Monoclonal antibodies against soybean and pea proteinase inhibitors: Characterization and applications for immunoassays in food processing and plant breeding

Monoclonal antibodies produced against protein‐type inhibitors from pea or soybean were used for investigations of the chemical and antigenic properties of trypsin and chymotrypsin inhibitors from pea. Cross‐reactivity studies revealed only the existence of Bowman‐Birk‐type inhibitors in pea. The inhibitors could be divided into at least two groups of iso‐inhibitors based on their characteristics in binding to different monoclonal antibodies produced against pea trypsin inhibitors or Bowman‐Birk inhibitor from soybean. The inhibitor contents in a series of pea extracts were measured in an ELISA‐based system using the different antibodies. Comparison with the inhibitor activity measured by traditional enzymatic analysis performed as microassay showed that only the inhibitor content of one of the two inhibitor groups correlated strongly with the trypsin inhibitor activity. The immunoassay was also shown to be suitable for measurement of inhibitor content after heat treatment as the results suggested that the two inhibitor groups contained equally heat resistant inhibitors.     

Liver cells respond to Aspergillus fumigatus with an increase in C3 secretion and C3 gene expression as well as an expression increase in TLR2 and TLR4

Fungal infections by molds like Aspergillus fumigatus are an increasing health problem which can be fatal in immuno-compromised patients. In healthy individuals, these infections are easily eliminated by the innate and acquired immune system. Complement factor 3 (C3) has a key place within the complement cascade and C3 RNA expression can therefore be used to monitor an impending immune response. Employing a liver cell line (HepG2) as a model system, we have examined their responses to A. fumigatus or beta-glucan, a major component of the fungal wall. C3 RNA expression was increased after stimulation with both LPS and A. fumigatus as well as after incubation with beta-glucan, although with different kinetics. C3 protein release into the supernatant followed an inverse bell-shaped curve when cells were incubated with A. fumigatus or beta-glucan while during LPS stimulation, the release was more stable. HepG2 cells also express Toll-like receptors (TLRs) and both for TLR2 and TLR4, an expression increase was found. These data demonstrate that liver cells are able to react specifically to a fungal pathogen without the help of Kupffer cells.     

Monitor unit calculations for range-modulated spread-out Bragg peak fields

We derive, from first principles, a model to predict the output factors for spread-out Bragg peak proton fields (SOBP). The model is based on the simple observation that the output factor is the ratio of SOBP plateau dose to the dose measured in the ionization reference chamber. The latter, in turn, equates to the entrance dose of the SOBP corrected for inverse square. We use a theoretical derivation of this ratio to establish the relationship between the output factor and the distal range and modulation width of the SOBP. In addition, the theoretical derivation reduces the dependence on the distal range and modulation width into a single factor r = (R - M)/M. We compare the theoretical derivation against measurements obtained at the Northeast Proton Therapy Facility for output factors for clinical fields. The agreement between measurements and prediction is 2.9%.     

The effect of physical and psychosocial loads on the trapezius muscle activity during computer keying tasks and rest periods

The overall aim was to investigate the effect of psychosocial loads on trapezius muscle activity during computer keying work and during short and long breaks. In 12 female subjects, surface electromyography (EMG) was recorded bilaterally from the upper trapezius muscle during a standardized one hand keying task—interspaced with short (30 s) and long (4 min) breaks—in sessions with and without a combination of cognitive and emotional stressors. Adding psychosocial loads to the same physical work did not increase the activity of the trapezius muscle on either the keying or the control side, both of which remained at median and static EMG activity levels of around 5% and 2.5% of the maximal voluntary electrical activity (EMG_max), respectively. The difference between the keying and the control side was significant; and further the control side activity was significantly increased above resting level. During both short and long breaks, exposure to psychosocial loads also did not increase the activity of the trapezius muscle either on the side of the keying or the control hand. Of note is that during long breaks the muscle activity of the keying side as well as that of the control side remained at the same level as during the short breaks, which was increased above resting level. This was to be seen from the static and the median EMG activity levels as well as gap times, the overall mean values being: 0.4%EMG_max, 1.1%EMG_max, and 50% in gap time, respectively. In conclusion: psychosocial loads are not solely responsible for increased non-postural muscle activity; and increasing the duration of breaks does not per se cause muscle relaxation.     

Global proteomics reveals insulin abundance as a marker of human islet homeostasis alterations

Aim

The variation in quality between the human islet samples represents a major problem for research, especially when used as control material. The assays assessing the quality of human islets used in research are non-standardized and limited, with many important parameters not being consistently assessed. High-throughput studies aimed at characterizing the diversity and segregation markers among apparently functionally healthy islet preps are thus a requirement. Here, we designed a pilot study to comprehensively identify the diversity of global proteome signatures and the deviation from normal homeostasis in randomly selected human-isolated islet samples.

Methods

By using Tandem Mass Tag 16-plex proteomics, we focused on the recurrently observed disparity in the detected insulin abundance between the samples, used it as a segregating parameter, and analyzed the correlated changes in the proteome signature and homeostasis by pathway analysis.

