Biosynthetic and regulatory role of lys9 mutants of Saccharomyces cerevisiae

Summary Derepression of lysine biosynthetic enzymes of Saccharomyces cerevisiae was investigated in lys9 auxotrophs which lack saccharopine reductase activity. Five enzymes (homocitrate synthase, homoisocitrate dehydrogenase, -aminoadipate aminotransferase, -aminoadipate reductase and saccharopine dehydrogenase) were constitutively derepressed in all lys9 mutants with up to eight-fold higher enzyme levels than in isogenic wild-type cells. Levels of these enzymes in lys2, lys14, and lys15 S mutants were the same or lower than those in wild-type cells. The regulatory property of lys9 mutants exhibited recessiveness to the wild-type gene in heterozygous diploids. Unlike the mating type effect, homozygous diploids resulting from crosses between lys9 auxotrophs exhibited even higher levels of derepressed enzymes than the haploid mutants. Addition of a higher concentration of lysine to the growth medium resulted in reduction of enzyme levels although they were still derepressed. These results suggest that lys9 mutants represent a lesion for the saccharopine reductase and may represent a repressor mutation which in the wild-type cells simultaneously represses unlinked structural genes that encode for five of the lysine biosynthetic enzymes.     

Lactobacillus acidophilus Induces a Strain-specific and Toll-Like Receptor 2–Dependent Enhancement of Intestinal Epithelial Tight Junction Barrier and Protection Against Intestinal Inflammation

Defective intestinal tight junction (TJ) barrier is an important pathogenic factor of inflammatory bowel disease. To date, no effective therapies that specifically target the intestinal TJ barrier are available. The purpose of this study was to identify probiotic bacterial species or strains that induce a rapid and sustained enhancement of intestinal TJ barrier and protect against the development of intestinal inflammation by targeting the TJ barrier. After high-throughput screening of >20 Lactobacillus and other probiotic bacterial species or strains, a specific strain of Lactobacillus acidophilus, referred to as LA1, uniquely produced a marked enhancement of the intestinal TJ barrier. LA1 attached to the apical membrane surface of intestinal epithelial cells in a Toll-like receptor (TLR)-2–dependent manner and caused a rapid increase in enterocyte TLR-2 membrane expression and TLR-2/TLR-1 and TLR-2/TLR-6 hetero-complex–dependent enhancement in intestinal TJ barrier function. Oral administration of LA1 caused a rapid enhancement in mouse intestinal TJ barrier, protected against a dextran sodium sulfate (DSS) increase in intestinal permeability, and prevented the DSS-induced colitis in a TLR-2– and intestinal TJ barrier–dependent manner. In conclusion, we report for the first time that a specific strain of LA causes a strain-specific enhancement of intestinal TJ barrier through a novel mechanism that involves the TLR-2 receptor complex and protects against the DSS-induced colitis by targeting the intestinal TJ barrier.

