Molecular characterization, tissue distribution and expression analysis of interleukin-12 receptor β2 chain in sheep

Agnathia-otocephaly is a rare, often lethal malformation characterized by absence or hypoplasia of the mandible, microstomia, hypoglossia/aglossia, and variable anterior midline fusion of the ears (melotia, synotia). Etiologies have been linked to both genetic and teratogenic factors and to date, a definitive, commonly identifiable cause has not been recognized. Mouse and human genetic studies have implicated OTX2 and PRRX1 as potential candidate genes for agnathia-otocephaly. In this study we report a sporadic case of agnathia-otocephaly complex with associated features of maldevelopment and examine the roles of OTX2 and PRRX1. The proband, a male born at 31 weeks, displayed severe micrognathia, microstomia, posteriorly-rotated and low set ears, and downward slanting palpebral fissures. Mutation analysis was performed after sequencing the entire coding regions of OTX2 and PRRX1 genes isolated from the proband and his parents. After thorough analysis, no DNA variations were detected. This suggests that mutations in different genes or environmental causes are responsible.     

Molecular characterization,tissue distribution and expression analysis of Interleukin-12 receptor β2 chain in sheep

Ovine β2 subunit of the interleukin (IL)-12 receptor (IL-12Rβ2) was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs). The complete coding sequence for ovine IL-12 Rβ2 was found to be 2586 nucleotides in length encoding 862-amino-acid residue protein. It showed 96.4% homology at the nucleotide level and 94.1% homology at the amino acid level with bovine IL-12 Rβ2. The ovine IL-12 Rβ2 subunit shares common structural and functional elements with their counterparts from the other species. Phylogenetic tree showed that ovine IL-12Rβ2 was clustered into the Artiodactyla group, together with those of cattle and pig, which was distinct from the other groups. Real-time RT-PCR was used to investigate expression of the IL-12Rβ2 in different tissues of sheep in order to determine the characterization of this receptor in tissue. Expression analysis showed that IL-12Rβ2 mRNA expression was detected at all the detected tissues with the exception of thymus.     

Molecular characterization of β-thalassemia in Azerbaijan

We have analyzed the -thalassemia mutations in 99 chromosomes of 49 adults with -thalassemia major and of one with Hb S--thalassemia, who are regular patients at a large hematology clinic in Bakü, Azerbaijan. A total of 20 different mutants were identified; three [frameshift at codon 8 (-AA); IVS-II-I (GA); IVS-I-110 (GA)] were present in about two-thirds of all chromosomes. Most alleles are the same as found in Mediterranean populations; a few have an Asian origin or come from Kurdistan, Lebanon, Saudi Arabia, or a black population. One mutant [frameshift at codons 82/ 83 (-G)] might be specific for the Azerbaijanian population. Nearly all patients were transfused, which made quantitation of Hb F impossible; high^G values were present in the Hb F of those patients whose -thalassemia chromosome carried the C T mutation at position — 158 in the promoter of the ^G-globin gene.     

Molecular characterization of β-thalassemia in Czechoslovakia

Summary We have identified different -thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific ³²P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The GA mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (GA), IVS-II-745 (CG), IVS-I-110 (GA), and codon 39 (CT); these were present in 9 additional families. The GT mutation at codon 121, known to cause Heinzbody -thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One -thalassemia allele was incompletely characterized. We observed in 2 families a TC mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism, -Thalassemia was rare; only one person carried the -^3.7 heterozygosity, and one other had a yet to be identified -thalassemia-1, while seven had the ^{anti 3.7} triplication.     

Molecular characterization of β-thalassemia in Hungary

We have identified seven different -thalassemia mutations and one -thalassemia determinant (the Sicilian type) in 32 members of 17 Hungarian families. The most common mutation is the IVS-I-1 (GA) change; its high frequency is comparable to that observed in neighboring Czechoslovakia. Additional mutations are of Mediterranean origin. One rare mutation (initiation codonATGGTG) was identified as an independent mutation because of the absence of known polymorphisms in the -globin gene. One new frameshift at codon 51 (-C) was observed in a single individual; hematological data were as expected for a °-thalassemia heterozygosity.     

β-Criptoxanthin stimulates bone formation and inhibits bone resorption in tissue culture in vitro

