reduced survival. Lung allograft SP-A mRNA expression appears to be associated with SP-A gene polymorphisms.

Donor surfactant protein D (SP-D) polymorphisms are associated with lung transplant outcome.

Chronic lung allograft dysfunction (CLAD) is the major factor limiting long-term success of lung transplantation. Polymorphisms of surfactant protein D (SP-D), an important molecule within lung innate immunity, have been associated with various lung diseases. We investigated the association between donor lung SP-D polymorphisms and posttransplant CLAD and survival in 191 lung transplant recipients consecutively transplanted. Recipients were prospectively followed with routine pulmonary function tests. Donor DNA was assayed by pyrosequencing for SP-D polymorphisms of two single-nucleotide variations altering amino acids in the mature protein N-terminal domain codon 11 (Met(11) Thr), and in codon 160 (Ala(160) Thr) of the C-terminal domain. CLAD was diagnosed in 88/191 patients, and 60/191 patients have died. Recipients of allografts that expressed the homozygous Met(11) Met variant of aa11 had significantly greater freedom from CLAD development and better survival compared to those with the homozygous Thr(11) Th variant of aa11. No significant association was noted for SP-D variants of aa160. Lung allografts with the SP-D polymorphic variant Thr(11) Th of aa11 are associated with development of CLAD and reduced survival. The observed genetic differences of the donor lung, potentially with their effects on innate immunity, may influence the clinical outcomes after lung transplantation.

Modified agar diffusion bioassay for better quantification of Nisaplin(®).

AIMS: To investigate the effect of different well sizes and pre-diffusion times at 4 °C, on the sensitivity, accuracy and precision of nisin quantification by agar diffusion bioassay. METHODS AND RESULTS: Nisin solution (0.625-125 µg ml(-1) ) was filled in wells (3.5 mm or 7 mm diameter) made on agar plates inoculated with Micrococcus luteus, followed by pre-diffusion (0, 24, 48 or 72 h), incubation and measurement of inhibition zone. Regression analysis indicated that wells with 3.5 mm diameter had smaller standard deviation and higher predictive accuracy, compared to wells with 7 mm diameter. Based on Tukey's test, pre-diffusion resulted in significantly different inhibition zones at different nisin concentrations. Pre-diffusion also improved sensitivity of the assay. Different regression models were considered to explore the relationship between inhibition zone and nisin concentration for different pre-diffusion times. A spline model was determined to be the best-fit model, and 48 h was the best pre-diffusion time. CONCLUSIONS: Wells with 3.5 mm diameter demonstrated higher accuracy for nisin quantification compared to wells with 7 mm diameter. 48 h was the best pre-diffusion time for nisin concentration in the range 0.625-125 µg ml(-1) . SIGNIFICANCE AND IMPACT OF THE STUDY: The findings from this study will be helpful in quantifying nisin and compounds with antimicrobial properties accurately over a wide range of concentrations using agar diffusion bioassay.

Effect of sucrose on physical properties of spray-dried whole milk powder.

Spray-dried whole milk powders were prepared from whole condensed milk with various sucrose concentrations (0%, 2.5%, 5%, 7.5%, and 10% w/w), and their glass transition temperature and some physical properties of importance in chocolate manufacture were evaluated. In milk powder samples, the glass transition temperature and free-fat content decreased in a nonlinear manner with sucrose addition. Moreover, increasing sucrose concentration reduced the formation of dents on the particle surface. Addition of sucrose in whole condensed milk increased linearly the apparent particle density and in a nonlinear manner the particle size of spray-dried milk powders. The particle size volume distribution of milk powders with the highest sucrose concentration differed from the log-normal distribution of the other samples due to the formation of large agglomerates. Neither vacuole volume, nor the amorphous state of milk powders was affected by sucrose addition.

Genetic variants of surfactant proteins A, B, C, and D in bronchopulmonary dysplasia.

BPD_28D (O2 dependency at 28 days of life) and BPD_36W (O2 dependency at 36 wks post-menstrual age) are diseases of prematurely born infants exposed to mechanical ventilation and/or oxygen supplementation. In order to determine whether genetic variants of surfactant proteins (SPs-A, B, C, and D) and SP-B-linked microsatellite markers are risk factors in BPD, we performed a family based association study using a Greek study group of 71 neonates (<30 wks gestational age) from 60 families with, 52 BPD_28D and 19 BPD_36W, affected infants. Genotyping was performed using newly designed pyrosequencing assays and previously published methods. Associations between genetic variants of SPs and BPD subgroups were determined using Transmission Disequilibrium Test (TDT) and Family Based Association Test (FBAT). Significant associations (p

Rare SP-A alleles and the SP-A1-6A(4) allele associate with risk for lung carcinoma.

