Identification of dehydro-ferulic acid-tyrosine in rye and wheat: evidence for a covalent cross-link between arabinoxylans and proteins

To monitor chemical reactions between ferulate and proteins during breadmaking, 8-(14)C-(E)-ferulic acid-(d-galactopyranose-6'-yl)ester was synthesized as a radiotracer and added to wheat and rye flour prior to breadmaking. Breads were lyophilized, extracted by means of a modified Osborne fractionation, and the radioactivity of the fractions was determined by scintillation analysis. The major portion of the radioactivity remained in the water-soluble fraction. However, a significant enrichment of the tracer was also detected in the prolamin and glutelin fractions in comparison to the control experiment. Separation of the prolamin fraction by RP-HPLC and scintillation measurement of the fractions gave evidence for a chemical modification of the tracer. To determine the structure of the reaction product, the prolamin fractions were completely hydrolyzed to free amino acids by means of an enzyme cocktail, and the digests were studied by LC-MS. In one fraction, a newly formed compound was detected. Comparison of its chromatographic and mass spectrometric behavior with a synthetic reference compound gave evidence that the newly identified compound was a dehydroferulic acid-tyrosine cross-link. It is likely that this cross-link represented a covalent linkage between arabinoxylans and cereal proteins. The newly identified cross-link was also identified in wheat and rye flour doughs, which had been prepared without addition of the ferulate tracer. The relative concentration of the dehydroferulic acid-tyrosine cross-link increased during wheat dough preparation.     

Identification of disulfide bonds in wheat gluten proteins by means of mass spectrometry/electron transfer dissociation

Disulfide bonds within gluten proteins play a key role in the breadmaking performance of wheat flour. In the present study, disulfide bonds of wheat gluten proteins were identified by using a new liquid chromatography-mass spectrometry (LC-MS) technique with alternating electron transfer dissociation (ETD)/collision-induced dissociation (CID). Wheat flour was partially hydrolyzed with thermolysin (pH 6.5, 37 °C, 16 h), and the digest was subjected to LC-MS with alternating ETD/CID fragmentation. Whereas CID provided peptide fragments with intact disulfide bonds, cleavage of disulfide bonds was preferred over peptide backbone fragmentations in ETD. The simultaneous observation of disulfide-linked and disulfide-cleaved peptide ions in the mass spectra not only provided distinct interpretation with high confidence but also simplified the conventional approach for determination of disulfide bonds, which often requires two separate experiments with and without chemical reduction. By application of the new method 14 cystine peptides were identified. Eight peptides confirmed previously established disulfide bonds within gluten proteins, and the other six cystine peptides were identified for the first time. One of the newly identified cystine peptides represented a "head-to-tail" cross-link between high molecular weight glutenin subunits. This type of cross-link, which has been postulated as an integral part of glutenin models published previously, has now been proven experimentally for the first time. From the six remaining cystine peptides interchain disulfide bonds between α-gliadins, γ-gliadins, and low molecular weight glutenin subunits were established.     

Changes of folates, dietary fiber, and proteins in wheat as affected by germination

Wheat kernels of the cultivar 'Tommi' were germinated for up to 168 h at 15, 20, 25, or 30 degrees C. Samples were taken at different stages of germination and were analyzed for the quantitative protein composition using an extraction/HPLC method, for folate vitamers using a stable isotope dilution assay, and for soluble, insoluble, and total dietary fiber using a gravimetric method. Gluten proteins were substantially degraded during germination. During the first stages of germination the degradation of glutenins was predominant, whereas longer germination times were required to degrade gliadins. The optimal temperature for gliadin degradation was 20 degrees C, and that for glutenin degradation was 25 degrees C. Omega5- and omega1,2-gliadins were less sensitive to proteolytic degradation than alpha- and gamma-gliadins, and LMW subunits of glutenin were less sensitive than HMW subunits. During germination a time- and temperature-dependent increase of total folate occurred. A maximum 3.6-fold concentration was obtained after 102 h of germination at 20 and 25 degrees C including 5-methyltetrafolate as the major vitamer. The concentration of dietary fiber remained constant or decreased during the first 96 h of germination. Prolonged germination times of up to 168 h led to a substantial increase of total dietary fiber and to a strong increase of the soluble dietary fiber by a factor of 3, whereas the insoluble fiber decreased by 50%.     

