Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia

Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE). We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency. We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency. The mutation, V94M, was found on both GALE alleles of this patient. This same mutation also was found in the homozygous state in two additional patients with generalized epimerase deficiency. The specific activity of the V94M-hGALE protein expressed in yeast was severely reduced with regard to UDP-galactose and partially reduced with regard to UDP-N-acetylgalactosamine. In contrast, two GALE-variant proteins associated with peripheral epimerase deficiency, L313M-hGALE and D103G-hGALE, demonstrated near-normal levels of activity with regard to both substrates, but a third allele, G90E-hGALE, demonstrated little, if any, detectable activity, despite near-normal abundance. G90E originally was identified in a heterozygous patient whose other allele remains uncharacterized. Thermal lability and protease-sensitivity studies demonstrated compromised stability in all of the partially active mutant enzymes.     

Characterization of a human digestive tract-specific calpain, nCL-4, expressed in the baculovirus system

Clinical experience has suggested that stressful life events and ongoing stressful illness, collectively termed 'social readjustment', may precipitate stroke. We investigated the association between a simple in-office evaluation of such stressors and stroke in an urban, multiethnic study population. Cases were patients from the Northern Manhattan Stroke Study with first ischemic stroke; controls were derived through random digit dialing with n:m matching for age, gender, and race-ethnicity. Social readjustment was measured through in-person interview using Amster and Krauss' Geriatric Social Readjustment Rating Scale (GSRRS), a one-time, 35-item, checklist type weighted questionnaire of stressful life events occurring in the previous 6 months. Conditional logistic regression was used to analyze the GSRRS and its quartiles as well as stressful events subgroups, adjusting for education, hypertension, cardiac disease, diabetes, and number of weekly visits as a measure of socialization. Six hundred and fifty-five cases of ischemic stroke and 1,087 controls were utilized. The mean age of the cases was 69.8 years, with 55.4% women, 51.0% Hispanics, 28.4% blacks, and 19.1% whites. GSRRS scores ranged from 0 to 812; the mean score was 205.5 for the cases and 206.2 for the controls. The analysis showed no association between stroke and a 20-point increase on the GSRRS (OR = 1.01, 95% CI = 0.99-1.01). There was also no effect for the second, third or highest versus lowest quartile. No association was found in age, gender or race-ethnic subgroups, or when analyzing negative events, severely threatening events, or ongoing stressful illnesses separately. While this study does not preclude social readjustment as a stroke risk factor, it suggests that the one-time assessment often done in the medical office setting has little relevance for stroke prevention planning.     

Characterization of two ELISAs for NGAL, a newly described lipocalin in human neutrophils

NGAL is a newly described member of the lipocalin protein family, secreted from specific granules of human neutrophils upon activation of the cells. Its ability to bind the bacterial chemotactic formylpeptide FMLP indicates, that NGAL may have modulatory effects on the immune response. We here describe monoclonal and polyclonal antibodies against NGAL, which can be used for Western blotting and immunohistochemistry, and furthermore describe two ELISAs using either exclusively the polyclonal anti-NGAL antibodies or the polyclonal and monoclonal antibodies in combination. The assays are equally specific, reproducible, accurate, and sensitive, with a detection limit of 32 ng/l. The antibodies and assays will be valuable tools in the future investigation of NGAL expression in inflammatory and malignant disorders and in the elucidation of the function of NGAL as a modulator of the inflammatory response.     

Characterization of a novel CC chemokine, HCC-4, whose expression is increased by interleukin-10

We have identified and characterized a human beta (CC) chemokine, designated HCC-4, that is most closely related to HCC-1 and which demonstrates chemotactic activity for monocytes. Northern analysis of multiple tissue blots and of activated monocytes mRNA shows expression of a 500-bp mRNA. A 1,500-bp mRNA was highly expressed in monocytes activated 12 hours in the presence of interleukin-10 (IL-10) but was absent in monocytes activated for only 1 hour regardless of the presence or absence of IL-10. The upregulation of expression in the presence of IL-10 is in contrast to the downregulatory effects of IL-10 on expression of most other chemokines. Recombinant HCC-4 demonstrated chemotactic activity for human monocytes and THP-1 monocyte cells but not for resting lymphocytes or neutrophils. HCC-4 also induced a Ca2+ flux in THP-1 cells that was desensitized by prior exposure to RANTES. Taken together, these data indicate that HCC-4 is a novel chemokine whose expression is uniquely upregulated by IL-10.     

Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas

Malignant astrocytomas, which are highly invasive, vascular neoplasms, compose the majority of nervous system tumors in humans. Elevated expression of fibroblast growth factors (FGFs) in astrocytomas has implicated the FGF family of mitogens in the initiation and progression of astrocyte-derived tumors. In this study, we demonstrated that human astrocytomas undergo parallel changes in FGF-receptor (FGFR) expression during their progression from a benign to a malignant phenotype. FGFR type 2 (BEK) expression was abundant in normal white matter and in all low-grade astrocytomas but was not seen in malignant astrocytomas. Conversely, FGFR type 1 (FLG) expression was absent or barely detectable in normal white matter but was significantly elevated in malignant astrocytomas. Malignant astrocytomas also expressed an alternatively spliced form of FGFR-1 (FGFR-1 beta) containing two immunoglobulin-like disulfide loops, whereas normal human adult and fetal brains expressed a receptor form (FGFR-1 alpha) containing three immunoglobulin-like disulfide loops. Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR-2 and a shift in expression from FGFR-1 alpha to FGFR-1 beta as they progressed from benign to malignant phenotype. These results suggest that differential expression and alternative splicing of FGFRs may be critical in the malignant progression of astrocytic tumors.     

p16(INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin's lymphomas

The molecular mechanisms underlying the pathogenesis of aggressive lymphomas and the histological transformation of indolent variants are not well known. To determine the role of p16(INK4a) gene alterations in the pathogenesis of non-Hodgkin's lymphomas (NHLs) and the histological progression of indolent variants, we have analyzed the expression, deletions, and mutations of this gene in a series of 112 NHLs. Hypermethylation of the gene was also examined in a subset of tumors with lack of protein expression but without mutations or deletions of the gene. p16(INK4a) gene alterations were detected in 3 out of 64 (5%) indolent lymphomas but in 16 out of 48 (33%) primary or transformed aggressive variants. In the low-grade tumors, p16(INK4a) alterations were detected in 1 (4%) chronic lymphocytic leukemia (hemizygous missense mutation), 1 (6%) follicular lymphoma (homozygous deletion), and 1 (5%) typical mantle cell lymphoma (homozygous deletion). The two later cases followed an aggressive clinical evolution. In the aggressive tumors, p16(INK4a) gene alterations were observed in 2 (29%) Richter's syndromes (2 homozygous deletions), 3 (33%) transformed follicular lymphomas (1 homozygous deletion and 2 nonsense mutations), 3 (43%) blastoid mantle cell lymphomas (2 homozygous and 1 hemizygous deletions), 5 (28%) de novo large-cell lymphomas (1 homozygous deletion and 4 hypermethylations), 2 lymphoblastic lymphomas (2 homozygous deletions), and 1 of 2 anaplastic large cell lymphomas (hypermethylation). Protein expression was lost in all tumors with p16(INK4a) alterations except in the typical chronic lymphocytic leukemia (CLL) with hemizygous point mutation. Sequential samples of the indolent and transformed phase of three cases showed the presence of p16(INK4a) deletions in the Richter's syndrome but not in the CLL component of two cases, whereas in a follicular lymphoma the deletion was present in both the follicular tumor and in the diffuse large-cell lymphoma. In conclusion, these findings indicate that p16(INK4a) gene alterations are a relatively infrequent phenomenon in NHLs. However, deletions, mutations, and hypermethylation of the gene with loss of protein expression are associated with aggressive tumors and they may also participate in the histological progression of indolent lymphomas.     

