Growth-dependent Changes Research Articles

Isolation and evaluation of erythroid progenitors in the livers of larval, froglet, and adult Xenopus tropicalis.

Xenopus liver maintains erythropoietic activity from the larval to the adult stage. During metamorphosis, thyroid hormone mediates apoptosis of larval-type erythroid progenitors and proliferation of adult-type erythroid progenitors, and a globin switch occurs during this time. In addition, the whole-body mass and the liver also change; however, whether there is a change in the absolute number of erythroid progenitors is unclear. To isolate and evaluate erythroid progenitors in the Xenopus liver, we developed monoclonal ER9 antibodies against the erythropoietin receptor (EPOR) of Xenopus. ER9 recognized erythrocytes, but not white blood cells or thrombocytes. The specificity of ER9 for EPOR manifested as its inhibitory effect on the proliferation of a Xenopus EPOR-expressing cell line. Furthermore, ER9 recognition was consistent with epor gene expression. ER9 staining with Acridine orange (AO) allowed erythrocyte fractionation through fluorescence-activated cell sorting. The ER9+ and AO-red (AOr)high fractions were highly enriched in erythroid progenitors and primarily localized to the liver. The method developed using ER9 and AO was also applied to larvae and froglets with different progenitor populations from adult frogs. The liver to body weight and the number of ER9+ AOrhigh cells per unit body weight were significantly higher in adults than in larvae and froglets, and the number of ER9+ AOrhigh cells per unit liver weight was the highest in froglets. Collectively, our results show increased erythropoiesis in the froglet liver and demonstrate growth-dependent changes in erythropoiesis patterns in specific organs of Xenopus.

Spatiotemporal 3D image registration for mesoscale studies of brain development

Comparison of brain samples representing different developmental stages often necessitates registering the samples to common coordinates. Although the available software tools are successful in registering 3D images of adult brains, registration of perinatal brains remains challenging due to rapid growth-dependent morphological changes and variations in developmental pace between animals. To address these challenges, we introduce CORGI (Customizable Object Registration for Groups of Images), an algorithm for the registration of perinatal brains. First, we optimized image preprocessing to increase the algorithm’s sensitivity to mismatches in registered images. Second, we developed an attention-gated simulated annealing procedure capable of focusing on the differences between perinatal brains. Third, we applied classical multidimensional scaling (CMDS) to align (“synchronize”) brain samples in time, accounting for individual development paces. We tested CORGI on 28 samples of whole-mounted perinatal mouse brains (P0–P9) and compared its accuracy with other registration algorithms. Our algorithm offers a runtime of several minutes per brain on a laptop and automates such brain registration tasks as mapping brain data to atlases, comparing experimental groups, and monitoring brain development dynamics.

Growth-dependent signals drive an increase in early G1 cyclin concentration to link cell cycle entry with cell growth.

Entry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 is thought to initiate cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that control cell growth and size. Together, the data are consistent with models in which Cln3 is a crucial link between cell growth and the cell cycle.

Growth dependent changes in protein efficiency and amino acid composition in cultured meagre Argyrosomus regius

The goal of this research was to determine the growth dependent changes in protein efficiency and amino acid composition of the cultured meagre Argyrosomus regius (Asso, 1801). The research was carried in net cages in a private company (Gokce Off-shore Sist.) in the Aegean Sea. Meagre of an initial weight of 8.22 ± 0.13 g reached to 373.96 ± 15.65 g in 420 days of production period. Average protein efficiency rate, protein store rate and protein consumption values of cultured meagre were 1.05 ± 0.11%, 11.49 ± 2.98% and 56.67 ± 14.52 g respectively. The total amino acid values varied between 14.97 ± 0.14 and 18.87 ± 0.10 g 100g–1, the statistical difference among the study periods was significant (p < 0.05). Most of the total essential (EAA), non-essential (NEAA), and branched‐chain amino acid values of cultured meagre were decreasing with increasing fish size but EAA/NEAA ratios were increasing. The findings of this study showed that the composition of amino acids and the consistency of meagre culture were adequate and appropriate for human consumption and nutrition.

