A critical appraisal of the role of triangulation in nursing research

Proopiomelanocortin (POMC) is the precursor for a number of biologically active peptides such as adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. It is well known that these peptides are involved in the stress response in fish as well as in mammals. We have cloned two different carp POMC cDNAs called, POMC-I and POMC-II. The nucleotide sequences of 955 bp for POMC-I and 959 bp for POMC-II share 93.5% identity in their cDNAs, and the deduced amino acid sequences (both 222 amino acids) are 91.4% identical. In the ACTH and beta-MSH domain, two amino acid substitutions are found, whereas alpha-MSH and beta-endorphin are identical. For beta-MSH, the serine replacement (in POMC-I) by a glycine (in POMC-II) results in a putative amidation site Pro-X-Gly for POMC-II. We used RT-PCR to show that both POMC mRNAs are expressed in the hypophysis, hypothalamus and other parts of the brain of a single fish. Furthermore, in a phylogenetic tree based on POMC sequences the divergence of carp POMC-I and -II from tetraploid animals (salmon, trout and xenopus) is demonstrated.     

A case report of multiple hepatic hilar cyst associated with hepatocellular carcinoma

Two types of cDNA encoding gonadotropin beta subunits (GTH beta) were isolated from a cDNA library prepared from pituitary gland of Baikal omul (Coregonus autumnalis migratorius Georgi). The nucleotide sequences of cDNA were determined. The CTHI beta and GTHII beta cDNAs code for polypeptides of 137 and 142 amino acids, respectively. Both of them include a putative signal peptide of 24 amino acids. The predicted amino acid structures of omul gonadotropins were compared with those of other vertebrate species.     

Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-1,4-glucanase

Five cellulose-binding polypeptides were detected in Cellulomonas fimi culture supernatants. Two of them are CenA and CenB, endo-beta-1,4-glucanases which have been characterized previously; the other three were previously uncharacterized polypeptides with apparent molecular masses of 120, 95, and 75 kDa. The 75-kDa cellulose-binding protein was designated endoglucanase D (CenD). The cenD gene was cloned and sequenced. It encodes a polypeptide of 747 amino acids. Mature CenD is 708 amino acids long and has a predicted molecular mass of 74,982 Da. Analysis of the predicted amino acid sequence of CenD shows that the enzyme comprises four domains which are separated by short linker polypeptides: an N-terminal catalytic domain of 405 amino acids, two repeated sequences of 95 amino acids each, and a C-terminal domain of 105 amino acids which is > 50% identical to the sequences of cellulose-binding domains in Cex, CenA, and CenB from C. fimi. Amino acid sequence comparison placed the catalytic domain of CenD in family A, subtype 1, of beta-1,4-glycanases. The repeated sequences are more than 40% identical to the sequences of three repeats in CenB and are related to the repeats of fibronectin type III. CenD hydrolyzed the beta-1,4-glucosidic bond with retention of anomeric configuration. The activities of CenD towards various cellulosic substrates were quite different from those of CenA and CenB.     

Isolation and characterization of human fast skeletal beta troponin T cDNA: comparative sequence analysis of isoforms and insight into the evolution of members of a multigene family

A cDNA encoding human fast skeletal beta troponin T (beta TnTf) has been isolated and characterized from a fetal skeletal muscle library. The cDNA insert is 1,000 bp in length and contains the entire coding region of 777 bp and 5' and 3' untranslated (UT) segments of 12 and 211 bp, respectively. The 3' UT segment shows the predicted stem-loop structure typical of eukaryotic mRNAs. The cDNA-derived amino acid sequence is the first available sequence for human beta TnTf protein. It is encoded by a single-copy gene that is expressed in a tissue-specific manner in fetal and adult fast skeletal muscles. Although the human beta TnTf represents the major fetal isoform, the sequence information indicates that this cDNA and the coded protein are quite distinct from the fetal and neonatal TnTf isoforms reported in other mammalian fetal muscles. The hydropathy plot indicates that human beta TnTf is highly hydrophilic along its entire length. The protein has an extremely high degree of predicted alpha-helical content involving the entire molecule except the carboxy-terminal 30 residues. Comparative sequence analysis reveals that the human beta TnTf shares a high level of sequence similarity in the coding region with other vertebrate TnTf and considerably reduced similarity with slow skeletal and cardiac TnT cDNAs. The TnT isoforms have a large central region consisting of amino acid residues 46-204 which shows a high sequence conservation both at the nucleotide and amino acid levels. This conserved region is flanked by the variable carboxy-terminal and an extremely variable amino-terminal segment. The tropomyosin-binding peptide of TnT, which is represented by amino acid residues 47-151 and also includes a part of troponin I binding region, is an important domain of this central segment. It is suggested that this conserved segment is encoded by an ancestral gene. The variable regions of vertebrate striated TnT isoforms reflect the subsequent addition and modification of genomic sequences to give rise to members of the TnT multigene family.     

