Label-free characterization of cancer-activated fibroblasts using infrared spectroscopic imaging.

Glandular tumors arising in epithelial cells comprise the majority of solid human cancers. Glands are supported by stroma, which is activated in the proximity of a tumor. Activated stroma is often characterized by the molecular expression of α-smooth muscle actin (α-SMA) within fibroblasts. However, the precise spatial and temporal evolution of chemical changes in fibroblasts upon epithelial tumor signaling is poorly understood. Here we report a label-free method to characterize fibroblast changes by using Fourier transform infrared spectroscopic imaging and comparing spectra with α-SMA expression in primary normal human fibroblasts. We recorded the fibroblast activation process by spectroscopic imaging using increasingly tissue-like conditions: 1), stimulation with the growth factor TGFß1; 2), coculture with MCF-7 human breast cancerous epithelial cells in Transwell coculture; and 3), coculture with MCF-7 in three-dimensional cell culture. Finally, we compared the spectral signatures of stromal transformation with normal and malignant human breast tissue biopsies. The results indicate that this approach reveals temporally complex spectral changes and thus provides a richer assessment than simple molecular imaging based on α-SMA expression. Some changes are conserved across culture conditions and in human tissue, providing a label-free method to monitor stromal transformations.

Temporal invariance and clonal uniformity of brain and cerebrospinal IgG, IgA, and IgM in multiple sclerosis.

Elevated cerebrospinal fluid (CSF) IgG and oligoclonal IgG bands on electrophoresis are valuable clinical markers for B cell proliferation in the brains of patients with multiple sclerosis (MS). Using two-dimensional electrophoresis, (2DE) we have established that the humoral immune response in MS brain is characterized by finite clonal complexity for the major Ig classes. An important question is whether this immune response is clonally stable or varies with time, related to the development of new lesions and random entry of B cells into the MS brain. To investigate this, we performed serial electrophoretic studies on CSF obtained from 19 patients with MS; the intervals ranged from 7 to 12 yr, with a mean of 8 yr. These analyses included studies of IgG, IgA, and IgM, and revealed that the humoral immune response in MS is clonally stable over long periods. Spontaneous fluctuations or reduction in CSF IgG levels by drugs did not qualitatively affect B cell clonal proliferation in MS brain, in that dominant bands and spots were not obliterated. It has been asserted that IgG synthesis in MS is nonsense antibody because the spectotypes of IgG isolated from different regions of MS brains differ. Factors other than clonal heterogeneity could account for differences found using one-dimensional analysis. B cell clonal products resolve into unique and well-resolved spots by 2DE; the method is uniquely suitable for analysis of restricted immune responses. Therefore, Ig were isolated from 11 regions of three MS brains and the 2DE patterns were compared. The similarity of the 2DE patterns indicate unequivocally that major clones are distributed uniformly although some clones are more prominent in some brain areas. IgA and IgM isolated from the same areas also showed similar patterns. Furthermore, the patterns of light and heavy chains in brain regions differed from serum but were similar to the autologous CSF, providing new evidence that CSF IgG in MS derives from synthesis in situ. Our results indicate that, once initiated, B cell clonal proliferation persists indefinitely and is little altered qualitatively at a clonal level over time, even when CSF IgG levels change or are altered by drugs. Our results are consistent with allotype and idiotype analysis of Ig production in MS and conflict with nonsense antibody proposals of the origin and nature of in situ synthesized Ig in MS.

Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A.

Methods published for the purification of P.II proteins from Neisseria gonorrhoea have been modified to allow the purification of class 5 proteins from Neisseria meningitidis serogroup A bacteria. The five class 5 protein electrophoretic variants detected within an epidemic in the Gambia (a, b, c, d, and e) and three other variants (f, g, and h) found within other isolates of the same clone in West Africa have been purified with yields of 6-28 mg. The NH2-terminal amino acid sequence for variant c differs from those of the other class 5 proteins, whereas the latter are very similar to the sequence predicted for two class 5 proteins from DNA analyses of serogroup C meningococci and determined for 8 P.II proteins from gonococci. Numerous other regulatory, chemical, and serological differences were found between the c protein and the other class 5 proteins such that we recommend that the class 5 proteins be subdivided into two subclasses. mAbs have been isolated that distinguish between these two protein subclasses and Western blotting with these antibodies enabled us to conclude that both protein subclasses were found in bacteria isolated from different epidemics and pandemics of the last 50 yr.

