The gene for trypsin inhibitor CMe is regulated in trans by the lys 3a locus in the endosperm of barley (Hordeum vulgare L.)

Summary A cDNA encoding trypsin inhibitor CMe from barley endosperm has been cloned and characterized. The longest open reading frame of the cloned cDNA codes for a typical signal peptide of 24 residues followed by a sequence which is identical to the known amino acid sequence of the inhibitor, except for an Ile/Leu substitution at position 59. Southern blot analysis of wheat-barley addition lines has shown that chromosome 3H of barley carries the gene for CMe. This protein is present at less than 2%–3% of the wild-type amount in the mature endosperm of the mutant Risø 1508 with respect to Bomi barley, from which it has been derived, and the corresponding steady state levels of the CMe mRNA are about I%. One or two copies of the CMe gene (synonym Itc1) per haploid genome have been estimated both in the wild type and in the mutant, and DNA restriction patterns are identical in both stocks, so neither a change in copy number nor a major rearrangement of the structural gene account for the markedly decreased expression. The mutation at the lys 3a locus in Risø 1508 has been previously mapped in chromosome 7 (synonym 5H). A single dose of the wild-type allele at this locus (Lys 3a) restores the expression of gene CMe (allele CMe-1) in chromosome 3H to normal levels.     

Wide distribution of a 36-megadalton plasmid among clinical and environmental Spanish isolates ofLegionella pneumophila serogroup 1

With the eventual goal of characterizingLegionella pneumophila serogroup 1 plasmids at the molecular level, we have analyzed the plasmid contents of 78 clinical and environmental Spanish isolates. After selection of a suitable alkaline lysis method, we detected plasmids with approximate molecular weights of 25, 36, 40, 61, 80, 85, 90, and 95 megadalton (MDal). Several factors (i.e., wide temporal and geographic distribution, high frequency in both clinical and environmental isolates, and apparent high copy number after subculturing) make the 36 MDal type IA plasmid an appropriate plasmid for further molecular studies.     

Treatment of acute otitis media: a controlled study of 142 children

Results of the use of ampicillin, penicillin G and symptomatic therapy in the treatment of acute otitis media in 142 children were compared. Antibiotic therapy conveyed significant benefit. No major differences were observed between penicillin and ampicillin, except in the age group under 3 years where ampicillin was associated with the best results. Ampicillin appears to be the drug of choice. Its superiority over symptomatic therapy was statistically significant. Long-term sequelae were not observed in any of the three treatment groups. The relative merits of erythromycin and ampicillin require further study.     

Isolation and promoter characterization of barley geneItr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutantlys3a endosperm

The geneItr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library ofHordeum vulgare L. The gene has no introns and presents in its 5-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the copia-like retro-transposon Bare-1. Functional analysis of theItr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system theItr1 promoter retains its endosperm specifity and thetrans-regulation mediated by theLys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer fullGUS expression and for endosperm specifity. In protoplasts derived from thelys3a mutant, Risø 1508,GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.Equal authours     

Effect of stress,adrenalectomy and changes in glutathione metabolism on rat kidney metallothionein content: comparison with liver metallothionein

Eighteen hours of immobilization stress, accompanied by food and water deprivation, increased liver metallothionein (MT) but decreased kidney MT levels. Food and water deprivation alone had a significant effect only on liver MT levels. In contrast, stress and food and water deprivation increased both liver and kidney lipid peroxidation levels, indicating that the relationship between MT and lipid peroxidation levels (an index of free radical production) is unclear. Adrenalectomy increased both liver and kidney MT levels in basal conditions, whereas the administration of corticosterone in the drinking water completely reversed the effect of adrenalectomy, indicating an inhibitory role of glucocorticoids on MT regulation in both tissues. Changes in glutathione (GSH) metabolism produced significant effects on kidney MT levels. Thus, the administration of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased kidney GSH and increased kidney MT content, suggesting that increased cysteine pools because of decreased GSH synthesis might increase kidney MT levels through an undetermined mechanism as it appears to be the case in the liver. However, attempts to increase kidney MT levels by the administration of cysteine or GSH were unsuccesful, in contrast to what is known for the liver. The present results suggest that there are similarities but also substantial differences between liver and kidney MT regulation in these experimental conditions.     

