Immediate and carryover effects of Gram-negative and Gram-positive toxin-induced mastitis on follicular function in dairy cows

This study compared immediate and carryover effects of mastitis induced by Gram-negative endotoxin (E. coli LPS) and Gram-positive exosecretions (Staph. aureus ex.) on preovulatory follicle function. Synchronized, uninfected cyclic lactating Holstein cows were treated with PGF_2α on day 6 of the cycle and 36 h later, a dose of either E. coli LPS (n = 8), S. aureus ex. (n = 10), or saline (n = 9) was administered into the mammary gland. Follicular fluids and granulosa cells were aspirated 6 h later from the preovulatory follicles and cows were treated with GnRH. This (cycle 1; immediate effect) was repeated three times (excluding the mammary injections) to induce three 7 d cycles (cycles 2, 3, and 4; carryover effect). E. coli LPS increased body temperature, plasma cortisol concentration, and somatic cell count (SCC), whereas S. aureus ex. induced a minor, subclinical elevation of SCC and slight rise (NS) in body temperature and cortisol concentration. Follicular estradiol, androstenedione, and progesterone concentrations in the E. coli LPS group decreased (P < 0.05) in cycle 1 to about 40%, 13%, and 35%, respectively, of control levels, whereas in the S. aureus ex. group, only estradiol decreased (P < 0.05), to 56% of control concentrations. In cycles 3 and 4, follicular steroids in the E. coli LPS group returned to control concentrations, whereas in the S. aureus ex. group, follicular concentrations of estradiol and androstenedione were lower (P < 0.10) than in controls. In the control group, the concentrations of all follicular and circulating steroids remained stable (P > 0.05) throughout the study. Follicle size was similar in all groups, but the S. aureus ex. treatment caused a decrease (P < 0.02) in the number of follicles developed in cycles 3 and 4. The mRNA expression of steroidogenic genes and LHCGR in the granulosa cells was not affected (P > 0.05) by either treatment during the study, except for a tendency toward lower (P < 0.1) expression in cycle 1 and lower (P < 0.05) expression in cycle 4 of the latter in the S. aureus ex. group. Strain levels, such as SCC and body temperature, following toxin injection correlated well with the magnitude of the immediate decline in follicular steroids. As is typical for Gram-negative clinical events, E. coli LPS-induced acute mastitis caused immediate, short-term, but not long-term impairment of follicular responses, whereas the Gram-positive S. aureus ex.-induced subclinical mastitis exhibited both immediate and carryover disruptive effects on preovulatory follicle function.     

A novel tumor-associated pancreatic glycoprotein is internalized by human dendritic cells and induces their maturation

Aberrant glycosylation or overexpression of cell-surface glycosylated tumor-associated Ags (TAA) distinguish neoplastic from normal cells. Interactions of TAA MUC1 and HER2/neu with dendritic cells (DC) preclude efficient processing, which impairs immune responses. It is thus important to define the mechanisms of interactions between DC and glycosylated TAA and their trafficking and processing for further T cell activation. In this work, we study interactions between DC and the oncofetal fucose-rich glycovariants of bile salt-dependent lipase (BSDL), expressed in pancreatic cancer tissues and referred to as pathological BSDL carrying the fucosylated J28 glycotope (pBSDL-J28) because it is characterized by the mAb J28. The expression of pBSDL-J28 was assessed by immunohistochemistry and quantified by confocal microscopy. Nontumoral pancreatic tissues and cells do not express pBSDL-J28. Using multidisciplinary approaches and functional studies, we provide the first evidence, to our knowledge, that this tumoral glycoprotein is rapidly internalized by human DC through macropinocytosis and endocytosis via mannose receptors and then transported to late endosomes for processing. Interestingly, pBSDL-J28 per se induced DC maturation with increased expression of costimulatory and CD83 molecules associated with cytokine secretion (IL-8 and IL-6). Surprisingly, DC retained their full ability to internalize Ags, making this maturation atypical. Finally, the allogeneic pBSDL-J28-treated DC stimulated lymphocyte proliferation. Besides, pulsing DC with pBSDL-J28 C-terminal glycopolypeptide and maturation with CD40L triggered CD4(+) and CD8(+) T cell proliferation. Therefore, interactions of pBSDL-J28, expressed on tumoral pancreatic tissue, with DC may lead to adequate Ag trafficking and processing and result in T cell activation.     