Next to cigarette smoking, genetic factors may contribute to lung cancer risk. Pulmonary surfactant components may mediate response to inhaled carcinogenic substances and/or play a role in lung function and inflammation. We studied associations between surfactant protein (SP) genetic variants and risk in lung cancer subgroups. Samples (n=308) were genotyped for SP-A1, -A2, -B, and -D marker alleles. These included 99 patients with small cell lung carcinoma (SCLC, n=31), or non-SCLC (NSCLC, n=68) consisting of squamous cell carcinoma (SCC, n=35), and adenocarcinoma (AC) (n=23); controls (n=99) matched by age, sex, and smoking status (clinical control) to SCLC and NSCLC; and 110 healthy individuals (population control). We found (a) no significant marker associations with SCLC, (b) rare SP-A2 (1A9) and SP-A1 (6A11) alleles associate with NSCLC risk when compared with population control, (c) the same alleles (1A9, 6A11) associate with risk for AC when compared with population (6A11) or clinical control (1A9), and (d) the SP-A1-6A4 allele (found in approximately 10% of the population) associates with SCC, when compared with population or clinical control. A correlation between SP-A variants and lung cancer susceptibility appears to exist, indicating that SP-A alleles may be useful markers of lung cancer risk.

Regularity of distribution of immunoreactive pulmonary surfactant protein A in rat tissues.

Existing data has shown that SP-A-like protein or mRNA is widely distributed in lamellar bodies such as tissues and mucosal surfaces. Using immunohistochemistry method with a polyclonal antibody against human SP-A, in this study we investigated distribution of immunoreactive pulmonary surfactant protein A (IR-SP-A) in a number of rat tissues. The SP-A-like immunoreactivity was found in alveolar, parenchyma, pleura of lung; myelin sheath of brain; epithelia of Bowman's capsule, glomerulus and renal tubules of kidney; epithelia of colon, stomach, duct of salivary gland, pharynx; and blood vessel wall and connective tissue of extracellular matrix. The positive signal was blocked by pre-absorbed SP-A antigen from recombinant or bronchoalveolar lavage (BAL). SP-A has long been considered as an important frontier host defense molecule which participates in immune and inflammatory regulation of lung. With every inhalation, small particles, viruses, bacteria, and antigens from environment are continuously deposited onto the vast pulmonary epithelial surface. While a proper host defense is required to protect the lung, an over-exuberant response can disrupt the appropriate balance between pro- and anti-inflammatory. Traditional Chinese medicine believes that body is an open system relevant to the external environment. The physical, chemical and biological environmental factors constantly affect the open system, and the body properly reacts to maintain homeostasis of body machinery. The Chinese traditional medicine scholars have thus hypothesized that 'Qi' (meaning air) is the communication way between the body and external environment. What is 'Qi'? The results from our study suggest that IR-SP-A is a candidate of 'Qi'. It is compatible with the sites, theoretically containing collagenous and lectin domain molecules, also compatible with the primary injury sites of some autoimmune diseases. SP-A may be as one of 'Qi' molecules mentioned in traditional Chinese medicine that trigger some of autoimmune diseases.

Structural analysis and lipid-binding properties of recombinant human surfactant protein a derived from one or both genes.

Surfactant protein A (SP-A) constitutes an important part of the innate immune defense in the lung. In humans there are two functional genes (SP-A1 and SP-A2). The functional importance of having two distinct chain types in human SP-A is undefined. Amino acid substitutions in the primary structure of the protein may have effects on structural stability or on activity. To address this issue, SP-A1, SP-A2, and coexpressed SP-A1/SP-A2 variants were in vitro expressed in insect cells, purified, and used for study. We found the following: (1) Human SP-A variants expressed in insect cells, derived from one gene (SP-A1 or SP-A2) or both genes, differ in the relative extent and heterogeneity of oligomerization. SP-A1 and SP-A2 exist in small oligomeric forms, whereas coexpressed SP-A1/SP-A2 products favor the formation of larger oligomers. (2) Circular dichroic and fluorescence spectroscopic studies identified structural differences between SP-A variants in the collagen domain, with SP-A2 being more stable than SP-A1 but not in the calcium binding region. Recombinant human SP-A variants expressed in insect cells exhibit a lower melting temperature compared to native human SP-A. Oligomerization does not increase the thermal stability of the collagen domain of coexpressed SP-A1/SP-A2. (3) The ability of SP-A to undergo self-aggregation and induce phospholipid and bacterial lipopolysaccharide aggregation is greater for SP-A2 than for coexpressed SP-A1/SP-A2, which in turn is greater than that observed for SP-A1. The presence of SP-A1 polypeptide chains in coexpressed products modulates functional capabilities of SP-A, which depend on both the collagen and globular domains.

