An immunodominant haptenic epitope of carbamazepine detected in serum from patients given long-term treatment with carbamazepine without allergic reaction

An anticarbamazepine antibody was detected in the serum of a patient with severe carbamazepine-induced serum sickness. We found that the patient's T cells and IgG antibody recognized an epitope which appeared in subjects showing an allergic reaction, as well as that in subjects who showed no allergic reaction, after long-term carbamazepine therapy. These results show that an anti-carbamazepine immune response does not occur in the majority of subjects who undergo long-term carbamazepine therapy without developing allergic symptoms, although the immunodominant haptenic epitope of carbamazepine is present in their sera.     

DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair

The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3′- or 5′-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5′ side of its binding region, a weak ssDNA-binding domain resides at the 3′ side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1–XPF on the DNA. With the 3′-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1–XPF, whereas the 5′-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.     

Cleavage of integrin by mu-calpain during hypoxia in human endometrial cells

PROBLEM: The distribution and activation of mu-calpain and possible cleavage of integrin in human endometrial cells under hypoxic condition were investigated. METHOD OF STUDY: Human endometrial epithelial and stromal cells were subjected to hypoxia, and subsequently used for immunostaining and western blot analysis. RESULTS: The proform of mu-calpain was detected in the cytoplasm of normal cells, and displayed a substantial decrease after hypoxia. Conversely, the active form of mu-calpain was not detected in normal cells, but was abundant after hypoxia. The cytoplasmic domain of integrin beta3 was also detected in the cytoplasm of endometrial cells. Western blot analysis confirmed that both the proform of mu-calpain and the integrin beta3 cytoplasmic domain decreased during hypoxia. CONCLUSIONS: Mu-calpain is activated in human endometrial cells during hypoxia and that subsequent cleavage of the integrin beta3 cytoplasmic domain may give some adverse effects to the function of human endometrium.     

Bam32 links the B cell receptor to ERK and JNK and mediates B cell proliferation but not survival

Bam32 is an adaptor protein recruited to the plasma membrane upon B cell receptor (BCR) crosslinking in a phosphoinositol 3-kinase (PI3K)-dependent manner; however, its physiologic function is unclear. To determine its physiologic function, we produced Bam32-deficient mice. Bam32(-/-) B cells develop normally but have impaired T-independent antibody responses in vivo and diminished responses to BCR crosslinking in vitro. Biochemical analysis revealed that Bam32 acts in a novel pathway leading from the BCR to MAPK/ERK Kinases (MEK1/2), MAPK/ERK Kinase Kinase-1 (MEKK1), extracellular signal-regulated kinase (ERK), and c-jun NH2-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (p38). This pathway appears to be initiated by hematopoietic progenitor kinase-1 (HPK1), which interacts directly with Bam32, and differs from all previously characterized BCR signaling pathways in that it is required for normal BCR-mediated proliferation but not for B cell survival.     

Polo-like kinase 1 expression in medullary carcinoma of the thyroid: its relationship with clinicopathological features.

OBJECTIVE: Polo-like kinase 1 (PLK1) is one of the serine-threonine kinases that contributes to cell mitosis and is regarded as a marker of cellular proliferation. However, its protein expression in human carcinoma has not been studied in depth. In this study, we investigated PLK1 expression in medullary thyroid carcinoma by means of immunohistochemistry. METHODS: We immunohistochemically investigated PLK1 expression in 67 cases of medullary thyroid carcinoma. RESULTS: The PLK1 expression level was elevated in 43 of the 67 cases (64.1%). Furthermore, the expression level was directly linked to lymph node metastasis, advanced stage and male sex. All patients who were negative for PLK1 expression are currently alive without tumor recurrence, while 6 of the 43 PLK1-positive patients showed recurrence and 3 have already died of this disease. CONCLUSIONS: These findings suggest that PLK1 expression significantly reflects aggressive characteristics of medullary thyroid carcinoma.     

Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia

Clostridium botulinum serotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.     

CD69-null mice protected from arthritis induced with anti-type II collagen antibodies

CD69, known as an early activation marker antigen on T and B cells, is also expressed on platelets and activated neutrophils, suggesting certain roles in inflammatory diseases. In order to address the role of CD69 in the pathogenesis of arthritis, we established CD69-null mice. CD69-null mice displayed a markedly attenuated arthritic inflammatory response when injected with anti-type II collagen antibodies. Cell transfer experiments with neutrophils, but not T cells or spleen cells, from wild-type mice into CD69-null mice restored the induction of arthritis. These results indicate a critical role for CD69 in neutrophil function in arthritis induction during the effector phase. Thus, CD69 would be a possible therapeutic target for arthritis in human patients.     

Activity-inducible protein Homer1a/Vesl-1S promotes redistribution of postsynaptic protein Homer1c/Vesl-1L in cultured rat hippocampal neurons

In cultured rat hippocampal neurons, overexpression of Homer1a/Vesl-1S, an inducible protein upregulated by seizure or long-term potentiation, caused a reduction of punctate distribution of a postsynaptic protein Homer1c/Vesl-1L, without significant decrease in its total amount. Clusters of F-actin were also decreased. Treatments of cells with BDNF or a proteasome inhibitor, which cause increase in the expression level of endogenous Homer1a, also resulted in the reduction of Homer1c puncta. These results indicate that the accumulation of Homer1a, either exogenously expressed or endogenously induced, caused redistribution and dispersion of postsynaptic clusters of Homer1c and F-actin, suggesting an important role of Homer1a in synaptic remodeling.     

ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats

Expansion of CAG trinucleotide repeats that encode polyglutamine is the underlying cause of at least nine inherited human neurodegenerative disorders, including Huntington's disease and spinocerebellar ataxias. PolyQ fragments accumulate as aggregates in the cytoplasm and/or in the nucleus, and induce neuronal cell death. However, the molecular mechanism of polyQ-induced cell death is controversial. Here, we show the following: (1) polyQ with pathogenic repeat length triggers ER stress through proteasomal dysfunction; (2) ER stress activates ASK 1 through formation of an IRE1-TRAF2-ASK1 complex; and (3) ASK1(-/-) primary neurons are defective in polyQ-, proteasome inhibitor-, and ER stress-induced JNK activation and cell death. These findings suggest that ASK1 is a key element in ER stress-induced cell death that plays an important role in the neuropathological alterations in polyQ diseases.