The effect of beta-cryptoxanthin, which is greatly present in fruits, has not been clarified so far on bone metabolism. The effect of beta-cryptoxanthin on bone formation and bone resorption was investigated in tissue culture in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-8)-10(-5) M beta-cryptoxanthin. The presence of beta-cryptoxanthin (10(-6) or 10(-5) M) caused a significant increase in calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues. These increases were completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. beta-Carotene (10(-6) or 10(-5) M) or xantine (10(-6) or 10(-5) M) had no effect on the diaphyseal and metaphyseal calcium contents. The bone-resorbing factors parathyroid hormone (1-34) (PTH; 10(-7) M) or prostaglandin E2 (PGE2; 10(-5) M) caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. The decrease in bone calcium content induced by PTH or PGE2 was completely inhibited by beta-cryptoxanthin (10(-8)-10(-6) M). In addition, beta-cryptoxanthin (10(-8)-10(-6) M) completely inhibited the PTH (10(-7) M)- or PGE, (10(-5) M)-induced increase in medium glucose consumption and lactic acid production by diaphyseal and metaphyseal tissues. The inhibitory effect of beta-cryptoxanthin (10(-7) M) on PTH (10(-7) M)- or PGE2 (10(-5) M)-stimulated decrease in the diaphyseal calcium content was significantly prevented in the presence of 10(-3) M vanadate, an inhibitor of protein tyrosine phosphatase. Vanadate (10(-3) M) did not have a significant effect on calcium content and lactic acid production in control bone tissues. The present study demonstrates that beta-cryptoxanthin has a direct stimulatory effect on bone formation and an inhibitory effect on bone resorption in tissue culture in vitro.     

Preparation,characterization and in vitro activity of a docetaxel–albumin conjugate

Docetaxel is one of the most effective anticancer drugs. However, the current formulation of docetaxel contains Tween 80 and ethanol as the solvent, which can cause severe side effects. Consequently, the development of new type of formulation of docetaxel with high efficiency and low side effects is a very important issue. In this study, we explored the covalent linking of docetaxel and albumin via one organic linker. 6-Maleimidocaproic acid was applied to link the C2′ hydroxyl group of docetaxel with the cysteine-34 of albumin to obtain 1:1 docetaxel–albumin conjugate. The synthesized conjugate can control the release of docetaxel in the bovine serum. Furthermore, in vitro cell cytotoxicity experiments indicated that the docetaxel–albumin conjugate have high activities for human prostate cancer cell line PC3 and human breast cancer cell line MCF-7. The present study provides a valuable strategy for further development of a new type of docetaxel–albumin prodrug.     

Molecular characterization of a ring X chromosome in a male with short stature

We report the molecular characterization of a ring X chromosome that was transmitted from a mother to a male who has short stature and minor dysmorphic features. This represents only the second reported ring X chromosome in a male. The ring is derived from breakage within the Xp pseudoautosomal region (PAR) and just proximal to the Xq PAR. The total amount of deleted material is 700-900 kb DNA and includes six known transcribed genes. Interestingly, SHOX, a gene implicated in short stature, is not deleted from the ring chromosome. Possible pathogenetic explanations for the patient's clinical features include insufficient dosage of deleted genes, a position effect on SHOX expression, and cell death during development because of ring chromosome nondisjunction. The findings are also relevant to observations made of "complete" ring chromosomes.     

Molecular characterization of β-thalassemia mutations in Egypt

Summary The relative frequency of different -thalassemia mutations and their association with -globin haplotypes were studied in patients from the Nile delta region, Egypt, by means of the polymerase chain reaction, oligonucleotide hybridization and restriction analysis. We found that 8 mutations account for 77% of -thalassemia chromosomes in this population, the commonest being IVS-1 nt 110, IVS-1 nt 6 and IVS-1 nt 1. Each mutation was associated with a specific haplotype, with the exception of IVS-1 nt 110, found on 3 different chromosomal backgrounds. Our data show that testing for the 8 detectable mutations makes feasible prenatal diagnosis in 65% of at risk couples and exclusion testing in an additional 25% of cases.     

Molecular cloning and expression characterization of a rice K+ channel β subunit

K⁺ channel proteins native to animal membranes have been shown to be composed of two different types of polypeptides: the pore-forming subunit and the subunit which may be involved in either modulation of conductance through the channel, or stabilization and surface expression of the channel complex. Several cDNAs encoding animal K⁺ channel subunits have been recently cloned and sequenced. We report the molecular cloning of a rice plant homolog of these animal subunits. The rice cDNA (KOB1) described in this report encodes a 36 kDa polypeptide which shares 45% sequence identity with these animal K⁺ channel subunits, and 72% identity with the only other cloned plant (Arabidopsis thaliana) K⁺ channel subunit (KAB1). The KOB1 translation product was demonstrated to form a tight physical association with a plant K⁺ channel subunit. These results are consistent with the conclusion that the KOB1 cDNA encodes a K⁺ channel subunit.Expression studies indicated that KOB1 protein is more abundant in leaves than in either reproductive structures or roots. Later-developing leaves on a rice plant were found to contain increasing levels of the protein with the flag leaf having the highest titer of KOB1. Leaf sheaths are known to accumulate excess K⁺ and act as reserve sources of this cation when new growth requires remobilization of K⁺. Leaf sheaths were found to contain higher levels of KOB1 protein than the blade portions of leaves. It was further determined that when K⁺ was lost from older leaves of plants grown on K⁺-deficient fertilizer, the loss of cellular K⁺ was associated with a decline in both KOB1 mRNA and protein. This finding represents the first demonstration (in either plants or animals) that changes in cellular K⁺ status may specifically alter expression of a gene encoding a K⁺ channel subunit.     