Models and tests of linkage and association studies of quantitative trait locus for multi-allele marker Loci.

In this paper, we explore models and tests for association and linkage studies of a quantitative trait locus (QTL) linked to a multi-allele marker locus. Based on the difference between an offspring's conditional trait means of receiving and not receiving an allele from a parent at marker locus, we propose three statistics T(m), T(m,row) and T(m,col) to test association or linkage disequilibrium between the marker locus and the QTL. These tests are composite tests, and use the offspring marginal sample means including offspring data of both homozygous and heterozygous parents. For the linkage study, we calculate the offspring's conditional trait mean given the allele transmission status of a heterozygous parent at the marker locus. Based on the difference between the conditional means of a transmitted and a nontransmitted allele from a heterozygous parent, we propose statistics T(parsi), T(satur), T(gen) and T(m,het) to perform composite tests of linkage between the marker locus and the quantitative trait locus in the presence of association. These tests only use the offspring data that are related to the heterozygous parents at the marker locus. T(parsi) is a parsimonious or allele-wise statistic, T(satur) and T(gen )are satured or genotype-wise statistics, and T(m,het) compares the row and column sample means for offspring data of heterozygous parents. After comparing the powers and the sample sizes, we conclude that T(parsi) has higher power than those of the bi-allele tests, T(satur), T(gen), and T(m,het). If there is tight linkage between the marker and the trait locus, T(parsi) is powerful in detecting linkage between the marker and the trait locus in the presence of association. By investigating the goodness-of-fit of T(parsi), we find that T(satur) does not gain much power compared to that of T(parsi). Moreover, T(parsi) takes into account the pattern of the data that is consistent with linkage and linkage disequilibrium. As the number of alleles at the marker locus increases, T(parsi) is very conservative, and can be useful even for sparse data. To illustrate the usefulness and the power of the methods proposed in this paper, we analyze the chromosome 6 data of the Oxford asthma data, Genetic Analysis Workshop 12.

Surfactant protein (SP) B associations and interactions with SP-A in white and black subjects with respiratory distress syndrome.

BACKGROUND: The etiology of respiratory distress syndrome (RDS) is multifactorial and/or multigenic. Surfactant protein A (SP-A) and/or SP-B genetic variants have been identified as risk or protection factors for RDS. METHODS: We genotyped subjects with and without RDS for the SP-B intron 4 size variants (invariant (inv), deletion (del), insertion (ins) and for four (-18 (A/C), 1013 (A/C), 1580 (C/T), 9306 (A/G)) SP-B single nucleotide polymorphisms (SNP), to study case-control associations in black and white subjects. We also determined whether specific SP-B variants interact with RDS susceptibility or protective SP-A variants to enhance or reduce risk for RDS. RESULTS: Based on odds ratio: (1) the SP-B intron 4 del variant in white subjects is more of an RDS risk factor for males and for subjects of 28 weeks

Family-based transmission disequilibrium test (TDT) and case-control association studies reveal surfactant protein A (SP-A) susceptibility alleles for respiratory distress syndrome (RDS) and possible race differences.

