Experimental chemotherapy with N'N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) in autochthonous neurogenic tumors of the rat transplacentally induced by ethylnitrosourea

Autochthonous neurogenic tumors of the rat induced by transplacental application of ethylnitrosourea were used for the first time to study their suitability as tumor models for experimental chemotherapy. Of 189 transplacentally treated rats, 87% developed neurogenic tumors. After the initial clinical diagnosis of a neurogenic tumor, additional malignant tumors often occurred. The mean number of neurogenic tumors from 62 untreated control rats increased from 1.0 per rat at the time of randomization to 1.2 as revealed by autopsy and 1.5 tumors by histological examinations. Out of all neurogenic tumors, tumors of the brain were observed in 31%, tumors of cranial nerves in 36% (90% tumors of trigeminal nerve), tumors of spinal cord in 21%, and tumors of peripheral nerves in 10%. The median survival time until natural death of 62 control rats was 228 days. Rats with tumors of peripheral nerves lived shortest, followed by rats with tumors of cranial nerves, tumors of the spinal cord, and brain tumors. Brain tumors were mainly astrocytomas and oligodendrogliomas. The survival time of untreated rats from randomization to natural death was longest for those with brain tumors, followed by tumors of peripheral nerves, cranial nerves, and tumors of the spinal cord. There was great variation in survival time from a few days to more than 6 months. To study the responsiveness to chemotherapy, 62 rats received BCNU as a single intravenous dose of 9 and later 10 mg/kg. Sixty-two untreated control rats had a median survival time of 36 days (95% confidence interval 26-52 days), the treated rats 43.5 days (26-62 days). The difference was not statistically significant. BCNU produced a remission or a no change of neurologic symptoms in 60% (37 out of 62) in comparison to 39% (24 out of 62) in the control group (p less than 0.05). The advantages and disadvantages of the present models are discussed. Due to methodical problems and the marginal response to BCNU, autochthonous neurogenic tumors of the rat are not suitable as models for chemotherapeutic studies.     

Homozygous deletions of methylthioadenosine phosphorylase (MTAP) are more frequent than p16INK4A (CDKN2) homozygous deletions in primary non-small cell lung cancers (NSCLC)

Homozygous deletions of the tumor suppressor gene p16INK4A and deficiency of methylthioadenosine phosphorylase (MTAP), both located on chromosome 9p21, have been independently reported in non-small cell lung cancer (NSCLC). To determine the frequency of co-deletion of these two genes, we investigated 50 samples of primary NSCLC using a quantitative PCR-ELISA. All specimens were fixed in formalin, paraffin embedded and stored until assayed. Histologic subtypes included 25 adenocarcinomas (50%), 21 squamous cell carcinomas (42%) and four large cell carcinomas (8%). Homozygous deletions of MTAP exon 8 could be detected in 19 of 50 NSCLC samples (38%). Adenocarcinoma (11 of 25, 44%) showed a higher deletion frequency than squamous cell carcinoma (six of 21, 29%). In contrast, homozygous p16INK4A deletions were detected in only nine of 50 (18%) samples using specific primers for p16INK4A exon 1alpha. No difference between the histological subtypes and p16INK4A deletion frequency was observed. We further investigated the ten samples with MTAP deletions but intact p16INK4A exon 1alpha with primers specific for p16INK4A exon 3, the exon nearest to MTAP exon 8. Interestingly, none of the ten samples had deletion of the p16INK4A exon 3 coding region. Fine mapping analysis performed in ten samples showed a frequent breakpoint between MTAP exon 4 and exon 5. In addition, p16 protein expression could not be detected in five out of six samples with intact p16 but deleted MTAP locus. These data show a high frequency of homozygous MTAP deletions in NSCLC which is associated with detectable co-deletion of p16INK4A in only half of the cases. This result suggests the existence either of another tumor suppressor gene telomeric of p16INK4A or of deletions involving 3'-untranslated (3'-UTR) regulatory regions of p16INK4A that can interfere with its expression or function.     

Microglia are more susceptible than macrophages to apoptosis in the central nervous system in experimental autoimmune encephalomyelitis through a mechanism not involving Fas (CD95)

Morphological studies have shown that macrophages and microglia undergo apoptosis in the central nervous system (CNS) in acute experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. To assess the relative levels of macrophage and microglial apoptosis, and the molecular mechanisms involved in this process, we used three-colour flow cytometry to identify CD45lowCD11b/c+ microglial cells and CD45highCD11b/c+ macrophages in the inflammatory cells isolated from the spinal cords of Lewis rats 13 days after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Simultaneously, we analyzed the DNA content of these cell populations to assess the proportions of cells undergoing apoptosis and in different stages of the cell cycle or examined their expression of three apoptosis-regulating proteins, i.e. Fas (CD95), Fas ligand (FasL) and Bcl-2. Microglia were highly vulnerable to apoptosis and were over-represented in the apoptotic population. Macrophages were less susceptible to apoptosis than microglia and underwent mitosis more frequently than microglia. The different susceptibilities of microglia and macrophages to apoptosis did not appear to be due to variations in Fas, FasL or Bcl-2 expression, as the proportions of microglia and macrophages expressing these proteins were similar, and were relatively high. Furthermore, in contrast to T cell apoptosis, apoptosis of microglia/macrophages did not occur more frequently in cells expressing Fas or FasL, or less frequently in cells expressing Bcl-2. These results indicate that the apoptosis of microglia and CNS macrophages in EAE is not mediated through the Fas pathway, and that Bcl-2 expression does not protect them from apoptosis. Expression of FasL by macrophages and microglia may contribute to the pathogenesis and immunoregulation of EAE through interactions with Fas+ oligodendrocytes and Fas+ T cells. The high level of microglial apoptosis in EAE indicates that microglial apoptosis may be an important homeostatic mechanism for controlling the number of microglia in the CNS following microglial activation and proliferation.     