Characterization of the gene encoding human sarcolipin (SLN), a proteolipid associated with SERCA1: absence of structural mutations in five patients with Brody disease

Sarcolipin (SLN) is a low-molecular-weight protein that copurifies with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase (SERCA1). Genomic DNA and cDNA encoding human sarcolipin (SLN) were isolated and characterized and the SLN gene was mapped to chromosome 11q22-q23. Human, rabbit, and mouse cDNAs encode a protein of 31 amino acids. Homology of SLN with phospholamban (PLN) suggests that the first 7 hydrophilic amino acids are cytoplasmic, the next 19 hydrophobic amino acids form a single transmembrane helix, and the last 5 hydrophilic amino acids are lumenal. The cytoplasmic and transmembrane sequences are not well conserved among the three species, but the lumenal sequence is highly conserved. Like SERCA1, SLN is highly expressed in rabbit fast-twitch skeletal muscle, but it is expressed to a lower extent in slow-twitch muscle and to an even lower extent in cardiac muscle, where SERCA2a and PLN are highly expressed. It is expressed in only trace amounts in pancreas and prostate. SLN and PLN genes resemble each other in having two small exons, with their entire coding sequences lying in exon 2 and a large intron separating the two segments. Brody disease is an inherited disorder of skeletal muscle function, characterized by exercise-induced impairment of muscle relaxation. Mutations in the ATP2A1 gene encoding SERCA1 have been associated with the autosomal recessive inheritance of Brody disease in three families, but not with autosomal dominant inheritance of the disease. A search for mutations in the SLN gene in five Brody families, four of which were not linked to ATP2A1, did not reveal any alterations in coding, splice junction or promoter sequences. The homozygous deletion of C438 in the coding sequence of ATP2A1 in Brody disease family 3, leading to a frameshift and truncation following Pro147 in SERCA1, is the fourth ATP2A1 mutation to be associated with autosomal recessive Brody disease.     

Spontaneous mutations in the env gene of the human immunodeficiency virus type 1 NDK isolate are associated with a CD4-independent entry phenotype

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.     

Identification and sequence of human PKY, a putative kinase with increased expression in multidrug-resistant cells, with homology to yeast protein kinase Yak1

The authors report a clinical case of endometriosis the abdomen rectum muscle, in woman 28 years old, after a cesarean section delivery. On the basis of literature on the topic, the following are taken into consideration, the incidence, the pathogenesis, the clinical characteristics of this kind of pathology and the aspects which might facilitate the diagnostic approach and correct therapeutic to be given or follow. Parietal endometriosis is an extremely rare disease with incidence in feminine population of 0.03-1%. The pathogenesis is still ill-known. Lack of the classical symptoms and the unusual site can make diagnosis difficult. Pathognomonics but not always present are the presence of tumescence palpable of the abdominal wall near or proximity of preceding surgical scar, the cyclic character of painful symptomatology, the augmentation of volume and the bleeding in period menstrual or premenstrual. The ultrasonography, the computerized axial tomography, the nuclear magnetic resonance can facilitate the preoperative diagnosis but they do not always furnish reports of certainty. The aspirate-needle in ultrasonography control can furnish one of orientation diagnosis. The diagnosis of certainty is founded on the histologic examination after biopsy or excision. The treatment of the abdominal wall endometriosis is surgically essential. The excision of tumescence, easy usually, it is the only means to obtain the definitive recovery. The medical therapy postoperative is adjuvant in the treatment of unrecognized pelvic centres of endometriosis.     

Functional defects of a muscle-specific calpain, p94, caused by mutations associated with limb-girdle muscular dystrophy type 2A

p94 (calpain3), a muscle-specific member of the calpain family, has been shown to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A), a form of autosomal recessive and progressive neuromuscular disorder. To elucidate the molecular mechanism of LGMD2A, we constructed nine p94 missense point mutants found in LGMD2A and analyzed their p94 unique properties. All mutants completely or almost completely lose the proteolytic activity against a potential substrate, fodrin. However, some of the mutants still possess autolytic activity and/or connectin/titin binding ability, indicating these properties are not necessary for the LGMD2A phenotypes. These results provide strong evidence that LGMD2A results from the loss of proteolysis of substrates by p94, suggesting a novel molecular mechanism leading to muscular dystrophies.     

Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes

Although oral electrolyte solutions (OES) replenish salts and water lost during diarrhea, present formulations do not address disturbances of the normal intestinal microbiota. Therefore, we evaluated the efficacy of an OES with and without fructooligosaccharide (FOS) for treatment of pigs with acute secretory diarrhea induced by cholera toxin. Before, during, and after diarrhea, bacteriologic evaluation was made of contents collected from the mid small intestine, cecum, and distal colon and mucosa scraped from the mid small intestine. Diarrhea caused significant declines in total bacterial counts of contents from all three regions, with less of an impact on bacteria associated with the mucosa. Although total bacterial counts recovered within 24 hr, regardless of treatment, densities of Enterobacteriaceae were higher in pigs treated with OES whereas those receiving FOS had more lactobacilli. Our results show that secretory diarrhea disturbs the normal densities and relative species abundance of the microbiota, with the influences more pronounced for contents relative to the mucosa, and that adding FOS to OES accelerates the recovery of bacteria perceived as beneficial while potentially slowing the recovery of pathogenic forms.     

Characterization of a gene for spinach CAP160 and expression of two spinach cold-acclimation proteins in tobacco

The cDNA sequence for CAP160, an acidic protein previously linked with cold acclimation in spinach (Spinacia oleracea L.), was characterized and found to encode a novel acidic protein of 780 amino acids having very limited homology to a pair of Arabidopsis thaliana stress-regulated proteins, rd29A and rd29B. The lack of similarity in the structural organization of the spinach and Arabidopsis genes highlights the absence of a high degree of conservation of this cold-stress gene across taxonomic boundaries. The protein has several unique motifs that may relate to its function during cold stress. Expression of the CAP160 mRNA was increased by low-temperature exposure and water stress in a manner consistent with a probable function during stresses that involve dehydration. The coding sequences for CAP160 and CAP85, another spinach cold-stress protein, were introduced into tobacco (Nicotiana tabacum) under the control of the 35S promoter using Agrobacterium tumefaciens-based transformation. Tobacco plants expressing the proteins individually or coexpressing both proteins were evaluated for relative freezing-stress tolerance. The killing temperature for 50% of the cells of the transgenic plants was not different from that of the wild-type plants. As determined by a more sensitive time/temperature kinetic study, plants expressing the spinach proteins had slightly lower levels of electrolyte leakage than wild-type plants, indicative of a small reduction of freezing-stress injury. Clearly, the heterologous expression of two cold-stress proteins had no profound influence on stress tolerance, a result that is consistent with the quantitative nature of cold-stress-tolerance traits.     

Cloning of a human cDNA for CTP-phosphoethanolamine cytidylyltransferase by complementation in vivo of a yeast mutant

CTP-phosphoethanolamine cytidylyltransferase (ET) is the enzyme that catalyzes the formation of CDP-ethanolamine in the phosphatidylethanolamine biosynthetic pathway from ethanolamine. We constructed a Saccharomyces cerevisiae mutant of which the ECT1 gene, putatively encoding ET, was disrupted. This mutant showed a growth defect on ethanolamine-containing medium and a decrease of ET activity. A cDNA clone was isolated from a human glioblastoma cDNA expression library by complementation of the yeast mutant. Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylethanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology with the yeast, rat, and human CTP-phosphocholine cytidylyltransferases, as well as yeast ET. The cDNA gene product was expressed as a fusion with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained here encodes human ET.     

IGFBP-2 expression in a human cell line is associated with increased IGFBP-3 proteolysis, decreased IGFBP-1 expression and increased tumorigenicity

