[Starvation and chemoreception in Antarctic benthic invertebrates].

Sensitivity (chemoreception) to different amino acids was studied in six invertebrate species: Serolis polita, Glyptonotus antarcticus, Abyssochromene plebs, Waldeckia obesa, Odontaster validus, and Sterechinus neumayeri. The sensitivity was estimated by the changes in basic metabolism (respiration rate). Starvation increased the sensitivity in all the species. The metabolism rates increased in the presence of L-glutamic acid in G. antarcticus, A. plebs, O. validus, and S. neumayeri. The serine and arginine amino acids had a significant impact on the metabolism of the necrophagous species S. polita and W. obesa. The chemical information may be mediated by means of L-glutamic acid via glutamate receptors, which can be blocked by kynurenic acid, as occurs in the experiments with G. antarcticus and A. plebs.

Somatostatin analogs.

Somatostatin is a hypothalamic peptide hormone that inhibits the secretion of growth hormone, glucagon, insulin, gastrin and secretin, and also plays a role in neural transmission. Because of its wide range of possible clinical applications hundreds of somatostatin analogs have been synthesized and bioassayed to date. This review gives a historical perspective, summarizing approximately 30 years of research on somatostatin. The main focus is on the structure-activity relationships and conformational studies of the last generation of somatostatin agonists and their selectivity for five somatostatin receptor subtypes. Achievements in the synthesis of nonpeptide somatostatin analogs, as well as the first somatostatin antagonists, are also discussed. Finally, the use of a cyclic somatostatin scaffold to design ligands for other G-protein-coupled receptors, such as opioid and melanocortin receptors, is mentioned.

A candidate prostate cancer susceptibility gene at chromosome 17p.

It is difficult to identify genes that predispose to prostate cancer due to late age at diagnosis, presence of phenocopies within high-risk pedigrees and genetic complexity. A genome-wide scan of large, high-risk pedigrees from Utah has provided evidence for linkage to a locus on chromosome 17p. We carried out positional cloning and mutation screening within the refined interval, identifying a gene, ELAC2, harboring mutations (including a frameshift and a nonconservative missense change) that segregate with prostate cancer in two pedigrees. In addition, two common missense variants in the gene are associated with the occurrence of prostate cancer. ELAC2 is a member of an uncharacterized gene family predicted to encode a metal-dependent hydrolase domain that is conserved among eukaryotes, archaebacteria and eubacteria. The gene product bears amino acid sequence similarity to two better understood protein families, namely the PSO2 (SNM1) DNA interstrand crosslink repair proteins and the 73-kD subunit of mRNA 3' end cleavage and polyadenylation specificity factor (CPSF73).

Human mitogen-activated protein kinase kinase 4 as a candidate tumor suppressor.

Mitogen-activated protein kinases function in signal transduction pathways that are involved in controlling key cellular processes in many organisms. A mammalian member of this kinase family, MKK4/JNKK1/SEK1, has been reported to link upstream MEKK1 to downstream stress-activated protein kinase/JNK1 and p38 mitogen-activated protein kinase. This mitogen-activated protein kinase pathway has been implicated in the signal transduction of cytokine- and stress-induced apoptosis in a variety of cell types. Here, we report that two human tumor cell lines, derived from pancreatic carcinoma and lung carcinoma, harbor homozygous deletions that eliminate coding portions of the MKK4 locus at 17p, located approximately 10 cM centromeric of p53. In addition, in a set of 88 human cancer cell lines prescreened for loss of heterozygosity, we detected two nonsense and three missense sequence variants of MKK4 in cancer cell lines derived from human pancreatic, breast, colon, and testis cells. In vitro biochemical assays revealed that, when stimulated by MEKK1, four of the five altered MKK4 proteins lacked the ability to phosphorylate stress-activated protein kinase. Thus, the incidence of coding mutations of MKK4 in the set of cell lines is 6 of 213 (approximately 3%). These findings suggest that MKK4 may function as a suppressor of tumorigenesis or metastasis in certain types of cells.

Localization of SMAD5 and its evaluation as a candidate myeloid tumor suppressor.

Acquired interstitial or complete losses of chromosome 5 are recurring anomalies associated with preleukemic myelodysplasia and acute myelogenous leukemia with a poor prognosis. Previous studies have delineated a potential myeloid tumor suppressor locus to a <2.4-Mb interval between the genes for IL9 and EGR1 on 5q31. In this report, we have localized the SMAD5 gene, a homologue of the tumor suppressor genes SMAD4/DPC-4 and SMAD2/JV18.1, to the minimal myeloid tumor suppressor locus and characterized its open reading frame and genomic organization. SMAD5 transcripts are readily detectable in hematolymphoid tissues and leukemic blasts. Absence of intragenic mutations in the remaining SMAD5 allele of leukemic patients and multiple solid tumor cell lines prescreened for loss of heterozygosity suggests that SMAD5 may not be a common target of somatic inactivation in malignancy.