Crystal structure of the outer membrane protein RcsF, a new substrate for the periplasmic protein-disulfide isomerase DsbC

The bacterial Rcs phosphorelay is a stress-induced defense mechanism that controls the expression of numerous genes, including those for capsular polysaccharides, motility, and virulence factors. It is a complex multicomponent system that includes the histidine kinase (RcsC) and the response regulator (RcsB) and also auxiliary proteins such as RcsF. RcsF is an outer membrane lipoprotein that transmits signals from the cell surface to RcsC. The physiological signals that activate RcsF and how RcsF interacts with RcsC remain unknown. Here, we report the three-dimensional structure of RcsF. The fold of the protein is characterized by the presence of a central 4-stranded β sheet, which is conserved in several other proteins, including the copper-binding domain of the amyloid precursor protein. RcsF, which contains four conserved cysteine residues, presents two nonconsecutive disulfides between Cys(74) and Cys(118) and between Cys(109) and Cys(124), respectively. These two disulfides are not functionally equivalent; the Cys(109)-Cys(124) disulfide is particularly important for the assembly of an active RcsF. Moreover, we show that formation of the nonconsecutive disulfides of RcsF depends on the periplasmic disulfide isomerase DsbC. We trapped RcsF in a mixed disulfide complex with DsbC, and we show that deletion of dsbC prevents the activation of the Rcs phosphorelay by signals that function through RcsF. The three-dimensional structure of RcsF provides the structural basis to understand how this protein triggers the Rcs signaling cascade.     

Identification of kinin-related peptides in the disease vector, Rhodnius prolixus

We have used an in silico approach to identify a gene from the blood-gorging vector, Rhodnius prolixus, that is predicted to produce an insect kinin prepropeptide. The prepropeptide is 398 amino acids in length and can potentially produce a large number of kinin-related peptides following post-translational processing. A comparison with other insect kinin precursor sequences demonstrates greatest conservation at the C-terminal region of the kinin peptides. Multiple peptides predicted from the kinin gene are phenotypically expressed in R. prolixus, as revealed by MALDI-TOF MS MS, including 12 kinins and one kinin precursor peptide (KPP). Six of these peptides are characterized by the typical insect kinin C-terminal motif FX₁X₂WGamide and five of these are also found as truncated forms. Five peptides were identified with an atypical, though similar, FX₁X₂WAamide C-terminus. There is also peptide with a C-terminal DDNGamide motif and a number of non-amidated peptides.     

Role for cohesin in the formation of a heterochromatic domain at fission yeast subtelomeres

Increasing evidence implicates cohesin in the control of gene expression. Here we report the first analysis of cohesin-dependent gene regulation in fission yeast. Global expression profiling of the mis4-367 cohesin loader mutant identified a small number of upregulated and downregulated genes within subtelomeric domains (SD). These 20- to 40-kb regions between chromosome arm euchromatin and telomere-proximal heterochromatin are characterized by a combination of euchromatin (methylated lysine 4 on histone H3/methylated Tysine 9 on histone H3 [H3K4me]) and heterochromatin (H3K9me) marks. We focused our analysis on the chromosome 1 right SD, which contains several upregulated genes and is bordered on the telomere-distal side by a pair of downregulated genes. We find that the expression changes in the SD also occur in a mutant of the cohesin core component Rad21. Remarkably, mutation of Rad21 results in the depletion of Swi6 binding in the SD. In fact, the Rad21 mutation phenocopied Swi6 loss of function: both mutations led to reduced cohesin binding, reduced H3K9me, and similar gene expression changes in the SD. In particular, expression of the gene pair bordering the SD was dependent both on cohesin and on Swi6. Our data indicate that cohesin participates in the setup of a subtelomeric heterochromatin domain and controls the expression of the genes residing in that domain.     

