Blood proteins of the silkworm Bombyx mori L

The qualitative and quantitative aspects of the proteins of the silkworm blood were studied by the technique of agarophoresis. The blood of larvae at the final stage revealed the presence of six different protein zones. Considerable differences in the patterns were observed at different stages of growth. There was an increase in the total nitrogen of the blood up to the 5th instar and then came a sudden decrease in the one-day old pupae. Nitrogen concentration was at its highest in egg 1 stage and the electrophoretic pattern closely corresponded to the final larval pattern. Results indicate to the involvement of silk glands in the synthesis and breakdown of a protein designated as protein 5.

Analysis and prediction of major blood proteins based on their amino acid and dipeptide composition

A method has been developed for predicting blood proteins using the SVM based machine learning approach. In this prediction method a two-step strategy was deployed to predict blood proteins and their subclasses. We have developed models of blood proteins and achieved the maximum accuracies of 90.57% and 91.39% with Matthews correlation coefficient (MCC) of 0.89 and 0.90 using single amino acid and dipeptide composition respectively. Furthermore, the method is able to predict major subclasses of blood proteins; developed based on amino acid (AC) and dipeptide composition (DC) with a maximum accuracy 90.38%, 92.83%, 87.41%, 92.52% and 85.27%, 89.07%, 94.82%, 86.31 for albumin, globulin, fibrinogen, and regulatory proteins respectively. All modules were trained, tested, and evaluated using the five-fold cross-validation technique.

Distribution of methylene chloride in human blood

Methylene chloride (dichloromethane) is widely used as a solvent for stripping of paint, as industrial cleaning agent, for coating of pills in the pharmaceutical industry, and in the decaffeination of coffee. There is “sufficient evidence for the carcinogenicity” of methylene chloride in animals and “inadequate evidence for its carcinogenity in humans”, according to IARC (IARC 1987; CEC 1990).

Determination of the ethylene oxide adduct S-(2-hydroxyethyl)cysteine by a fluorometric HPLC method in albumin and globin from human blood

A new method has been developed for the quantification of 2-hydroxyethylated cysteine resulting as adduct in blood proteins after human exposure to ethylene oxide, by reversed-phase HPLC with fluorometric detection. The specific adduct is analysed in albumin and in globin. After isolation of albumin and globin from blood, acid hydrolysis of the protein and precolumn derivatisation of the digest with 9-fluorenylmethoxycarbonylchloride, the levels of derivatised S-hydroxyethylcysteine are analysed by RP-HPLC and fluorescence detection, with a detection limit of 8 nmol/g protein. Background levels of S-hydroxyethylcysteine were quantified in both albumin and globin, under special consideration of the glutathione transferase GSTT1 and GSTM1 polymorphisms. GSTT1 polymorphism had a marked influence on the physiological background alkylation of cysteine. While S-hydroxyethylcysteine levels in "non-conjugators" were between 15 and 50 nmol/g albumin, "low conjugators" displayed levels between 8 and 21 nmol/g albumin, and "high conjugators" did not show levels above the detection limit. The human GSTM1 polymorphism had no apparent effect on background levels of blood protein 2-hydroxyethylation.

Genetic diversity of Yunnan local pig breeds inferred from blood protein electrophoresis

Protein electrophoresis was used to examine the blood protein polymorphism in Yunnan local pig breeds, i.e., the Saba pig, Dahe pig, and Diannan small-ear pig breeds, Of 38 genetic loci surveyed 9 were found to be polymorphic. The percentage of polymorphic loci (P) varies from 0.1875 to 0.2121, and the mean individual heterozygosity (H) varies front 0.0712 to 0.1027 in three pig breeds. The results indicate that blood protein polymorphism in Yunnan pig breeds is high. Yunnan local pig breeds have a wealth of genetic diversity at the level of blood proteins.

Acid-labile protein-adducted heterocyclic aromatic amines in human blood are not viable biomarkers of dietary exposure: A systematic study.

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of protein-rich foods. HCA residues adducted to blood proteins have been postulated as biomarkers of HCA exposure. However, the viability of quantifying HCAs following hydrolytic release from adducts in vivo and correlation with dietary intake are unproven. To definitively assess the potential of labile HCA-protein adducts as biomarkers, a highly sensitive UPLC-MS/MS method was validated for four major HCAs: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx). Limits of detection were 1e5 pg/ml plasma and recoveries 91e115%. Efficacy of hydrolysis was demonstrated by HCA-protein adducts synthesised in vitro. Plasma and 7-day food diaries were collected from 122 fasting adults consuming their habitual diets. Estimated HCA intakes ranged from 0 to 2.5 mg/day. An extensive range of hydrolysis conditions was examined for release of adducted HCAs in plasma. HCA was detected in only one sample (PhIP, 9.7 pg/ml), demonstrating conclusively for the first time that acid-labile HCA adducts do not reflect dietary HCA intake and are present at such low concentrations that they are not feasible biomarkers of exposure. Identification of biomarkers remains important. The search should concentrate on stabilised HCA peptide markers and use of untargeted proteomic and metabolomic approaches.

