Pseudohypoparathyroidism type Ia: two new heterozygous frameshift mutations in exons 5 and 10 of the Gsα gene

Pseudohypoparathyroidism type Ia (PHP-Ia) is a hereditary disease characterized by resistance to PTH and other hormones that act via cAMP. Patients have deficient activity of Gs, the subunit of the G protein, which couples hormone receptors to stimulation of adenylate cyclase. We describe two new mutations discovered in two sporadic patients with PHP-Ia. Using genomic DNA, we have amplified exons 2–13 of the Gs gene (GNAS1) by PCR, and sequenced the resulting products. Both patients had Albright's hereditary osteodystrophy, resistance to multiple hormones, and deficient Gs activity. In the first patient, a deletion of a C in exon 5 at codon 115 was found. In the second patient, an insertion of a C in exon 10 at codon 267 was detected. Both these heterozygous mutations cause frameshift, and predict decreased production of Gs. This report adds two new Gs mutations to the known ten mutations recently described.     

Role ofDrosophila TRP in inositide-mediated Ca2+ entry

Inositol lipid signaling relies on an InsP₃-induced Ca²⁺ release from intracellular stores and on extracellular Ca²⁺ entry, which takes place when the Ca²⁺ stores become depleted of Ca²⁺. This interplay between Ca²⁺ release and Ca²⁺ entry has been termed capacitative Ca²⁺ entry and the inward current calcium release activated current (CRAC) to indicate gating of Ca²⁺ entry by Ca²⁺-store depletion. The signaling pathway and the gating mechanism of capacitative Ca²⁺ entry, however, are largely unknown and the molecular participants in this process have not been identified. In this article we review genetic, molecular, and functional studies of wild-type and mutantDrosophila photoreceptors, suggesting that thetransient receptor potential mutant (trp) is the first putative capacitative Ca²⁺ entry mutant. Furthermore, several lines of evidence suggest that thetrp gene product TRP is a candidate subunit of the plasma membrane channel that is activated by Ca²⁺ store depletion.     

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformation

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10⁵ transformants/g DNA) was achieved. Correspondence to: J. Nevera     

Epidermal growth factor,phorbol esters,and aurintricarboxylic acid are survival factors for MDA-231 cells exposed to adriamycin

Summary The ability of epidermal growth factor (EGF), insulinlike growth factor-1 (IGF-1), insulin, 12-O-tetradecanoylphorbol-13-acetate (TPA), and aurintricarboxylic acid (ATA) to protect the human breast cancer cell line MDA-231 from death induced by the anticancer drug adriamycin was investigated. Cell death was induced in the MDA-231 cells either by a short-time exposure to a high dose of adriamycin (2 μg · ml⁻¹ · 1 h⁻¹) and further culturing in the absence of the drug, or by continuous exposure to a low dose of adriamycin (0.3μg/ml). Cell death was evaluated after 48 h of incubation by several techniques (trypan blue dye exclusion, lactic dehydrogenase activity, cellular ATP content, transmission electron microscopy, and DNA fragmentation). EGF, TPA, and ATA, each at an optimal concentration of 20 ng/ml, 5 ng/ml, and 100μg/ml respectively, substantially enhanced survival of cells exposed either to a high or low dose of adriamycin. Neither IGF-1 nor insulin, each at concentrations of 20 ng/ml, had an effect on cell survival. The three survival factors enhanced protein synthesis in the untreated cells and attenuated the continuous decrease in protein synthesis in the adriamycin-treated cells. Moreover, the three survival factors protected the MDA-231 cells from death in the absence of protein synthesis (cycloheximide 30μg/ml). These results suggest that EGF, TPA, and ATA promote survival of adriamycin pretreated cells by at least two mechanisms: enhancement of protein synthesis and by a protein synthesis independent process, probably a posttranslational modification effect.     