Specificity of the H-2 L(d)-restricted cytotoxic T-lymphocyte response to the mouse hepatitis virus nucleocapsid protein

Cytotoxic T lymphocytes provide protection against persistent infection of the central nervous system by the JHM strain of mouse hepatitis virus. In BALB/c (H-2d) mice, the dominant response is directed against an Ld-restricted peptide in the nucleocapsid protein (APTAGAFFF). Characterization of the fine specificity of this response revealed that the predicted anchor residues at positions 2 and 9 were the most critical for class I binding. Amino acids at positions 7 and 8 were identified as T-cell receptor contact residues. Virus-induced cytotoxic T lymphocytes to other Ld motif-containing nucleocapsid peptides were not detected, despite the identification of two epitopes with reduced Ld affinity. These data suggest that mutations within four residues of the dominant epitope could contribute to the persistence of the JHM strain of mouse hepatitis virus.     

Minor groove binding of a bis-quaternary ammonium compound: the crystal structure of SN 7167 bound to d(CGCGAATTCGCG)2

The X-ray crystal structure of the complex between the synthetic antitumour and antiviral DNA binding ligand SN 7167 and the DNA oligonucleotide d(CGCGAATTCGCG)2 has been determined to an R factor of 18.3% at 2.6 A resolution. The ligand is located within the minor groove and covers almost 6 bp with the 1-methylpyridinium ring extending as far as the C9-G16 base pair and the 1-methylquinolinium ring lying between the G4-C21 and A5-T20 base pairs. The ligand interacts only weakly with the DNA, as evidenced by long range contacts and shallow penetration into the groove. This structure is compared with that of the complex between the parent compound SN 6999 and the alkylated DNA sequence d(CGC[e6G]AATTCGCG)2. There are significant differences between the two structures in the extent of DNA bending, ligand conformation and groove binding.     

X-SCID B cell responses to interleukin-4 and interleukin-13 are mediated by a receptor complex that includes the interleukin-4 receptor alpha chain (p140) but not the gamma c chain

We previously reported that patients with mild to moderate airflow limitation have a lower exercise capacity than age-matched controls with normal lung function, but the mechanism of this reduction remains unclear (1). Although the reduced exercise capacity appeared consistent with deconditioning, the patients had altered breathing mechanics during exercise, which raised the possibility that the reduced exercise capacity and the altered breathing mechanics may have been causally related. Reversal of reduced exercise capacity by an adequate exercise training program is generally accepted as evidence of deconditioning as the cause of the reduced exercise capacity. We studied 11 asymptomatic volunteer subjects (58 +/- 8 yr of age [mean +/- SD]) selected to have a range of lung function (FEV1 from 61 to 114% predicted, with a mean of 90 +/- 18% predicted). Only one subject had an FEV1 of less than 70% predicted. Gas exchange and lung mechanics were measured during both steady-state and maximal exercise before and after training for 30 min/d on 3 d/wk for 10 wk, beginning at the steady-state workload previously determined to be the maximum steady-state exercise level that subjects could sustain for 30 min without exceeding 90% of their observed maximal heart rate (HR). The training workload was increased if the subject's HR decreased during the training period. After 10 wk, subjects performed another steady-state exercise test at the initial pretraining level, and another maximal exercise test. HR decreased significantly between the first and second steady-state exercise tests (p < 0.05), and maximal oxygen uptake (VO2max) and ventilation increased significantly (p < 0.05) during the incremental test, indicating a training effect. However, the training effect did not occur in all subjects. Relationships between exercise parameters and lung function were examined by regression against FEV1 expressed as percent predicted. There was a significant positive correlation between VO2max percent predicted and FEV1 percent predicted (p < 0.02), and a negative correlation between FEV1 and end-expiratory lung volume (EELV) at maximal exercise (p < 0.03). There was no significant correlation between FEV1 and maximal HR achieved during exercise; moreover, all subjects achieved a maximal HR in excess of 80% predicted, suggesting a cardiovascular limitation to exercise. These data do not support the hypothesis that the lower initial VO2max in the subjects with a reduced FEV1 was due to deconditioning. Although increased EELV at maximal exercise, reduced VO2max and a reduced VO2max response with training are all statistically associated with a reduced FEV1, there is no direct evidence of causality.     

