Three-step monoclonal antibody tumor targeting in carcinoembryonic antigen-positive patients.

We describe a method to postlabel, in vivo, biotinylated monoclonal antibodies pretargeted onto tumor deposits when most of the non-tumor-bound antibodies have already been cleared as avidin-bound complexes. The application of this principle to tumor detection by immunoscintigraphy was tested in 20 patients with histologically documented cancer and increased circulating carcinoembryonic antigen levels. One mg of biotinylated anti-carcinoembryonic antigen monoclonal antibody (FO23C5) was administered i.v. (first step). After 3 days, 4-6 mg of cold avidin were injected i.v. (second step), followed 48 h later by 0.2-0.3 mg of a biotin derivative labeled with 111In (2-3 mCi) (third step). No evidence of toxicity was observed. Whole body radioactivity distribution was measured in five patients at various intervals postinjection by the conjugate counting technique. Tumors and metastases were detected in 18 of 19 patients (the remaining patient was a true negative) within 3 h after administration of 111In-biotin by planar or single photon emission tomography imaging. At the time of imaging, tumor/blood pool ratio was 5.5 +/- 3.2, and tumor/liver ratio was 6.7 +/- 3.9. Blood clearance of 111In-biotin was multiexponential, with the fast component having a t1/2 of 5 +/- 3 min. Urinary excretion of radioactivity over 3 h was 63.5 +/- 4.9% of the injected dose. Radioactivity at 3 h was 6.5 +/- 1.8% in blood, 1.6 +/- 0.3% in the kidney, and 2.4 +/- 0.6% in the liver. This approach represents an improvement in immunoscintigraphic techniques for tumor localization. The potential use for radioimmunotherapy is discussed.

Purification and molecular properties of the AMP-activated pyruvate kinase from Escherichia coli.

The AMP-activated pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Escherichia coli has been purified 200 times through a three-step procedure which gives a homogeneous preparation with a specific activity of 110. The enzyme appears to be a tetramer of molecular weight 190 000. Subunits (molecular weight 51 000) show a single amino-terminal amino acid (serine) and appear as a single band in polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The enzyme crystallizes in conditions of reduced dielectric constant of the solvent in the pH range 6.5-7.5. Kinetic and regulatory properties of the purified enzyme are similar to those described for crude preparations of the enzyme.

Two forms of pyruvate kinase in Escherichia coli. A comparison of chemical and molecular properties.

The two forms of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) present in Escherichia coli have been purified from the same cultures and crystallized. A modified procedure for the purification of type I pyruvate kinase is described. Molecular weight, subunit structure, amino acid composition, NH2-terminal amino acid, maps of tryptic peptides and conditions for crystallization have been determined for the two forms. A comparison of these data shows that the two forms are different proteins, each being a tetramer of identical subunits.

The intron-containing L3 ribosomal protein gene (RPL3): sequence analysis and identification of U43 and of two novel intronic small nucleolar RNAs.

Isolation and sequencing of bovine and human intron-containing L3 ribosomal protein genes are here reported. They exhibit very similar organisation, both comprising 10 exons and nine introns. A polymorphic locus, involving a 19-bp deletion, was found in intron 6 of the human gene. The frequency of the two alleles has been estimated in 200 haploid genomes. In bovine and human genes intron sequences are rather different, except for limited regions, located in corresponding positions, which show a surprisingly high degree of identity. All these regions contain conserved features defining the box C/D class of small nucleolar RNAs. Demonstration is given that U43 small nucleolar RNA is encoded within the first intron of both bovine and human genes. Single nucleotide sequences, encoding two novel species of small nucleolar RNAs (U82, U83a and U83b), are located in introns 3, 5 and 7. Their expression has been investigated and a possible role of these molecules in 2'-O-ribose methylation of rRNAs is discussed.

cDNA sequence for bovine ribosomal protein L3 carrying a bipartite nuclear targeting motif, identified also in many other ribosomal proteins.

A full-length cDNA encoding bovine ribosomal protein L3 was isolated and sequenced. The deduced protein sequence comprises 403 amino acids and shows a high level of identity with the other known mammalian L3 proteins. Southern blot analysis of bovine genomic DNA suggests that the bovine genome contains at least 4 copies of the L3 gene. A single hybridisation band of about 1.3 kb is detectable by Northern blot analysis. Within the amino acid sequence, two potential nuclear targeting sequences were detected: one at the N-terminal end and the other, consisting in a bipartite motif (amino acids 341 to 358), present and not previously noticed also in the other known mammalian L3 proteins. A search on all the available mammalian ribosomal proteins revealed the presence of this bipartite motif in many of these proteins.