Alcohol, amines, and alkaloids: a possible biochemical basis for alcohol addiction.

Tetrahydropapaveroline is a benzyltetrahydroisoquinoline alkaloidderivative of the biogenic amine, dopnmine. Alcohol, by way of its primary metabolite, acetaldehyde, competitively inhibits nicotinamide-adenine Sinucleotide-linked aldehyde dehydrogenase and augments the formation of tetrahydropapaveroline in vitro. The limited capacity of brain to oxidize aldehydes may be of pharmacological importance because it facilitates the production of tetrahydropapaveroline in the presence of drugs which inhibit this enzyme.

E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor.

The tumor suppressor KLF6 is a member of the Krüppel-like family of transcription factors, which has been implicated in the pathogenesis of several human carcinomas. Uncovering the transcriptional targets relevant for its tumorigenic properties, including cellular proliferation and invasion, will be essential to understanding possible mechanisms by which KLF6 and its antagonistic splice form, KLF6-SV1, regulate this development. To begin defining possible metastatic-related pathways, we analysed the effect of KLF6 dysregulation on a recognized suppressor of cellular invasion, E-cadherin. Targeted KLF6 reduction in an ovarian cancer cell line, SKOV-3, resulted in a 50% reduction of E-cadherin expression (P<0.01) and conversely, KLF6-SV1 silencing upregulated E-cadherin approximately fivefold (P<0.0001). These changes resulted from KLF6 directly transactivating the E-cadherin promoter as demonstrated by luciferase promoter assay and chromatin immunoprecipitation (ChIP). KLF6-mediated changes in E-cadherin levels were accompanied by downstream changes in both the subcellular localization of beta-catenin and c-myc expression levels. Moreover, and consistent with these experimental findings, patient-derived epithelial ovarian tumors with low KLF6 and high KLF6-SV1 expression ratios had significantly decreased E-cadherin expression (P<0.0001). These combined findings highlight the E-cadherin pathway as a novel and functionally important mediator by which changes in KLF6 and KLF6-SV1 can directly alter ovarian tumor invasion and metastasis.

Dual implication of 2',3'-cyclic nucleotide 3' phosphodiesterase as major autoantigen and C3 complement-binding protein in the pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is characterized by intra-blood-brain barrier immunoglobulin synthesis that persists lifelong. Subcellular fractionation and two-dimensional electrophoresis were used in conjunction with immune precipitation and immunoblotting to identify antigenic determinants for this immunoglobulin. We report that 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein associated with oligodendrocyte/myelin membranes, also present in lymphocytes and retina, is one major target for the humoral response. Antibodies to CNP are detected in sera of 74% of MS patients. The antibodies are IgM and are present in serum in high titer as well as in cerebrospinal fluid. The antibody response is temporally persistent, consistent with systemic immune activation and persistent antigenic stimulation. Moreover, CNP is isolated as an immune complex from MS brain. CNP is expressed as two isoforms, with CNPII identical to CNPI but with a 20-amino acid extension at the amino terminus of CNPII; however, the antibody response is exclusively restricted to CNPI. In contrast, both isoforms bind the C3 complement, providing a plausible mechanism in MS central nervous system (CNS) for opsonization of myelin membrane CNP, mediated via the C3 receptor, and phagocytosis of CNP-Ig immune complexes, mediated by membrane Ig Fc receptors of macrophages and CNS microglia.

A regulatory element is characterized by purine-mediated and cell-type-specific gene transcription.

