Historical connectivity, contemporary isolation and local adaptation in a widespread but discontinuously distributed species endemic to Taiwan, Rhododendron oldhamii (Ericaceae).

Elucidation of the evolutionary processes that constrain or facilitate adaptive divergence is a central goal in evolutionary biology, especially in non-model organisms. We tested whether changes in dynamics of gene flow (historical vs contemporary) caused population isolation and examined local adaptation in response to environmental selective forces in fragmented Rhododendron oldhamii populations. Variation in 26 expressed sequence tag-simple sequence repeat loci from 18 populations in Taiwan was investigated by examining patterns of genetic diversity, inbreeding, geographic structure, recent bottlenecks, and historical and contemporary gene flow. Selection associated with environmental variables was also examined. Bayesian clustering analysis revealed four regional population groups of north, central, south and southeast with significant genetic differentiation. Historical bottlenecks beginning 9168-13,092 years ago and ending 1584-3504 years ago were revealed by estimates using approximate Bayesian computation for all four regional samples analyzed. Recent migration within and across geographic regions was limited. However, major dispersal sources were found within geographic regions. Altitudinal clines of allelic frequencies of environmentally associated positively selected outliers were found, indicating adaptive divergence. Our results point to a transition from historical population connectivity toward contemporary population isolation and divergence on a regional scale. Spatial and temporal dispersal differences may have resulted in regional population divergence and local adaptation associated with environmental variables, which may have played roles as selective forces at a regional scale.

Genetic diversity and biogeography of Cunninghamia konishii (Cupressaceae), an island species in Taiwan: a comparison with Cunninghamia lanceolata, a mainland species in China.

Luanta-fir (Cunninghamia konishii), an endemic to Taiwan, is an outcrossing, long-lived conifer. Populations of C. konishii are generally fragmented due to a once high intensity of timber exploitation. C. konishii and Cunninghamia lanceolata are two sibling taxa constituting derivative-progenitor species relationship. The amount of genetic variations within and between 11 and 10 populations of C. konishii and C. lanceolata, respectively, were assessed using amplified fragment length polymorphism (AFLP) markers in this report. Three AFLP primer pairs generated a total of 357 and 226 markers for C. konishii and C. lanceolata samples, of which 56.1 and 65.3% are polymorphic, respectively. Analysis of molecular variance indicates a 4.78% variation between C. konishii and C. lanceolata. A relatively high value of genetic variation (24.60%) was apportioned between the populations of C. konishii. In contrast, a lower divergence value (12.21%) between populations was found for C. lanceolata. The population with the highest genetic diversity was found in Nantou County, which concurred with the results of many other tree species investigated in Taiwan. The estimates of the number of migrants between populations (Nm), obtained from population pair-wise PhiST, suggest that gene flow in C. konishii is efficient in some adjacent populations but is restricted in the rest. Individual UPGMA tree, generated based on AFLP markers, suggests six evolutionary lineages for C. konishii. All evolutionary lineages of C. konishii were derived from C. lanceolata. In conclusion, the migration patterns of Cunninghamia from mainland China may have been established following multiple sources, migrant-pools, long-distance dispersal events, and via different directions.

Effect of insulin stimulation on the proliferation and death of Chinese hamster ovary cells.

The effect of environmental and genetic parameters on cell death was studied in Chinese hamster ovary cell cultures. Experiments were performed using an anchorage-dependent CHO cell line expressing gamma-IFN, and a second cell line obtained by transfection of the previous one with bcl-2. In serum-free medium the two cell lines showed a considerable degree of growth control entering quiescence while maintaining high viabilities. The addition of transferrin did not have any effect but insulin addition allowed cells arrested by serum withdrawal to reenter the cell cycle. However, insulin supplementation also resulted in cell death, which was possible to avoid through bcl-2 overexpression or in the presence of serum. We propose that the expression of c-myc, shown to be induced by insulin, plays an important role in the cell death observed after insulin addition in an inappropriate environment, deficient in protective factors. This hypothesis is supported by measurements of c-myc expression and cell cycle distribution.