Primary structure of the kinase domain region of rabbit skeletal and cardiac muscle titin

A 2.3 kb region of rabbit cardiac and skeletal muscle titin has been cloned. The cDNA sequences of the two tissues are identical and show 91% identity on the nucleotide level with the corresponding region of human cardiac muscle titin. On the amino acid level the identity is 96% and similarity is 98%. Alignment of predicted amino acid sequences of several homologous kinase domains reveals that the rabbit titin kinase has all the necessary elements of an active catalytic domain and carries a potential regulatory region on its C-terminal end. The distance of the 2.3 kb contig from the 3' end of the message was determined to be 5.7 kb in both tissues using oligonucleotide directed RNase H cleavage of titin mRNAs. This is essentially identical with the length of the fully sequenced human cardiac titin C-terminal end. It therefore appears unlikely that there are major tissue specific differences in this 8 kb cDNA region which encodes the C-terminus of rabbit skeletal and cardiac titin.     

Mouse prolactin receptor gene: genomic organization reveals alternative promoter usage and generation of isoforms via alternative 3'-exon splicing

In rodents, the prolactin receptor is expressed as multiple isoforms with identical extracellular and membrane-proximal region sequences but with different 3' sequences, encoding different cytoplasmic regions, and different 5' untranslated region (UTR) sequences. These divergent sequences could be the result of multiple prolactin receptor genes or of a single gene which displays alternative promoter usage and 3'-exon splicing. To investigate the molecular basis for these observations, we have cloned and determined the organization of the mouse prolactin receptor gene. Genomic DNA cloning allowed the arrangement of promoters 1A, 1B, and 1C to be determined. 5'-RACE-PCR from mouse liver identified two novel 5' prolactin receptor sequences, indicating that the gene has at least five different promoters, four of which are active in liver. The remaining nonvariable 5' UTR is encoded by a separate exon (exon 2), while a further 11 coding exons follow, the last 4 of which are alternatively spliced to produce the four isoforms of the receptor. Functional units were found to be exon specific. Thus, the multiple prolactin receptor isoforms are the product of a single gene of >120 kb which displays multiple promoter usage and 3'-exon splicing.     

Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.     

Production of biologically active recombinant tilapia insulin-like growth factor-II polypeptides in Escherichia coli cells and characterization of the genomic structure of the coding region

Insulin-like growth factor-II (IGF-II) is a fetal growth factor in humans, but has not been clearly identified in fish up to now. For a detailed understanding of the physiological response of fish IGF-II, the first step was to clone tilapia IGF-II cDNA from the brain cDNA library, coding the region of genomic DNA, and also expressing tilapia IGF-II polypeptides from Escherichia coli. Tilapia cDNA sequences total 1,977 bp, and predicted nucleotide sequences and amino acid sequences of tilapia share 77.9% and 90.7% homology identity with rainbow trout IGF-II, respectively. The genomic structure of the tilapia prepro-IGF-II coding region is very difficult to sequence in mammals and birds. The cloned tilapia IGF-II gene coding region appears much more complex than in other vertebrates. In tilapia IGF-II, the first coding exon I encoding part of the signal peptide sequence is 25 amino acids shorter than the first coding exon of mammals and birds. The other 23 amino acids of the signal peptide, and the first amino acids of the B domain and C domain are encoded by tilapia coding exon 2. The C, A, and D domains, and the first 20 amino acids of the E peptide are encoded by tilapia coding exon 3. The other E peptides and the 3' untranslated region (UTR) region are encoded by tilapia coding exon 4. These data show that the IGF-II genes have significantly differing structures in vertebrate evolution, and there are differences of interrupting introns in the IGF-I genomic structure compared with mammals. To obtain recombinant biologically active polypeptides, tilapia IGF-II B-C-A-D domains were amplified using the polymerase chain reaction (PCR), then ligated with glutathione S-transferase (GST, pGEX-2T vector). Tilapia recombinant IGF-II protein was purified and characterized in E. coli. The fusion protein was also digested with thrombin and appeared as a recombinant IGF-II polypeptide single band with a molecular mass of 7 kD. The recombinant tilapia IGF-II protein biological function was measured by stimulation of [3H]thymidine incorporation. The assay concentration was set up from 0 to 120 nM to stimulate tilapia ovary cell line (TO-2) significantly to uptake thymidine. The results suggest that the recombinant IGF-II protein was dose dependent.     

Identification of a novel, tissue-specific calpain htra-3; a human homologue of the Caenorhabditis elegans sex determination gene

We cloned a novel human gene encoding a tissue-specific calpain, termed htra-3, which is highly homologous to the tra-3 sex determination gene of Caenorhabditis elegans. The predicted htra-3 polypeptide had similarity to the calpain large subunits in domain organization throughout domains I to III, but the sequences of domain IV lacked calcium-binding motifs. Northern blot analysis revealed high expression in the colon, small intestine and testis. Radiation hybrid mapping localized the htra-3 gene to chromosome 11q14 (2.53cR apart from WI-3895). Western blot analysis demonstrated that the approximately 73-kDa htra-3 protein was transiently expressed in COS-7 cells. These observations, together with the genetic information in C.elegans, suggest a unique function for htra-3 protein.     

