Quantitative analysis of yeast gene function using competition experiments in continuous culture

One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10-90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments is continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype.     

Development of a green fluorescent protein reporter for a yeast genotoxicity biosensor

A reporter system, constructed for a laboratory screen for new genes involved in DNA repair in the brewer's yeast Saccharomyces cerevisiae, has been developed for use in a genotoxicity biosensor. The strain produces green fluorescent protein (yEGFP) when DNA damage has occurred. yEGFP is codon optimised for yeasts. The reporter does not respond to chemicals which delay mitosis, and responds appropriately to the genetic regulation of DNA repair. Data is presented which demonstrate strain improvements appropriate to biosensor technology: improved signal to noise ratio, ease of data collection and uncomplicated material handling.     

3H]Rilmenidine-labelled imidazoline-receptor binding sites co-localize with [3H]2-(benzofuranyl)-2-imidazoline-labelled imidazoline-receptor binding sites and monoamine oxidase-B in rabbit, but not rat, kidney

The distribution and relative densities of imidazoline-receptor binding sites (I-RBS) and monoamine oxidase (MAO)-A and -B enzyme(s) in rat and rabbit kidney were compared autoradiographically using fixed nanomolar concentrations of [3H]rilmenidine and [3H]2-(benzofuranyl)-2-imidazoline ([3H]2-BFI) to label I-RBS, and [3H]RO41-1049 and [3H]RO19-6327 to label MAO-A and -B isoenzymes, respectively. In rat kidney, high densities of I-RBS labelled by [3H]rilmenidine were observed in the cortex and outer stripe (120-280 fmol/mg tissue), in contrast to low I-RBS densities labelled by [3H]2-BFI (<4 fmol/mg). A relatively high density of [3H]RO41-1049 binding to MAO-A enzyme was present in all regions of the rat kidney (160-210 fmol/mg) compared with a low density of [3H]RO19-6327 binding to MAO-B (< 25 fmol/mg). Comparison of MAO-A and -B distributions with that of [3H]rilmenidine-labelled I-RBS strongly suggests a lack of association in rat kidney. Similarly, the extremely low densities of [3H]2-BFI-labelled I2-RBS in rat kidney contrasts with the density of MAO-A, but is consistent with the low density of MAO-B. Rabbit kidney cortex and outer stripe contained high relative densities of [3H]rilmenidine-labelled I-RBS (200-215 fmol/mg) and [3H]2-BFI-labelled I2-RBS (45-60 fmol/mg) with lower densities in the inner stripe and inner medulla (< or = 100 and 30 fmol/mg respectively). A high density of MAO-A binding was observed in the inner stripe (515 fmol/mg) with lower levels in the cortex and outer stripe (100-240 fmol/mg), while high densities of MAO-B binding were observed in the cortex and outer stripe (290-450 fmol/mg) with lower levels in the inner stripe (65 fmol/mg). The correlation between the localization of [3H]rilmenidine-labelled I-RBS and [3H]RO19-6327-labelled MAO-B in rabbit kidney (r = 0.87, P = 0.057) suggest that [3H]rilmenidine may label a binding site co-existent with MAO-B, but not MAO-A (n.s.), in this tissue, but rilmenidine did not inhibit [3H]RO41-1049 or [3H]RO19-6327 binding. The distribution of [3H]2-BFI-labelled I2-RBS overlapped the combined distributions of both MAO-A and -B isoenzymes, suggesting that [3H]2-BFI may label sites on both enzymes in the rabbit, but [3H]2-BFI binding only correlated with [3H]RO19-6327 (r = 0.84, P = 0.07), not [3H]RO41-1049 binding (n.s.). Moreover, 2-BFI only inhibited [3H]RO19-6327, not [3H]RO41-1049 binding. These data are consistent with reports that I2-RBS are located on MAO-B and allosterically influence the catalytic site. The relationship of [3H]rilmenidine- and [3H]2-BFI-labelled I-RBS and the identity of non-MAO-associated [3H]rilmenidine-labelled I-RBS requires further investigation.     

Dermacentor hunteri (Acari: Ixodidae): seasonal variation in questing adults and on-host juvenile stages, and host associations and feeding behavior of larvae and nymphs

Dermacentor hunteri Bishopp is the only completely desert adapted tick in the Nearctic realm, and chiefly parasitizes desert bighorn sheep (Ovis canadensis Shaw) as an adult. The remainder of its life history has been unknown. We conducted field investigations in the Sonoran desert of the temporal and spatial variation of adult host-seeking ticks and of the host associations of juvenile ticks. Additionally, the feeding success of juvenile ticks was assessed in the laboratory. Adult ticks were found in significant numbers only in plateau and rocky slopes habitats, chiefly during the period from January to June. Questing adults were not found in July and August, and they were present in small numbers from September through December. Juvenile stages were found only on desert woodrats, Neotoma lepida Thomas (larvae and nymphs), and cactus mice, Peromyscus eremicus Baird (larvae only), in March, May, and early June. In the laboratory; both larvae and nymphs fed on N. lepida, but only larvae fed on P. maniculatus bairdii (Wagner). We concluded that the life history of D. hunteri may be constrained by the co-distribution of desert bighorn, desert woodrats, and perhaps cactus mice; and that adults oversummer either on desert bighorn or sequestered in favorable microclimates off the host.     

