Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases

Tay-Sachs and Sandhoff diseases are autosomal recessive neurodegenerative diseases resulting from the inability to catabolize GM2 ganglioside by beta-hexosaminidase A (Hex A) due to mutations of the alpha subunit (Tay-Sachs disease) or beta subunit (Sandhoff disease) of Hex A. Hex B (beta beta homodimer) is also defective in Sandhoff disease. We previously developed mouse models of both diseases and showed that Hexa-/- (Tay-Sachs) mice remain asymptomatic to at least 1 year of age while Hexb-/- (Sandhoff) mice succumb to a profound neurodegenerative disease by 4-6 months of age. Here we find that neuron death in Hexb-/- mice is associated with apoptosis occurring throughout the CNS, while Hexa-/- mice were minimally involved at the same age. Studies of autopsy samples of brain and spinal cord from human Tay-Sachs and Sandhoff diseases revealed apoptosis in both instances, in keeping with the severe expression of both diseases. We suggest that neuron death is caused by unscheduled apoptosis, implicating accumulated GM2 ganglioside or a derivative in triggering of the apoptotic cascade.      

The role of recombination signal sequences in the preferential joining by deletion in DH-JH recombination and in the ordered rearrangement of the IgH locus

The bias favoring deletion over inversion in DH-JH rearrangement has been known for years, but the underlying mechanism has yet to be fully defined. It has been suggested that the ratio of deletion/inversion is determined by the combined effect of two factors: (i) the relative strengths of 5' and 3' recombination signal sequences (RSS) of a DH segment, and (ii) the efficiency with which the deletional product (one joint) forms relative to the inversional product (two joints). In this study, we analyzed for the first time the effect of factor 1 alone on the biased 3' RSS utilization in DH-JH joining by using deletional plasmids in an extrachromosomal substrate V(D)J recombination assay. It was found that the 3' RSS and associated coding end (12 bp) mediate recombination more efficiently than the 5' RSS/coding end DH-JH plasmids. These results demonstrate that the effect of the RSS/coding end alone can account, at least partially, for the predominant deletion in DH-JH recombination. The potential effect of the relative strength of RSS and associated coding end on the ordered rearrangement of DH-JH followed by VH to DH-JH was also assessed. When recombination frequencies of D-->J (3' DH to J3) were compared with frequencies of V-- >D (VHPJ14 to 3' DH or VHOX2 to 3' DH), it was found that V-->D joining was, if anything, more efficient than D-->J joining. Therefore, if all three segments were accessible, RSS/coding end effects would not contribute to the ordered rearrangement of the IgH locus.      

Mechanisms of enhanced prevalence of asthma and atopy in developed countries

Atopy — a T helper 2 cell driven hypersensitivity to innocuous antigens (allergens) which causes most cases of asthma — is of complex genetic and environmental origins. There is compelling epidemiological evidence for a rise in atopic disease in ‘westernised’ communities. The changing pattern of microbial exposure in early childhood is suggested to be the principal candidate mechanism for this rise.     

Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation

The tumour suppressor gene PTEN , which maps to 10q23.3 and encodes a 403 amino acid dual specificity phosphatase (protein tyrosine phosphatase; PTPase), was shown recently to play a broad role in human malignancy. Somatic PTEN deletions and mutations were observed in sporadic breast, brain, prostate and kidney cancer cell lines and in several primary tumours such as endometrial carcinomas, malignant melanoma and thyroid tumours. In addition, PTEN was identified as the susceptibility gene for two hamartoma syndromes: Cowden disease (CD; MIM 158350) and Bannayan-Zonana (BZS) or Ruvalcaba-Riley-Smith syndrome (MIM 153480). Constitutive DNA from 37 CD families and seven BZS families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37 (81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven) of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%) BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation, R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small group of BZS families. However, genotype-phenotype analysis inthe group of CD families revealed two possible associations worthy of follow-up in independent analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system, thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families.      

The salivary microbiota as a diagnostic indicator of oral cancer: A descriptive,non-randomized study of cancer-free and oral squamous cell carcinoma subjects

Background  

The purpose of the present investigation was to determine if the salivary counts of 40 common oral bacteria in subjects with an oral squamous cell carcinoma (OSCC) lesion would differ from those found in cancer-free (OSCC-free) controls.     

Release of ATP induced by hypertonic solutions in Xenopus oocytes

ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 m m ). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water.     

Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells

 This study uses a new strategy to investigate the hypothesis that, of the various Ca²⁺ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca²⁺ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o). So, at low [Ca²⁺]_o the K⁺-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type Ca²⁺ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca²⁺ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca²⁺ channel blockade); in high [Ca²⁺]_o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca²⁺ channel blockade) or 3 μM furnidipine (L-type Ca²⁺ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca²⁺]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at both Ca²⁺ concentrations. The results suggest that Q-type Ca²⁺ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca²⁺ that enters the cell through a Ca²⁺ channel located near to chromaffin vesicles will saturate the K⁺ secretory response at both [Ca²⁺]_o, i.e. 0.5 mM and 5 mM. In contrast, Ca²⁺ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca²⁺]_o. Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997     

The development of mouse APECED models provides new insight into the role of AIRE in immune regulation

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy is a rare recessive autoimmune disorder caused by a defect in a single gene called AIRE (autoimmune regulator). Characteristics of this disease include a variable combination of autoimmune endocrine tissue destruction, mucocutaneous candidiasis and ectodermal dystrophies. The development of Aire-knockout mice has provided an invaluable model for the study of this disease. The aim of this review is to briefly highlight the strides made in APECED research using these transgenic murine models, with a focus on known roles of Aire in autoimmunity. The findings thus far are compelling and prompt additional areas of study which are discussed.