Use of electrospray mass spectrometry for mass determination of grape (Vitis vinifera) juice pathogenesis-related proteins: a potential tool for varietal differentiation

Methods based on liquid chromatography-mass spectrometry (LC-MS) and protein trap mass spectrometry (trap-MS) were developed to determine the complement of pathogenesis-related (PR) proteins in grape juice. Trap-MS was superior to LC-MS in terms of simplicity, rapidity, and sensitivity. Proteins with a wide range of masses (13--33 kDa) were found in the juices of 19 different varieties of grape (Vitis vinifera) and were identified as mostly PR-5 type (thaumatin-like) and PR-3 type (chitinases) proteins. Although the PR proteins in juices of grapes are highly conserved, small consistent differences in molecular masses were noted when otherwise identical proteins were compared from different varieties. These differences persisted through different harvest years and in fruits grown in different Australian locations. With the definition of four different masses for PR-5 proteins (range = 21,239--21,272 Da) and nine different masses of PR-3 proteins (range = 25,330--25,631 Da) and using statistical analysis, the methods developed could be used for varietal differentiation of grapes grown in several South Australian locations on the basis of the PR protein composition of the juice. It remains to be seen whether this technology can be extended to grapes grown worldwide and to wine and other fruit-derived products to assist with label integrity to the benefit of consumers.     

Physical demands and low-back injury risk among children and adolescents working on farms

Little information currently exists regarding the risk of low-back disorders among youth who perform physically demanding farm activities. Thus, afield study was conducted in which children and adolescents who engage in farm work were recruited, fitted with a lumbar motion monitoring system, and then observed performing their usual chores. The lumbar motion monitor was used to record the trunk movements required while youth were performing routine manual material handling tasks on a farm. Workplace factors and motions from both males and females were recorded on over 40 farm tasks, such as feeding animals, lifting bales of hay and straw, and other miscellaneous farm chores. Although the sample size and number of observations in this study were small, the results showed that the magnitude of several work-related factors, such as weight and horizontal moment arm, and trunk motions for many farm activities were equal to or greater than those associated with high injury risk jobs previously assessed in industrial workplaces. In this study, we quantified the physical demands of tasks performed by children and adolescents on farms. In addition, the specific farm chores more likely to load the spines of youth and thereby contribute to musculoskeletal injury were identified.     

Thaumatin-like proteins and chitinases, the haze-forming proteins of wine, accumulate during ripening of grape (Vitis vinifera) berries and drought stress does not affect the final levels per berry at maturity

Thaumatin-like proteins and chitinases, which are pathogenesis-related (PR) proteins, were the major soluble protein components of grapes from five cultivars of Vitis vinifera. This dominance of PR proteins was apparent at berry softening (véraison) and then throughout berry development for the Muscat of Alexandria, Sultana, and Shiraz cultivars and in the berries of the Sauvignon Blanc and Pinot Noir cultivars examined at commercial maturity. The M(r) of the major thaumatin-like protein from Muscat of Alexandria grapes was 21 272, and those of the three major chitinases from this cultivar, ChitB, ChitC, and ChitD, were 25 588, 25 410, and 25 457, respectively. The vines in the study were irrigated and showed no obvious signs of disease. Shiraz vines that had not been irrigated throughout the season were clearly water stressed, but had levels of PR proteins in the berry similar to vines that had been fully irrigated. It appears that the production of PR proteins that cause protein instability in wines by grapes may be little influenced by environmental conditions.     

Infection with foot-and-mouth disease virus (FMDV) induces a natural killer (NK) cell response in cattle that is lacking following vaccination

Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFN-γ). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a disease of cloven-hoofed animals and remains a threat to livestock industries throughout the world. IL-2 stimulation of PBMC resulted in poor killing of human K562 cells, which are often used as NK target cells, while lysis of the bovine BL3.1 cell line was readily detected. Depletion of NKp46-expressing cells revealed that 80% of the killing induced by IL-2 could be attributed to NKp46⁺ cells. In order to characterize the response of NK cells against FMDV in vivo, we infected groups of cattle with three different strains of the virus (A24 Cruzeiro, O1 Manisa, O Hong Kong) and evaluated the cytolytic ability of NK cells through the course of infection. We consistently observed a transient increase in cytolysis, although there was variation in magnitude and kinetics. This increase in cytolysis remained when CD3⁺ cells were removed from the preparation of lymphocytes, indicating that cytolysis was independent of MHC-T cell receptor interaction or γδ T cell activation. In contrast, animals monitored following vaccination against FMDV did not exhibit any increase in NK killing. These data suggest that NK cells play a role in the host immune response of cattle against FMDV, and contrast with the suppression of NK activity previously observed in swine infected with FMDV.     