Introgression of Lilium rubellum Baker chromosomes into L. longiflorum Thunb.: a genome painting study of the F1 hybrid, BC1 and BC2 progenies.

Interspecific hybrids between Lilium longiflorum (L, 2n = 2x = 24) and Lilium rubellum (R, 2n = 2x = 24) were produced with the aim of transferring desirable horticultural traits from L. rubellum to L. longiflorum. All F1 hybrids (LR, 2n = 2x = 24) and BC1 individuals (LLR, 2n = 3x = 36) were phenotypically uniform for plant height, flowering time, leaf shape and flower colour. The BC1 plants were, in spite of their triploid nature, fertile and could be used as a female parent in backcrossings with autotetraploid L. longiflorum (LLLL, 2n = 4x = 48). Twelve BC2 individuals were obtained and three of them were selected for further chromosome analysis. As L. longiflorum and L. rubellum chromosomes were indistinguishable in the hybrids, genomic in-situ hybridization (GISH) was applied to establish the parentage of the chromosomes of the F1 hybrids and the BC1 and BC2 progenies. GISH confirmed the LLRR constitution of the doubled amphimonoploid (allodiploid), and the LLR constitution of all BC1 plants. The three selected BC2 plants were, as expected, aneuploid, containing three complete sets of L. longiflorum chromosomes and six, seven or eight L. rubellum chromosomes, respectively. However, L/R translocation or recombinant chromosomes could not be demonstrated in the mitotic metaphase complements of the F1, BC1 and BC2 plants. In spite of the high frequencies of homoeologous recombination in the F1 hybrids (LR) pollen was found to be sterile in all cases. At metaphase I of the pollen mother cells of the BC1 plants, genome painting did not reveal any cases of homoeologous pairing and recombination between L and R chromosomes. This lack of exchange between homoeologous chromosome segments indicates complete preferential pairing of the L and R chromosomes in the F1 (amphidiploid) and BC1 plants. It seems that the preferential pairing in the F1 and BC1 hybrids hinder the introgression of the chromosome segments or species-specific genes into the recipient for breeding purposes.

Design of metabolic feed controllers: application to high-density fermentations of Pichia pastoris.

High-density cultures of the methylotrophic yeast Pichia pastoris were found to exhibit oscillatory metabolic behavior when fed methanol under closed-loop operations using a dissolved oxygen-based bioreactor feed controller (DOstat). This behavior, if left unattended, led to the irreversible loss of culture productivity, 1 to 2 days after growth on methanol commenced, presumably through the accumulation of incompletely oxidized intermediates. To provide insights into how fermentation operation conditions and strain variations might contribute to this phenomenon a theoretical study was initiated. In this article, a simple mathematical model of the closed-loop DOstat is developed and analyzed with the goal of deriving theoretical stability criteria applicable to the design of metabolic feed controllers during high-cell-density fermentations. The model consists of a system of differential, integral, and algebraic equations describing the biological process and the components of the standard proportional-integral (PI) feedback controller. Inputs into the process model include metabolic pathway information, oxidative metabolism stoichiometry, and substrate uptake kinetics. Frequency-response analysis and the Bode stability criterion are applied to derive controller stability criteria with particular emphasis on elucidating the role(s) that model parameters, both biological and operational, have on system stability. The results of this analysis are used to construct a closed-loop fermentation-operating diagram relating fermentation operating parameters to the oxidative capacity of the culture. The utility of this analysis is demonstrated through the application of the results to the design and stabilization of the DOstat during high-density fermentations of P. pastoris growing on methanol or glycerol. From this analysis, it is possible to conclude that, when the rate of oxygen transfer approaches in magnitude the rate of oxygen utilization, the potential for controller destabilization is greatest. Under these conditions, the region of parameter space associated with stable controller operations is further constrained. Because the only system-specific experimental inputs into the model are the routinely measured residual substrate and dissolved oxygen concentrations, the framework presented should provide simple and practical theoretical guidelines relevant to the design of similar industrial fermentation feed controllers independent of the specifics of the biological system at hand.