Liver histology of an afibrinogenemic patient with the Bbeta-L353R mutation showing no evidence of hepatic endoplasmic reticulum storage disease (ERSD); comparative study in COS-1 cells of the intracellular processing of the Bbeta-L353R fibrinogen vs. the ERSD-associated gamma-G284R mutant.

BACKGROUND: Type I fibrinogen deficiencies (hypofibrinogenemia and afibrinogenemia) are rare congenital disorders characterized by low or unmeasurable plasma fibrinogen antigen levels. Their genetic bases are represented by mutations within the three fibrinogen genes. Among the 11 reported missense mutations, a few have been characterized by expression studies and found to have an impaired fibrinogen assembly and/or secretion. Histopathological analyses were previously reported in two hypofibrinogenemic cases with discernible hepatic disease, revealing that both underlying mutations (gamma-Gly284Arg and gamma-Arg375Trp) were associated with hepatic fibrinogen endoplasmic reticulum storage disease (ERSD). OBJECTIVE: The objective of this study was to investigate the liver histology in an afibrinogenemic patient, homozygous for the Bbeta-Leu353Arg mutation, and to study the intracellular processing of the mutant protein. PATIENTS AND METHODS: Liver histology was evaluated by light microscopy, electron microscopy and immunocytochemistry. Intracellular processing of mutant fibrinogen was analyzed by pulse-chase labeling and immunoprecipitation experiments. Messenger RNA levels were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The histopathological characterization of the liver showed no signs of fibrinogen accumulation, a difference from the previously reported findings in two hypofibrinogenemic kindreds with ERSD. To evaluate whether the Bbeta-Leu353Arg mutation and the ERSD-associated gamma-Gly284Arg mutation affected intracellular fibrinogen trafficking differently, both mutant proteins were expressed in COS-1 cells. Bbeta-Leu353Arg led to a more severe secretion defect, but no differences that could explain phenotype-genotype correlation were found in the intracellular processing. Endoglycosidase-H analysis demonstrated a secretion block before translocation to the Golgi medial stacks. Real-time RT-PCR studies showed normal levels of the Bbeta mRNA in the patient's liver. CONCLUSIONS: The results confirm that Bbeta-Leu353Arg is associated with impaired fibrinogen secretion, but not with hepatic ERSD.

Crystal structure of apo-avidin from hen egg-white.

The three-dimensional structure of hen egg-white apo-avidin, crystallized in a tetragonal crystal form, has been refined to a crystallographic R-factor of 0.164 (for the 6390 observed reflections in the 10.0 to 2.8 A resolution range). As in the case of holo-avidin, from which starting atomic co-ordinates were derived, the functional tetramer shows 2-pseudo 22 molecular symmetry. Each promoter is organized in an eight-stranded antiparallel orthogonal beta-barrel, with extended loop regions, which define the biotin binding pocket in the protomer core. In the absence of biotin the binding site is only partly occupied by water molecules. The structure of the binding site residues, as observed in apo-avidin, is highly complementary to that of the incoming biotin molecule, accounting for prompt and specific recognition. A crystal lattice contact may play a role in stabilizing the conformation of one protein loop, part of the biotin-binding pocket.

Three-dimensional structure of the tetragonal crystal form of egg-white avidin in its functional complex with biotin at 2.7 A resolution.

The three-dimensional structure of hen egg-white avidin, crystallized in a tetragonal crystal form, has been solved at 2.7 A resolution by molecular replacement methods. After refinement the crystallographic R-factor is 16.8%, for the 7255 reflections in the 10.0 to 2.7 A resolution range. The asymmetric unit contains two avidin polypeptide chains (M(r) 2 x 15,600), which build up the functional tetramer through a crystallographic 2-fold axis parallel to the c unit cell direction. The avidin tetramer has almost exact 222 molecular symmetry; the three possible dimers display quite distinct packing interfaces. Each protomer is organized in an eight-stranded antiparallel orthogonal beta-barrel, with extended loop regions. The avidin binding site within each promoter is located in a deep pocket, at the center of the barrel, displaying both hydrophobic and polar residues for recognition of the tightly bound vitamin. Two Trp residues, Trp70 and Trp97, and Phe79 are in close contact with biotin. Moreover, the binding pocket is partly closed in its outer rim by residue Trp110 of a neighboring subunit. Once bound, biotin is almost completely buried in the protein core, with the exception of the valeryl side-chain carboxylate group which is exposed to solvent, hydrogen bonds to residues Ala39, Thr40 and Ser75, and triggers the formation of a network of hydrogen bonded water molecules. Modeling of synthetic biotin analogues allows us to rationalize functional data available for the binding of these compounds, and to analyze them in terms of biotin recognition mechanism. Hen egg-white avidin shows clear structural homology to streptavidin, from Streptomyces avidinii, but significant deviations can be observed in some regions.