Antigen-specific proliferation and activation of peripheral blood mononuclear cells from Mycobacterium bovis-infected reindeer

To evaluate antigen-specific proliferative and activation-associated responses from Mycobacterium bovis-infected reindeer, blood mononuclear cells from M. bovis- (n = 10) and non-infected reindeer (n = 4) were stimulated with a recombinant early secretory antigenic target-6 and culture filtrate protein-10 fusion protein (rESAT6:CFP10), M. bovis purified protein derivative, pokeweed mitogen, or medium alone and evaluated by flow cytometry using dye tracker analysis and cell surface marker staining. gammadelta TCR+ and CD8+ cells, but not CD4+ cells, from M. bovis-infected reindeer proliferated in response to specific antigen stimulation. Expression (i.e., mean fluorescence intensity) of CD44 was increased and CD62L decreased on proliferative as compared to non-proliferative fractions in antigen- and mitogen-stimulated cultures. In response rESAT6:CFP10 stimulation, MHC II fluorescence intensity was increased on CD4+, gammadelta TCR+, CD172a+, and IgM+ cells from infected reindeer as compared to that of non-stimulated cells from the same reindeer. Recombinant ESAT6:CFP10 stimulation also induced expansion of a CD172a+, MHC II+ population within mononuclear cell cultures from M. bovis-infected reindeer. Despite a moderate challenge dose and extended duration of incubation, experimental infection of reindeer was generally limited to lymph nodes draining the inoculation site, suggestive of host resistance to progressive disease. Present in vitro findings, therefore, may be predictive of host responses by reindeer that limit progression to disseminated disease.     

X-Chromosomal Monosomy (77, XO) in a Doberman Pinscher With Gonadal Dysgenesis

A 6-month-old stunted female Doberman Pinscher was found to have a 77,X0 chromosomal complement. The ovaries were small, consisting primarily of interstitial-type cells and solid epithelial cords. The dam, sire, and a male littermate had normal karyotypes.     

Acute thallium toxicosis in a dog.

A Doberman Pinscher was evaluated for acute onset of gastroenteritis, characterized by anorexia, hematemesis, and hematochezia. The dog had ingested mole bait containing thallium 2 days prior to admission. Thallium toxicosis was confirmed by detection of thallium in the urine, using colorimetric analysis. The dog responded well to administration of antibiotics, fluids administered IV, warm-water enemas, and oral administration of activated charcoal slurries.     

Intracranial Lesions in Dogs With Hemangiosarcoma

A retrospective analysis of 85 dogs with hemangiosarcoma (HSA) that underwent complete necropsy, including gross examination of the brain, was conducted. Grossly identifiable intracranial lesions were present in 17 dogs. Twelve of 85 dogs (14.2%) had brain metastases. Four of 85 dogs (4.7%) had hemorrhagic lesions and/or ischemic necrosis without identifiable tumor. One dog had a primary central nervous system tumor. Signs of intracranial disease were present in six of 85 dogs (7.1%) with HSA; four had brain metastases and two had nonneoplastic lesions. Metastases had a propensity for cerebrum and gray matter. Dogs with brain metastases had more widely disseminated disease than dogs without brain metastases (P less than 0.001). Dogs with pulmonary metastases were at greater risk for developing brain metastases than dogs without pulmonary metastases (odds ratio = 8.31). Although thoracic radiography accurately identified ten of 12 dogs (83%) with pulmonary metastases, too few cases were available to assess the applicability/accuracy of thoracic radiography in predicting the presence or absence of brain metastases in dogs with malignancy and signs of intracranial disease.