Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).     

Genetic variability in the European minnow,Phoxinus phoxinus (L.)

An electrophoretic study of genetic variation at 11 loci was performedfor a population of European minnows, Phoxinus phoxinus (L.). Ten loci, EST-1 ^*, EST-2 ^* EST-3 ^*,GPD-1 ^*,GPD-2 ^*,GPI-1 ^*,GPI-2 ^*,MPI ^*,6PGD ^* and PGM ^* were polymorphic. IDH ^*wasmonomorphic. The mean number of heterozygotic loci over all 176 fish was 3.05 ± 0.104(SE). Observed mean heterozygosity was 0.28±0.058(SE) and expected mean heterozygosity was 0.27±0.054(SE). EST-2 ^*, EST-3 ^* andPGM ^* were not in Hardy-Weinberg equilibrium. Length,condition, parasite numbers or male breeding characters, i.e. red colorationand tubercles, were not influenced by single enzyme loci.     

Detection and characterization of denitrifying bacteria from a permanently ice-covered Antarctic Lake

Denitrifying bacterial strains were isolated from Lake Bonney,apermanently ice-covered and chemically stratified lake in theMcMurdo dry valley region of Antarctica, using complex mediaat4 °C. Three strains, identified as denitrifiers bytheirability to produce nitrous oxide using nitrate or nitrite as arespiratory substrate, were characterized as to theirtemperatureand salinity optima for aerobic growth in batch culture; allthreewere psychrophilic and moderately halophilic. Maximum growthratesof near 0.024 h^–1 were measured for all three strains.Growthrates projected to occur at in situ temperature andsalinityimply generation times on the order of 100 h. Species specificpolyclonal antisera were prepared against two of the strains,ELB17 (from the east lobe of the lake at 17 m) and WLB20 (fromthewest lobe at 20 m). Both strains were subsequently detectedandenumerated in the lake using the antisera. ELB17 was presentinboth lobes below the chemocline, while WLB20 was present inthewest lobe below the chemocline but only in surface waters oftheeast lobe. These distributions are related to the observedchemicaldistributions which imply the occurrence of denitrification inthewest lobe of the lake and not in the east lobe.     

The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase, is a member of the T-box family.

The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase (IleRS), contains a long mRNA leader region. This region exhibits many of the features of the gram-positive synthetase gene family, including the T box and leader region terminator and antiterminator. The terminator was shown to be functional in vivo, and readthrough increased during growth in the presence of mupirocin, an inhibitor of IleRS activity. The S. aureus ileS leader structure includes several critical differences from the other members of the T-box family, suggesting that regulation of this gene in S. aureus may exhibit unique features.     

Shell variation in sympatric freshwater Lymnaea peregra and L. ovata (Gastropoda: Lymnaeidae)

Snails of the genus Lymnaea are morphologically variable and their taxonomy is unclear. In particular, the forms peregra and ovata , distinguished by shell shape, are often considered variants of the same species, L. peregra. We studied a rare situation in a Swiss mountain lake where both forms are sympatric. First, we found that the forms shows complete separation for a number of allozymes. Second, we examined the response of the two snail forms to infection by the trematode Diplostomum phoxini . For the ovata form, there was a transitory, 10% increase in the growth of infected snails compared to uninfected controls. The peregra form showed no gigantism and had higher parasite-induced mortality. Third, we assessed differences between the forms in reproduction under different environmental conditions. Density negatively influenced egg production to the same degree in both forms. However, decreasing water levels, characteristic of part of the lake studied, led to a 30% decrease in egg production for the ovata form, but only a 10% decrease for the peregra form. These differences are discussed in relation to the microdistributions of the snails in the lake and we conclude that the two forms are almost certainly separate species.     

The summer zooplankton community at South Georgia: biomass,vertical migration and grazing

Zooplankton abundance and biomass were determined during January 1990 at two stations to the north-west of South Georgia using a Longhurst Hardy Plankton Recorder (LHPR). At both shelf and oceanic station sites, zooplankton biomass, (excluding Euphausia superba), was found to be ca. 13 g dry mass m^–2. Copepods and small euphausiids dominated the catches. These estimates are over 4 times higher than values generally reported for the Southern Ocean and may reflect firstly, the high productivity of the study area, secondly, the time of year, summer, when biomass for many species is maximal, and thirdly, the high sampling efficiency of the LHPR. Principal components analysis disclosed similarities and differences between adjacent depth strata in terms of abundance, biomass and species composition. At both stations most variability occurred in the mixed layer (0–60 m) and thermocline (60–120 m) with depth horizons below this being more homogeneous. Diel migrations were observed for most taxa with abundance increasing in the mixed layer at night. At the oceanic station, species and higher taxa belonging to the mesopelagic community were generally well spread throughout this domain and, with the exception of Pleuromamma robusta and Metridia curticauda, showed little evidence of migration. The grazing impact of the epipelagic community (copepods and small euphausiids) was estimated to remove 3–4% of the microbial standing stock day^–1 and a conservative 25% and 56% of daily primary production at the oceanic and shelf stations respectively.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Transfer and expression of the human multiple drug resistance gene as potential human gene therapy

The human multiple drug resistance (MDR) gene has been used as a model for human gene transfer which could lead to human gene therapy. MDR is a transmembrane protein which pumps a number of toxic substances out of cells including several drugs used in cancer chemotherapy. Normal bone marrow cells express low levels of MDR and are particularly sensitive to the toxic effects of these drugs. There are two general applications of MDR gene therapy: (1) to provide drug-resistance to the marrow of cancer patients receiving chemotherapy, and (2) as a selectable marker which when co-transferred with a non-selectable gene such as the human beta globin gene can be used to enrich the marrow for cells containing both genes. We demonstrate efficient transfer and expression of the human MDR gene in a retroviral vector into live mice and human marrow cells including CD34⁺ cells isolated from marrow and containing the bulk of human hematopoietic progenitors. MDR gene transduction corrects the sensitivity of CD34⁺ cells to taxol, an MDR drug substrate, and enriches the marrow for MDR-transduced cells. The MDR gene-containing retroviral supernatant used has been shown to be safe and free of replication-competent retrovirus. Because of the safety of the MDR retroviral supernatant, and efficient gene transfer into mouse and human marrow cells, a phase 1 clinical protocol for MDR gene transfer into cancer patients has been approved to evaluate MDR gene transfer and expression in human marrow.     

Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.