A key cause of respiratory distress syndrome (RDS) in the prematurely born infant is deficiency of pulmonary surfactant, a lipoprotein complex. Both low levels of surfactant protein A (SP-A) and SP-A alleles have been associated with RDS. Using the candidate gene approach, we performed family-based linkage studies to discern linkage of SP-A to RDS and identify SP-A susceptibility or protective alleles. Moreover, we performed case-control studies of whites and blacks to detect association between RDS and SP-A alleles. Transmission disequilibrium test (TDT) analysis revealed that the frequency of transmission (from parent to the offspring with RDS) of alleles 6A(2) and 1A(0) and of 1A(0)/6A(2) haplotype in RDS was increased, whereas transmission of alleles 1A(5) and 6A(4) and of haplotype 1A(5)/6A(4) was decreased. Extended TDT analysis further strengthened the observations made. The case-control studies showed that in whites or blacks with RDS the frequencies of specific genotypes, 1A(0) and 6A(2) or 1A(0), were increased, respectively, but the frequency of specific 6A(3) genotypes was increased in certain white subgroups and decreased in blacks. Regression analysis revealed gestational age (GA) and 6A(3) genotypes are significant factors in blacks with RDS. In whites with RDS, GA and antenatal steroids are important factors. The data together indicate linkage between SP-A and RDS; certain SP-A alleles/haplotypes are susceptibility (1A(0), 6A(2), 1A(0)/6A(2)) or protective (1A(5), 6A(4), 1A(5)/6A(4)) factors for RDS. Some differences between blacks and whites with regard to SP-A alleles may exist.

Surfactant protein gene A, B, and D marker alleles in chronic obstructive pulmonary disease of a Mexican population.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation. It is most likely the result of complex interactions of environmental and genetic factors. Because pulmonary surfactant components play important roles in normal lung function, innate host defence, and inflammation in the lung, this study investigated the hypothesis that the surfactant protein genes are involved in certain cases of COPD. Genotype analysis of surfactant protein (SP)-A, SP-B, SP-B-linked microsatellite, and SP-D marker alleles was performed in patients with COPD (n=97) and smoker (n=82) or nonsmoker (n=99) controls. Univariate and multiple logistic regression analyses were performed. The regression analysis results between COPD and smokers revealed several COPD susceptibility alleles (AA62_A, B1580_C, D2S388_5), based on an odds ratio (OR >2.5). The predictive ability of this model for developing COPD is good (c=0.926). Allele-allele (B1580_C and D2S388_5) and allele-environment (i.e. smoking) interactions were detected. When smoker controls were compared to nonsmoker controls, marker D2S388 5 appeared to be smoking-independent (p=0.874), whereas marker alleles AA62_A (p=0.045) and B1580_5 (p=0.007) were smoking-dependent. Males were at higher risk (OR=6.05, p=0.001), and smoking (>50 packs x yr(-1)) increased risk (OR=5.38, p=0.007). Males and alleles of loci flanking SP-B were associated with more severe cases (forced expiratory volume in one second/forced vital capacity < or = 40%). The present results indicate that the surfactant protein alleles may be useful in chronic obstructive pulmonary disease by either predicting the disease in a subgroup and/or by identifying disease subgroups that may be used for therapeutic intervention. These observations should now be confirmed in a larger study, designed according to strict epidemiological criteria.

Both human SP-A1 and Sp-A2 genes are expressed in small and large intestine.

The human SP-A locus consists of two functional genes and one pseudogene, and SP-A is shown to play a role in local host defense and the regulation of inflammation in lung. Because the intestine, like the lung, is constantly exposed to foreign and potentially harmful substances, we investigated the hypothesis that both human SP-A genes are expressed in intestine. We demonstrate that both SP-A genes are expressed in human small and large intestine. The presence of SP-A mRNA in human intestine was detected by reverse transcription polymerase chain reaction (RT-PCR), Northern blot analysis, and immunohistochemistry. The size of intestinal SP-A mRNA is the same as that in human lung, but the level of expression, compared with that in the lung, is very low in both the small and large intestine. Immunohistochemical analysis revealed positive reactivity for SP-A in a subgroup of epithelial cells in the intestine. Expression of both SP-A1 and SP-A2 genes was established by gene-specific PCR amplification, PCR-based converted RFLP discrimination, and direct sequencing of RT-PCR products. We speculate that SP-A in the intestine plays a role in local host defense and inflammation.

Response surface modeling for the inactivation of Escherichia coli O157:H7 on green peppers (Capsicum annuum L.) by chlorine dioxide gas treatments.

