Severe combined immune deficiency due to a homozygous 3.2-kb deletion spanning the promoter and first exon of the adenosine deaminase gene

We have investigated the structural gene for adenosine deaminase (ADA) in a female infant with ADA deficiency associated severe combined immune deficiency (ADA-SCID) disease and her family by DNA restriction-fragment-length analysis. In this family a new ADA-specific restriction-fragment-length variant was detected, which involves a 3.2-kb deletion spanning the ADA promoter as well as the first exon. It was found that the patient, who was born to a consanguineous couple, was homozygous and both her parents and her brother were heterozygous for the deletion. No ADA-specific mRNA could be detected by hybridization in fibroblasts derived from this patient. Thus the patient was established to be homozygous for a true null ADA allele. In the light of the apparently normal development of most tissues except the lymphoid tissue the above finding directly questions the classification of ADA as a 'housekeeping' enzyme.     

A point mutation in the 5′ splice region of intron 7 causes a deletion of exon 7 in adenosine deaminase mRNA

An adenosine deaminase (ADA;EC 3.5.4.4)-deficient B lymphoblastoid cell line BAD05 derived from a Japanese patient with severe combined immunodeficiency was characterized. As previously reported, one allele of BAD05 expresses undetectable ADA mRNA, and the other allele produces an aberrant mRNA without exon 7. Genomic ADA DNA of BAD05 spanning from a portion of exon 6 to a portion of exon 8 was amplified by PCR. The amplified fragments were cloned into a vector, and 8 clones were isolated and sequenced. The analytical result showed a single base change of G to A at the invariant 5′ GT of intron 7 of ADA gene in one allele of BAD05, which accounts for the elimination of exon 7 during splicing. © 1993 Wiley-Liss, Inc.     

Ehrlich ascites tumour cells become refractory to alpha-difluoromethylornithine at a certain stage of growth

When Ehrlich ascites tumour cells are induced to proliferate by serum stimulation, the ornithine decarboxylase (ODC) activity increases rapidly and reaches two to three peaks during the first 24 h. Inhibition of the first peak in ODC activity (occurring at 4 h) by adding alpha-difluoromethylornithine (DFMO) within 2 h of serum stimulation, results in maximal growth inhibition. Under these conditions, similar degrees of polyamine depletion are achieved. When DFMO is added 3 h after seeding, however, enough polyamines have already accumulated during the initial burst in ODC activity to reduce the antiproliferative effect of the drug. The antiproliferative effect is further reduced when DFMO is added 6 h after seeding. When DFMO is added 23 h after seeding, i.e. after maximal accumulation of polyamines, there is no inhibition of cell proliferation. These findings are important to consider both when designing experimental as well as clinical regimens for this drug.     

Molecular cloning and mRNA expression analysis of ornithine decarboxylase antizyme 2 in ovarian follicles of the Sichuan white goose (Anser cygnoides)

The ornithine decarboxylase antizyme 2 (OAZ2) gene is a member of the antizyme gene family. Antizymes play pivotal roles in various cellular pathways, including polyamine anabolism and apoptosis. The molecular structure and expression profile of the OAZ2 in goose ovarian follicles have not been reported. In this study, the OAZ2 cDNA sequence of the Sichuan white goose was cloned (Anser cygnoides), and phylogenetic and structural analyses of the OAZ2 were performed. The expression profiling of OAZ2 mRNA in goose ovarian follicles was examined using quantitative real-time PCR. The sequence analysis showed that the 756 bp OAZ2 sequence contained two overlapping open reading frames (ORF). ORF1 was 99 bp in length, and encoded a 32 aa polypeptide. ORF2 was 477 bp in length, and encoded a 158 aa polypeptide. The frameshift site that initiates the translation of ORF2 was located at nucleotide position 97 in the OAZ2. The analysis of OAZ2 mRNA expression in hierarchical follicles showed that the level of OAZ2 mRNA was higher in the SWF and F2 follicular stages than that in the ovarian stroma (P < 0.05). The lowest level of OAZ2 expression was detected in the ovarian stroma. These results suggest that the highly conserved frameshift region plays an important role in sustaining the function of OAZs. Furthermore, the significantly higher level of OAZ2 mRNA in the SWF stage indicates that OAZ2 may be involved in recruiting hierarchical follicles. Our results also suggest that OAZ2 may augment the effects of OAZ1 in follicle development.     