Backbone Cyclization of the C-terminal Part of Substance P. Part 1: The Important Role of the Sulphur in Position 11

Novel backbone-to-side chain and backbone-to-backbone cyclic analogues of substance P (SP) were prepared by solid-phase synthesis and screened for biological activity. An analogue containing a thioether- lactam ring between positions 9 and 11 showed an EC₅₀ value of 20nM toward the neurokinin 1 (NK-1) and was inactive toward the NK-2 and NK-3 receptors. On the other hand, in a multiple backbone cyclic peptide library of similar analogues, in which the sulphur was excluded from the ring, very low activity was detected. The activity was re-evaluated and was found to be even lower (EC₅₀=0.11 mM ) than the previously published data. These results indicate that the thioether moiety has a crucial role in receptor activation. The results also show tolerance of the NK-1 receptor, but not NK-2 or NK-3, to cyclization of the C-terminal portion of the SP_6–11 hexapeptide.     

Parietochloris incisa comb. nov. (Trebouxiophyceae, Chlorophyta)

A coccoid green alga, Myrmecia incisa Reisigl, was isolated from the soil of Mt Tateyama, Japan. Electronmicroscopy revealed that the organism has pyrenoids sparsely covered with starch segments and traversed by many parallel thylakoid membranes, and zoo-spores with counterclockwise basal body orientation. Due to the presence of these features, we have proposed a reclassification of M. incisa into the genus Parietochloris, Trebouxiophyceae.     

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l^–1 sucrose, 1 g l^–1 yeast extract and 0.05 mg l^–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l^–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - Km kanamycin - NPTII neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid - BM basal medium     

SINGLE-CELL PIGMENTATION OF PORPHYRA LINEARIS ANALYZED BY FOURIER TRANSFORM MULTI-PIXEL SPECTROSCOPY AND IMAGE ANALYSIS1

The novel method of Fourier transform multi-pixel spectroscopy was used for the nondestructive analysis of and comparison of pigmentation in different regions of live thalli of the red alga Porphyra linearis. Because the thallus in this alga consists of a monolayer of nonoverlapping cells, we were able to analyze the pigmentation of single cells by combining light absorbance with natural fluorescence data. From the image of each cell in the vegetative male and female reproductive and holdfast regions, more than 4 ± 10⁴ fluorescence and absorbance spectra were obtained. Specific pigments in the different regions were localized by the use of a software program of similarity mapping followed by image construction. The reconstructed images revealed subcellular localization of each pigment according to specific spectroscopic fingerprints. The results showed that the vegetative and female reproductive cell types had a significantly higher content of phycoerythrin than of phycocyanin, and quite similar chlorophyll a levels. Most of the holdfast cells were poorly pigmented, but had more chlorophyll a than phycoerythrin or phycocyanin. The male reproductive cells contained only traces of pigments. Thus, by using Fourier transform multipixel spectroscopy, we were able to characterize the pigmentation of different regions of the thallus and follow the distribution patterns of the different pigments on the subcellular level along the differentiation gradient of the alga.     

A defective conditioned behavior and a defective adenylate cyclase in theDrosophila mutantrutabaga

Summary TheDrosophila X-linked mutantrutabaga (Duerr and Quinn 1982) fails to perform normally in olfactory conditioning paradigms, in spite of being able to sense odorants and shock (Figs. 1–3).rut is capable of forming an association between shock and odorant, but memory decays rapidly; the memory of the mutant following intensive training resembles that of normal flies following very brief training (Fig. 4).rut flies also display in vitro a defective adenylate cyclase activity (Fig. 6). The enzyme in the mutant is responsive to stimulation by a putative neurotransmitter and by a guanyl nucleotide (Fig. 8) but the activity is lower than normal even in the presence of forskolin (Fig. 8) and MnATP (Fig. 9), suggesting that the lesion is closely associated with the function of the catalytic subunit.rut/ + heterozygotes are semi-recessive with regard to both the behavioral defect and the biochemical defect (Figs. 5, 7). The behavioral and the biochemical lesions detected inrut flies are discussed in light of current molecular models of learning.     

Transforming activity of plasmid and chromosomal DNA inEscherichia coli

An auxotrophic strain ofEscherichia coli with therecB recC sbcB genotype was transformed by chromosomal DNA of the prototrophic strain and by plasmid DNA carrying genes for antibiotic resistance (R1drd 19). The donor plasmid DNA obtained by cell lysis in the presence of Triton X-100 and subsequent centrifugation in a caesium chloride-ethidium bromide gradient was shown to have a circulaf molecule and to retain its completeness after penetration into the recipient. Experiments with mixtures or plasmid and chromosomal DNA indicate a competition between these two DNA types during the transformation reaction in the given system.