Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

P-factor,a developmental hormone of Penicillium cyclopium?

From the mycelium of Penicillium cyclopium a biologically active fraction (P-factor) was isolated, which increases conidiation and the formation of the benzodiazepine alkaloids cyclopenin and cyclopenol. Its activity was determined by measuring the increase of alkaloid formation in strain SM 72. On a preparative scale P-factor preparations were obtained from fermenter-grown hyphae of mutant dev 63 by extraction with water at 120°. P-factor is strongly hydrophilic but it is not a protein. It was active if added during conidiospore germination and early growth phase, causing an acceleration of protein biosynthesis. The action on alkaloid biosynthesis and sporulation is indirect and resembles that of a developmental hormone.     

Bilayer orientation of membrane-bound NH2 groups across microsomal vesicles and their role in the function of gastric K+-stimulated adenosine triphosphatase

The transversal distribution of the free NH₂ groups associated with phosphatidyl ethanolamine and the intrinsic membrane proteins of the purified pig gastric microsomes was quantitated and their relations to the function of the gastric K⁺-stimulated ATPase was investigated. Three different chemical probes such as 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (FDNB), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) were used for the study. The structure-function relationship of the membrane NH₂ groups was studied after modification with the probes under various conditions and relating the inhibition of the K⁺-stimulated ATPase to the ATPase-dependent H⁺ accumulation by the gastric microsomal vesicles. TNBS (2 mm) inhibits nearly completely the K⁺-stimulated ATPase and the vesicular dye accumulation, both in presence and absence of valinomycin plus K⁺. Both the K⁺-ATPase and dye uptake were largely (about 50%) protected against TNBS inhibition if the treatment with TNBS was carried out in presence of 2 mm ATP. TNBS and FDNB labeled 70% of the total microsomal PE; the intra- and extravesicular orientation being 48 and 22%, respectively. The presence or absence of ATP did not have any effect on the TNBS labeling of microsomal PE. ATP, however, significantly (P < 0.05) reduced the labeling of protein-bound NH₂ groups of gastric microsomes by TNBS. The intra- and extravesicular orientation of the protein NH₂ groups were 60 and 40%, respectively. Eighteen percent of the total protein-NH₂ appeared to be associated with the K⁺-stimulated ATPase; the rest being associated with non-ATPase proteins of the microsomes. About half (50%) of the total free NH₂ groups of the K⁺-stimulated ATPase were exposed to the vesicle exterior and were found to play critical roles in gastric ATPase function. The generation of florescence after MDPF conjugation of gastric microsomes was largely (50%) inhibited by ATP. ATP also protected completely the MDPF inhibition of gastric K⁺-stimulated ATPase and dye uptake.     

Increased Expression of β-Amyloid Protein Precursor and Microtubule-Associated Protein τ During the Differentiation of Murine Embryonal Carcinoma Cells

Expression of the genes encoding the beta/A4 amyloid protein precursor (APP) and microtubule-associated protein tau was studied in an embryonal carcinoma cell line (P19) that differentiates in vitro into cholinergic neurons after treatment with retinoic acid. Expression of APP increased 34- (mRNA) and 50-fold (protein) during neuronal differentiation; APP-695 accounted for most of this increase. These remarkable increases in APP expression coincided with a proliferation of neuronal processes and with an increase in content of tau mRNA. Moreover, subsequent decreases in the levels of APP and tau mRNA coincided with the onset of the degeneration of the neuronal processes. Immunocytochemical staining suggested that greater than 85% of the P19-derived neurons are cholinergic and that APP is present in the neuronal processes and cell bodies. These results suggest that APP may play an important role in construction of neuronal networks and neuronal differentiation and also indicate that this embryonal carcinoma cell line provides an ideal model system to investigate biological functions of APP and the roles of APP and tau protein in development of Alzheimer's disease in cholinergic neurons.     

THE INORGANIC CARBON REQUIREMENTS OF CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) FOR CELL DEVELOPMENT IN SYNCHRONOUS CULTURES1

The time in the cell cycle when CO₂ provision was required for cell development and division was determined in synchronous cultures of Chlamydomonas segnis Ettl bubbled with air (0.03% CO₂) or air enriched with 5% CO₂ under continuous light at 25°C and pH 7. Provision of CO₂ (% in air v/v) during the G₁-phase was found to be essential for the completion of the cell cycle. There was no demand for CO₂ supply throughout the S-phase and mitosis. Using cultures adapted to CO₂ concentrations ranging from 0.03 to 5% in air, the apparent CO₂ concentration (K_m) required for the cells to develop during the G_-1-phase and to attain one half the maximal rates of photo-synthetic O₂ evolution was calculated as 0.05%. This value increased to 0.1 and 0.5% during the S-phase. For total protein and carbohydrate accumulation, which would reflect inorganic carbon (CO₂+ HCO₃^?) assimilation, the K_m (% CO₂) were ca. 0.1 and 0.14 throughout the cell cycle, respectively. The CO₂ concentration at which the cells exhibited the shortest generation time (6.7 h) was 0.1%. These results showed that during development, cells photosynthesizing (evolving O₂) at maximal rates but accumulating protein and carbohydrate at one half the maximal rates or less would complete their vegetative life cycle in the shortest time.     

