Broomrape (Orobanche) seed germination inhibitors from plant roots

Two species (tomato and cucumber) which are not hosts to Orobanche crenata but which are hosts to other species of Orobanche not only failed to produce the compound required to trigger O. crenata to germinate but produced germination inhibitors which stopped germination even in the presence of a suitable stimulant. This suggested the possibility of using germination inhibitors to control at least some species of Orobanche. The question whether host species produce inhibitors as well as stimulants has not however been resolved.     

Intermediate sucrose octa-acetate sensitivity suggests a third allele at mouse bitter taste locus Soa and Soa-Rua identity

Mice have been characterized as either tasters or non-tastersof the bitter compound sucrose octa-acetate(SOA). However, 11of 17 supposedly non-taster inbred strains were found to avoid1 mM SOA. All 17 strains were indifferent to 0.1 mM SOA. Tasterstrains avoided both concentrations. The intermediate phenotypewas dubbed demitaster. A consistent phenotypic dominance orderwas found in crosses among both inbred and outbred strains (taster> non-taster > demitaster). Demitasters were found (withtasters) in an outbred strain showing monogenic segregationfor SOA avoidance. This, plus monogenic segregation in a back-crossof taster to demitaster inbred strains, suggested a third alleleat the Soa locus (Soa^c). Demitaster allelism was supported bythe strong associations found in 15 strains between the threeSOA phenotypes and HindIII restriction fragment patterns forthe closely linked Prp (proline rich protein) loci. SOA demitasterstrains were also intermediate in raffinose undeca-acetate (RUA)avoidance. Furthermore, B6.SW-Soa² congenic mice avoided notonly SOA, but RUA and eight other acetylated sugars. A previouslyproposed separate RUA-sensitivity gene (Rua) thus appeared tobe redundant.     

In vitro synthesis of vitamin D-3 by cultured human keratinocytes and fibroblasts: action spectrum and effect of AY-9944

With delineation of the photochemical events occurring in the skin after ultraviolet exposure, there has been increased interest in the skin's role in the vitamin D-3-endocrine system. We provide here in vitro conditions for the generation of both labelled (from [3H]acetate) and unlabelled vitamin D-3 in cultures of human keratinocytes and fibroblasts. Sterol precursors and photoproducts in irradiated and non-irradiated cultures are identified by co-chromatography, ultraviolet absorbance spectra, thermal conversion characteristics of previtamin D-3 and mass spectrometry. Because the conversion of 7-dehydrocholesterol to cholesterol is more efficient in vitro than in vivo, the specific delta 7 inhibitor, AY-9944, was added in non-toxic doses to modulate 7-dehydrocholesterol content. Both cell types were equally capable of generating photoproducts, depending on the amount of 7-dehydrocholesterol present. The 290 +/- 5 and 295 nm filters were much more efficient than the 305 nm filter for generating previtamin D-3 and vitamin D-3 in fibroblasts. In contrast, the 305 nm filter was as efficient as the 290 +/- 5 and 295 nm filters in keratinocytes, where it yielded previtamin D-3, with much less lumisterol and tachysterol than appeared with the shorter-wavelength filters. The amount of lumisterol and tachysterol versus previtamin D-3 formed in both cell types was dependent on the total energy applied, with lower energies (less then 1 J/cm2) favoring previtamin D-3 over the other photoproducts. The use of cultured cells provides a system whereby the regulation of vitamin D-3 synthesis by extracutaneous factors can be studied in a homogeneous setting.     

Induction of T4 DNA ligase in a recombinant strain of Escherichia coli

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed-batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature-sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30 degrees C followed by an induction phase which was initiated by shifting the temperature to 42 degrees C. In the fed-batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42 degrees C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed-batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.     