Temporal pattern of blood volume change in cerebral infarction: evaluation with dynamic contrast-enhanced T2*-weighted MR imaging.

OBJECTIVE: The purposes of this study were to evaluate the temporal pattern of blood volume change in cerebral infarction and to provide a guideline in the interpretation of blood volume data, which are known to vary according to the stage of infarction. SUBJECTS AND METHODS: Thirty-three patients with large middle cerebral infarctions were examined one to three times (one time in 20 patients, two times in eight patients, and three times in five patients) after the onset of stroke by dynamic contrast-enhanced T2*-weighted MR imaging and MR angiography. A total of 54 infarctions (29 in an acute stage [up to 7 days], 15 in a subacute stage [8-21 days], and 10 in a chronic stage [22-35 days]) were included. After blood volume maps were created, blood volume ratios (blood volume of the infarcted region divided by blood volume of corresponding contralateral region) were compared at different stages. Likewise, findings on MR angiography were compared at different stages. RESULTS: Mean blood volume ratios in each stage of infarction were 0.46 in the acute stage, 1.48 in the subacute stage, and 0.73 in the chronic stage (p < .001). Recanalization of occluded arteries occurred in 21% of infarctions in the acute stage and 80% in the subacute stage. Infarctions with recanalization had higher blood volume ratios than did those without recanalization (p < .001). A biphasic pattern of blood volume ratios was found in 13 patients who underwent at least two MR examinations: increased blood volume in the subacute stage and decreased blood volume in the chronic stage, regardless of recanalization (p < .01). CONCLUSION: Blood volume that initially decreases in cerebral infarction increases in the subacute stage, reflecting reperfusion hyperemia. Blood volume decreases again in the chronic stage. The time interval between onset of stroke and MR examination must be considered for correct interpretation of blood volume data in cerebral infarction at various stages.

Growth factor and bcl-2 mediated survival during abortive proliferation of hybridoma cell line.

Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment.

Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation.

Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.

Flow cytometric study of differentiating cultures of Bacillus subtilis.

We report on 1) the development of a flow cytometry-based technique for detecting beta-galactosidase in differentiating cultures of Bacillus subtilis and 2) the application of this technique in the study of early developmental gene expression. The problems associated with generating detectable signals (despite the small size of B. subtilis cells) have been overcome using the fluorogenic substrate 5-octanolyaminofluorescein di-beta-D-galactopyranoside (C8-FDG). Additionally, to control for background fluorescence during the staining process, we included a control population in the C8-FDG staining mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26. The distinct emission spectra of C8-fluorescein and PKH26 allow nonspecific C8-FDG staining in this control population to be monitored using two-color analysis. This technique has been applied in the study of developmental gene expression in sporulating cultures of B. subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations. One population is induced for expression of early sporulation genes, which is determined using lacZ fusions, whereas the other remains uninduced. These results have allowed us to understand better the patterns of gene expression exhibited by wild-type and mutant cultures early during the development process of spore formation.

Studies of transcriptional state heterogeneity in sporulating cultures of bacillus subtilis.

Using a flow cytometry-based technique for detecting p-galactosidase. lacZ fusions have been used to study the pattern of gene expression exhibited by stationary phase cultures of Bacillus subtilis that have initiated the developmental pathway of spore formation. We have found that within such cultures there exist two distinct cell types: one that has induced the developmental program of gene expression and one that has not. This heterogeneity among transcriptional states is shown to be established early during the stationary phase and plays a significant role in influencing later stationary phase events. Additionally, when this technique is used to study gene expression in mutants that display altered patterns of gene expression, we are able to conclude that the cellular apparatus responsible for sensing and transducing stationary phase developmental signals represents a bottleneck that controls the expression of certain early stationary phase genes. These findings provide a demonstration of the utility of single cell measurements of gene expression and indicate that unless culture heterogeneity is properly taken into account, standard measurements of gene expression may not provide information suitable for the analysis of events occurring at the cellular level. (c) 1995 John Wiley & Sons, Inc.