Intestinal epithelial tight junctions (TJs) are the apical-most junctional complexes and act as a functional and structural barrier against the paracellular permeation of harmful luminal antigens, which promote intestinal inflammation.¹ The increased intestinal permeability caused by defective intestinal epithelial TJ barrier or a leaky gut is an important pathogenic factor that contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) and other inflammatory conditions of the gut, including necrotizing enterocolitis and celiac disease.^2,3 Clinical studies in patients with IBD have found that a persistent increase in intestinal permeability after clinical remission is predictive of poor clinical outcome and early recurrence of the disease, whereas normalization of intestinal permeability correlates with a sustained long-term clinical remission.4, 5, 6 Accumulating evidence has found that a defective intestinal TJ barrier plays an important role in exacerbation and prolongation of intestinal inflammation in IBD. Currently, no effective therapies that specifically target the tightening of the intestinal TJ barrier are available.Intestinal microbiota play an important role in modulating the immune system and in the pathogenesis of intestinal inflammation.⁷ Patients with IBD have bacterial dysbiosis in the gut, characterized by a decrease in bacterial diversity and an aberrant increase in some commensal bacteria, which are an important factor in the pathogenesis of intestinal inflammation.^8,9 Normal microbial flora of the gastrointestinal tract consists both of bacteria that are known to have beneficial effects (probiotic bacteria) on intestinal homeostasis and bacteria that could potentially have detrimental effects on gut health (pathogenic bacteria).¹⁰ The modulation of intestinal microflora affects the physiologic and pathologic states in humans and animals. For example, fecal transplantation from healthy, unaffected individuals to patients with refractory Clostridium difficile colitis is curative in up to 94% of the treated patients, and transfer of stool microbiome from obese mice induces obesity in previous lean mice, whereas transfer of microbiome from lean mice preserves the lean phenotype.11, 12, 13 The beneficial effects of gut microbiota are host and bacterial species-specific.¹⁴ Although multiple studies indicate that some commensal bacteria play a beneficial role in gut homeostasis by preserving or promoting the intestinal barrier function, because of conflicting reports, it remains unclear which probiotic species cause a persistent predictable enhancement in the TJ barrier and could be used to treat intestinal inflammation by targeting the TJ barrier. For example, some studies suggest that Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, or Lactobacillus rhamnosus cause a modest enhancement in the intestinal epithelial TJ barrier, whereas others have found minimal or no effect of these probiotic species on the intestinal TJ barrier.15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 The major aim the current study was to perform a high-throughput screening of Lactobacillus and other bacterial species to identify probiotic species that induce a rapid, predictable, and marked increase in the intestinal epithelial TJ barrier and protect against the development of intestinal inflammation by preserving the intestinal TJ barrier.In the studies described herein, most of the probiotic species tested (>20 species or strains) had a modest or minimal effect on intestinal TJ barrier function. L. acidophilus uniquely caused a rapid and marked increase in intestinal TJ barrier function. Further analysis indicated that the effect of L. acidophilus was strain-specific, limited to a specific strain of L. acidophilus, and did not extend to other L. acidophilus strains. The L. acidophilus enhancement of the intestinal TJ barrier was mediated by live bacterial-enterocyte interaction that involved Toll-like receptor (TLR)-2 heterodimeric complexes on the apical membrane surface of intestinal epithelial cells. Our animal studies also found that L. acidophilus causes a marked enhancement in mouse intestinal barrier function and protects against the dextran sodium sulfate (DSS)–induced colitis by preserving and augmenting the mouse intestinal barrier function in a strain-specific manner.     

Apoptosis in endometrial glandular and stromal cells in women with and without endometriosis

BACKGROUND: The aetiology of endometriosis is unknown. Ectopic dissemination of the endometrial cells gives origin to endometriotic lesions, but occurs in women with and without endometriosis. It has been suggested that increased ectopic cell survival facilitates their implantation. The objectives of this study were to evaluate endometrial apoptosis in women with endometriosis according to: (i) cyclic changes, (ii) glandular and stromal contribution, and (iii) stage of the disease. METHODS: The subjects were women undergoing diagnostic laparoscopy and endometrial biopsies for suspected endometriosis. Spontaneous apoptosis was evaluated using TdT-mediated dUTP-biotin nick end-labelling (TUNEL) assay. Apoptotic cells per 10 mm(2) (apoptotic index) in an area of 10-50 mm(2) in 5 microm endometrial tissue sections were counted and location of these cells was recorded. RESULTS: The apoptotic index in glandular epithelium was lower in endometriosis than controls (26.0 +/- 5.5 versus 51.2 +/- 9.7, P = 0.03) but not in the stroma (36.3 +/- 6.4 versus 48.4 +/- 11.3, NS). In controls, apoptosis was highest during the late secretory/menstrual and early proliferative phases and cyclic variability was apparent. In endometriosis, this cyclic variability was lost. There was a trend toward decreased apoptosis with increasing stage of the disease, but the differences lacked statistical significance. CONCLUSIONS: Spontaneous apoptosis is decreased in the endometrial glands in women with endometriosis, especially during late secretory/menstrual and early proliferative phases of the cycle. This may indicate increased viability of endometrial cells shed during menses, facilitating their ectopic survival and implantation.     