Purines and purine nucleotides were found to affect transcription of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in whole nuclei isolated from intestinal mucosa of adult rats fed a purine- and purine nucleotide-free diet. Nuclear run-on transcription assays, performed on whole nuclei from different tissues and cell types, identified an intestine-specific decrease in the overall incorporation of [alpha-32P]UTP in HPRT transcripts from intestinal epithelial cell nuclei when exogenous purines or purine nucleotides were omitted from either the diet or culture medium. Using a 990-base-pair genomic fragment that contains the 5'-flanking region from the HPRT gene, we generated plasmid constructs with deletions, transfected the DNA into various cell types, and assayed for chloramphenicol acetyltransferase (CAT) reporter activity in vitro. We determined that an element upstream from the putative transcriptional start site is necessary to maintain the regulatory response to purine and nucleotide levels in cultured intestinal epithelial cells. These results were tissue and cell type specific and suggest that in the absence of exogenous purines, the presence of specific factors influences transcriptional initiation of HPRT. This information provides evidence for a mechanism by which the intestinal epithelium, which has been reported to lack constitutive levels of de novo purine nucleotide biosynthetic activity, could maintain and regulate the salvage of purines and nucleotides necessary for its high rate of cell and protein turnover during fluctuating nutritional and physiological conditions. Furthermore, this information may provide more insight into regulation of the broad class of genes recognized by their lack of TATA and CCAAT box consensus sequences within the region proximal to the promoter.

Immunoglobulin heavy chain associated protein in multiple sclerosis cerebrospinal fluid.

Immunoglobulins G, A, and M (IgG, IgA and IgM) were isolated from multiple sclerosis (MS) cerebrospinal fluid (CSF) and sera by Protein A-Sepharose (PAS) affinity chromatography or using solid-phase immunoabsorbents. An isoelectric point heterogeneous protein with apparent mol. wt of 74,000-80,000 was seen consistently when CSF Igs were immunoaffinity purified but was never seen when PAS was used for Ig purification. Control experiments exclude the binding of this protein non-specifically either to Sepharose or to Igs or to other CSF proteins. Analysis of purified Igs under non-reducing conditions leads to some reduction in staining pattern indicating that a portion of the protein may be disulfide linked to itself or to other CSF proteins. Immunoblot analysis of CSF Igs separated on 2-DE gels is consistent with a portion of the total CSF Ig protein existing as "free" heavy (H)-chains. PAS requires dimeric Fc fragment of Ig for binding; the differences in 2-DE gels of PAS and immunoaffinity purified Igs may be due to interaction of the 74,000-80,000 protein with "free" H-chain, which nevertheless, still leaves this complex available for binding by anti-H chain antisera. It has been previously suggested that the cytotoxicity of "free" H-chains is abrogated in the absence of the complementary L-chains by H-chain binding proteins; the protein reported here has features similar to these proteins reported previously and may be involved in some way in regulating Ig production in the MS central nervous system.

Complete amino acid sequence of the epidermal desmoglein precursor polypeptide and identification of a second type of desmoglein gene.

The amino acid sequence of the precursor to desmoglein, a major desmosomal cadherin, has been determined from a cDNA clone from bovine muzzle epithelium, and the transcription start site, i.e., the beginning of the approximately 7.6 kb mRNA, identified by primer extension analysis. The precursor segment of 49 amino acids starts with a relatively hydrophobic stretch of 17 amino acids, conforming to the typical features of signal peptides, displays no sequence homology to the corresponding portion of other cadherins. The isolation of the complete cDNA has allowed the cloning of a desmoglein cDNA construct, which under the control of the human beta-actin promoter, was successfully used in cell transfection. In addition, a major N-glycosylation site has been identified by lectin affinity chromatography and amino acid sequencing at amino acid position 61, i.e., in the middle of the first extracellular domain. In the course of these studies we have identified, in colon carcinoma and other simple epithelial cells, another kind of desmoglein which by partial cDNA-derived sequence and by Southern blotting is clearly the product of a different gene. This suggests that there are multiple desmogleins which can be differentially expressed in various epithelia.

Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules.