Crystallization of hen egg-white avidin in a tetragonal form.

Hen egg-white avidin has been crystallized at pH 5.7 from ammonium sulfate solutions. The crystals belong to the tetragonal space group P4(2)2(1)2, with unit cell edges a = b = 79.6 A, c = 84.3 A. Assuming a molecular weight of 15,600 per avidin monomer, this crystal form is compatible with the presence of a dimer in the asymmetric unit, and is suitable for a crystallographic structural investigation at high resolution.

Severe factor V deficiency: exon skipping in the factor V gene causing a partial deletion of the C1 domain.

BACKGROUND: Severe factor V (FV) deficiency is a rare coagulation disorder, characterized by very low or unmeasurable plasma levels of functional and immunoreactive FV. Among rare inherited coagulopathies, FV deficiency is the least characterized from a molecular point of view (only 12 mutations have been reported). OBJECTIVES: The aim of this work was to investigate, at the molecular level, the pathogenetic mechanisms responsible for a case of severe FV deficiency. PATIENTS AND METHODS: A 19-year-old Iranian man showing unmeasurable FV activity and severely reduced FV antigen level in plasma was studied. Mutation screening was performed by sequencing. The effect of the identified mutation was investigated both at the mRNA and at the protein level. RESULTS: Molecular analysis of the factor V (FV) gene identified a novel homozygous A-->T transversion at position + 3 of the donor splice site of intron 19 (IVS19 + 3A-->T). Production of mutant mRNA in HeLa cells demonstrated that this mutation causes the entire exon 19 to be skipped from the FV mRNA. The mutant processed transcript codes for a deleted FV, lacking the first 24 amino acids of the C1 domain. Expression of the mutant FV protein in COS-1 cells showed that the deleted protein was synthesized but not secreted; moreover, the intracellular amount of deleted FV was reduced compared to wild type, suggesting intracellular degradation of mutant FV. CONCLUSIONS: This work reports the molecular characterization of the first mutation causing a partial deletion in the FV molecule, resulting in a severe impairment of protein secretion.

Identification of a glucocorticoid response element in the human gamma chain fibrinogen promoter.

The effect of the synthetic glucocorticoid hormone dexamethasone on human gamma chain fibrinogen gene expression was examined. The whole promoter region of 3.8 kb of this gene and progressive 5'-deletions were inserted into a promoterless expression vector, upstream of the luciferase gene and transiently transfected into the human hepatoma HepG2 cells, in the presence or in the absence of dexamethasone stimulation. Deletion analysis allowed to identify a region located between -1359 and -954 bp upstream from the transcription start site, involved in hormone inducibility. On the basis of a computer-assisted analysis, a putative GRE was found in this region at bases -1116 to -1102. Specific point mutations eliminating this putative GRE led to complete loss of glucocorticoid inducibility, thus indicating its functional role. Binding of the rat glucocorticoid receptor to this site was demonstrated by mobility-shift assays.

SER252PHE and 776INS3 mutations in the CHRNA4 gene are rare in the Italian ADNFLE population.

41 patients (19 sporadic and 22 familial) affected by autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) were analyzed for the presence of two mutations (Ser252Phe, 776ins3) in the CHRNA4 gene, reported to be associated with this disease. Electroclinical findings of sporadic forms were indistinguishable from familial ones. In none of the patients, these mutations were found by dot blot analysis with allele specific oligonucleotides. These data, obtained on the largest group so far studied, suggest the rarity of the reported mutations.

Refined mapping of CHRNA3/A5/B4 gene cluster and its implications in ADNFLE.

The chromosome 15q24 region, containing the CHRNA3/A5/B4 gene cluster, coding for the alpha3, alpha5 and beta4 subunits of neuronal nicotinic acetylcholine receptors, has been reported to be linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in one family. However, nor the gene nor the mutation involved have been identified. We report the refined mapping of CHRNA3/A5/B4 cluster. Segregation analyses of CHRNA3/A5/B4 polymorphisms in families showing recombinations for 15q24 G¿en¿ethon STR markers allowed to position the cluster in a 0.6 cM interval, between STRs D15S1027 and D15S1005. This location is external to the 15q24-ADNFLE-linked region, therefore excluding the involvement of this cluster in the pathogenesis of ADNFLE in the 15q24-linked family. Moreover, these data provide more precise information for further linkage studies.