An alternatively-spliced exon in the 5'-UTR of human ALAS1 mRNA inhibits translation and renders it resistant to haem-mediated decay

Haem controls its own synthesis in non-erythroid cells primarily by regulation of ALAS1 mRNA stability. Alternative splicing of human ALAS1 generates two mRNAs with different 5'-UTRs: a major one, where exon 1B is omitted, and a minor form containing exon 1B. We show that, unlike the major ALAS1 mRNA, the minor form was resistant to haem-mediated decay. Furthermore, we demonstrate that the ALAS1 5'-UTR alone did not confer haem-mediated decay upon a heterologous mRNA and the inclusion of exon 1B inhibited translation. These data suggest that translation of ALAS1 mRNA itself might be required for destabilisation in response to haem.     

Mitochondrial DNA depletion and fatal infantile hepatic failure due to mutations in the mitochondrial polymerase γ (POLG) gene: a combined morphological/enzyme histochemical and immunocytochemical/biochemical and molecular genetic study

Combined morphological, immunocytochemical, biochemical and molecular genetic studies were performed on skeletal muscle, heart muscle and liver tissue of a 16‐months boy with fatal liver failure. The pathological characterization of the tissues revealed a severe depletion of mtDNA (mitochondrial DNA) that was most pronounced in liver, followed by a less severe, but still significant depletion in skeletal muscle and the heart. The primary cause of the disease was linked to compound heterozygous mutations in the polymerase γ (POLG) gene (DNA polymerase γ; A467T, K1191N). We present evidence, that compound heterozygous POLG mutations lead to tissue selective impairment of mtDNA replication and thus to a mosaic defect pattern even in the severely affected liver. A variable defect pattern was found in liver, muscle and heart tissue as revealed by biochemical, cytochemical, immunocytochemical and in situ hybridization analysis. Functionally, a severe deficiency of cytochrome‐c‐oxidase (cox) activity was seen in the liver. Although mtDNA depletion was detected in heart and skeletal muscle, there was no cox deficiency in these tissues. Depletion of mtDNA and microdissection of cox‐positive or negative areas correlated with the histological pattern in the liver. Interestingly, the mosaic pattern detected for cox‐activity and mtDNA copy number fully aligned with the immunohistologically revealed defect pattern using Pol γ, mtSSB‐ and mtTFA‐antibodies, thus substantiating the hypothesis that nuclear encoded proteins located within mitochondria become unstable and are degraded when they are not actively bound to mtDNA. Their disappearance could also aggravate the mtDNA depletion and contribute to the non‐homogenous defect pattern.     

Silencing CDR1as enhances the sensitivity of breast cancer cells to drug resistance by acting as a miR‐7 sponge to down‐regulate REGγ

In our study, we aimed to investigate the role of CDR1as during competitive inhibition of miR‐7 in the regulation of cisplatin chemosensitivity in breast cancer via regulating REGγ. RT‐qPCR was applied to detect the expression of CDR1as and miR‐7 in breast cancer tissues, breast cancer cell lines and corresponding drug‐resistant cell lines. The correlation between CDR1as and miR‐7 and between miR‐7 and REGγ was evaluated. MCF‐7‐R and MDA‐MB‐231‐R cells were selected followed by transfection of a series of mimics, inhibitors or siRNA. The effect of CDR1as on the half maximal inhibitor concentration (IC50), cisplatin sensitivity and cell apoptosis was also analysed. Furthermore, a subcutaneous xenograft nude mouse model was established to further confirm the effect of CDR1as on the chemosensitivity of breast cancer to cisplatin in vivo. Immunohistochemical staining was conducted to test the Ki‐67 expression in nude mice. A positive correlation was found between the drug resistance and CDR1as expression in breast cancer. CDR1as could increase the resistance of breast cancer cells to cisplatin. miR‐7 expression was low, while REGγ was highly expressed in MCF‐7‐R and MDA‐MB‐231‐R cells. CDR1as competitively inhibited miR‐7 and up‐regulated REGγ. Overexpression of miR‐7 could reverse the enhanced sensitivity of silenced CDR1as to drug‐resistant breast cancer cells. Additionally, in vivo experiments demonstrated that CDR1as mediated breast cancer occurrence and its sensitivity to cisplatin. Silencing CDR1as decreased Ki‐67 expression. Silencing CDR1as may inhibit the expression of REGγ by removing the competitive inhibitory effect on miR‐7 and thus enhancing the sensitivity of drug‐resistant breast cancer cells.     