Molecular cloning of cDNA encoding the alpha unit of CNGC gene from human fetal heart

Cyclic nucleotide-gated ion channels (CNGCs) play crucial roles in visual and olfactory signal transduction. As a first step to explore the presence of a CNGC gene in human heart, we cloned a human heart CNGC gene. The sequence consists of 111 bp 5' non-coding region and a 2064 bp open reading frame which is followed by a 459 bp 3' non-coding region. The predicted protein consists of 688 amino acids with a short highly charged segment rich in lysine and glutamate. Sequence comparison indicates that the human heart cDNA is almost identical to the retinal rod photo receptor CNGC cDNA. However, the human cardiac cDNA is lacking a 205 bp Alu fragment in the 5'-uncoding region, has a glutamic acid residue at amino acid position 129, and has a replacement of glutamic acid with a lysine residue at amino acid position 99. Data obtained with northern blot analysis confirm the presence of RNA for the CNGC alpha chain. This channel might play a role in cyclic nucleotide-mediated cellular processes, such as the inotropic effect in the heart.     

Molecular characterization and expression of two putative protective excretory secretory proteins of Haemonchus contortus

It has been shown that vaccination with two low molecular mass excretory secretory (ES) antigens of 15 and 24 kDa, respectively, afforded a substantial degree of protection against Haemonchus contortus to sheep. In vitro cultivation of the parasite usually yields a limited amount of these proteins and therefore, recombinant DNA technology was employed to clone the cDNAs encoding the ES proteins of interest and to express them in a convenient vector system. The N-terminal amino acid sequences of the two ES products were determined. Specific 5' primers were used in combination with an oligo (dT) 3' primer to amplify the appropriate cDNAs by polymerase chain reaction (PCR). A lambda ZAPII cDNA library was constructed from mRNA of L5 larvae and subsequently screened with the PCR products. The full length clone of the 15 kDa ES protein coded for a 17.2 kDa precursor molecule of 148 amino acids with a signal peptide of 30 amino acids. The full length clone of the 24 kDa ES protein coded for a 24.6 kDa precursor protein of 222 amino acids with a leader sequence of 19 residues. The expression of both ES products appeared to be developmentally regulated; mRNA encoding occurs only in the parasitic life stages. A cDNA of each ES protein was sub-cloned, without the leader sequence, into a pQE9 expression vector. Both purified recombinant proteins were recognized by sera from H. contortus hyperimmunised sheep as judged by immunoblot analysis, suggesting that antigenic determinants were also present on the recombinant proteins.     

Caenorhabditis elegans has two isozymic forms, CE-1 and CE-2, of fructose-1,6-bisphosphate aldolase which are encoded by different genes

Two distinct types of cDNAs for fructose-1,6-bisphosphate (FBP) aldolase, Ce-1 and Ce-2, have been isolated from nematode Caenorhabditis elegans, and the respective recombinant aldolase isozymes, CE-1 and CE-2, have been purified and characterized. The Ce-1 and Ce-2 are 1282 and 1248 bp in total length, respectively, and both have an open reading frame of 1098 bp, which encodes 366 amino acid residues. The entire amino acid sequences deduced from Ce-1 and Ce-2 show a high degree of identity to one another and to those of vertebrate and invertebrate aldolases. The highest sequence diversity was found in the carboxyl-terminal region that corresponds to one of the isozyme group-specific sequences of vertebrate aldolase isozymes that play a role in determining isozyme-specific functions. Southern blot analysis suggests that CE-1 and CE-2 are encoded by different genes. Concerning general or kinetic properties, CE-2 is quite different from CE-1. CE-1 exhibits unique characteristics which are not identical to any aldolase isozymes previously reported, whereas CE-2 is similar to vertebrate aldolase C. These results suggest that CE-2 might preserve the properties of a progenitor aldolase with a moderate preference for FBP over fructose 1-phosphate (F1P) as a substrate, whereas CE-1 evolved to act as an intrinsic enzyme that exhibits a much broader substrate specificity than dose CE-2.     

Validation of the indicators of abuse (IOA) screen

The ephrins are a family of ligands that bind to Eph family receptor tyrosine kinases, and have been implicated in axon guidance and other patterning processes during vertebrate development. We describe here the identification and characterization of murine ephrin-B3. The cDNA encodes a 340 amino acid transmembrane molecule, most closely related to the two other known transmembrane ligands, ephrin-B1 and ephrin-B2. In addition to homology in their extracellular receptor binding domains, these transmembrane ligands share striking homology between their cytoplasmic domains, with 31 of the last 34 amino acids of ephrin-B3 being identical to ephrin-B2, suggesting functional interactions of the cytoplasmic tail. While most Eph family ligands are promiscuous in their interactions with Eph receptors, binding studies with the five receptors known to bind other transmembrane ligands only revealed a high affinity interaction of ephrin-B3 with EphB3, with a dissociation constant of approximately 1 nM. In situ hybridization of mouse embryos showed ephrin-B3 is expressed prominently at the dorsal and ventral midline of the neural tube, particularly in the floor plate, a structure with key functions in patterning the nervous system. The isolation of this ligand may help to elucidate the molecular basis of patterning activities at the neural tube midline.