Use of activated clotting time for monitoring anticoagulation during cardiopulmonary bypass in infants and children with congenital heart disease

The use of a fixed dosage schedule was compared with the use of activated clotting time (ACT) for determining heparin and protamine dosages during and after cardiopulmonary bypass disease. Use of the ACT resulted in a statistically significant increase in heparin dosage and a statistically significant reduction of postoperative blood loss. With ACT use, chest tubes were retained for a shorter period of time, and the incidence of serious postoperative hemorrhage was reduced from 44% to 18%. These results confirm the superiority of the ACT method for monitoring intraoperative anticoagulation in pediatric patients with congenital heart disease.     

Finite element analysis of active Eustachian tube function.

The inability to open the collapsible Eustachian tube (ET) has been related to the development of chronic otitis media. Although ET dysfunction may be due to anatomic and/or mechanical abnormalities, the precise mechanisms by which these structural properties alter ET opening phenomena have not been investigated. Previous investigations could only speculate on how these structural properties influence the tissue deformation processes responsible for ET opening. We have, therefore, developed a computational technique that can quantify these structure-function relationships. Cross-sectional histological images were obtained from eight normal adult human subjects, who had no history of middle ear disease. A midcartilaginous image from each subject was used to create two-dimensional finite element models of the soft tissue structures of the ET. ET opening phenomena were simulated by applying muscle forces on soft tissue surfaces in the appropriate direction and were quantified by calculating the resistance to flow (R(v)) in the opened lumen. A sensitivity analysis was conducted to determine the relative importance of muscle forces and soft-tissue elastic properties. Muscle contraction resulted in a medial-superior rotation of the medial lamina, stretching deformation in the Ostmann's fatty tissue, and lumen dilation. Variability in baseline R(v) values correlated with tissue size, whereas the functional relationship between R(v) and a given mechanical parameter was consistent in all subjects. ET opening was found to be highly sensitive to the applied muscle forces and relatively insensitive to cartilage elastic properties. These computational models have, therefore, identified how different tissue elements alter ET opening phenomena, which elements should be targeted for treatment, and the optimal mechanical properties of these tissue constructs.     

Enantiopure ethyl 2,3-dibromopropionate: Enantioselective synthesis vs preparative HPLC enantioseparation of racemate on multigram scale

Racemic ethyl 2,3-dibromopropionate, commercially available at low price, is a key intermediate used in the synthesis of several heterocycle fragments, which are present in many biologically active compounds. Surprisingly, the enantiomers are not commercially available and have never been described in the literature. In this work, we undertook two different strategies to obtain these enantiomers, which are enantioselective synthesis and preparative HPLC enantioseparation of commercially available racemate on multigram scale. The first strategy has proved inadequate because racemization occurred during the synthesis (ee ≈ 9-50%). Conversely, the second strategy produced a very good enantioseparation of commercially available racemate (ee > 99.5% for both enantiomers) on multigram scale.     

Mapping QTLs for 15 morpho-metric traits in Arabidopsis thaliana using Col-0 × Don-0 population

Genome wide quantitative trait loci (QTL) mapping was conducted in Arabidopsis thaliana using F2 mapping population (Col-0 × Don-0) and SNPs markers. A total of five linkage groups were obtained with number of SNPs varying from 45 to 59 per linkage group. The composite interval mapping detected a total of 36 QTLs for 15 traits and the number of QTLs ranged from one (root length, root dry biomass, cauline leaf width, number of internodes and internode distance) to seven (for bolting days). The range of phenotypic variance explained (PVE) and logarithm of the odds ratio of these 36 QTLs was found be 0.19–38.17% and 3.0–6.26 respectively. Further, the epistatic interaction detected one main effect QTL and four epistatic QTLs. Five major QTLs viz. Qbd.nbri.4.3, Qfd.nbri.4.2, Qrdm.nbri.5.1, Qncl.nbri.2.2, Qtd.nbri.4.1 with PVE > 15.0% might be useful for fine mapping to identify genes associated with respective traits, and also for development of specialized population through marker assisted selection. The identification of additive and dominant effect QTLs and desirable alleles of each of above mentioned traits would also be important for future research.