The e subunit gene of murine F1F0-ATP synthase. Genomic sequence, chromosomal mapping, and diet regulation

Genomic sequences encoding murine Lfm1, whose predicted protein sequence is 96% and 98% similar to bovine and rat F1F0-ATP synthase e subunits (respectively), have been amplified from BALB/cByJ DNA, cloned, and sequenced. The 1.1-kilobase gene has 3 introns and 4 exons, and its coding sequence differs by two nucleotides compared to the previously published BALB/cHnn Lfm1 cDNA sequence. A PstI restriction site polymorphism in intron 2 between C57BL/6J and Mus spretus was used to map this gene to Chromosome 5 near D5Mit9. Related sequences were mapped on Chromosomes 8, 11, and 2 unlinked loci on Chromosome 2 using Southern blot analyses with the 1. 1-kilobase gene as probe. Previous studies from this laboratory indicated that the Lfm1/e subunit was regulated by the level of dietary fat and carbohydrate. Northern hybridization analyses demonstrated that e subunit mRNA abundance showed statistically significant differences (p < 0.025) between hearts of BALB/c mice fed 3% and those fed 20% corn oil for 2 weeks and in liver (p < 0. 05) from the same animals. Significant differences were also observed in hepatic and heart mRNA expression at different times after eating in animals subjected to a fast/refeed regimen. The implications of the high degree of sequence similarity to the e subunit for rat and bovine F1F0-ATP synthase and its regulation by diet are discussed.     

Strategic planning for postacute care

The author examines workable approaches and benefits of using strategic planning to prepare for future postacute care.     

SLASH: a program for analysing the functional groups in molecules

The authors examined the impact of a number of job stressors, including sexual harassment and gender-based discrimination, on female construction workers' level of job satisfaction and psychological and physical health. Results from a telephone survey with 211 female laborers indicated that having responsibility for others' safety and having support from supervisors and male coworkers was related to greater job satisfaction. Increased reported psychological symptoms were also related to increased responsibility, as well as skill underutilization, experiencing sexual harassment and gender-based discrimination from supervisors and coworkers, and having to overcompensate at work. Perceptions of overcompensation at work and job uncertainty were positively associated with self-reports of insomnia. Finally, sexual harassment and gender discrimination were positively related to reports of increased nausea and headaches.     

Structure of alpha-lytic protease complexed with its pro region

While the majority of proteins fold rapidly and spontaneously to their native states, the extracellular bacterial protease alpha-lytic protease (alphaLP) has a t(1/2) for folding of approximately 2,000 years, corresponding to a folding barrier of 30 kcal mol(-1). AlphaLP is synthesized as a pro-enzyme where its pro region (Pro) acts as a foldase to stabilize the transition state for the folding reaction. Pro also functions as a potent folding catalyst when supplied as a separate polypeptide chain, accelerating the rate of alphaLP folding by a factor of 3 x 10(9). In the absence of Pro, alphaLP folds only partially to a stable molten globule-like intermediate state. Addition of Pro to this intermediate leads to rapid formation of native alphaLP. Here we report the crystal structures of Pro and of the non-covalent inhibitory complex between Pro and native alphaLP. The C-shaped Pro surrounds the C-terminal beta-barrel domain of the folded protease, forming a large complementary interface. Regions of extensive hydration in the interface explain how Pro binds tightly to the native state, yet even more tightly to the folding transition state. Based on structural and functional data we propose that a specific structural element in alphaLP is largely responsible for the folding barrier and suggest how Pro can overcome this barrier.     

Quality assurance of serial tomotherapy for head and neck patient treatments

PURPOSE: A commercial serial tomotherapy intensity-modulated radiation therapy (IMRT) treatment planning (Peacock, NOMOS Corp., Sewickley, PA) and delivery system is in clinical use. The dose distributions are highly conformal, with large dose gradients often surrounding critical structures, and require accurate localization and dose delivery. Accelerator and patient-specific quality assurance (QA) procedures have been developed that address the localization, normalization, and delivery of the IMRT dose distributions. METHODS AND MATERIALS: The dose distribution delivered by serial tomotherapy is highly sensitive to the accuracy of the longitudinal couch motion. There is also an unknown sensitivity of the dose distribution on the dynamic mutlileaf collimator alignment. QA procedures were implemented that assess these geometric parameters. Evaluations of patient positioning accuracy and stability were conducted by exposing portal films before (single exposure) and after (single or double exposure) treatments. The films were acquired with sequential exposures using the largest available fixed multileaf portal (3.36 x 20 cm2). Comparison was made against digitally reconstructed radiographs generated using independent software and appropriate beam geometries. The delivered dose was verified using homogeneous cubic phantoms. Radiographic film was used to determine the localization accuracy of the delivered isodose distributions, and ionization chambers and thermoluminescent dosimetry (TLD) chips were used to verify absolute dose at selected points. Ionization chamber measurements were confined to the target dose regions and TLD measurements were obtained throughout the irradiated volumes. Because many more TLD measurements were made, a statistical evaluation of the measured-to-calculated dose ratio was possible. RESULTS: The accelerator QA techniques provided adequate monitoring of the geometric patient movement and dynamic multileaf collimator alignment and positional stability. The absolute delivered dose as measured with the ionization chamber varied from 0.94 to 0.98. Based on these measurements, the delivered monitor units for both subsequent QA measurements and patient treatments were adjusted by the ratio of measured to calculated dose. TLD measurements showed agreement, on average, with the ionization chamber measurements. The distribution of TLD measurements in the high-dose regions indicated that measured doses agreed within 4.2% standard deviation of the calculated doses. In the low-dose regions, the measured doses were on average 5% greater than the calculated doses, due to a lack of leakage dose in the dose calculation algorithm. CONCLUSIONS: The QA system provided adequate determination of the geometric and dosimetric quantities involved in the use of IMRT for the head and neck. Ionization chamber and TLD measurements provided accurate determination of the absolute delivered dose throughout target volumes and critical structures, and radiographic film yielded precise dose distribution localization verification. Portal film acquisition and subsequent portal film analysis using 3.36 x 20 cm2 portals proved useful in the evaluation of patient immobilization quality. Adequate bony landmarks were imaged when carefully selected portals were used.     

Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection

The title compounds (6-9) were prepared and evaluated for serotonin 5-HT4 agonistic activity in in vitro tests. Introducing a propyl or allyl group at the 3-position of benzamide caused only a slight enhancement of agonistic activity. Construction of the benzo[b]furan skeleton and 2,3-dihydrobenzo[b]furan skeleton caused a significant enhancement of the activity. 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2-methylbenzo[b]furan-7-carboxamide (7b) hemifumarate was as potent as cisapride. 4-Amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dihydro-2-methylbenzo[b]furan-7-carboxamide (8a) hemifumarate, 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dihydro-2-ethylbenzo[b]furan-7-carboxamide (8c) hemifumarate, and 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dimethylbenzo[b]furan-7-carboxamide (8d) hemifumarate were more potent than cisapride. Furthermore, 8a hemifumarate was free from dopamine D1, D2, serotonin 5-HT1, 5-HT2 and muscarine M1, M2 receptor binding activity in the in in vitro tests. On the other hand, construction of the indole skeleton caused a remarkable decrease in activity.     

Dietary omega-3 and omega-9 fatty acids uniquely enhance allograft survival in cyclosporine-treated and donor-specific transfusion-treated rats

BACKGROUND: Both laboratory and clinical studies have shown that dietary lipids may affect immunologic responses. This study was conducted to compare different classes of long-chain unsaturated fatty acids for their effect on allograft survival in animals receiving a donor-specific transfusion and a short course of low-dose cyclosporine (CsA). METHODS: Heterotopic ACI strain cardiac allografts were transplanted to Lewis strain rat recipients given diets with different lipid composition. In experiment 1, animals received CsA for 14 days and different diets were enriched with lipids with high concentrations of omega-3, omega-6, or omega-9 fatty acids. In experiment 2, animals received CsA for only 8 days and different diets were enriched with corn oil (omega-6), canola oil (omega-3 and omega-9), fish oil (omega-3) or a mixture of sunflower oil and fish oil (omega-3 and omega-9). RESULTS: In experiment 1, animals receiving the diet with 30% sunflower oil had the best allograft survival (200+/-42 days vs. 53+/-8 days for regular chow plus donor-specific transfusion and CsA, P<0.05). In experiment 2, diets containing canola oil (a mixture of omega-3 and omega-9 fatty acids) were associated with the best survival (P=0.0011 vs. regular chow). CONCLUSION: Dietary omega-3 and omega-9 fatty acids both enhanced cardiac allograft survival in a stringent rat strain combination. Canola oil is a convenient oil for administering both alpha-linoleic acid (omega-3) and oleic acid (omega-9) in a palatable form for human consumption. Further investigation of the potential usefulness of lipids in transplant therapy is warranted.     

Olfactory function in young adolescents with Down's syndrome

Decreased ability to smell is present in adults with Down's syndrome, many of whom are known to have brain pathology analogous to that seen in Alzheimer's disease. Because olfactory loss is well documented in Alzheimer's disease, the question arises whether young adolescents with Down's syndrome, who have no clear Alzheimer's disease-like neuropathology, also exhibit olfactory dysfunction. To consider this issue, standardised tests of odour discrimination and identification were administered to 20 young adolescents with Down's syndrome (mean age (SD) 13.89 (1.98) years) and their test scores were compared with 20 mentally retarded and 20 non-mentally retarded control subjects matched to the patients with Down's syndrome on the basis of cognitive ability. No significant differences in olfactory function were found among the three study groups. These findings, along with those from studies of olfactory function in older patients with Down's syndrome, suggest that Down's syndrome related olfactory dysfunction occurs only at ages when Alzheimer's disease-like pathology is present.     

Activation of the mas oncogene involves coupling to human alphoid sequences

We have shown previously that mouse NIH3T3 cells transfected with DNA from a human ovarian carcinoma were rendered tumourigenic by an activated mas oncogene in four independent transfection experiments. In all cases the 5'-noncoding region was rearranged in comparison to the original ovarian tumour DNA. We now report that in all four transfectants the newly acquired sequences consist of human centromeric alpha satellite repeat DNA. In at least three transfectants the alphoid DNA originates from the centromere of chromosome three. Analysis of the sequences of the recombination site in one transfectant revealed that a homologous sequence of five base pairs (CAGCA) is present in both parental strands, and might thus have contributed to the recombinational event. To establish a conclusive role for alphoid DNA in the activation of mas, we performed a co-transfection experiment in NIH3T3 cells with cloned alphoid DNA and the mas coding sequence. We show that the transfectants expressing a transformed phenotype contain amplified mas linked to alphoid DNA. NIH3T3 cells transfected with plasmids that contained alphoid sequences cloned directly upstream of the mas coding sequence, and injected into nude mice, gave rise to tumours with amplified mas sequences (7/7). In six of these tumours the alphoid sequences were amplified as well. Our data suggest a novel mechanism of oncogene activation: recombination with normal alphoid repeat DNA resulting in amplification of the oncogene.