Toxic Immunoglobulin Light Chain Autoantibodies are Associated with a Cluster of Severe Complications in Older Adult Type 2 Diabetes.

AIMS: To assess neuronal depolarization evoked by autoantibodies in diabetic depression compared to depolarization evoked by autoantibodies in control patients. To determine whether a subset of severe (late-onset) diabetic complications may be mediated in part by toxic immunoglobulin light chains that may increase in diabetic nephropathy. METHODS: Protein-A eluates from plasma of 21 diabetic depression patients and 37 age-matched controls were tested for depolarization in hippocampal or immature neurons. Subsets of depolarizing or non-depolarizing autoantibodies were tested for neurite outgrowth inhibition in N2A neuroblastoma cells or the ability to modulate Ca2+ release in HL-1 atrial cardiomyocytes or in endothelial cells. The stability of depolarizing autoantibodies was investigated by heat treatment (56°C × 30 minutes) or following prolonged exposure to the pro-protein convertase, furin. Gel filtration of active depolarizing autoantibodies was performed to determine the apparent molecular mass of peak neurotoxicity associated with the autoantibodies. RESULTS: Diabetic depression (n = 21) autoantibodies caused significantly greater mean depolarization in neuroblastoma cells (P < 0.01) compared to autoantibodies in diabetic (n = 15) or non-diabetic (n = 11) patients without depression. Depolarizing autoantibodies caused significantly more (P=0.011) inhibition of neurite outgrowth in neuroblastoma cells than non-depolarizing autoantibodies (n = 10) and they evoked sustained, global intracellular Ca2+ release in atrial cardiomyocytes or in endothelial cells. A subset of older diabetic patients suffering with a cluster of nephropathy, non-ischemic cardiomyopathy and/or depression demonstrated the presence of stable light chain dimers having apparent MW of 46 kD and associated with peak neurotoxicity in neuroblastoma cells. CONCLUSION: These data suggest that autoantibodies in older adult diabetic depression cause long-lasting depolarization in hippocampal neurons including adult dentate gyrus neural progenitor cells. The autoantibodies may impair adult dentate gyrus neurogenesis associated with treatment-refractory depression via several mechanisms including suppression of neurite outgrowth, and alteration of membrane excitability. Stable, toxic light chain autoantibody components may contribute to a cluster of severe (late-onset) complications characterized by dysfunction in highly vascularized tissues.

Temperature structure functions in the Bolgiano regime of thermal convection.

We measure temperature fluctuations in the Rayleigh-Bénard apparatus, which is a closed cylindrical container with the bottom wall heated and the top wall cooled. The aspect ratio, which is the diameter-to-height ratio of the apparatus, is unity. The Rayleigh number is 1.5 x 10(11). The working fluid is cryogenic helium gas. We compute temperature structure functions up to order 16, and use extended self-similarity to obtain scaling exponents in the Bolgiano regime. In contrast to passive scalars, the scaling exponents tend not to saturate with the order of the structure function, suggesting the absence of ramplike structures in temperature traces of convective motion.

The dynamics of bone marrow stromal cells in the proliferation of multipotent hematopoietic progenitors by substance P: an understanding of the effects of a neurotransmitter on the differentiating hematopoietic stem cell.

Communication within the hematopoietic-neuroendocrine-immune axis is partly mediated by neurotransmitters (e.g. substance P, SP) and cytokines. SP mediates neuromodulation partly through the stimulation of bone marrow (BM) progenitors. This study shows that SP, through the neurokinin-1 receptor, stimulates the proliferation of primitive hematopoietic progenitors: cobblestone-forming cells (CAFC, CD34+). This effect is optimal when macrophage is included within the fibroblast support. Indirect induction of IL-1 could be important in the proliferation of CAFC colonies by SP. Phenotypic and functional studies suggest that SP might directly interact with the CD34+/CD45(dim) population. These studies indicate that SP can initiate a cascade of biological responses in the BM stroma and stem cells to stimulate hematopoiesis.

Congenital ovine neuronal ceroid lipofuscinosis--a cathepsin D deficiency with increased levels of the inactive enzyme.

We recently showed that a form of neuronal ceroid lipofuscinosis (NCL) in white Swedish landrace sheep is caused by a missense mutation in the cathepsin D gene resulting in complete inactivation of the enzyme. Despite the lack of cathepsin D activity, the brains of the cathepsin D deficient sheep showed strongly increased staining for cathepsin D in immunohistochemistry. By Western blotting, a 5-10 fold increase in the level of cathepsin D was confirmed. These results indicate that the missense mutation in congenital NCL sheep results in the synthesis of an inactive yet stable cathepsin D.