Generation of an integrated transcription map of the BRCA2 region on chromosome 13q12-q13.

An integrated approach involving physical mapping, identification of transcribed sequences, and computational analysis of genomic sequence was used to generate a detailed transcription map of the 1. 0-Mb region containing the breast cancer susceptibility locus BRCA2 on chromosome 13q12-q13. This region is included in the genetic interval bounded by D13S1444 and D13S310. Retrieved sequences from exon amplification or hybrid selection procedures were grouped into physical intervals and subsequently grouped into transcription units by clone overlap. Overlap was established by direct hybridization, cDNA library screening, PCR cDNA linking (island hopping), and/or sequence alignment. Extensive genomic sequencing was performed in an effort to understand transcription unit organization. In total, approximately 500 kb of genomic sequence was completed. The transcription units were further characterized by hybridization to RNA from a series of human tissues. Evidence for seven genes, two putative pseudogenes, and nine additional putative transcription units was obtained. One of the transcription units was recently identified as BRCA2 but all others are novel genes of unknown function as only limited alignment to sequences in public databases was observed. One large gene with a transcript size of 10.7 kb showed significant similarity to a gene predicted by the Caenorhabditis elegans genome and the Saccharomyces cerevisiae genome sequencing efforts, while another contained a motif sequence similar to the human 2',3' cyclic nucleotide 3' phosphodiesterase gene. Several retrieved transcribed sequences were not aligned into transcription units because no corresponding cDNAs were obtained when screening libraries or because of a lack of definitive evidence for splicing signals or putative coding sequence based on computational analysis. However, the presence of additional genes in the BRCA2 interval is suggested as groups of putative exons and hybrid selected clones that were transcribed in consistent orientations could be localized to common physical intervals.

The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds.

Breast carcinoma is the most common malignancy among women in developed countries. Because family history remains the strongest single predictor of breast cancer risk, attention has focused on the role of highly penetrant, dominantly inherited genes in cancer-prone kindreds (1). BRCA1 was localized to chromosome 17 through analysis of a set of high-risk kindreds (2), and then identified four years later by a positional cloning strategy (3). BRCA2 was mapped to chromosomal 13q at about the same time (4). Just fifteen months later, Wooster et al. (5) reported a partial BRCA2 sequence and six mutations predicted to cause truncation of the BRCA2 protein. While these findings provide strong evidence that the identified gene corresponds to BRCA2, only two thirds of the coding sequence and 8 out of 27 exons were isolated and screened; consequently, several questions remained unanswered regarding the nature of BRCA2 and the frequency of mutations in 13q-linked families. We have now determined the complete coding sequence and exonic structure of BRCA2 (GenBank accession #U43746), and examined its pattern of expression. Here, we provide sequences for a set of PCR primers sufficient to screen the entire coding sequence of BRCA2 using genomic DNA. We also report a mutational analysis of BRCA2 in families selected on the basis of linkage analysis and/or the presence of one or more cases of male breast cancer. Together with the specific mutations described previously, our data provide preliminary insight into the BRCA2 mutation profile.

Reduced-size antagonists of luteinizing hormone-releasing hormone active in vitro.

A series of reduced-size analogs of LHRH was designed with the length varying from nine to two amino acids. These compounds were tested in vitro for the LH suppression in cultured rat pituitary cells treated with 1 ng of LHRH. The best analogs were also tested in vivo for their antiovulatory activity in rats. It appeared that terminal amino acids as well as the presence of Arg or ILys in the sequence are both crucial for the antagonism. The most potent antagonist in this series was a heptapeptide, AcDNal-Ser-Tyr-DNal-Leu-Arg-ProNHEt, which completely inhibited LH release at the dose 0.1 microgram and inhibited ovulation at 1000 micrograms/rat. For fragments shorter than heptapeptide the inhibition of LH release was observed at the dose 100 micrograms of the analog.

Antide B, an antagonist of LHRH with cis-3-(4-pyrazinylcarbonylaminocyclohexyl)alanine in position 5.

