Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra-centrifugation. The antigen showed little cross-reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross-reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

Catalytic properties and mode of action of three endo-beta-glucanases from Talaromyces emersonii on soluble beta-1,4- and beta-1,3;1,4-linked glucans

In this paper, we present the first detailed analysis of the modes of action of three purified, thermostable endo-beta-D-glucanases (EG V-VII) against a range of soluble beta-linked glucans. Studies indicated that EG V-VII, purified to homogeneity from a new source, the thermophilic fungus Talaromyces emersonii, are strict beta-glucanases that exhibit maximum activity against mixed-link 1,3;1,4-beta-D-glucans. Time-course hydrolysis studies of 1,4-beta-D-glucan (carboxymethylcellulose; CMC), 1,3;1,4-beta-D-glucan from barley (BBG) and lichenan confirmed the endo-acting nature of EG V-VII and verified preference for 1,3;1,4-beta-D-glucan substrates. The results suggest that EG VI and EG VII belong to EC 3.2.1.6, as both enzymes also exhibit activity against 1,3-beta-glucan (laminaran), in contrast to EG V. Although cellobiose, cellotriose and glucose were the main glucooligosaccharide products released, the range and relative amount of each product was dependent on the particular enzyme, substrate and reaction time. Kinetic constants (Km, Vmax, kcat and kcat/Km) determined for EG V-VII with BBG as substrate yielded similar Km and Vmax values for EG V and EG VI. EG VII exhibited highest affinity for BBG (Km value of 9.1 mg ml(-1)) and the highest catalytic efficiency (kcat/Km of 12.63 s(-1) mg(-1) ml).     

YtjE from Lactococcus lactis IL1403 Is a C-S lyase with alpha, gamma-elimination activity toward methionine

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.     

Heterologous Production of Methionine-γ-Lyase from Brevibacterium linens in Lactococcus lactis and Formation of Volatile Sulfur Compounds

The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-γ-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade l-methionine as well as other sulfur-containing compounds such as l-cysteine, l-cystathionine, and l-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.Methionine (Met) catabolism plays a major role in cheese flavor development. Met is believed to be the precursor of numerous diverse and quantitatively minor volatile sulfur compounds (VSCs) (38) which make important contributions to the overall flavor that are typical to different cheeses (7). Most of these compounds are derived from the degradation of Met to methanethiol (MTL), giving rise to a variety of compounds such as dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and S-methylthioesters (41).The Met biosynthetic and catabolic pathways leading to MTL vary among bacteria (36), as do the enzymes involved and the amount of MTL produced during cheese ripening (15). The most direct pathway occurs via l-methionine γ-elimination of Met to MTL, α-ketobutyrate, and ammonia. This l-methionine γ-elimination activity has been shown to be quite efficient in Brevibacterium linens (21), while its presence has been suggested in several other cheese surface bacteria such as Micrococcus luteus, Arthrobacter sp., Corynebacterium glutamicum, and Staphylococcus equorum (9). In B. linens, this activity is catalyzed by an l-methionine-γ-lyase (MGL), which has been previously purified and characterized (16). Moreover, disruption of the mgl gene encoding the enzyme has been shown to almost eliminate this strain''s considerable capacity to produce VSCs (2). In lactococci, which produce only limited amounts of VSCs (15), this reaction is catalyzed by cystathionine lyases (β- or γ-) which are responsible for the simultaneous deamination and demethylthiolation of Met to MTL (1, 11, 18, 22); recently, a new C-S lyase (YtjE) with α,γ-elimination activity that degrades Met into MTL has been characterized in our laboratory (30). Unfortunately, C-S lyases display relatively low activities toward Met, limiting their capacities to produce VSCs. Another route for Met catabolism involving a two-step mechanism initiated by an aminotransferase has also been identified in lactococci (8, 23) and other cheese-ripening bacteria (3, 9), which leads to the formation of α-keto-γ-methylthiobutyric acid which is subsequently converted to MTL; this route, however, produces only limited amounts of MTL.In recent years, numerous studies have pursued the control and/or diversification of VSCs primarily by means of using selected cheese-ripening microorganisms or combinations of them (4, 7, 25). A few studies have focused on engineering lactic acid bacteria (LAB) with enhanced VSC-producing abilities by increasing cystathionine lyase activities (22, 27). In this respect, a Lactococcus lactis strain engineered to overproduce cystathionine β-lyase was shown to produce larger quantities of VSCs with Met as a substrate compared to the wild-type strain (22). However, this study also showed no significant difference in VSC production between the wild-type strain and a cystathionine β-lyase-knockout variant, implying that other enzymes may play a more prominent role in the conversion of Met.The aim of this study was to develop LAB with improved capability to produce MTL and other related VSCs as a more efficient strategy to enhance cheese flavor development. We describe the bioengineering of food-grade L. lactis strains to produce MGL, which has been reported to play a major role in the catabolism of Met to MTL in B. linens, a good producer of VSCs (2). The enzyme activity of the recombinant MGL was confirmed, and its role in the production of VSCs by recombinant L. lactis was also investigated.     

In Vivo Identification and Characterization of CD4+ Cytotoxic T Cells Induced by Virulent Brucella abortus Infection

CD4⁺ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4⁺ T cells (CD4⁺ CTL) during Brucella abortus infection. These CD4⁺ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4⁺ CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4⁺ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4⁺ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.     

Chemical structure of the core oligosaccharide of aerotolerant Campylobacter jejuni O:2 lipopolysaccharide

The structure of the core oligosaccharide of aerotolerant Campylobacter jejuni 0:2 lipopolysaccharide was determined and found to contain 3-deoxy-D-manno-octulosonic acid (Kdo), L-glycero-D-manno-heptose (LD-Hep), D-galactose, D-glucose, and phosphorylethanolamine (PEtn). Based on 1H, 13C and 31P NMR spectroscopic studies including 2D COSY, TOCSY, ROESY and heteronuclear 1H-31P and HMQC experiments it was established that the oligosaccharide has the following structure: [structure: see text].