Four regimes of decaying grid turbulence in a finite channel.

Attenuation of second sound in helium II has been used to observe up to 6 orders of magnitude of decaying vorticity displaying four distinctly different regimes of decaying grid turbulence in a finite channel. A purely classical spectral model for homogeneous and isotropic turbulence describes most of the decay of helium II vorticity in the temperature range 1.2

A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration.

The neuronal ceroid lipofuscinoses (NCLs) constitute a group of neurodegenerative storage diseases characterized by progressive psychomotor retardation, blindness and premature death. Pathologically, there is accumulation of autofluorescent material in lysosome-derived organelles in a variety of cell types, but neurons in the central nervous system appear to be selectively affected and undergo progressive death. In this report we show that a novel form of NCL, congenital ovine NCL, is caused by a deficiency in the lysosomal aspartyl proteinase cathepsin D. A single nucleotide mutation in the cathepsin D gene results in conversion of an active site aspartate to asparagine, leading to production of an enzymatically inactive but stable protein. This results in severe cerebrocortical atrophy and early death, providing strong evidence for an important role of cathepsin D in neuronal development and/or homeostasis.

Neurokinin-1 expression and co-localization with glutamate and GABA in the hypothalamus of the cat.

Recent behavioral studies using pharmacological techniques have demonstrated that the high affinity substance P (SP) receptor, neurokinin-1 receptor (NK-1), in the medial hypothalamus could be important in mediating defensive rage behavior in the cat. These observations prompted us to use molecular techniques to determine the distribution of NK-1 in the hypothalamus and in other regions of the forebrain relevant to the control of rage behavior. We cloned a 650 bp fragment of the cat NK-1 cDNA. Partial DNA sequence analyses of this fragment indicate 90% homology with the human cDNA. By in situ hybridization (ISH), we showed that NK-1 mRNA was localized in the cytoplasm but not nuclei of cat forebrain neurons. Furthermore, NK-1 mRNA was co-localized in neurons that displayed positive immunolabeling for glutamate or GABA. Moderate labeling was visualized in the anterior medial hypothalamus which receives significant SP input via the stria terminalis from the medial amygdala. Strong labeling was also observed in the basal amygdaloid complex. The functional significance of this labeling pattern is suggested from the observation that both the medial and basal complex of amygdala serve as powerful modulators of defensive rage behavior. Weaker labeling was seen over the posterior medial and lateral hypothalamus. The distribution of NK-1 in the hypothalamus was matched by that of SP-immunoreactive axons and pre-terminals that were observed in the hypothalamus. The overall findings provide anatomical evidence to show that the high affinity SP receptor, NK-1, is linked to glutamate and GABA neurons in the anterior medial hypothalamus and further suggests its likely role in the regulation of feline aggression.

Physiological and molecular characterization of the Na+/Ca2+ exchanger in human platelets.

In this report, we have demonstrated that Na+/Ca2+ exchanger activity in a human megakaryocytic cell line (CHRF-288 cells) is K+ dependent, similar to the properties previously described for Na+/Ca2+ exchange activity in human platelets. With the use of RT-PCR techniques and mRNA, the exchanger expressed in CHRF-288 cells was found to be identical to that expressed in human retinal rods. Northern blot analysis of the mRNA for the human retinal rod exchanger in CHRF-288 cells revealed a major transcript at 5.8 kb with two minor bands at 4.9 and 6.8 kb. mRNA for the retinal rod exchanger was also identified in human platelets. Using Ba2+ influx as a measure of Na+/Ca2+ exchange activity in human platelets, we have demonstrated that exchange activity is driven by the transmembrane gradient for K+ as well as that for Na+. We propose that the K+ dependence of the platelet Na+/Ca2+ exchanger could make platelets especially sensitive to daily fluctuations in salt intake.

Mutational analysis of the defective protease in classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder.

The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G-->C transversion in the invariant AG of a 3' splice junction, found in 38 of 115 alleles, and a C-->T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg-->His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.

Structural organization and sequence of CLN2, the defective gene in classical late infantile neuronal ceroid lipofuscinosis.

Mutations in the CLN2 gene result in classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disease. In this report, we present the complete sequence of the human CLN2 gene and define its physical relationship with two other genes that have been previously mapped to chromosome 11p15. The CLN2 gene consists of 13 exons and 12 introns and spans 6.65 kb. By S1 mapping and primer extension, the 5'-terminus of the CLN2 mRNA was mapped to 32 nucleotides upstream of the proposed initiation codon. A number of other elements were found to be located in close proximity to CLN2, including the gene encoding transcription factor TAFII30, the gene encoding intregrin-linked kinase, and an approximately 914-bp fragment that is 82% identical to antithrombin III. In addition, an EST cDNA clone that is transcribed on the strand opposite to CLN2 and that overlaps a portion of the CLN2 gene was identified. Finally, a set of primer pairs are presented for the amplification of the coding sequences, putative promoter, and splice junctions of the CLN2 gene. Taken together, this information will facilitate the molecular analysis of and genetic testing for classical LINCL.