Synthesis,characterization, and in vitro and in vivo evaluation of a novel pectin–adriamycin conjugate

Adriamycin (ADM) has been widely used in the treatment of many types of solid malignant tumor. However, cardiotoxicity, multidrug resistance and a short half-life in vivo are significant problems that limit its clinical application. To resolve these problems, a novel pectin–adriamycin conjugate (PAC) was synthesized by attaching ADM to low-methoxylated pectin via an amide linkage. The ADM content and weight-average molecular weight (Mw) of PAC were greater than 25% (w/w) and 50,360 g/mol, respectively. PAC was highly stable in plasma, but 33.2% of ADM was released from PAC after incubation for 30 h with lysosomes derived from rat liver. PAC was distributed uniformly in the cytoplasm of most A549 cells and accumulated in the nucleus of a few A549 cells after incubation for 30 h. At concentrations equivalent to 0.125–1.000 μg of ADM/mL, PAC did not inhibit the growth of either A594 or B16 cells to the same extent as free ADM or a mixture of ADM and pectin. Interestingly, at all concentrations, PAC inhibited the growth of 2780cp cells in vitro significantly more effectively than ADM or the mixture of ADM and pectin. The anticancer effect of PAC in vivo was evaluated with C57BL/6 mice bearing pulmonary metastases of B16 cells. Compared with ADM and the mixture of ADM and pectin, PAC suppressed tumor growth significantly and prolonged the mean survival time of the B16-inoculated mice. PAC has great potential for development as a tumor targeting polymer-drug.     

Molecular characterization of two types of 22 kilodalton α-zein genes in a gene cluster in maize

Summary Five genes of the -zein subfamily four (SF4) are located in a 56 kb genomic region of the maize inbred line W22. Their nucleotide and deduced amino acid sequences have been determined. The sequences define two types of -zein SF4 genes — type 1 (T1) and type 2 (T2). The single T1 -zein SF4 gene codes for an -zein protein with a M_r of about 22 000. This is the first -zein SF4 gene sequenced that contains no early in-frame stop codons in its coding sequence. The four T2 -zein SF4 genes in this cluster contain one or two early in-frame stop codons. In addition, our T1 and T2 genes differ markedly in the base sequences of their distal 5 non-translated flanking regions. The nucleotide and the deduced amino acid sequences of these two types of -zein SF4 genes are similar ( > 90 %) to one another and to all known -zein SF4 genes and cDNAs. Of the known W22 -zein SF4 genes, only one in six does not contain an early in-frame stop codon. If the number of -zein SF4 genes is 15–20, then we estimate that only about 4 of the W22 -zein SF4 genes are without in-frame early stop codons.     

Mechanical evaluation of a tissue-engineered zone of calcification in a bone–hydrogel osteochondral construct

The objective of this study was to test the hypothesis that mechanical properties of artificial osteochondral constructs can be improved by a tissue-engineered zone of calcification (teZCC) at the bone–hydrogel interface. Experimental push-off tests were performed on osteochondral constructs with or without a teZCC. In parallel, a numerical model of the osteochondral defect treatment was developed and validated against experimental results. Experimental results showed that the shear strength at the bone–hydrogel interface increased by 100% with the teZCC. Numerical predictions of the osteochondral defect treatment showed that the shear stress at the bone–hydrogel interface was reduced with the teZCC. We conclude that a teZCC in osteochondral constructs can provide two improvements. First, it increases the strength of the bone–hydrogel interface and second, it reduces the stress at this interface.     

Identification and characterization of a copper-binding site in αA-crystallin

Previous studies have shown that both αA- and αB-crystallins bind Cu2+, suppress the formation of Cu2+-mediated active oxygen species, and protect ascorbic acid from oxidation by Cu2+. αA- and αB-crystallins are small heat shock proteins with molecular chaperone activity. In this study we show that the mini-αA-crystallin, a peptide consisting of residues 71-88 of αA-crystallin, prevents copper-induced oxidation of ascorbic acid. Evaluation of binding of copper to mini-αA-crystallin showed that each molecule of mini-αA-crystallin binds one copper molecule. Isothermal titration calorimetry and nanospray mass spectrometry revealed dissociation constants of 10.72 and 9.9 μM, respectively. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid interaction with mini-αA-crystallin was reduced after binding of Cu2+, suggesting that the same amino acids interact with these two ligands. Circular dichroism spectrometry showed that copper binding to mini-αA-crystallin peptide affects its secondary structure. Substitution of the His residue in mini-αA-crystallin with Ala abolished the redox-suppression activity of the peptide. During the Cu2+-induced ascorbic acid oxidation assay, a deletion mutant, αAΔ70-77, showed about 75% loss of ascorbic acid protection compared to the wild-type αA-crystallin. This difference indicates that the 70-77 region is the primary Cu2+-binding site(s) in human native full-size αA-crystallin. The role of the chaperone site in Cu2+ binding in native αA-crystallin was confirmed by the significant loss of chaperone activity by the peptide after Cu2+ binding.