Insulin-like growth factors (IGF-I and -II) play an active role in cell proliferation. In biological fluids, they are non-covalently bound to high-affinity binding proteins (IGFBPs), at least 6 species of which have been identified to date, but with poorly defined functions. One of these IGFBPs, IGFBP-2, is secreted by most cell lines and appears to be involved in cell proliferation. A human epidermoid carcinoma cell line, KB 3.1, which produces IGFBP-1 and -3 and small amounts of IGFBP-4, but no IGFBP-2, was stably transfected with an expression vector comprising IGFBP-2 complementary DNA (cDNA), whose expression was placed under the control of the constitutive and ubiquitous cytomegalovirus promoter. After an s.c. injection of these IGFBP-2-expressing KB 3.1 cells into nude mice, tumours developed more quickly than in controls, they were 3 to 4 times larger and grew about 3 times as fast. Concomitant with IGFBP-2 expression in these tumours, were a decrease in IGFBP-1 expression and an increase in IGFBP-3 proteolysis, both of which increase the bioavailability of the IGF-II produced by the cells. The increased IGFBP-3 proteolysis most probably resulted from amplified expression of tissue-type plasminogen activator (t-PA) and depression of its inhibitor (PAI-I) observed in IGFBP-2-expressing xenografts. Our findings suggest that IGFBP-2 plays a role in this model of experimental tumorigenesis via a mechanism that remains unclear, but appears to involve increased protease activity and IGF-II bioavailability.     

Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.     

Characterization of VPS41, a gene required for vacuolar trafficking and high-affinity iron transport in yeast

Mutations in the yeast gene VPS41 give rise to poor growth on low iron medium, severe alterations in vacuolar morphology, and cause the missorting of membranous and soluble vacuolar proteins. Our studies predict that VPS41 encodes a hydrophilic protein of 992 amino acids that contains no obvious signal sequence or hydrophobic domains. The deduced Vps41p sequence contains a domain rich in glutamic and aspartic residues, as well as a domain with resemblance to a region of clathrin heavy chain. We have also identified and sequenced putative VPS41 homologues from Caenorhabditis elegans, plants, and humans. The VPS41 homologues (but not the yeast VPS41 itself) contain a conserved cysteine-rich RING-H2 zinc finger at their COOH termini. Biochemical experiments suggest that VPS41 functions in post-Golgi protein processing: the deletion mutant exhibits defective high affinity transport due to impaired Fet3p activity and also exhibits defects in the processing and sorting of multiple vacuolar hydrolases.     

Characterization of hemoglobin adducts from a 4, 4'-methylenedianiline metabolite evidently produced by peroxidative oxidation in vivo

4,4'-Methylenedianiline (MDA) is a widely used mutagenic and carcinogenic industrial chemical. It is also a metabolite of 4, 4'-methylenediphenyl diisocyanate (MDI), which is used in the manufacturing of polyurethane foams. Biomonitoring of MDA, like other aromatic amines, is mainly carried out by GC/MS measurement of cysteine adducts in Hb from the nitroso metabolite, released by alkaline hydrolysis. In the present study it was investigated whether the formation of Hb adducts from non-nitroso metabolites of MDA can be used for the dosimetry of MDA. The study was carried out by treatment of mice with MDA and tritiated MDA or deuterated MDA and by identification of their products of reaction with Hb, after enzymatic hydrolysis of the globin and enrichment of the adducts. The main adduct, about 50% of the total amount of MDA associated with Hb, was characterized by MS and was shown to be a reaction product of MDA and the amino group of N-terminal valine in Hb, the derived structure being 1-[(4-imino-2, 5-cyclohexadien-1-ylidene)methyl]benzene-4-azo-2-isovaleric acid. It is likely that this quinonoid MDA imine adduct to valine was formed by an attack of a metabolite formed through peroxidative oxidation of MDA, in analogy with earlier observed oxidation of some other aromatic amines, e.g., benzidine. The reactive intermediate is suggested to be [(4-imino-2, 5-cyclohexadien-1-ylidene)methyl]-4-aminobenzene. The formation of the adduct was confirmed by incubating MDA with valine methyl ester in vitro in the presence of H2O2 and lactoperoxidase. Further, the same adduct was detected in MDI-exposed and control rats, the level in the exposed animals being about 60 times higher than in the controls. This study indicates that, at least in the mouse, extrahepatic peroxidative metabolism is an important pathway for the bioactivation of MDA, possibly leading to a genotoxic reactive intermediate. This study also demonstrates the usefulness of Hb adduct analysis for the identification of reactive intermediates in vivo.