The poor penetration of topical burn agent through burn eschar on a porcine burn model [Letter to the Editor]

We read with great interest the article entitled “Enhancing drugs absorption through third-degree burn wound eschar” by Manafi et al. [1]. The authors addressed the concern of poor penetration of topically applied anti-microbials through burn eschar and detailed the improvement of this penetration by penetration enhancers. Here, we would like to report the poor penetration of a topical agent into the viable deep dermal layer under burn eschar on a porcine burn model [2]. In burn treatment, a common practice is the topical application of either anti-microbial products or wound enhancing agents. While the activity of anti-microbial products is designed to fight against microbes on the wound surface but with the least toxicity to viable tissue, wound enhancing agents need to reach the viable tissue layer under the burn eschar. Many studies have reported the accelerated healing of superficial burn wounds and skin graft donor sites by the topical application of exogeneous growth factors [3]. It is well known that the efficacy of the penetration of a topical agent on intact skin mostly depends on the molecular size of the product [4] and [5]. While burn injury destroys this epidermal physiological barrier, the coagulated burn tissue layer on the burn wound surface makes it difficult for topical agents to reach viable tissue....

Fetuin-A : a major fetal serum protein that promotes "wound closure" and scarless healing [Letter to the Editor]

Burn-wound healing is a dynamic, interactive process involving a number of cellular and molecular events and is characterized by inflammation, granulation tissue formation, re-epithelialization, and tissue remodeling (Greenhalgh, 2002; Linares, 2002). Unlike incisional-wound healing, it also requires extensive re-epithelialization due to a predominant horizontal loss of tissue and often heals with abnormal scarring when burns involve deep dermis. The early mammalian fetus has the remarkable ability to regenerate normal epidermis and dermis and to heal dermal incisional wounds with no signs of scarring. Extensive research has indicated that scarless healing appears to be intrinsic to fetal skin (McCallion and Ferguson, 1996; Ferguson and O’Kane, 2004). Previously, we reported a fetal burn model, in which 80-day-old ovine fetuses (gestation¼ 145–153 days) healed deep dermal partial thickness burns without scars, whereas postnatal lambs healed equal depth burns with significant scarring (Cuttle et al., 2005; Fraser et al., 2005). This burn model provided early evidence that fetal skin has the capacity to repair and restore dermal horizontal loss, not just vertical injuries.

European aspects of standard setting in occupational hygiene and medicine

Occupational standards concerning the allowable concentrations of chemical compounds in the ambient air of workplaces have been established in several countries at national levels. With the integration of the European Union, a need exists for establishing harmonized Occupational Exposure Limits. For analytical developments, it is apparent that methods for speciation or fractionation of carcinogenic metal compounds will be of increasing practical importance for standard setting. Criteria of applicability under field conditions, cost-effectiveness, and robustness are practical driving forces for new developments. When the European Union issued a list of 62 chemical substances with Occupational Exposure Limits in 2000, 25 substances received a 'skin' notation. The latter indicates that toxicologically significant amounts may be taken up via the skin. Similar notations exist on national levels. For such substances, monitoring concentrations in ambient air will not be sufficient; biological monitoring strategies will gain further importance in the medical surveillance of workers who are exposed to such compounds. Proceedings in establishing legal frameworks for a biological monitoring of chemical exposures within Europe are paralleled by scientific advances in this field. A new aspect is the possibility of a differential adduct monitoring, using blood proteins of different half-life or lifespan. This technique allows differentiation between long-term mean exposure to reactive chemicals and short-term episodes, for example, by accidental overexposure. For further analytical developments, the following issues have been addressed as being particularly important: New dose monitoring strategies, sensitive and reliable methods for detection of DNA adducts, cytogenetic parameters in biological monitoring, methods to monitor exposure to sensitizing chemicals, and parameters for individual susceptibilities to chemical toxicants.

Saliva proteome research: current status and future outlook

Human saliva harbours proteins of clinical relevance and about 30% of blood proteins are also present in saliva. This highlights that saliva can be used for clinical applications just as urine or blood. However, the translation of salivary biomarker discoveries into clinical settings is hampered by the dynamics and complexity of the salivary proteome. This review focuses on the current status of technological developments and achievements relating to approaches for unravelling the human salivary proteome. We discuss the dynamics of the salivary proteome, as well as the importance of sample preparation and processing techniques and their influence on downstream protein applications; post-translational modifications of salivary proteome and protein: protein interactions. In addition, we describe possible enrichment strategies for discerning post-translational modifications of salivary proteins, the potential utility of selected-reaction-monitoring techniques for biomarker discovery and validation, limitations to proteomics and the biomarker challenge and future perspectives. In summary, we provide recommendations for practical saliva sampling, processing and storage conditions to increase the quality of future studies in an emerging field of saliva clinical proteomics. We propose that the advent of technologies allowing sensitive and high throughput proteome-wide analyses, coupled to well-controlled study design, will allow saliva to enter clinical practice as an alternative to blood-based methods due to its simplistic nature of sampling, non-invasiveness, easy of collection and multiple collections by untrained professionals and cost-effective advantages.