Identification of four novel polymorphisms in the Aalpha and gamma fibrinogen genes and analysis of association with plasma levels of the protein.

Four novel polymorphisms were identified in the fibrinogen gene cluster. Three of them were localized in the promoter regions of the Aalpha-chain (alpha -128 C/G, alpha -58 G/A) or the gamma-chain (gamma -239 A/G) gene, while the remaining one was identified in intron 9 of the gamma-chain gene (gamma 7792 C/T). Genotype distributions for these polymorphisms were analyzed in 200 healthy Italian individuals and were in Hardy-Weinberg equilibrium. Since high levels of plasma fibrinogen have been associated with an increased risk of cardiovascular disease and genetic variations have been evaluated as thrombotic risk predictors, we analyzed their role in determining the plasma levels of this protein. Owing to the low frequency of the rare allele of alpha -128 C/G and gamma -239 A/G polymorphisms, association with plasma fibrinogen levels was investigated for only alpha -58 G/A and gamma 7792 C/T. We also investigated in the same population two previously identified polymorphisms in the fibrinogen gene cluster (alpha TaqI and beta -455 G/A) chosen for their widely studied association with plasma fibrinogen levels. In the multivariate linear regression analysis, no statistically significant association with plasma fibrinogen levels was found.

Comparison of sequence of cDNA clone with other genomic and cDNA sequences for human C-reactive protein.

A clone for C-reactive protein (CRP) has been isolated from a human liver cDNA library; this clone harbors a plasmid, pC81, which has an insert of 1631 bp. When compared to genomic and cDNA sequences published to date now, pC81 has revealed homologies and differences that might help to clarify the structure of this gene and the presence of allelic variants in man.

Culture techniques for human keratinocytes.

The two main approaches developed for in vitro culture of human keratinocytes are reviewed and discussed. The older technique is based on the use of a serum-containing medium and of a feeder-layer of lethally irradiated mouse fibroblasts; the second relies upon serum-free media, in the absence of a feeder-layer. While the former technique is most widely used for the preparation of epithelial sheets for grafting, the latter is best suited for studies on the biological properties of keratinocytes.

Monovalent cations requirement of the fructose 1,6-bisphosphate-activated pyruvate kinase from E. coli.

The fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli has been purified by a simplified procedure, which gives a homogeneous enzyme in approximately half the working time required by other methods and is suitable for large scale preparations. The activity of the enzyme is strictly dependent on the presence of monovalent cations. Enzyme activity is elicited by K+ and NH4+, but not by Na+. Homotropic cooperativity is displayed in the activation by K+ and NH4+ and heterotropic effects are reciprocally exerted by monovalent cations and other ligands, such as phosphoenolpyruvate and fructose 1,6-bisphosphate. The allosteric nature of such interactions is suggested by changes in heat stability of the enzyme induced by K+ and fructose 1,6-bisphosphate. NH4+, but not K+, at high concentrations, cause an inhibition of enzyme activity.

Fructose 1,6-bisphosphate-activated pyruvate kinase from E. coli: ligand promoted conformational changes.

The ligand-dependent susceptibility to heat inactivation and to tryptic digestion and the intrinsic fluorescence of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli were investigated in the absence and in the presence of physiological ligands. With respect to the enzyme alone, binding of the allosteric activator fructose 1,6-bisphosphate makes the protein sensitive to tryptic attack and thermolabile, while binding of phosphoenolpyruvate and Mg2+, but not of either ligand separately, induces in the enzyme a highly thermostable conformation, the attainment of which does not require an ordered binding sequence of the two ligands. The apparent loosening of the enzyme structure induced by fructose bisphosphate suggests that the activation it exerts at low phosphoenolpyruvate concentration might be due to an increased accessibility of substrate to the active site.

Polymorphism of the human gamma chain fibrinogen gene.

Nucleotide sequence comparisons of the published cDNAs and genomic sequences coding for the gamma-chain of human fibrinogen revealed several differences. We isolated two independent human cDNA clones, coding for part of this protein and compared their sequences, which are identical with the published relevant data. Our sequence allowed us to solve a conflict for aminoacid 88. All the remaining differences resulting from this comparison occurred in the third position of a codon and did not change codon properties or restriction sites. The level of polymorphism of this gene is discussed, taking into account also the nucleotide differences among all the published relevant data.