Up-regulation of hepatic LDL receptor gene expression by monatepil, a novel calcium antagonist, in high cholesterol diet-fed Japanese monkeys.

The action of the novel antihypertensive calcium antagonist monatepil on the hepatic LDL receptor was investigated at the gene expression level to clarify the mechanism of its hypolipidemic effect. In cholesterol-fed control monkeys, the LDL receptor mRNA level decreased to approximately 30% of that in the normal diet-fed monkeys. However, the administration of monatepil increased LDL receptor mRNA to normal levels (three- to fourfold stimulation). It is suggested that monatepil raises the number of LDL receptors in liver tissue and that this increase may accelerate the removal of plasma LDL. Treatment with prazosin, an alpha 1-adrenoceptor antagonist, also increased the LDL receptor mRNA level, but this restorative effect was much weaker than that of monatepil, suggesting that mechanisms additional to its alpha 1-adrenoceptor blocking activity are involved in the hypolipidemic effect of monatepil.

[Brachial plexus block with a nerve stimulator and "around the needle" catheter technique].

Brachial plexus block using a nerve stimulator is an accurate procedure. But prolonged analgesic effect can not be obtained. Therefore we used "around the needle" catheter technique to have a long analgesic effect. A 20-gauge, 5-inch intravenous catheter (Angiocath) was threaded over a 23-gauge, 10-cm needle (Pole). We used axillary approach. The cathode of the nerve stimulator (NS-2CA, Professional Instrument company) is connected to the needle, and its anode is connected to the electrode on the surface of the skin. When the needle is introduced and advanced, 1 mA of electric current is applied for nerve stimulation. When the muscle twitch is obtained, a plastic cannula is then threaded off the needle into the axillary sheath. An infusion tube and three way stopcock is connected to the cannula. From 1988 to 1991 we had 31 cases. The success rate is about 90%. If we stimulate other nerves in the same sheath, it is not necessary to seek aimed one. But the musculocutaneous nerve is the only exception, because it may be stimulated outside the neurovascular sheath.

Cloning and sequencing of the cDNA for human monocyte chemotactic and activating factor (MCAF).

cDNA clones having a nucleotide sequence encoding a human monocyte chemotactic and activating factor (MCAF) were isolated and sequenced. The amino acid sequence deduced from the nucleotide sequence reveals the primary structure of the MCAF precursor to be composed of a putative signal peptide sequence of 23 amino acid residues and a mature MCAF sequence of 76 amino acid residues. The amino acid sequence of MCAF showed 25-55% homology with other members of an inducible cytokine family, including macrophage inflammatory protein and some putative polypeptide mediators known as JE, LD78, RANTES and TCA-3. This suggests that MCAF is a member of family of factors involved in immune and inflammatory responses.

Complete nucleotide sequence of the gene for human interleukin 1 alpha.

The chromosomal gene for human interleukin 1 alpha (IL-1 alpha) was isolated from a human genomic DNA library by using as a probe cloned human IL-1 alpha cDNA. Complete nucleotide sequence of about 12 kilobase pairs (kbp) long was determined and the structure and organization of this gene were elucidated. This gene contains seven exons and six introns. The first exon encodes the 5'-untranslated region. Most of the prepeptide portion of the precursor polypeptide is encoded by the next three exons, and the mature form of IL-1 alpha is encoded by the remaining three exons. The last exon also encodes the intronless 3'-untranslated region. The fourth intron as well as both 5'- and 3'-flanking regions contains the sequence of an Alu family member. In the middle part of the last intron, a 46-bp sequence with a unique structure is repeated five times in a head to tail manner. These repeats are flanked by the regions containing alternative purine and pyrimidine tracts. In the 5'-flanking region, immediately upstream of the putative TATA box, the 16-bp sequence highly homologous to the binding site of the adenovirus 2 major late promoter transcription factor is identified. The nucleotide sequence reported here showed only one nucleotide substitution in the 3'-untranslated exon in comparison with the nucleotide sequence of the cDNA from HL-60, a promyelocytic leukemia cell line, previously cloned and sequenced in our laboratories.

Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor.

DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1.

Efficient production of a human tumour necrosis factor in Escherichia coli.

To examine the effect of altering the nucleotide sequence of the Shine-Dalgarno (SD) region and the spacer sequence between the SD sequence and the AUG translation start signal, several plasmids were constructed which directed the synthesis of mature human tumour necrosis factor (TNF) under the control of the E. coli trp leader promoter. We found that the presence of the SD sequence, AAGGAGGT, which is complementary to the 3' end of 16S rRNA, gave the higher translational efficiency, and also, the presence of the spacer sequence which consists of only A and T residues raised the production of TNF 2-3 fold. The levels of expression of TNF were elevated over 20-fold by the alteration of the SD and spacer sequences and amounted to 20% of the total cellular proteins or approx. 5 X 10(6) molecules of TNF per cell.

Primary structure of gamma subunit precursor of calf-muscle acetylcholine receptor deduced from the cDNA sequence.

Clones carrying cDNA sequences for the gamma subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has revealed the primary structure of this polypeptide, which consists of 519 amino acids including a hydrophobic prepeptide of 22 amino acids. The gamma subunit of the calf muscle acetylcholine receptor, like the alpha subunit of the calf as well as the human muscle receptor, shares features characteristic of all four subunits of the Torpedo electroplax receptor, such as the putative disulphide bridge corresponding to that in the alpha subunit proposed as being in close proximity to the acetylcholine binding site and the four putative, hydrophobic transmembrane segments M1-M4. Thus, the calf gamma subunit molecule apparently exhibits the same transmembrane topology as proposed for the fish receptor subunits. The degree of sequence homology between the calf and Torpedo gamma subunits (56%) is lower than that between the alpha subunits of the two species (81%). Some regions of the gamma subunit molecule, including the region encompassing the putative disulphide bridge and the region containing the putative transmembrane segments M1, M2 and M3, are relatively well conserved between the two species.

Sequence requirement for transcription in vitro of the human corticotropin/beta-lipotropin precursor gene.

Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned human and bovine corticotropin/beta-lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5'-deletion mutants of a fusion gene that contains the 5'-flanking sequence and capping site of the human corticotropin/beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the 'TATA box' present in this region seems to be the main promoter element for transcription of the human corticotropin/beta-lipotropin precursor gene in the HeLa cell-free system. We have also developed a transcription system in vitro from the corticotropin-producing mouse pituitary tumor cell line AtT-20 in culture. Deletion mapping of the fusion gene promoter has indicated that the 'TATA box' region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT-20 cell-free system, but hardly in the HeLa cell-free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT-20 cell extract is responsible for the modulation of transcription of the human corticotropin/beta-lipotropin precursor gene.

Isolation and characterization of the mouse corticotropin-beta-lipotropin precursor gene and a related pseudogene.

Two mouse genomic DNA sequences homologous with human corticotropin-beta-lipotropin precursor gene sequences have been cloned. One of them represents the functional corticotropin-beta-lipotropin precursor gene, which exhibits a structural organization similar to those of its bovine and human counterparts. The other represents a pseudogene that corresponds to the functional mouse gene sequence encoding the carboxy-terminal 143 amino acid residues (including corticotropin and beta-lipotropin) and the 3'-untranslated region.

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.

Structural analysis of repetitive DNA sequences in the bovine corticotropin-beta-lipotropin precursor gene region.

Repetitive DNA sequences in the bovine corticotropin-beta-lipotropin precursor gene region have been mapped and subjected to nucleotide sequence analysis. Two of the four repetitive DNA segments found are located in the 5'-flanking region, and one each within the intervening sequences. Each repetitive DNA segment contains one to three highly homologous unit sequences with an approximate length of 120 base pairs. All the unit sequences are flanked on the 3' side by tandem repeats. There are about 10(5) copies of the repetitive DNA in the bovine genome. Comparison of the bovine repetitive sequences with those of other mammalian species reveals the presence of a homologous segment of approximately 40 base pairs. This segment and the region preceding it in the bovine repetitive DNA exhibit sequence homology with the region encompassing the origin of DNA replication in papovaviruses.