Several LHRH antagonists with trans-3-(4-pyrazinylcarbonylaminocyclohexyl)alanine (trans-PzACAla) in the position 5 were synthesized and their antiovulatory activity was compared with the activity of the analogs containing cis-PzACAla in this position. In all cases cis-isomer produced more potent analogs. Introduction of cis-PzACAla in the position 5 of Antide gave Antide B which completely inhibits ovulation at a dose of 0.5µg/rat. Antide B releases negligible histamine (ED50 = 104µg/mL), and has excellent solubility in water. Also, an improved synthesis of cis-PzACAla is reported, involving the hydrogenation of 4-aminophenylalanine on a rhodium catalyst to give the desired cis-isomer with a 53% yield.

Novel, potent luteinizing hormone-releasing hormone antagonists with improved solubility in water.

A series of luteinizing hormone-releasing hormone antagonists with new substitutions in position 6 or positions 5 and 6 that included lysine acylated at the epsilon-amino group with different heterocyclic carboxylic acids or amino-substituted heterocyclic carboxylic acids was synthesized. These novel analogs wee synthesized on a solid-phase support via the acylation of lysine residue in otherwise protected resin-bound peptides. All analogs were tested in the rat antiovulatory assay (AOA) and the best of them in in vitro histamine release assay. Introduction of lysine acylated with amino-substituted heterocyclic carboxylic acids yielded several water-soluble antagonists with good therapeutic ratio (high AOA to low histamine releasing activity). The best antagonist in terms of activity, histamine release, and solubility was nictide: NAcDNal-DCpa-DPal-Ser-PicLys-D(6ANic)-Orn- Leu-ILys-Pro-DAlaNH2 (6ANic = 6-aminonicotinoyl).

New, highly active antagonists of LHRH with acylated lysine and p-aminophenylalanine in positions 5 and 6.

A series of antagonists of the luteinizing hormone releasing hormone (LHRH) with substitutions in position 5 and/or 6 that included acylated lysine or p-aminophenylalanine were synthesized, characterized and tested for antiovulatory activity (AOA) in rats, and histamine releasing activity. Some of these antagonists were considerably more soluble at neutral pH than antagonists like Antide. Of 37 new antagonists, the best physico-chemical and biological properties were found for the following two analogs: NAcDNal-DCpa-DPal-Ser-PicLys-D(PicSar)Lys- Leu-ILys-Pro-DAlaNH2 (named Sartide) and NAcDNal-DCpa-DPal-Ser-Tyr-D(PicSar)Lys-Leu-IL ys-Pro-DAlaNH2. They are both soluble in water, inhibit ovulation completely at 0.5 microgram per rat, and have ED50 values for histamine release of about 30 micrograms/mL.

The structural features of effective antagonists of the luteinizing hormone releasing hormone.

The structure-activity data of 6 years on 395 analogs of the luteinizing hormone releasing hormone (LHRH) have been studied to determine effective substituents for the ten positions for maximal antiovulatory activity and minimal histamine release. The numbers of substituents studied in the ten positions are as follows: (41)(1)-(12)(2)-(12)(3)-(5)(4)-(47)(5)-(52)(6)-(16)(7)-(18)(8)-(4)(9)-(8)(10). In position 1, DNal and DQal were effective with the former being more frequently the better substituent. DpClPhe was uniquely effective in position 2. Positions 3 and 4 are very sensitive to change. D3Pal in position 3 and Ser in position 4 of LHRH were in the best antagonists. PicLys and cPzACAla were the most successful residues in position 5 with cPzACAla being the better substituent. Position 6 was the most flexible and many substituents were effective; particularly DPicLys. Leu(7) was most often present in the best antagonists. In position 8, Arg was effective for both antiovulatory activity and histamine release; ILys was effective for potency and lesser histamine release. Pro(9) of LHRH was retained. DAlaNH2 (10) was in the best antagonists.

Effective antagonists of luteinizing hormone releasing hormone modified at position one.

The amino acid, D-2-naphthylalanine, has been used by many investigators as a substituent for position one of antagonists of LHRH. We have newly designed substituents for position one in which the carboxy groups of 2-naphthoic acid, 3-quinoline- and 2-quinoxaline-carboxylic acids are linked to the five amino acids, DAla, DThr, DNVal, DSer, and Gly. The substituents in positions 2-10 were DpClPhe(2), DPal(3), Ser(4), PicLys(5), DPicLys(6), Leu(7), ILys(8), Pro(9), DAlaNH2 (10).Remarkably, DThr, acylated on the amino group by 3-quinolinecarboxylic acid or by 3-quinoxalinecarboxylic acid, and introduced into position one of a relatively potent antagonist, gave a new class of antagonists of LHRH, which released as little histamine as yet recorded, and yet possessed reasonable antiovulatory activity and greatly improved solubility.These structure-activity results advance the basic knowledge of understanding the structural features of such decapeptides which cause antiovulatory activity and histamine release.