Monoclonal antibodies to the constitutive desmosomal glycoprotein desmoglein were characterized whose epitopes are located intracellularly, i.e., in the cytoplasmic portion of this molecule, and contribute to the structure of the desmosomal plaque. Using one of these antibodies (DG3.10), a peptide was isolated from a proteolytic digest of desmoglein purified from isolated bovine muzzle demosomes, and its amino acid sequence was determined. In comparisons of this sequence with the amino acid sequence of desmoglein as deduced from the sequence of cDNA clones from the same tissue, encompassing most of approximately 7.6 kb mRNA and the complete coding region of 959 residues (calculated molecular weight approximately 102,400), the DG3.10 epitope was identified in a region starting 163 amino acids before the carboxy terminus in the first of four consecutive repeats of a homologous element of 29 +/- 1 amino acids. This topological information, together with the identification of a single hydrophobic region of sufficient length to provide a transmembrane segment and of several extended regions showing high sequence homology to various cadherins, has allowed the construction of a model of the molecular organization of desmoglein. We conclude that desmoglein is a member of the cadherin family of cell adhesion glycoproteins which is characterized by an unusually long cytoplasmic domain which exceeds those of the cadherins by more than 275 amino acids, contains special repetitive elements and spans the desmosomal plaque at least once.

Does the amount of endurance exercise in combination with weight training and a very-low-energy diet affect resting metabolic rate and body composition?

Effects of large (LA; 400 min/wk) and moderate (MA; 200 min/wk) amounts of endurance exercise in combination with weight training (3 d/wk) were compared with the effects of no exercise (C) in 23 obese females after a 12-wk, 3360-kJ/d very-low-energy diet (VLED). The LA group lost 6.5 kg more weight, mainly as fat (6.4 kg), than the C group (P < 0.05). No measurable differences were found among groups for decreases in resting metabolic rate (-729 to -1233 kJ/d; NS) or fat-free mass (-2.9 to -3.9 kg; NS). No improvements in aerobic capacity were achieved with the addition of exercise to a VLED (-0.079 to -0.037 L/min; NS). Strength indexes were improved (+16 to +5 kg; P < 0.05) or maintained with exercise (-3 kg; NS) whereas a loss (-9.3 kg; P < 0.05) or maintenance (+4.5 kg; NS) was found for VLED alone. Large amounts of endurance exercise in combination with weight training added to a VLED appear to improve weight and fat loss compared with a VLED alone.

Left-to-right transfer of language dominance: a case study.

Partial recovery from aphasia was documented in an individual rendered hemiplegic and globally aphasic by embolic infarction in the distribution of the left middle cerebral artery. Computed tomography showed total destruction of the classical left hemisphere language areas, indicating that the right hemisphere was responsible for the improved linguistic function. This observation is consistent with right hemisphere language capacity demonstrated after left hemispherectomy or commissurotomy. Right hemisphere language function may underlie much of the recovery from aphasia after injury of the left hemisphere.

Autosomal dominant chorea-acanthocytosis with polyglutamine-containing neuronal inclusions.

BACKGROUND: The term chorea-acanthocytosis describes a heterogeneous group of neurodegenerative disorders with variable clinical features and modes of inheritance. The characteristic acanthocytic appearance of red blood cells is attributed to abnormalities of a membrane protein, band 3, although the relationship between this and the neurodegenerative process has yet to be determined. OBJECTIVE: To describe features of phenotype, inheritance, and neuropathological findings in a family with this disorder. METHODS: Clinical and hematologic evaluations were performed on all available family members and neuropathological examination was performed on one case. RESULTS: Autosomal dominant inheritance was evident, with variable clinical features of chorea or parkinsonism, marked cognitive changes, but no seizures or peripheral neurologic abnormalities. Abnormalities of band 3 were demonstrated on gel electrophoresis of red blood cell membranes. Neuropathological examination revealed severe neuronal loss of the caudate-putamen and intranuclear inclusion bodies in many areas of the cerebral cortex. These inclusion bodies were immunoreactive for ubiquitin, expanded polyglutamine repeats, and torsinA. CONCLUSIONS: This family extends the genetic spectrum of chorea-acanthocytosis to include autosomal dominant inheritance, possibly due to expanded trinucleotide repeats. Intraneuronal inclusion bodies have recently been associated with a wide range of inherited neurodegenerative disorders and may provide a clue to etiopathogenesis, in addition to potentially indicating a function of torsinA.