Truncation of Sp1 transcription factor by myeloblastin in undifferentiated HL60 cells.

When HL60 cells are exposed to 1,25-dihydroxyvitamin D3 (1,25D3), they undergo changes approximating the phenotype of the monocyte. Little is known, however, about the regulation and the mechanisms of this transition. It was previously noted that DNA binding by the Sp1 transcription factor in nuclear extracts of HL60 cells is profoundly altered when these cells are induced to differentiate by 1,25D3. In the present study, we show that in untreated HL60 cells only a truncated, approximately 30-kDa Sp1 fragment, encompassing the C-terminal region, binds to the GC element-containing DNA. Full-length 105-kDa Sp1 protein cannot be detected in these cells, although reverse transriptase-polymerase chain reaction reveals the presence of both 5' and 3' ends of Sp1 mRNA. Following treatment with 10(7) M 1,25D3 for 96 hr or in cells made resistant to 1,25D3 or to 1-beta-D-arabinocytosine, the Sp1 protein can be demonstrated. After an exposure to purified myeloblastin, a serine protease, purified recombinant Sp1 protein and extracts of 1,25D3-treated cells show a pattern of DNA binding similar to the pattern seen using extracts of untreated HL60 cells, indicating that the Sp1 protein is a target for myeloblastin. Because myeloblastin is present in naive HL60 cells and is downregulated during their differentiation, inhibition of proteolysis of these transcription factors seems to provide a mechanism through which differentiating HL60 cells can acquire a new repertoire of gene expression, perhaps for the maintenance of the differentiated phenotype.

Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region.

SETTING: Five IS6110 chromosomal insertion sites were characterized in the multidrug resistant Mycobacterium tuberculosis 'W' strain. OBJECTIVE: To use insertion site probes to study the phylogenetic distribution of IS6110 in the M. tuberculosis genome. DESIGN: A total of 722 M. tuberculosis isolates, previously genotyped using the standard IS6110 Southern blot hybridization methodology, were re-hybridized with the Region A insertion site probe and representative strains were further hybridized with the Region B and C probes. Strains were grouped on the basis of having IS6110 insertions in these different regions and their relatedness was further compared by sequencing the IS6110 insertion sites. RESULTS: The insertion site probes revealed that the collection of Chinese isolates previously grouped as the Beijing strain family shared IS6110 insertions in common with the W and other genotypic group 1 strains. Unexpectedly, we found that IS6110 integrated at least 10 independent times between the dnaA and dnaN genes encoding deoxyribonucleic acid replication proteins. CONCLUSIONS: IS6110 insertion site mapping is able to identify genetic relatedness among a collection of M. tuberculosis clinical strains representing the breadth of species diversity. The mapping data indicate that IS6110 insertion sites are not always random.

Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.

Differential gene expression in SV40-mediated immortalization of human fibroblasts.

Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A lambda cDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is reduced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of J4-4 and J4-3 strongly suggest that they are involved in the process of immortalization and/or can serve as target genes for assessing regulators of gene expression in this process.

Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.

p53 gene aberrations in non-small-cell lung carcinomas from a smoking population.

We examined 46 non-small-cell lung carcinomas (NSCLCs) for the presence of p53 mutations in exons 4-9, positive p53 immunostaining and loss of heterozygosity (LOH) in the TP53 locus. p53 mutations were detected in 13 tumour samples (28.3%), whereas overexpression of the p53 protein was found in 30 of 45 (67%) samples. Allelic loss was found in 9 of 38 (23.6%) informative cases. The statistical analysis revealed no significant correlation between p53 mutations and clinicopathological data, although mutations appear to occur more frequently in squamous cell carcinomas (7 of 18) than in adenocarcinomas (2 of 15). All but three individuals in this study group smoked. In contrast to previous reports, we found a higher prevalence of GC-->AT transitions than of GC-->TA transversions, as expected in a smoking population. A trend was found between p53-positive immunostaining and a history of heavy smoking (76-126 pack-years) and was inversely correlated with allelic deletion (LOH) at the TP53 locus. Eight of the 12 NSCLCs containing p53 mutations also had concomitant p53 overexpression, and it is of specific note that three of the four tumours containing p53 'mutations' with no overexpression of the p53 protein had either insertions or deletions in the p53 gene. No correlation was found between p53 mutations and fractional allele loss or ras mutations. p53 mutations in this Merseyside population in the UK do not appear to be as common as in other reports for NSCLC and exhibit predominance of GC-->AT transitions preferentially at non-CpG sites, suggesting that other carcinogens in addition to those in tobacco smoke may be involved in NSCLC in the Merseyside area of the UK.