Construction of Novel Shuttle Vectors for Use between Moderately Halophilic Bacteria andEscherichia coli

Shuttle vectors useful for the genetic manipulation of several moderately halophilic bacteria have been constructed. These vectors are based on the minimal replicon of pCM1, a cryptic plasmid fromChromohalobacter marismortui,combined with the useful properties of pUC18 plasmid (i.e., small size, high copy number, multiple cloning sites,lacZfragment), as well as with the trimethoprim resistance gene as a selection marker for moderate halophiles. These vectors can be efficiently transferred by RP4-mediated conjugation fromEscherichia colito the moderate halophilesChromohalobacter marismortui, Deleya halophila, Halomonas elongata, Halomonas subglaciescola,andVolcaniella eurihalina.     

Copper distribution in the normal human brain

Copper concentration was determined in samples from 38 areas of 7 normal human brains. The grey matter contained higher concentrations of copper than the white matter. Identical areas of the grey and white matter of the cerebral cortex showed significant differences between individuals. In the caudate nucleus the highest concentrations of copper were found in the tail followed by the body and the head, respectively. A negative linear regression between age and brain copper levels was demonstrated.     

Isolation of a mitochondrial factor from rat liver which potentiates the inactivation of glutamate dehydrogenase by lysosomes

Rat liver glutamate dehydrogenase (L-glutamate: NAD(P) oxidoreductase, deaminating) E.C. 1.4.1.3.) is inactivated by the mitochondrial matrix in combination with lysosomal preparations. Neither lysosomal or mitochondrial matrix extracts per se inactivate the enzyme appreciably under the conditions used. Fractionation of the matrix indicates that a low molecular weight factor is responsible for the potentiation of inactivation of glutamate dehydrogenase by lysosomes. Its absorption spectrum and chromatographic behaviour suggest that this factor is NADP.     

High‐throughput analysis of vocalizations reveals sex‐specific changes in Fmr1 mutant pups

There have been several reports that individuals with Fragile X syndrome (FXS) and animal models of FXS have communication deficits. The present study utilized two different call classification taxonomies to examine the sex‐specificity of ultrasonic vocalization (USV) production on postnatal day (PD8) in the FVB strain of Fmr1 knockout (KO) mice. One classification protocol requires the investigator to score each call by hand, while the other protocol uses an automated algorithm. Results using the hand‐scoring protocol indicated that male Fmr1 KO mice exhibited longer calls (P = .03) than wild types on PD8. Male KOs also produced fewer complex, composite, downward, short and two‐syllable call‐types, as well as more frequency steps and chevron call‐types. Female heterozygotes exhibited no significant changes in acoustic or temporal aspects of calls, yet showed significant changes in call‐type production proportions across two different classification taxonomies (P < .001). They exhibited increased production of harmonic and frequency steps calls, as well as fewer chevron, downward and short calls. According to the second high‐throughput analysis, female heterozygotes produced significantly fewer single‐type and more multiple‐type syllables, unlike male KOs that showed no changes in these aspects of syllable production. Finally, we correlated both scoring methods and found a high level of correlation between the two methods. These results contribute further knowledge of sex differences in USV calling behavior for Fmr1 heterozygote and KO mice and provide a foundation for the use of high‐throughput analysis of neonatal USVs.     

Escherichia coli rimM and yjeQ null strains accumulate immature 30S subunits of similar structure and protein complement

Assembly of the Escherichia coli 30S ribosomal subunits proceeds through multiple parallel pathways. The protein factors RimM, YjeQ, RbfA, and Era work in conjunction to assist at the late stages of the maturation process of the small subunit. However, it is unclear how the functional interplay between these factors occurs in the context of multiple parallel pathways. To understand how these factors work together, we have characterized the immature 30S subunits that accumulate in ΔrimM cells and compared them with immature 30S subunits from a ΔyjeQ strain. The cryo-EM maps obtained from these particles showed that the densities representing helices 44 and 45 in the rRNA were partially missing, suggesting mobility of these motifs. These 30S subunits were also partially depleted in all tertiary ribosomal proteins, particularly those binding in the head domain. Using image classification, we identified four subpopulations of ΔrimM immature 30S subunits differing in the amount of missing density for helices 44 and 45, as well as the amount of density existing in these maps for the underrepresented proteins. The structural defects found in these immature subunits resembled those of the 30S subunits that accumulate in the ΔyjeQ strain. These findings are consistent with an “early convergency model” in which multiple parallel assembly pathways of the 30S subunit converge into a late assembly intermediate, as opposed to the mature state. Functionally related factors will bind to this intermediate to catalyze the last steps of maturation leading to the mature 30S subunit.