Results

In this pilot study, we showed that insulin protein abundance is a predictor of human islet homeostasis and quality. This parameter is independent of other quality predictors within their acceptable range, thus being able to further stratify islets samples of apparent good quality. Human islets with low amounts of insulin displayed changes in their metabolic and signaling profile, especially in regard to energy homeostasis and cell identity maintenance. We further showed that xenotransplantation into diabetic hosts is not expected to improve the pre-transplantation signature, as it has a negative effect on energy balance, antioxidant activity, and islet cell identity.

Conclusions

Insulin protein abundance predicts significant changes in human islet homeostasis among random samples of apparently good quality.     

Bladder Squamous Cell Carcinomas Express Psoriasin and Externalize it to the Urine

Purpose

To report a single biomarker, psoriasin (Mr 11.0 kd, pI 6.2), a calcium binding protein which is expressed largely by stratified squamous epithelia and is externalized to the urine of bladder squamous cell carcinoma (SCC) bearing patients.

Materials and Methods

Protein expression profiles of SCCs obtained immediately after surgery were analyzed by two-dimensional gel electrophoresis and Coomassie blue staining. Protein identity was determined by microsequencing and immunoblotting. Protein expression in cryosections was studied by immunofluorescence.

Results

Four patients with SCC were identified from 100 samples of patients with suspected transitional cell carcinoma (TCC). The protein profiles of the 4 SCCs (56-1, grade III, T4; 181-1, grade I, T3; 219-1, grade III, T3 and 239-1, grade not determined, T2-4) resembled that of keratinocytes, suggesting that these cells express an early developmental pattern of gene expression. Besides expressing markers characteristic of keratinizing stratified squamous epithelia, the SCCs exhibited psoriasin, a protein externalized to the medium by keratinocytes. Immunohistochemistry of 3 of the SCCs with psoriasin antibodies showed that the positive cells were confined chiefly to the “squamous pearls.” The presence of psoriasin in the urine of the 4 SCC patients was demonstrated by two-dimensional gel immunoblotting. Similar analysis of 43 urines from patients with bladder tumors other than SCC revealed 7 positives, some of which may reflect squamous differentiation. Analysis of the urine of 13 control individuals (12 males matched by age and a 42-year-old female) revealed 2 positives. Immunoblotting of the SCC patients' serum proteins with psoriasin antibodies failed to reveal the protein.

Conclusion

The results point towards psoriasin, alone or as part of a biomarker profile, as a potential marker for the noninvasive follow-up of patients with SCC.     

Towards a Comprehensive Database of Proteins From the Urine of Patients With Bladder Cancer

Purpose

To establish a comprehensive two-dimensional database of proteins from the urine of patients with bladder cancer.

Materials and Methods

Urines dialized against distilled H₂ O were freeze-dried and subjected to isoelectrofocusing two-dimensional gel electrophoresis. Coomassie brilliant blue stained dry gels were scanned and analyzed with the PDQUEST software. Proteins were identified by one or more of the following techniques: microsequencing (44 proteins), mass spectrometry, comigration and immunoblotting.

Results

The urine protein database, which includes all the polypeptides detected in the urines of 50 patients, lists 339 proteins (including variants) of which 124 have been identified. Of these, psoriasin, the psoriasis-associated fatty acid binding protein 5, the gelsolin fragments and prostaglandin D2 synthetase have not been previously described.

Conclusions

The database provides a solid basis for further studies aiming at identifying tumor markers in the urine that may serve as prognostic factors in bladder cancer.     

The design of an environmentally relevant mixture of persistent organic pollutants for use in in vivo and in vitro studies

Amongst the substances listed as persistent organic pollutants (POP) under the Stockholm Convention on Persistent Organic Pollutants (SCPOP) are chlorinated, brominated, and fluorinated compounds. Most experimental studies investigating effects of POP employ single compounds. Studies focusing on effects of POP mixtures are limited, and often conducted using extracts from collected specimens. Confounding effects of unmeasured substances in such extracts may bias the estimates of presumed causal relationships being examined. The aim of this investigation was to design a model of an environmentally relevant mixture of POP for use in experimental studies, containing 29 different chlorinated, brominated, and perfluorinated compounds. POP listed under the SCPOP and reported to occur at the highest levels in Scandinavian food, blood, or breast milk prior to 2012 were selected, and two different mixtures representing varying exposure scenarios constructed. The in vivo mixture contained POP concentrations based upon human estimated daily intakes (EDIs), whereas the in vitro mixture was based upon levels in human blood. In addition to total in vitro mixture, 6 submixtures containing the same concentration of chlorinated + brominated, chlorinated + perfluorinated, brominated + perfluorinated, or chlorinated, brominated or perfluorinated compounds only were constructed. Using submixtures enables investigating the effect of adding or removing one or more chemical groups. Concentrations of compounds included in feed and in vitro mixtures were verified by chemical analysis. It is suggested that this method may be utilized to construct realistic mixtures of environmental contaminants for toxicity studies based upon the relative levels of POP to which individuals are exposed.