Cloning and sequence analysis of a cDNA for the α-globin mRNA of carp, Cyprinus carpio

A cDNA for α-globin mRNA of the carp, Cyprinus carpio, was cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2 (1982) 161–170) and its complete nucleotide sequence was determined. The 5′ non-coding region contained 23 nucleotides. Following this region, there was an open reading frame encoded with an α-globin polypeptide consisting of 142 amino acids. The 3′ non-coding region was 88 nucleotides in length, including two copies of the hexanucleotide AATAAA and a poly(A) site of the GC dinucleotide. There were 16 discrepancies between the reported amino acid sequence of the carp α-globin chain and the amino acid sequence predicted from the DNA sequence of the clone. The possible explanations for these differences in amino acid sequence are discussed.     

Biochemical and immunological characterization of a glycosylated alpha-fucosidase from the invertebrate Unio: interaction of the enzyme with its in vivo binding partners

Mammalian alpha-fucosidase (EC 3.2.1.51) is a lysosomal enzyme that catalyzes the removal of fucose residues from glycosphingolipids and its absence in humans results in a rare metabolic disorder called fucosidosis. Among the invertebrates in the molluscs (Unio) two forms of the enzyme have been reported, a 68 kDa non-glycosylated form and a 56 kDa glycosylated form. The glycosylated form has been purified from the seminal fluid of Unio [Biochem. Biophys. Res. Commun. 234 (1997) 54]. In the present study, the 56 kDa glycosylated form has been purified to homogeneity from the whole body tissue of Unio using a series of chromatographic steps. The purified enzyme migrated as a single protein species in 10% SDS-PAGE. Antibodies to the purified enzyme were raised in a rabbit in order to study its biochemical and immunological properties. The purified enzyme is a glycoprotein that exhibits strong binding to Con A-Sepharose gel and can be deglycosylated by PNGase F enzyme suggesting it to be N-glycosylated. The enzyme has been shown to specifically interact with the mannose 6-phosphate receptor protein (MPR 300) purified from goat and Unio. This specific interaction is discussed in view of its possible in vivo binding partners.     

Identification of the putative tumor suppressor Nit2 as ω-amidase, an enzyme metabolically linked to glutamine and asparagine transamination

The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is ω-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated ω-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are α-ketoglutaramate and α-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of α-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating α-keto-γ-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess α-ketoglutaramate may be neurotoxic. Moreover, α-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of ω-amidase is to remove potentially toxic intermediates by converting α-ketoglutaramate and α-ketosuccinamate to biologically useful α-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of ω-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology.     

NMR chemical shift mapping of the binding site of a protein proteinase inhibitor: changes in the (1)H, (13)C and (15)N NMR chemical shifts of turkey ovomucoid third domain upon binding to bovine chymotrypsin A(alpha)

The substrate-like inhibition of serine proteinases by avian ovomucoid domains has provided an excellent model for protein inhibitor-proteinase interactions of the standard type. 1H,15N and 13C NMR studies have been undertaken on complexes formed between turkey ovomucoid third domain (OMTKY3)2 and chymotrypsin A(alpha) (Ctr) in order to characterize structural changes occurring in the Ctr binding site of OMTKY3. 15N and 13C were incorporated uniformly into OMTKY3, allowing backbone resonances to be assigned for OMTKY3 in both its free and complex states. Chemical shift perturbation mapping indicates that the two regions, K13-P22 and N33-A40, are the primary sites in OMTKY3 involved in Ctr binding, in full agreement with the 12 consensus proteinase-contact residues of OMTKY3 defined previously on the basis of X-ray crystallographic and mutational analysis. Smaller chemical shift perturbations in selected other regions may result from minor structural changes on binding. Through-bond 15N-13C correlations between P1-13C' and P1'-15N in two-dimensional H(N)CO and HN(CO) NMR spectra of selectively labeled OMTKY3 complexed with Ctr indicate that the scissile peptide bond between L18 and E19 of the inhibitor is intact in the complex. The chemical shifts of the reactive site peptide bond indicate that it is predominantly trigonal, although the data are not inconsistent with a slight perturbation of the hybridization of the peptide bond toward the first tetrahedral state along the reaction coordinate.     