家禽体外寄生虫的控制

编者按:虱子、恙虫、家禽壁虱和鸡蚤是家禽业中常见的体外寄生虫.虽然禽舍苍蝇不是寄生虫,但是备受家禽生产者的关注,尤其对于笼养蛋鸡生产者.这些体外寄生虫对家禽业的危害日益得到重视.本专题就上述几种体外寄生虫的生活习性和药物控制措施进行总结,希望为广大读者提供借鉴.     

In vitro anti‐tubulin effects of mebendazole and fenbendazole on canine glioma cells

Benzimidazole anthelmintics have reported anti‐neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC₅₀) (±SD) obtained from performing the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay after treating J3T, G06‐A, and SDT‐3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550 ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC₅₀ showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required.     

Following the track of “Híbrido de Timor” origin by cytogenetic and flow cytometry approaches

The supposedly first plant of the coffee cultivar “Híbrido de Timor” (HT) was found in 1927, being denoted as HT CIFC 4106. According to different researchers, this plant originated from a natural interspecific hybridation between Coffea arabica (4x = 44) and Coffea canephora (2x = 22). From HT CIFC 4106, other HT accessions were obtained and employed to establish germplasm banks in some countries. As HT has been widely used in Coffea breeding programs, this study aimed to characterize different HT accessions with regard to ploidy, nuclear DNA content and base composition. Based on these data, the ploidy of HT CIFC 4106 was determined, suggesting that this accession is an allotriploid formed from reduced reproductive cell of C. canephora and of C. arabica. All HT CIFC 4106 plants exhibited the same 2C-value, AT% and chromosome number, showing that vegetative propagation has enabled the multiplication and germplasm conservation of this cytotype since 1927. Further five analyzed HT accessions showed distinct nuclear 2C-value and AT%. Since HT CIFC 4106 has been considered the first HT, it is suggested that aneuploid reproductive cells of this HT originated the other plants. Considering that HT accessions are used in the development of C. arabica cultivars, the findings of this study are important for the design of strategies to obtain new cultivars for breeding programs. Moreover, these data represent the first step to understand the origin and genome evolution of the HT.     

A case-control study of the effects of nephrolithiasis in cats with chronic kidney disease

OBJECTIVE: To determine whether nephrolithiasis was associated with an increase in mortality rate or in the rate of disease progression in cats with naturally occurring stage 2 (mild) or 3 (moderate) chronic kidney disease. DESIGN: Retrospective case-control study. ANIMALS: 14 cats with stage 2 (mild) or 3 (moderate) chronic kidney disease (7 with nephroliths and 7 without). PROCEDURES: All cats were evaluated every 3 months for up to 24 months. Possible associations between nephrolithiasis and clinicopathologic abnormalities, incidence of uremic crises, death secondary to renal causes, and death secondary to any cause were evaluated. RESULTS: There were no clinically important differences in biochemical, hematologic, or urinalysis variables between cats with and without nephroliths at baseline or after 12 and 24 months of monitoring. No associations were detected between nephrolithiasis and rate of disease progression, incidence of uremic crises, or death. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that in cats with mild or moderate chronic kidney disease, nephrolithiasis was not associated with an increase in mortality rate or in the rate of disease progression. Findings support recommendations that cats with severe kidney disease and nephrolithiasis be managed without surgery.     

Keratitis and periocular lesions associated with equine herpesvirus‐3 in a 3‐month‐old filly

A 3‐month‐old Quarter Horse filly presented with corneal ulceration in the right eye with extensive coalescing periocular ulcerations, erosions, and cutaneous crusts. Similar periocular lesions were present around the left eye, on the gingival mucosa, and on the cutaneous and mucosal surfaces of the lips. Based on the severity of the filly's corneal lesions, expense and duration of treatment, euthanasia was elected. Histological post mortem examination revealed numerous hyperplastic and/or dysplastic epithelial cells adjacent to areas of ulceration and erosion with intranuclear viral inclusion bodies. Equine herpesvirus‐3 (EHV‐3) was identified by polymerase chain reaction from the right cornea and lip. The virus was isolated from the right cornea, right eyelid and lip. The dam presented with multifocal to coalescing perineal vesicles. EHV‐3 was confirmed by polymerase chain reaction from the vulvar lesions and the mare recovered spontaneously. This is the first case of EHV‐3 corneal infection reported in horses and emphasises that EHV‐3 should be included as a differential diagnosis for vesicular lesions involving the equine periocular and oronasal epithelium.