DNA sequences required for transcription in vivo of the human corticotropin-beta-lipotropin precursor gene.

The cloned human corticotropin-beta-lipotropin precursor gene, when joined with an SV40 vector and introduced into COS monkey cells, is transcribed from its own promoter. The DNA sequences required for promoter function have been identified by using 5' deletion mutants of the fusion gene AT which contains the 5'-flanking sequence and capping site of the human corticotropin-beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The deletion of the sequence located between 53 and 59 bp upstream of the capping site enhances the transcription approximately 3-fold, while the deletion of the TATA box region abolishes the transcription.

Isolation and characterization of the bovine corticotropin/beta-lipotropin precursor gene.

The entire bovine corticotropin/beta-lipotropin precursor gene has been isolated as a set of overlapping genomic DNA fragments which extend over a length of approximately 17000 base pairs. Restriction mapping of the cloned DNA fragments and nucleotide sequence analysis of the whole mRNA-coding segments and their surrounding regions have established that the corticotropin/beta-lipotropin precursor gene is approximately 7300-base-pairs long and contains two intervening sequences; one with an approximate length of 4000 base pairs is located within the segment encoding the 5'-untranslated region of the mRNA, and the other with an approximate length of 220 base pairs interrupts the protein-coding sequence near the signal peptide region. Sequence analysis of more than 200 base pairs preceding the proximal end of the corticotropin/beta-lipotropin precursor gene has revealed a 'Hogness box' and a variant of the model sequence d(G-G-TC-C-A-A-T-C-T) as well as palindrome structures as observed in other eukaryotic genes. Furthermore, some sequence similarities in the 5'-flanking region are found between the corticotropin/beta-lipotropin precursor gene and the mouse alpha-globin and beta-globin genes, all of which are negatively regulated by glucocorticoids. At least four homologous repetitive sequences are distributed at 3000-5000-base-pair distances in the corticotropin/beta-lipotropin precursor gene region; two such sequences are located in the 5'-flanking region, and one within each intervening sequence. Blot hybridization analysis of bovine pituitary nuclear RNA has indicated that the entire corticotropin/beta-lipotropin precursor gene is transcribed into a primary hnRNA product, which is then spliced to form the mature mRNA.

The protein-coding sequence of the bovine ACTH-beta-LPH precursor gene is split near the signal peptide region.

The pituitary hormones corticotropin (ACTH) and beta-lipotropin (beta-LPH) are formed from a large common precursor. Recently, we have elucidated the whole primary structure of the bovine ACTH-beta-LPH precursor (designated alternatively as preproopiocortin) by determining the nucleotide sequence of cloned DNA complementary to the mRNA coding for the precursor protein. The amino acid sequence assigned has disclosed a characteristic repetitive structure of the ACTH-beta-LPH precursor. The repetitive units of the precursor protein each contain a melanotropin (MSH) sequence (alpha-, beta- or gamma-MSH) as well as other peptide components such as beta-endorphin and corticotropin-like intermediate lobe peptide (CLIP). The repetitive units as well as their peptide components are each bounded by paired basic amino acid residues, which apparently represent the sites of proteolytic processing. Several studies have confirmed the translational initiation site and protein structure assigned (see also ref. 11 and refs therein). In view of the recent knowledge about the organization of eukaryotic genes (see refs 12, 13 for reviews), it would be of particular interest to investigate the relationship between the repetitive structure of the ACTH-beta-LPH precursor containing different functional components and the arrangement of the protein-coding sequence in its gene. We have now isolated and characterized bovine genomic DNA fragments encoding this precursor protein and have demonstrated that the protein sequence is encoded by two non-consecutive DNA segments. An intron (intervening sequence) of approximately 2.2 kilobase pairs separates the smaller exon (mRNA-coding sequence), which contains the gene sequence encoding the signal peptide, from the larger exon, which contains the gene sequence for most of the protein structure, including the known biologically active component peptides.