Effects of a kappa-opioid agonist on adrenocorticotropic and diuretic function in man

Although kappa-opiate receptors represent an important fraction of the total opiate receptor capacity in human brain their endocrine function is unknown. We determined the effects of a kappa-opiate receptor agonist on the secretion of vasopressin, ACTH and cortisol and on diuresis. The racemic benzomorphan kappa agonist MR 2033 or its opiate active (-)-isomer, MR 2034, inhibited the release of cortisol and ACTH in 12 trials in a naloxone reversible manner; plasma levels of vasopressin were not altered. The (+)-isomer, MR 2035, did not affect the secretion of cortisol or ACTH. Surprisingly, in five other subjects large increases were observed in vasopressin, ACTH and cortisol following the kappa-agonist, which were probably elicited indirectly by aversive effects of the opioid. The subjects in whom vasopressin release was not altered by MR 2033 and MR 2034 displayed large decreases in urine osmolality which were not antagonized by naloxone. The opiate inactive (+)-isomer, MR 2035, caused no diuretic response. Subjects in whom vasopressin release was stimulated did not show decreases in urine osmolality indicating that vasopressin is capable of antagonizing the diuretic action of the kappa-agonist. Our data show that a kappa-agonist inhibits secretion of cortisol and ACTH by acting at stereospecific opiate receptors and elicits diuresis by acting at stereospecific, but naloxone-insensitive non-classical opioid receptors. These data support the concept that different types of kappa-receptors can be distinguished in man.     

Stacking Interactions of nucleobases: NMR-Investigations I. Selfassociation of N6,N9-Dimethyladenine and N6-Dimethyl-N9-Ethyladenine

The selfassociation of N⁶,N⁹-dimethyladenine and N⁶-dimethyl-N⁹-ethyladenine has been studied by means of NMR technique. The thermodynamic quantities have been calculated using an isodesmic NMR model with three NMR parameters (the monomer shift _M and two complex shifts ₂ and ₃).The dependence of the thermodynamic quantities on the NMR parameters is discussed. Special attention is given to the determination of _M and its temperature dependence.Calculations with ₃=2· ₂ and _M taken independently of temperature result in an average entropy _S =–17.9±1.8 e.u. for N⁶,N⁹-dimethyladenine and _S =–16.7±1.7 e.u. for N⁶-dimethyl-N⁹-ethyladenine and in an average enthalpy _H=–7.2±0.6 kcal· mol^–1 for both substances investigated.Part of the Ph.D. Theses of W. Schimmack and H. Sapper.Dedicated to Professor Dr. A. Schraub on the occasion of his 65th birthday.     

Distinct RB1 gene mutations with low penetrance in hereditary retinoblastoma

The interfamilial diversity in penetrance and expressivity of hereditary retinoblastoma was investigated in 29 families. By using a simple parameter for estimating the severity of the disease (diseased-eye-ratio), we were able to identify four families with a discrete low-penetrance phenotype. The underlying genetic defect was identified in three families. One family has a 3-bp deletion in exon 16 that results in the deletion of Asn⁴⁸⁰. In two further unrelated families, the identical missense mutation at codon 661 in exon20 (CGG to TGG, Arg to Trp) was identified. These mutations are distinct from the majority of retinoblastoma gene alterations, as they do not result in the disruption of the gene product. We propose that reduced penetrance of retinoblastoma is the result of a residual function of these alleles in retinoblastoma precursor cells.     

The spectrum of RB1 germ-line mutations in hereditary retinoblastoma.

We have searched for germ-line RB1 mutations in 119 patients with hereditary retinoblastoma. Previous investigations by Southern blot hybridization and PCR fragment-length analysis had revealed mutations in 48 patients. Here we report on the analysis of the remaining 71 patients. By applying heteroduplex analysis, nonisotopic SSCP, and direct sequencing, we detected germ-line mutations resulting in premature termination codons or disruption of splice signals in 51 (72%) of the 71 patients. Four patients also showed rare sequence variants. No region of the RB1 gene was preferentially involved in single base substitutions. Recurrent transitions were observed at most of the 14 codons within the RB1. No mutation was observed in exons 25-27, although this region contains two CGA codons. This suggests that mutations within the 3'-terminal region of the RB1 gene may not be oncogenic. When these data were combined with the results of our previous investigations, mutations were identified in a total of 99 (83%) of 119 patients. The spectrum comprises 15% large deletions, 26% small length alterations, and 42 % base substitutions. No correlation between the location of frameshift or nonsense mutations and phenotypic features, including age at diagnosis, the number of tumor foci, and manifestation of nonocular tumors was observed.     

The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.     

“On A Piece of Chalk”-Updated1

ABSTRACT. Many advances have been made in our knowledge of the biology of foraminifera over the past several decades. Fine structural, biophysical, and molecular biological studies have shown that the most prominent components of their distinctive bidirectional granuloreliculopods are bundles of micro tubules linked by crossbridges to each other, as well as to membrane-bound organelles and the plasma membrane. the microtubules ratchet past each other as dynein transduces the free energy of ATP to produce pseudopodal movements. In spite of the fact that there are over 40,000 described species of living and fossil species of foraminifera, there have been many recent exciting discoveries of new species and groups. New casting techniques are providing us with greater understanding of the complexities and functional aspects of form in the group. Significant advances are being made in understanding the distribution and energetics of deep-sea forms. Larger and planktonic foraminifera are the hosts for a particularly diverse range of endosymbiotic algae, including dinoflagellates, chlorophytes, unicellular rhodophytes, and diatoms. Chloroplast husbandry also occurs. Significant research effort has been expended yielding us considerable insight into various aspects of the endosymbiotic phenomenon. A unified conceptual framework has been drawn to help us understand the life cycle options found in foraminifera.     

A stem-loop "kissing" model for the initiation of recombination and the origin of introns

Mutations which improve the efficiency of recombination should affect either the proteins which mediate recombination or their substrate, DNA itself. The former mutations would be localized to a few sites. The latter would be dispersed. Studies of hybridization between RNA molecules have suggested that recombination may be initiated by a homology search involving the "kissing" of the tips of stem loops. This predicts that, in the absence of other constraints, mutations which assist the formation of stem loops would be favored. From comparisons of the folding of normal and shuffled DNA sequences, I present evidence for an evolutionary selection pressure to distribute stem loops generally throughout genomes. I propose that this early pressure came into conflict with later local pressures to impose information concerning specific function. The conflict was accommodated by permitting sections of DNA concerned with a specific function to evolve in dispersed segments. Traces of the conflict seem to be present in some modern intron-containing genes. Thus, introns may have allowed the interspersing of selectively advantageous stem loops in coding regions of DNA.      

A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.     

A highly multiplexed quantitative phosphosite assay for biology and preclinical studies

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.