Controversies in surgical pathology: minimal involvement of sentinel lymph node in breast carcinoma: prevailing concepts and challenging problems

Sentinel lymph node biopsy has attained "standard of care' status in the management of breast carcinoma. However, the pathological interpretation and clinical consequences of "minimally involved' sentinel lymph nodes remain controversial. Herein, we present some of the complex and challenging pathological problems inherent in this evolving setting. Clearly, at least some of our current concepts regarding "minimally involved' sentinel lymph nodes need reappraisal.     

Fine-needle aspiration of a primary mediastinal large B-cell lymphoma: a case report with cytologic, histologic, and flow cytometric considerations

Fine-needle aspiration (FNA) cytology and immunophenotyping by flow cytometry (FCM) are increasingly being used for diagnosing and subclassifying lymphoma in the REAL/WHO classification. Herein, we report a case of primary mediastinal large B-cell lymphoma (PMBL), a subtype of diffuse large B-cell lymphoma in the WHO classification, diagnosed by FNA cytology in conjunction with FCM. This, to our knowledge, has not previously been reported. A 57-yr-old woman presented with bilateral axillary lymphadenopathy and intermittent shortness of breath. CT scan revealed a 5-cm anterior mediastinal mass and mediastinal lymphadenopathy. Endoscopic ultrasound-guided FNA of a 4.5-cm subcarinal lymph node showed medium to large atypical lymphocytes with scant to moderate finely vacuolated cytoplasm. Nuclei were enlarged, cleaved, noncleaved, lobulated, and hyperchromatic. The background showed lymphoglandular bodies. Malignant large cell lymphoma was cytologically diagnosed. FCM, performed on a portion of the FNA specimen, demonstrated large B cells devoid of surface immunoglobulin expression, the characteristic immunophenotype of PMBL. The histologic diagnosis was PMBL. Touch-imprint cytology of the histologic specimen showed large cells with a narrow rim of clear cytoplasm and prominent outer cell border. Nuclear features were similar to the FNA specimen. In the presence of a mediastinal mass, FNA cytology in conjunction with FCM can effectively diagnose PMBL in the appropriate clinical setting.     

Effect of Dietary Essential Amino Acid Limitations upon the Susceptibility to Salmonella typhimurium and the Effect Upon Humoral and Cellular Immune Responses in Mice

We investigated the effects of dietary essential amino acid limitations on the susceptibility of mice to Salmonella typhimurium infections and on humoral and cellular immune (cell-mediated immune) responses of mice. Mice fed synthetic diets limited (significantly less than optimum concentration) in a single essential amino acid (leucine, isoleucine, valine, or lysine) for 3 weeks after they were weaned exhibited significantly enhanced susceptibility to S. typhimurium infection, as evidenced by the higher levels of mortality and spread of the bacterial cells in their livers and spleens compared with mice fed the control diet. Compared with mice fed the control diet, mice fed the diet limited in leucine had a lower ability to clear S. typhimurium cells from the peritoneal cavity 5 min after intraperitoneal injection, whereas mice fed the diet limited in lysine had a greater ability. The in vivo phagocytosis and in vitro bactericidal kinetics against S. typhimurium cells by peritoneal macrophages were not significantly different in the control group and the groups of mice fed experimental diets. Certain experimental groups exhibited significantly lower resistance and antibody response against S. typhimurium SL3770 on day 5 after immunization with heat-killed S. typhimurium SL3770. On day 8 after immunization, the levels of serum antibody against S. typhimurium in the mice fed the experimental diets were comparable to the levels in mice fed the control diet. However, the levels of serum transferrin and complement C3 were significantly lower in mice fed certain experimental diets. The cellular immune capacities of mice fed any of the experimental diets were not impaired compared with the capacities of mice fed the control diet, as measured by spleen cell responsiveness to phytohemagglutinin and the ability to clear infecting Listeria monocytogenes cells from livers and spleens.     