Electron transfer and catalytic activity of nitric oxide synthases. Chimeric constructs of the neuronal, inducible, and endothelial isoforms

The nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of .NO synthesis, cytochrome c reduction, and NADPH utilization and in the Ca2+ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of .NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca2+ independence of inducible NOS requires interactions of calmodulin with both the calmodulin binding motif and the flavoprotein domain, and (d) the effects of tetrahydrobiopterin and L-arginine on electron transfer rates are mediated exclusively by heme domain interactions.     

Radio-frequency tissue ablation of VX2 tumor nodules in the rabbit lung

Familial hypothalamic diabetes insipidus is an autosomal dominant disorder characterized by deficient vasopressin synthesis. Different point mutations in the vasopressin-neurophysin (VP-NP) precursor gene have been found in affected families. In a Dutch kindred, a single G to T transversion in the NP-encoding exon B of one allele converts the highly conserved glycine 17 to a valine residue. In order to examine whether this point mutation affects the processing and transport of the VP-NP precursor, the normal (HV2) and mutant (MT6) vasopressin cDNAs were stably expressed in the mouse pituitary cell line AtT20. The normal precursor was correctly glycosylated and processed, and NP was detected in the culture medium. Secretion of NP was stimulated by 8-bromo-cAMP, indicating that the normal precursor was targeted to the regulated secretory pathway. In contrast, the mutant precursor was synthesized, but processing and secretion were dramatically reduced. The mutant precursor was core-glycosylated but remained endoglycosidase H-sensitive, suggesting that the protein did not reach the trans-Golgi network. These results were supported by immunocytochemical studies. In HV2 cells, NP derived from the precursor was concentrated in the tips of the cell processes where secretory granules accumulate. In MT6 cells, NP staining was restricted to the endoplasmic reticulum (ER) as determined by colocalization with an ER-resident protein, BiP. These results suggest that the mutation within the conserved part of NP alters the conformation of the precursor and thus triggers its retention in the ER.     

Role of saphenous vein surgery in the treatment of venous ulceration

We present 19 ray lengthenings in 14 patients done with a small external fixator. In six cases the thumb was lengthened and in the other 13 cases, other digital rays. The most frequent reason for lengthening was an amputation sustained in a work accident. All the lengthenings were done by an osteotomy and subsequent gradual distraction with a small external fixator. The mean lengthening achieved was 20 mm (range, 0-32). An iliac-crest graft was needed in nine cases, corrective osteotomy in five cases and a deepening of the web in the six cases of thumb lengthening. In five cases an additional technique was needed to achieve bony consolidation. We have analysed the functional results and the ability to perform activities of daily living and resume employment. Although most of the patients had multiples injuries, the results have been very favourable, achieving a very high level of patient satisfaction.     

Cochlear implantation in the deaf-blind

Due to its strong tendency to crystallize, the glass properties of mannitol cannot be measured directly. However, because mannitol can exist in a fully or partially amorphous state in drug formulations, it is important to determine the glass properties of mannitol. We obtained the glass properties of mannitol by introducing a small amount of sorbitol, an isomer of mannitol, to delay the onset of crystallization. Extrapolation to zero sorbitol concentration yielded the following properties for the mannitol glass: Tg onset = 10.7(o)C, Tg midpoint= 12.6( o)C, Tg end = 18.4 degreesC and DeltaCp = 1.27 J/g/K. In addition, we estimated the following parameters of the mannitol glass from the width of glass transition using the results of Moynihan (J. Am. Ceram. Soc. 1993, 76, 1081) and Angell (J. Phys. Chem. 1994, 98, 13780): DeltaH (at Tg onset) = 103 kcal/mol, D = 11, and T0 = 222 K. The value of T0 is consistent with the Kauzmann temperature TK (236 K) obtained calorimetrically. The properties of the mannitol glass may be useful for predicting the behavior of amorphous mixtures containing mannitol.     

Differences in urologist and patient assessments of health related quality of life in men with prostate cancer: results of the CaPSURE database

Premenstrual syndrome (PMS) is characterized by distressing somatic and behavioral symptoms that develop after ovulation, reach a maximum during the premenstrual days, and disappear within 4 days after the onset of menstruation. Corpus luteum formation is necessary for the presence of symptoms, but the role of luteal hormones is unclear. The aim of this work was to investigate the relationship between sex hormone serum concentrations and premenstrual symptom severity in patients with PMS. Mental and physical symptoms were marked on a validated visual analog scale by 30 PMS patients every evening. Daily blood samples were taken in the luteal phase and in most of the follicular phase. Estradiol, progesterone, FSH, and LH were analyzed. Symptom severity was calculated as the number of negative symptoms expressed per day and as summarized scores of negative ratings. Based on premenstrual hormone concentrations and using the median split method, patients were divided into groups with high and low hormone levels. The pattern of expressed symptoms and summarized scores during the menstrual cycle was similar for the 2 groups. High concentration of luteal-phase estradiol and LH were related to the severity of negative premenstrual symptoms.