Surgery for lung cancer: age alone is not a contraindication.

A retrospective analysis of the clinical features, operative procedures, postoperative complications and subsequent survival of 70 (50 male) elderly patients undergoing surgery for lung cancer compared with 74 (53 male) younger patients treated at the same hospital during the same period was performed, to determine if elderly people with lung cancer are less likely to benefit from and/or tolerate surgery. The elderly group had to wait longer for operation (p = 0.001) and were more likely to have pre-existing disease (p = 0.019). In contrast, they had fewer recognised postoperative complications (p = 0.032) and there was no difference between the two groups in perioperative mortality and subsequent survival. Surgical treatment of localised lung cancer represents the best chance for cure and this study suggests that age should not be a consideration in the decision to operate or not. The patient's general state of health should be assessed and management decisions based on individual status rather than on age.

The intracellular domain of the second chain of the interferon-gamma receptor is interchangeable between species.

In this report we show that the mouse interferon (IFN)-gamma R1 and IFN-gamma R2 subunits expressed in hamster cells are capable of rendering the cells sensitive to mouse IFN-gamma as measured by induction of class I MHC antigens and the activation of the transcription factor Stat1 alpha. However, these cells showed no antiviral protection in response to IFN-gamma when challenged with vesicular stomatitis virus (VSV) but limited protection when challenged with encephalomyocarditis virus (EMCV). Furthermore, the cytoplasmic domains of the IFN-gamma R2 subunits, like the cytoplasmic domains of the IFN-gamma R1 chains, can be interchanged between species with no loss of biologic activity, demonstrating that the species-specific interaction of the IFN-gamma R1 and IFN-gamma R2 chains involves only the extracellular domains of the two proteins.

Fractional allele loss data indicate distinct genetic populations in the development of non-small-cell lung cancer.

Allelic imbalance or loss of heterozygosity (LOH) has been widely used to assess genetic instability in tumours, and high LOH on chromosome arms 3p, 9p and 17p has been considered to be a common event in non-small-cell lung cancer (NSCLC). We have investigated allelic imbalance in 45 NSCLCs using 92 microsatellite markers on 38 chromosome arms. LOH of 38% was observed on 3p using nine markers, 58% on 9p using 15 markers and 38% on 17p using five markers. Fractional allele loss (FAL) has been calculated for each tumour (FAL is the number of chromosome arms showing LOH/number of informative chromosome arms) and a median FAL value of 0.09 was obtained in the 45 NSCLCs studied. The LOH data were examined on the basis of FAL scores: low FAL (LFAL) (0.00-0.04), medium FAL (MFAL) (0.05-0.13) and high FAL (HFAL) (0.14-0.45) based symmetrically around the median FAL value of 0.09. Tumours with HFAL values showed a very clear polarisation of the LOH data on chromosome arms 3p, 9p and 17p, such that 80% showed loss on 3p, 80% on 9p and 73% on 17p. These incidences of LOH were significantly higher than would be expected, since overall genetic instability in these HFAL tumours ranged from 14% to 45% LOH. Nine of the 14 patients in the LFAL group were found to have no LOH on 3p, 9p or 17p, but five of these had LOH at other sites: i.e. LOH on 5p, 5q, 8p, 13q, 16q and 19q. These results indicate that LFAL patients form a new subset of NSCLC tumours with distinct molecular-initating events, and may represent a discrete genetic population.

Cost effectiveness of detecting Barrett's cancer.

BACKGROUND: Screening Barrett's oesophagus is controversial owing to a large variation in the reported incidence of neoplastic change and lack of evidence that screening improves tumour prognosis. AIMS: To determine the incidence of Barrett's cancer, its cost of detection, and stage of disease at time of diagnosis. PATIENTS AND METHODS: Data from our surveillance programme have been reviewed to assess the incidence of malignant change, tumour stage at diagnosis, and the cost per cancer detected. RESULTS: 166 patients had annual endoscopic surveillance. Six patients (five men) developed cancer-an incidence of one cancer per 59 male and 167 female patient-years of follow up. The screened group had a significantly earlier stage than a control group of unscreened cancers (p < 0.05). The cost of detecting one cancer was Pounds 14 868 for men and Pounds 42 084 for women. CONCLUSIONS: The cost of screening for Barrett's cancer is high but may be justified on the basis of the high incidence of detecting early stage disease.