Human haplotype block sizes are negatively correlated with recombination rates

The International Haplotype Map ("HapMap") Project is motivated, in part, by the belief that the organization of the human genome, the mechanics of recombination, and the population-level behavior of alleles at adjacent loci should allow researchers to parse the genome into small segments, or "blocks," that show strong linkage disequilibrium (LD) between alleles at loci within those segments. The discovery and evidence for these blocks is to be based solely on the observed LD strength and patterns between alleles at adjacent loci throughout the genome. Although there are many factors that contribute to LD strength, we assessed the correlation between block structure, in terms of length and percentage of the genome assembled into blocks within a region, and recombination rate obtained from two independent sources. We found evidence of a striking negative correlation between the average recombination rate and average block length, suggesting that recombination rate is a strong contributor to haplotype block structure within the genome. We discuss the potential implications of this negative correlation in the context of the organization, properties, and potential ubiquity of a block-like structure in the human genome.     

Interleukin-1beta increases urine flow rate and inhibits protein expression of Na(+)/K(+)-ATPase in the rat jejunum and kidney.

The effect of interleukin-1beta (IL-1beta) on urine flow rates and Na(+)/K(+)-ATPase activity and expression was studied in rat intestinal and renal epithelia. The cytokine produced a significant diuretic effect and increased urine flow rate by around 10-fold compared with the control. This effect was considered to be secondary to the well-documented natriuretic effect of the cytokine described in the literature. On the other hand, we have shown previously that IL-1beta inhibits glucose absorption from the jejunum. As sodium excretion and glucose absorption are both dependent on Na(+)/K(+)-ATPase activity, the effect of the cytokine on the renal and intestinal pump was investigated. IL-1beta inhibited, in a dose-dependent manner, the activity of Na(+)/K(+)-ATPase in villus and crypt jejunal cells and in medullary and cortical kidney cells. Western blot analysis revealed a decrease in the protein expression of the enzyme, which was confirmed by the radiolabeled ouabain binding assay. The results suggest that the diuretic and natriuretic effect of IL-1beta and its inhibitory effect on glucose absorption are all due to downregulation of the Na(+)/K(+) pump in the kidney and jejunum.     

Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro

Entry of opsonized pathogens into phagocytes may benefit or, paradoxically, harm the host. Opsonization may trigger antimicrobial mechanisms such as reactive oxygen or nitric oxide (NO) production but may also provide a safe haven for intracellular replication. Brucellae are natural intramacrophage pathogens of rodents, ruminants, dogs, marine mammals, and humans. We evaluated the role of opsonins in Brucella-macrophage interactions by challenging cultured murine peritoneal macrophages with Brucella melitensis 16M treated with complement- and/or antibody-rich serum. Mouse serum rich in antibody against Brucella lipopolysaccharide (LPS) (aLPS) and human complement-rich serum (HCS) each enhanced the macrophage uptake of brucellae. Combinations of suboptimal levels of aLPS (0. 01%) and HCS (2%) synergistically enhanced uptake. The intracellular fate of ingested bacteria was evaluated with an optimal concentration of gentamicin (2 microg/ml) to control extracellular growth but not kill intracellular bacteria. Bacteria opsonized with aLPS and/or HCS grew equally well inside macrophages in the absence of gamma interferon (IFN-gamma). Macrophage activation with IFN-gamma inhibited replication of both opsonized and nonopsonized brucellae but was less effective in inhibiting replication of nonopsonized bacteria. IFN-gamma treatment of macrophages with opsonized or nonopsonized bacteria enhanced NO production, which was blocked by N(G)-monomethyl L-arginine (MMLA), an NO synthesis inhibitor. MMLA also partially blocked IFN-gamma-mediated bacterial growth inhibition. These studies suggest that primary murine macrophages have limited ability to control infection with B. melitensis, even when activated by IFN-gamma in the presence of highly opsonic concentrations of antibody and complement. Additional cellular immune responses, e.g., those mediated by cytotoxic T cells, may play more important roles in the control of murine brucellosis.