The two-exon gene of the human forkhead transcription factor FREAC-2 (FKHL6) is located at 6p25.3

Subcutaneous metastases from clear cell endometrial carcinoma are an uncommon event and tumor implantations are rarely found with diagnostic imaging techniques. The nodular form is the most frequent type of subcutaneous metastasis from genital system tumors, even though plaque-like and infiltrative forms have also been reported. We report the first case of subcutaneous metastasis from clear cell endometrial carcinoma whose progression from the early nodular to the lymphangitic infiltrative form was studied with computed tomography (CT). Differential diagnostic problems are discussed.     

Rapid protein sequencing by tandem mass spectrometry and cDNA cloning of p20-CGGBP. A novel protein that binds to the unstable triplet repeat 5'-d(CGG)n-3' in the human FMR1 gene

The autonomous expansion of the unstable 5'-d(CGG)n-3' repeat in the 5'-untranslated region of the human FMR1 gene leads to the fragile X syndrome, one of the most frequent causes of mental retardation in human males. We have recently described the isolation of a protein p20-CGGBP that binds sequence-specifically to the double-stranded trinucleotide repeat 5'-d(CGG)-3' (Deissler, H., Behn-Krappa, A., and Doerfler, W. (1996) J. Biol. Chem. 271, 4327-4334). We demonstrate now that the p20-CGGBP can also bind to an interrupted repeat sequence. Peptide sequence tags of p20-CGGBP obtained by nanoelectrospray mass spectrometry were screened against an expressed sequence tag data base, retrieving a clone that contained the full-length coding sequence for p20-CGGBP. A bacterially expressed fusion protein p20-CGGBP-6xHis exhibits a binding pattern to the double-stranded 5'-d(CGG)n-3' repeat similar to that of the authentic p20-CGGBP. This novel protein lacks any overall homology to other known proteins but carries a putative nuclear localization signal. The p20-CGGBP gene is conserved among mammals but shows no homology to non-vertebrate species. The gene encoding the sequence for the new protein has been mapped to human chromosome 3.     

Episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6) due to CAG repeat expansion in the CACNA1A gene on chromosome 19p

Point mutations of the CACNA1A gene coding for the alpha 1A voltage-dependent calcium channel subunit are responsible for familial hemiplegic migraine (FHM) and episodic ataxia type 2 (EA2). In addition, expansions of the CAG repeat motif at the 3' end of the gene, smaller than those responsible for dynamic mutation disorders, were found in patients with a progressive spinocerebellar ataxia, named SCA6. In the present work, the analysis of two new families with small CAG expansions of the CACNA1A gene is presented. In one family, with a clinical diagnosis of EA2, a CAG23 repeat allele segregated in patients showing different interictal symptoms, ranging from nystagmus only to severe progressive cerebellar ataxia. No additional mutations in coding and intron-exon junction sequences in disequilibrium with the CAG expansion were found. In the second family, initially classified as autosomal dominant cerebellar ataxia of unknown type, an inter-generational allele size change showed that a CAG20 allele was associated with an EA2 phenotype and a CAG25 allele with progressive cerebellar ataxia. These results show that EA2 and SCA6 are the same disorder with a high phenotypic variability, at least partly related to the number of repeats, and suggest that the small expansions may not be as stable as previously reported. A refinement of the coding and intron-exon junction sequences of the CACNA1A gene is also provided.     

Commentary: The Torrance Tests of Creative Thinking already overcome many of the perceived weaknesses that Silvia et al.'s (2008) methods are intended to correct.

Commentary on an article by P. J. Silvia et al. (see record 2008-05954-001) which discusses the topic of divergent thinking. Silvia et al.'s (2008) primary motivations for exploring and proposing their subjective scoring method are their perceived deficiencies of current divergent thinking tests. First, scores on divergent thinking tests frequently correlate highly with general intelligence. Second, the scoring of divergent thinking tests has changed little since the 1960s. Third, the necessity of instructing people to be creative prior to taking divergent thinking tests is integral to obtaining useful responses and needs to be reaffirmed. Fourth, and finally, the problems posed by uniqueness scoring--confounding with fluency, ambiguity of rarity, and the seeming penalty imposed on large samples--need to be addressed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.     

Presenilin 1 mutations linked to familial Alzheimer's disease increase the intracellular levels of amyloid beta-protein 1-42 and its N-terminally truncated variant(s) which are generated at distinct sites

Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid beta-protein (A beta) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuroblastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or delta exon 10). The induction of mutated PS1 increased the intracellular levels of two distinct A beta species ending at residue 42 that were likely to be A beta1-42 and its N-terminally truncated variant(s) A beta x-42. The induction of mutated PS1 resulted in a higher level of intracellular A beta1-42 than of intracellular A beta x-42, whereas extracellular levels of A beta1-42 and A beta x-42 were increased proportionally. In addition, the intracellular generation of these A beta42 species in wt and mutated PS1-induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular A beta x-42 versus inhibition of intracellular A beta1-42 generation. These data strongly suggest that A beta x-42 is generated in a proximal Golgi, whereas A beta1-42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific gamma-secretase cleavage that occurs in the normal beta-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to beta-secretase cleavage or (b) in the distinct sites where A beta x-42 and A beta1-42 are generated.