O-acetylation and de-O-acetylation of sialic acids in human colorectal carcinoma.

A decrease in the level of O-acetylated sialic acids observed in colorectal carcinoma may lead to an increase in the expression of sialyl Lewis(X), a tumor-associated antigen, which is related to progression of colorectal cancer to metastasis. The underlying mechanism for this reduction is, however, not fully understood. Two enzymes are thought to be primarily responsible for the turnover of O-acetyl ester groups on sialic acids; sialate-O-acetyltransferase (OAT) and sialate-O-acetylesterase (OAE). We have previously reported the characterization of OAT activity from normal colon mucosa, which efficiently O-acetylates CMP-Neu5Ac exclusively in the Golgi apparatus prior to the action of sialyltransferase. In this report we describe the identification of a lysosomal and a cytosolic OAE activity in human colonic mucosa that specifically hydrolyses 9-O-acetyl groups on sialic acid. Utilizing matched resection margin and cancer tissue from colorectal carcinoma patients we provide strong evidence suggesting that the level of O-acetylated sialic acids present in normal and diseased human colon may be dependent on the relative activities of OAT to lysosomal OAE. Furthermore, we show that the level of free cytosolic Neu5,9Ac2 in human colon is regulated by the relative activity of the cytosolic OAE.     

Molecular cloning and sequence analyses of calcium/calmodulin- dependent protein kinase II from fetal and adult human brain – Sequence analyses of human brain calcium/calmodulin-dependent protein kinase II

The aims of this study were to characterize specific mRNAs and the expression pattern for isoforms of calcium/calmodulin-dependent protein kinase II (CaMKII) in the human brain. We cloned and sequenced the CaMKII and subunit cDNAs, and used them to study the CaMKII expression in human brain. Four distinct isoforms of CAMKII were isolated. Two of them were characterized as CaMKII and subunits. The other two showed similar nucleotide sequences, but one had a 33-bp insertion relative to the subunit, and the other had a 75-bp deletion relative to the subunit. These alterations are located within the variable regions. These two isoforms were characterized as CaMKII _B and _e. Northern blot analysis showed that a 4.4-kb messenger RNA for the isoform and a 3.9-kb messenger RNA for the isoform were expressed in both human fetal and adult brain to different degrees. The results indicate that CaMKII expression is developmentally regulated. The CaMKII isoform expression was confirmed in human fetal and adult brain using RT-PCR with specific primers, which flanked the CaMKII variable regions. The CaMKII , _B, , and _e isoforms were characterized in both human fetal and adult brain.     

Combined evaluation of LC3B puncta and HMGB1 expression predicts residual risk of relapse after adjuvant chemotherapy in breast cancer

In spite of adjuvant chemotherapy, a significant fraction of patients with localized breast cancer (BC) relapse after optimal treatment. We determined the occurrence of cytoplasmic MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3B)-positive puncta, as well as the presence of nuclear HMGB1 (high mobility group box 1) in cancer cells within surgical BC specimens by immunohistochemistry, first in a test cohort (152 patients) and then in a validation cohort of localized BC patients who all received adjuvant anthracycline-based chemotherapy (1646 patients). Cytoplasmic LC3B⁺ puncta inversely correlated with the intensity of SQSTM1 staining, suggesting that a high percentage cells of LC3B⁺ puncta reflects increased autophagic flux. After setting optimal thresholds in the test cohort, cytoplasmic LC3B⁺ puncta and nuclear HMGB1 were scored as positive in 27.2% and 28.6% of the tumors, respectively, in the validation cohort, while 8.7% were considered as double positive. LC3B⁺ puncta or HMGB1 expression alone did not constitute independent prognostic factors for metastasis-free survival (MFS) in multivariate analyses. However, the combined positivity for LC3B⁺ puncta and nuclear HMGB1 constituted an independent prognostic factor significantly associated with prolonged MFS (hazard ratio: 0.49 95% confidence interval [0.26–0.89]; P = 0.02), and improved breast cancer specific survival (hazard ratio: 0.21 95% confidence interval [0.05–0.85]; P = 0.029). Subgroup analyses revealed that within patients with poor-prognosis BC, HMGB1⁺ LC3B⁺ double-positive tumors had a better prognosis than BC that lacked one or both of these markers. Altogether, these results suggest that the combined positivity for LC3B⁺ puncta and nuclear HMGB1 is a positive predictor for longer BC survival.     

Pregnancy allows the transfer and differentiation of fetal lymphoid progenitors into functional T and B cells in mothers

T lymphocytes of fetal origin found in maternal circulation after gestation have been reported as a possible cause for autoimmune diseases. During gestation, mothers acquire CD34+CD38+ cells of fetal origin that persist decades. In this study, we asked whether fetal T and B cells could develop from these progenitors in the maternal thymus and bone marrow during and after gestation. RAG-/--deficient female mice (Ly5.2) were mated to congenic wild-type Ly5.1 mice (RAG+/+). Fetal double-positive T cells (CD4+CD8+) with characteristic TCR and IL-7R expression patterns could be recovered in maternal thymus during the resulting pregnancies. We made similar observations in the thymus of immunocompetent mothers. Such phenomenon was observed overall in 12 of 68 tested mice compared with 0 of 51 controls (p=0.001). T cells could also be found in maternal spleen and produced IFN-gamma in the presence of an allogenic or an Ag-specific stimulus. Similarly, CD19+IgM+ fetal B cells as well as plasma Igs could be found in maternal RAG-/- bone marrow and spleen after similar matings. Our results suggest that during gestation mothers acquire fetal lymphoid progenitors that develop into functional T cells. This fetal cell microchimerism may have a direct impact on maternal health.     

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6′‐phosphate phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose‐specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6‐phospho‐α‐glucosidase, which in B. subtilis hydrolyses maltose 6′‐P into glucose and glucose 6‐P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6‐P into glucose 1‐P and glucose 6‐P. However, purified MalP phosphorolyses maltose but not maltose 6′‐P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6′‐P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1‐P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6′‐P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6′‐P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1‐P. MapP therefore connects PTS‐mediated maltose uptake to maltose phosphorylase‐catalysed metabolism. Dephosphorylation assays with a wide variety of phospho‐substrates revealed that MapP preferably dephosphorylates disaccharides containing an O‐α‐glycosyl linkage.     

Deficient tryptophan catabolism along the kynurenine pathway reveals that the epididymis is in a unique tolerogenic state

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway. Intriguingly, IDO is constitutively and highly expressed in the mammalian epididymis in contrast to most other tissues where IDO is induced by proinflammatory cytokines, such as interferons. To gain insight into the role of IDO in the physiology of the mammalian epididymis, we studied both wild type and Ido1(-/-)-deficient mice. In the caput epididymis of Ido1(-/-) animals, the lack of IDO activity was not compensated by other tryptophan-catabolizing enzymes and led to the loss of kynurenine production. The absence of IDO generated an inflammatory state in the caput epididymis as revealed by an increased accumulation of various inflammation markers. The absence of IDO also increased the tryptophan content of the caput epididymis and generated a parallel increase in caput epididymal protein content as a consequence of deficient proteasomal activity. Surprisingly, the lack of IDO expression had no noticeable impact on overall male fertility but did induce highly significant increases in both the number and the percentage of abnormal spermatozoa. These changes coincided with a significant decrease in white blood cell count in epididymal fluid compared with wild type mice. These data provide support for IDO playing a hitherto unsuspected role in sperm quality control in the epididymis involving the ubiquitination of defective spermatozoa and their subsequent removal.