A unique alpha chain in hemoglobin of “Skive” DanishMus musculus

The primary structures of the alpha chains in hemoglobins from three stocks of mice with theHba ^w2,Hba ^w3, andHba ^w4 haplotypes were determined to establish whether the tentative alpha-chain assignments based on the results of isoelectric focusing patterns were correct. TheseHba haplotypes were identified in laboratory descendants of feral mice captured in different parts of the world. Hemoglobin from Centreville, Maryland,Mus musculus domesticus (Hba ^w2) contains equal amounts of alpha chains 1 and 3. Hemoglobin from CzechMus musculus musculus (Hba ^w4) contains equal amounts of alpha chains 3 and 4. Amino acid analysis of the alpha-globins of Skive DanishMus musculus musculus (Hba ^w3) establishes that its hemoglobin is comprised of about one-third alpha chain 2 as expected plus a greater amount of a unique alpha chain that has not been described previously. This unique alpha chain has glycine at position 25, isoleucine at position 62, and serine at position 68; it is called chain 7. It may represent an intermediate in the evolution of genes that code for chain 2 (which has glycine, valine, and serine at positions 25, 62, and 68, respectively) and chain 4 (which has valine, isoleucine, and serine at positions 25, 62, and 62, respectively).This research was sponsored jointly by the National Institutes of Environmental Health Sciences under Contract 1-ES-55078 and by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.     

Sequence of a full-length cDNA for rat lung beta-galactoside-binding protein: primary and secondary structure of the lectin

A full-length cDNA for rat lung beta-galactoside lectin (subunit Mr approximately 14,000, lectin 14K) was cloned and the nucleotide sequence determined. The deduced amino acid sequence agrees with the amino acid composition and direct amino acid sequence analysis of purified rat lung lectin peptides. We found that the amino-terminal alanine is blocked with an acetyl group. Comparison of the amino acid sequence with other proteins shows a high degree of homology only with other vertebrate lectin sequences, supporting the suggestion that these lectins may constitute a unique class of vertebrate proteins. The amino acid composition and sequence of lectin peptides, the sequence of lectin cDNA, and isoelectric focusing of purified lectin indicate that rat lung lectin 14K is composed predominantly of a single protein. In addition, rat uterus lectin 14K was found to be the same protein as that present in lung. We characterized the secondary and tertiary structure of rat lung lectin 14K by circular dichroism, by analytical ultracentrifugation, and by computer analysis of its primary structure. Results of these experiments suggest that lectin 14K is primarily a hydrophilic protein with an asymmetric, elongated structure consisting of approximately equal amounts of alpha helix, beta sheet, beta turn, and random coil. We found that Cys-2 and Cys-130 react most rapidly with iodoacetamide; one or both of these residues may be primarily responsible for the thiol requirement of lectin activity.     

Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3

Primary cultures of neonatal human foreskin keratinocytes converted 25-hydroxyvitamin D in high yield to a metabolite with the chromatographic behavior of 1,25-dihydroxyvitamin D3. The identity of this metabolite as 1,25-dihydroxyvitamin D3 was confirmed both by its potency in displacing 1,25-dihydroxyvitamin D3 in the chick cytosol receptor assay and by mass spectral analysis. These results suggest that 1,25-dihydroxyvitamin D3 may be formed in the epidermis to regulate vitamin D production by the epidermis and to provide an alternative to 1,25-dihydroxyvitamin D3 production by the kidneys.     

Oxidation and chemical modification of lung beta-galactoside-specific lectin.

Galaptins are small, soluble, lectins with a specificity for beta-galactose residues. Many galaptins are inactivated by atmospheric oxygen and are protected by disulphide-reducing reagents. We find that each subunit of rat lung galaptin contains one residue of tryptophan and six of cysteine. Oxygen inactivates rat lung galaptin by oxidation of the cysteine residues. During oxidation, the normal dimeric structure is maintained and all disulphide bonds are formed within individual subunits. Exogenous thiols protect against inactivation, but oxidized thiols accelerate inactivation. Human lung fibroblast galaptin is almost completely inactivated within 1 h in tissue culture medium at 37 degrees C. Alkylation of native rat lung galaptin with iodoacetate or ethyleneimine causes substantial loss of activity. The dimeric galaptin structure is maintained. In contrast, alkylation with iodoacetamide yields carboxamidomethyl-galaptin, which is fully active and stable to atmospheric oxygen in the absence of disulphide-reducing reagents. This derivative is very useful for studies of galaptin properties and function.