Gene expression profiling reveals a downregulation in immune-associated genes in patients with AS

Objective: To identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) compared with healthy individuals. Methods: RNA was extracted from PBMCs collected from 18 patients with active disease and 18 gender-matched and age-matched controls. Expression profiles of these cells were determined using microarray. Candidate genes with differential expressions were confirmed in the same samples using quantitative reverse transcription-PCR (qRT-PCR). These genes were then validated in a different sample cohort of 35 patients with AS and 18 controls by qRT-PCR. Results: Microarray analysis identified 452 genes detected with 485 probes which were differentially expressed between patients with AS and controls. Underexpression of NR4A2, tumour necrosis factor AIP3 (TNFAIP3) and CD69 was confirmed. These genes were further validated in a different sample group in which the patients with AS had a wider range of disease activity. Predictive algorithms were also developed from the expression data using receiver-operating characteristic curves, which demonstrated that the three candidate genes have ∼80% power to predict AS according to their expression levels. Conclusions: The findings show differences in global gene expression patterns between patients with AS and controls, suggesting an immunosuppressive phenotype in the patients. Furthermore, downregulated expression of three immune-related genes was confirmed. These candidate genes were also shown to be strong predictive markers for AS.

Crystal structure of a beta-prism II lectin from Remusatia vivipara

The crystal structure of a beta-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4 A. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the ``monocot mannose-binding'' lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.

Chronic cough in children: bronchoalveolar lavage findings

Isolated chronic cough in childhood is a common complaint. Although the symptom cough is included in the definition of clildhood asthma, there is debate as to whether the majoritv of these children have asthma. The authors studied children with isolated chronic cough looking for evidence of airway inflammation typical of asthma, with increased numbers of airway eosinophils as assessed from bronchoalveolar lavage (BAL).

The investigations were carried out on 23 children (median age: 6.7 yrs; range: 1.7-12.75 yrs), attending the Royal Belfast Hospital for Sick Children for elective surgery, who also had a chronic unexplained cough. Written informed consent was obtained from the parent(s) and a nonbronchoscopic BAL was performed. BAL samples were analysed for total and differential white cell counts and also for the inflammatory mediators, eosinophil cationic protein (ECP) and histamine. Results were compared with a group of normal nonatopic children and also a group of atopic asthmatic children, who had been recruited for other studies on airway inflammation.

There was a small but statistically significant increase in BAL percentage eosinophils in the children with chronic cough compared with nonasthmatic controls (0.28% versus 0.10%, p=0.03). However, the children with cough had lower percentage eosinophils than the atopic asthmatic controls (0.28% versus 0.66%, p=0.01). Three out of 23 children with chronic cough had BAL eosinophils greater than the normal upper 95% reference interval in BAL. There was a small but statistically significant increase in percentage neutrophils in the children with cough compared with the nonasthmatic controls (5.85% versus 3.21%, p=0.03). Four out of the 23 children had BAL neutrophils greater than the normal upper 95% reference interval in BAL.

The authors conclude that only a minority of children with chronic unexplained cough have asthmatic-type airway inflammation. It is speculated that the increased percentage neutrophils in bronchoalveolar lavage from children with cough could relate to underlying persistent airways infection.

Non-enzymatic glycosylation

Experience with the use of glycosylated haemoglobin throughout the 1980s has confirmed its uniqueness and usefulness as an objective index of long-term glycaemia in diabetes mellitus, and has enabled the definition of realistic and achievable targets for outpatient management. Measurement of glycosylated serum proteins yields information over a much shorter time-scale which may be particularly useful in diabetic pregnancy. The formation of advanced glycosylation end-products may provide a link between hyperglycaemia and chronic diabetic complications. Therapeutic inhibition or the promotion of alternative metabolic pathways, to yield inert glycosylated products, represents an innovative approach to the problem of preventing these complications.

Glycosylation of low density lipoprotein in patients with type 1 (insulin-dependent) diabetes:correlations with other parameters of glycaemic control

Glycosylation of low density lipoproteins obtained from 16 patients with Type 1 (insulin-dependent) diabetes and from 16 age-, sex-, and race-matched controls, was determined. The diabetic patients were normolipaemic and were in good or fair glycaemic control. Eleven patients performed home blood glucose monitoring. Glycosylation of low density lipoproteins in the diabetic patients was significantly higher (p less than 0.001) than in the control subjects, and was significantly correlated with haemoglobin A1c, (p less than 0.01), glycosylation of plasma proteins, (p less than 0.001), and mean home blood glucose, (p less than 0.01). This study confirms that, in diabetic patients, increased glycosylation of low density lipoprotein occurs to an extent which correlates closely with other commonly used indices of glycaemic control.