Vegetative propagation of adult Eucalyptus grandis X urophylla and comparison of growth between micropropagated plantlets and rooted cuttings.

Methods for the production of micropropagated plantlets and rooted cuttings were developed and used to vegetatively multiply adult Eucalyptus grandis X urophylla. Rooting success was less than 5% when cuttings excised from twigs of 3-year-old trees were used. The rooted cuttings were grown in the greenhouse as explant- or cutting-donors and maintained at a height of 30 to 100 cm by trimming back periodically. Good rooting success (95%) of cuttings was obtained for epicormic shoots produced from donor plants after trimming 5 times. Explants of both apical and axillary buds taken from the donor plants produced multiple shoots when cultured in vitro. In vitro multiple shoot production was optimal on MS medium containing 0.1 mg/l BA and 0.01 mg/l NAA averaging 13.7 shoots per explant in a 40-day culture period. Shoot elongation was accelerated on a modified MS medium containing half strength potassium nitrate and sucrose. Elongated shoots excised at approximately 1.5 cm in length were successfully rooted on media with NAA or IBA concentrations ranging from 0.1 to 10 mg/l. Root formation was optimal on medium consisting of full strength MS basal macro elements and vitamins, half strength micro elements, 1% sucrose and supplemented with 0.3 mg/l IBA. In the field test, no significant differences were found in tree height and DBH between micropropagated plantlets and rooted cuttings at 1 and 3 years old, with the exception at 2 years old. A considerable difference arose between the 2 types of vegetative propagules in physiological response to flowering, caused by dissimilar degrees of rejuvenation.

Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation.

Early during endospore formation in the bacterium Bacillus subtilis, two distinct cell types are formed. The initiation of this developmental pathway requires several physiological conditions (e.g., nutrient deprivation) and is controlled by the Spo0A transcription factor. We have found that in a culture of sporulating cells, there are two subpopulations, one that has initiated the developmental program and activated the expression of early developmental genes and one in which early developmental gene expression remains uninduced. We measured the expression of developmental (spo) genes in single cells of B. subtilis by using spo-lacZ fusions. Cells containing a spo-lacZ fusion were stained with a dye that fluoresces upon hydrolysis by beta-galactosidase, and the fluorescence in individual cells was measured with a flow cytometer. For Spo+ cells, we found that the proportion of the population expressing early developmental genes correlates well with the fraction of the population that eventually produces spores. In addition, mutations that cause a decrease in the amount of activated (phosphorylated) Spo0A transcription factor cause a decrease in the size of the subpopulation expressing early developmental genes that are directly activated by Spo0A approximately P. Again, the size of the subpopulation correlates well with the fraction of cells that produce spores. These results indicate that a threshold level of activated Spo0A (Spo0A approximately P) or of a component of the phosphorylation pathway must accumulate to induce sporulation gene expression and that most of the cells that are able to induce the expression of early genes that are directly activated by Spo0A approximately P go on to produce mature spores.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of hemodynamic change before extubation--lidocaine through modified endotracheal tube.

Twenty females, aged 31 to 49 years, scheduled for abdominal total hysterectomy were randomly divided into two groups in this study. An epidural catheter was placed at T11-12 before general anesthesia. All patients receive the combination of epidural anesthesia and general anesthesia for the operation and relief of pain postoperatively. The modified endotracheal tube we used is shown in Fig. 1. For patients in group I (Lidocaine group), 2 mL 4% lidocaine solution was injected through the catheter to desensitize the tracheal mucosa around the cuff after the surgeon had removed the uterus. In group II (Control group), no special management was made. All patients were not extubated until they were considered to be awake. Systolic blood pressure at three and one minute before extubation and pulse rate recorded at one minute before extubation showed in patients of group I were statistically smoother than those recorded in group II (p less than 0.01). All patients had gag reflex just after awake extubation.