Subacute sclerosing panencephalitis: measles virus matrix protein nucleic acid sequences detected by in situ hybridization.

Subacute sclerosing panencephalitis (SSPE) is characterized by a hyperimmune state toward the polypeptides of measles virus except the matrix (M) protein. Using cloned (3H)-labeled complementary DNA probes for in situ hybridization, we found the M protein and nucleocapsid (NP) protein nucleotide sequences in glial cells and neurons of cryostat sections from two SSPE brains. In one SSPE brain, M protein was lacking, but the other measles polypeptides were present. IgG and IgM antibodies eluted from that brain lacked antibodies to M protein, but antibodies to other measles polypeptides were present. In SSPE brain, the viral M-protein defect is not a deletion of the M gene, but rather a block in gene expression.

Effects of hypoxia and acute osmotic stress on intermediary metabolism in Leishmania promastigotes.

This study further explores the effects of hypoxia and acute osmotic stress on intermediary metabolism of Leishmania major and Leishmania donovani. Late log phase promastigotes were washed and incubated with glucose as the sole exogenous carbon source, and rates of glucose consumption and product formation were measured as a function of osmotic strength (610, 305, and 167 mOsm kg-1) and pO2 (95, 10, and 0% O2) in the presence of 5% CO2. Very mild hypoxia dramatically altered flux through the pathways of intermediary metabolism and increased the rates of production of the major metabolites, thus confirming the presence of a low-affinity O2 sensor which was active under all osmolalities tested. The data also require that as pO2 is lowered towards anoxia an endogenous carbohydrate source is mobilized. Under aerobic conditions, acute hypo-osmotic stress had little effect on product formation, whereas acute hyperosmotic stress altered metabolism in a manner similar to mild hypoxia, with the exception of decreasing the rates of acetate and succinate production. It was also shown in L. donovani promastigotes that the effects of anoxia and hyperosmolality were not additive. Thus, separate sensors with partially overlapping actions are involved in the metabolic responses to hypoxia and hyperosmolality. There was no apparent species-specificity for the responses to pO2 and osmotic stress. Uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone caused changes in metabolite flux patterns which differed from the changes caused by either hypoxia or acute osmotic stress, while rotenone and calcium ionophore A23187 had no significant effects. The identity of the sensors responsive to pO2 and osmolality, and the mechanisms by which they regulate flux through the pathways of intermediary metabolism, require further study.

Isoelectric point restriction of cerebrospinal fluid and serum IgG antibodies to measles virus polypeptides in multiple sclerosis.

Thirty consecutive isoelectric point (pI)-discrete IgG fractions were isolated from multiple sclerosis (MS) cerebrospinal fluid (CSF) and used to immune precipitate measles virus (MV) polypeptides. Most basic fractions were enriched in activity against nucleocapsid protein (NP), and to a lesser extent against hemagglutinin (H) protein; intermediate fractions were enriched in activity against H and fusion (F) proteins; and more anodic pI fractions were almost exclusively enriched in activity against the large (L) protein of MV. In MS there are marked differences between CSF and autologous serum in regard to antibody activity to MV. In contrast, there were similar profiles of antibody response to MV proteins in SSPE CSF and serum.

Impact of preexisting statin use on adhesion molecule expression in patients presenting with acute coronary syndromes.

Of 147 patients admitted with acute coronary syndromes, 17 were taking statins at the time of presentation. These were matched with 17 subjects not taking statins. We found that statin therapy was associated with lower levels of sP-selectin, a marker of platelet and vascular endothelial activation. This provides further insight into the extralipid effect of statins in clinical practice and may help explain the greater-than-expected benefits of statin therapy in ischemic heart disease.

Helicobacter pylori serology in patients with angiographically documented coronary artery disease.

We studied the relation between angiographically defined coronary artery disease and serologic evidence of Helicobacter pylori infection in 488 patients undergo ing elective coronary angiography. There was no association between Helicobacter pylori infection and coronary artery disease (odds ratio 1.3, 95% confidence interval 0.83 to 2.16).