Improved <Emphasis Type="Italic">p</Emphasis>-hydroxybenzoate production by engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12 by using a mixed-substrate feeding strategy

The key precursors for p-hydroxybenzoate production by engineered Pseudomonas putida S12 are phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), for which the pentose phosphate (PP) pathway is an important source. Since PP pathway fluxes are typically low in pseudomonads, E4P and PEP availability is a likely bottleneck for aromatics production which may be alleviated by stimulating PP pathway fluxes via co-feeding of pentoses in addition to glucose or glycerol. As P. putida S12 lacks the natural ability to utilize xylose, the xylose isomerase pathway from E. coli was introduced into the p-hydroxybenzoate producing strain P. putida S12palB2. The initially inefficient xylose utilization was improved by evolutionary selection after which the p-hydroxybenzoate production was evaluated. Even without xylose-co-feeding, p-hydroxybenzoate production was improved in the evolved xylose-utilizing strain, which may indicate an intrinsically elevated PP pathway activity. Xylose co-feeding further improved the p-hydroxybenzoate yield when co-fed with either glucose or glycerol, up to 16.3 Cmol% (0.1 g p-hydroxybenzoate/g substrate). The yield improvements were most pronounced with glycerol, which probably related to the availability of the PEP precursor glyceraldehyde-3-phosphate (GAP). Thus, it was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways. Moreover, this approach opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks such as lignocellulose that typically has a high content of both glucose and xylose and (crude) glycerol.     

Genomics of an endemic cystic fibrosis Burkholderia multivorans strain reveals low within-patient evolution but high between-patient diversity

Burkholderia multivorans is a member of the Burkholderia cepacia complex (Bcc), notorious for its pathogenicity in persons with cystic fibrosis. Epidemiological surveillance suggests that patients predominantly acquire B. multivorans from environmental sources, with rare cases of patient-to-patient transmission. Here we report on the genomic analysis of thirteen isolates from an endemic B. multivorans strain infecting four cystic fibrosis patients treated in different pediatric cystic fibrosis centers in Belgium, with no evidence of cross-infection. All isolates share an identical sequence type (ST-742) but whole genome analysis shows that they exhibit peculiar patterns of genomic diversity between patients. By combining short and long reads sequencing technologies, we highlight key differences in terms of small nucleotide polymorphisms indicative of low rates of adaptive evolution within patient, and well-defined, hundred kbps-long segments of high enrichment in mutations between patients. In addition, we observed large structural genomic variations amongst the isolates which revealed different plasmid contents, active roles for transposase IS3 and IS5 in the deactivation of genes, and mobile prophage elements. Our study shows limited within-patient B. multivorans evolution and high between-patient strain diversity, indicating that an environmental microdiverse reservoir must be present for this endemic strain, in which active diversification is taking place. Furthermore, our analysis also reveals a set of 30 parallel adaptations across multiple patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the cystic fibrosis lung.     

Reduction of starch granule size by expression of an engineered tandem starch-binding domain in potato plants

Granule size is an important parameter when using starch in industrial applications. An artificial tandem repeat of a family 20 starch-binding domain (SBD2) was engineered by two copies of the SBD derived from Bacillus circulans cyclodextrin glycosyltransferase via the Pro-Thr-rich linker peptide from Xyn10A from Cellulomonas fimi. SBD2 and a single SBD were introduced into the amylose-free potato mutant, amf, using appropriate signal sequences. The accumulation of SBD2 into transgenic starch granules was much higher than that of SBD. In a number of transformants, particularly amfSS3, the starch granules were much smaller than in control plants. The amfSS3 mean granule size was 7.8 microm, compared with 15.2 microm in the control, whereas other starch properties were unaltered. This new starch combines the advantage of the high purity of potato starch with that of the small granule size of other crop species, such as cassava, taro and wheat. This starch may find application in the manufacture of biodegradable plastic films. Both genes were also expressed in Escherichia coli and the affinity for soluble starch of the purified recombinant proteins was determined. SBD2 had an approximately 10-fold higher affinity for starch than SBD, indicating that the two appended SBDs act in synergy when binding to their target polysaccharide ligand.