Screening of microorganisms producing α-methylserine hydroxymethyltransferase, purification of the enzyme, gene cloning, and application to the enzymatic synthesis of α-methyl-l-serine

Through the screening of microorganisms capable of utilizing α-methylserine, three representative strains belonging to the bacterial genera Paracoccus, Aminobacter, and Ensifer were selected as potent producers of α-methylserine hydroxymethyltransferase, an enzyme that catalyzes the interconversion between α-methyl-l-serine and d-alanine via tetrahydrofolate. Among these strains, Paracoccus sp. AJ110402 was selected as the strain exhibiting the highest α-methylserine hydroxymethyltransferase activity. The enzyme was purified to homogeneity from a cell-free extract of this strain. The native enzyme is a homodimer with apparent molecular mass of 85 kDa and contains 1 mol of pyridoxal-5′-phosphate per mol of the subunit. The K_m for α-methyl-l-serine and tetrahydrofolate was 0.54 mM and 73 μM, respectively. The gene from Paracoccus sp. AJ110402 encoding α-methylserine hydroxymethyltransferase was cloned and expressed in Escherichia coli. Sequence analysis revealed an open reading frame of 1278 bp, encoding a polypeptide with a calculated molecular mass of 46.0 kDa. Using E. coli cells as whole-cell catalysts, 9.7 mmol of α-methyl-l-serine was stereoselectively obtained from 15 mmol of d-alanine and 13.2 mmol of formaldehyde.     

Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in lambda repressor.

In the N-terminal domain of lambda repressor, the Asp 14 side chain forms an intrahelical, hydrogen bond/salt bridge with the Arg 17 side chain and a tertiary hydrogen bond with the Ser 77 side chain. By measuring the stabilities to urea denaturation of the wild-type N-terminal domain and variants containing single, double, and triple alanine substitutions at positions 14, 17, and 77, the side-chain interaction energies, the coupling energy between interactions, and the intrinsic effects of each wild-type side chain on protein stability have been estimated. These studies indicate that the Asp 14-Arg 17 and Asp 14-Ser 77 interactions are stabilizing by roughly 0.8 and 1.5 kcal/mol, respectively, but that Asp 14, by itself, is destabilizing by roughly 0.9 kcal/mol. We also show that a peptide model of alpha-helix 1, which contains Asp 14 and Arg 17, forms a reasonably stable, monomeric helix in solution and responds to alanine mutations at positions 14 and 17 in the fashion expected from the intact protein studies. These studies suggest that it is possible to view the stability effects of mutations in intact proteins in a hierarchical fashion, with the stability of units of secondary structure being distinguishable from the stability of tertiary structure.     

Immobilization of the glycosidases: α- -arabinofuranosidase and β- -glucopyranosidase from Aspergillus niger on a chitosan derivative to increase the aroma of wine. Part II

The purpose of this paper was to study the immobilization of two glycosidases, α- -arabinofuranosidase (EC 3.2.1.55) and β- -glucopyranosidase (EC 3.2.1.21), contained in a commercial preparation and purified as reported in Part I. The procedure which proved to be the best is simple and inexpensive to perform, employing the chitosan derivative, glyceryl-chitosan, especially synthesized and characterized, as a support. The glycosidases were adsorbed on this support and cross-linked with glutaraldehyde to prevent them from being released into the wine. Subsequent reduction of the biocatalyst with sodium borohydride allowed for improved stability. Finally, the immobilized glycosidases were compared with free ones in terms of optimum pH and temperature, stability over time, and kinetics parameters (K_m and V_max) after which they were employed for aromatizing a model wine solution containing aromatic precursors.