The effects of chlorine dioxide (ClO2) gas concentration (0.1 to 0.5 mg/liter), relative humidity (RH) (55 to 95%), treatment time (7 to 135 min), and temperature (5 to 25 degrees C) on inactivation of Escherichia coli O157:H7 on green peppers were studied using response surface methods. A four-factor, central, composite, rotatable design was used. The microbial log reduction was measured as a response. A direct membrane-surface-plating method with tryptic soy agar and sorbitol MacConkey agar was used to resuscitate and enumerate ClO2-treated E. coli O157:H7 cells. The statistical analysis and the predictive model developed in this study suggest that ClO2 gas concentration, treatment time, RH, and temperature all significantly (P < 0.01) increased the inactivation of E. coli O157:H7. ClO2 gas concentration was the most important factor, whereas temperature was the least significant. The interaction between ClO2 gas concentration and RH indicated a synergistic effect. The predictive model was validated, and it could be used to determine effective ClO2 gas treatments to achieve a 5-log reduction of E. coli O157:H7 on green peppers.

Human surfactant protein A (SP-A) variants: why so many, why such a complexity?

Human surfactant protein A (SP-A) exhibits extensive complexity at several levels: genetic, transcript (splicing), protein, and composition and size of protein oligomers. Its multiple and important roles in innate host defense, regulation of inflammation, and in aspects of pulmonary surfactant may have necessitated such a complexity from an evolutionary point of view. Moreover, understanding of such a complexity may be useful in the study of disease pathogenesis and the development of disease diagnostics and/or therapeutics.

Surfactant protein A and B genetic variants and respiratory distress syndrome: allele interactions.

The contribution of multiple genetic components in disease pathogenesis is relevant to both diseases of multifactorial and/or multigenic etiology such as the respiratory distress syndrome (RDS) and to diseases where a single gene has been identified as the disease-causing gene. An example of the latter is cystic fibrosis (CF) where the disease-causing gene has been clearly identified as the CF transmembrane conductance regulator gene, but genetic variants of the mannose binding protein and surfactant protein A have been associated with disease severity in CF. The overall rationale for considering genetic contribution to disease pathogenesis is based on the premise that all diseases or deaths (except perhaps those resulting from trauma) have a genetic component. The difference in genetic contribution among various diseases is the percent contribution and the number of factors that make this contribution. Therefore, if the number of genetic contributors is small and the percentage of genetic contribution is high it may be less challenging to identify such factors. In this paper we summarize allele associations and discuss allele interaction of the surfactant protein genes in relation to RDS (the term allele and genetic variant will be used interchangeably).

A point of view: quantitative and qualitative imbalance in disease pathogenesis; pulmonary surfactant protein A genetic variants as a model.

The high degree of similarity at the molecular level, between humans and other species, has provided the rationale for the use of a variety of species as model systems in research, resulting in enormous advances in biological sciences and medicine. In contrast, the individual variability observed among humans, for example, in external physique, organ functionality and others, is accounted for, by only a fraction of 1% of differences at the DNA level. These small differences, which are essential for understanding disease pathogenesis, have posed enormous challenges in medicine, as we try to understand why patients may respond differently to drugs or why one patient has complications and another does not. Differences in outcome are most likely the result of interactions among genetic components themselves and/or the environment at the molecular, cellular, organ, or organismal level, or the macroenvironment. In this paper: (1) we consider some issues for multifactorial disease pathogenesis; (2) we provide a review of human SP-A and how the knowledge gained and the characteristics of the hSP-A system may serve as a model in the study of disease with multifactorial etiology; and (3) we describe examples where hSP-A has been used in the study of disease.

Polymorphisms of human SP-A, SP-B, and SP-D genes: association of SP-B Thr131Ile with ARDS.

An allele association study of 19 polymorphisms in surfactant proteins SP-A1, SP-A2, SP-B, and SP-D genes in acute respiratory distress syndrome (ARDS) was carried out. Trend-test analysis revealed differences (p < 0.05) in the frequency of alleles for some of the microsatellite markers flanking SP-B, and for one polymorphism (C/T) at nucleotide 1580 [C/T (1580)], within codon 131 (Thr131Ile) of the SP-B gene. The latter determines the presence or absence of a potential N-linked glycosylation site. Multivariate analysis revealed significant differences only for the C/T (1580) polymorphism. When the ARDS population was divided into subgroups, idiopathic (i.e., pneumonia, etc.) or exogenic (i.e., trauma, etc.), significant differences were observed for the C/T (1580), for the idiopathic ARDS group, and the frequency of the C/C genotype was increased in this group. Based on the odds ratio, the C allele may be viewed as a susceptibility factor for ARDS. Although the expression of both C and T alleles occurs in heterozygous individuals, it is currently not known whether these alleles correspond to similar levels of SP-B protein. These data suggest that SP-B or a linked gene contributes to susceptibility to ARDS.