Evaluation of the tetrodotoxin uptake ability of pufferfish Takifugu rubripes tissues according to age using an in vitro tissue slice incubation method

The tetrodotoxin (TTX) uptake ability of pufferfish Takifugu rubripes tissues and its growth-associated changes were investigated using an in vitro tissue slice incubation method. Tissue slices prepared from the liver, skin, and intestine of a non-toxic cultured adult T. rubripes (20 months old) and incubated with incubation buffer containing 25 μg/mL TTX for 1–48 h showed a time-dependent increase in the TTX content in all tissues. The TTX contents of the skin and intestine slices were comparable to or slightly higher than that of the liver slices, with a similar transition pattern, suggesting similar TTX uptake ability among the skin, intestine, and liver. The TTX uptake ability of the liver and intestine did not differ significantly between young (8 months old) and adult (20 months old) fish, but the skin slices of young fish took up approximately twice as much TTX as that of adult fish, suggesting that the TTX uptake ability of the skin is involved in the growth-dependent changes in the toxin distribution inside the body in T. rubripes. To estimate the TTX uptake pathway in each tissue, an immunohistochemical technique was used to observe temporal changes in the intra-tissue microdistribution of TTX during incubation. The findings suggested that TTX is transferred and accumulates from pancreatic exocrine cells to hepatic parenchymal cells in the liver, from connective tissues to basal cells in the skin, and from villi epithelial cells via the lamina propria to the muscle layer in the intestine.

Growth Dependent Changes in Pressure Sensing Walkway Data for Turkeys.

Genetic selection for rapidly growing turkeys has created an unfavorable consequence impacting the skeletal system resulting in long bone distortions. These distortions have resulted in locomotor problems, gait abnormalities, leg weakness, or lameness issues. These effects raise welfare concerns along with animal agriculture inefficiency in the form of lost product. The purpose was to determine baseline gait and force distribution in visibly unimpaired growing turkey hens. Hendrix commercial turkey hen poults (n = 100) were placed on pine wood shavings providing 0.78 m2 per bird with ad libitum access to feed and water at the MSU Poultry Farm. Fifty hens were randomly selected at 5 weeks and identified with a leg band to ensure longitudinal data collection. The turkeys were walked across a pressure-sensing walkway (PSW, Tekscan, Boston, MA) and weighed at 5, 6, 8, and 10 weeks of age. PSW collected data on gait length, gait time, step force and step length, and the statistics were analyzed with SAS. Both temporospatial data, including step time and step length, and kinetic data, including peak downward force, and vertical impulse, were recorded. Body weight increased linearly with age (P < 0.001), demonstrating a typical growth pattern. Gait cycle time and peak vertical force (PVF) all displayed no difference between right and left sides, indicating that the hens had no detectable gait abnormalities. Gait velocity increased with age (P = 0.02) suggesting hens' growth impacted their gait velocity. The gait cycle time (P < 0.01) did not correspond with age. PVF increased linearly with age (P < 0.01) from 6 weeks (2.23 kg) to 10 weeks of age (5.91 kg). PVF/kg body weight (P < 0.01) increased from 6 weeks of age (96.9% BW) to 8 weeks of age (106%BW). Overall, the birds were not lame and some data was influenced by the hen's adjustment to the materials or stage of growth; in contrast, some temporospatial data did not coincide with age. The PSW could be used to detect locomotor issues in commercially produced turkey hens providing another tool for assessing well-being.

Suberized transport barriers in Arabidopsis, barley and rice roots: From the model plant to crop species

Water is the most important prerequisite for life and plays a major role during uptake and transport of nutrients. Roots are the plant organs that take up the major part of water, from the surrounding soil. Water uptake is related to the root system architecture, root growth, age and species dependent complex developmental changes in the anatomical structures. The latter is mainly attributed to the deposition of suberized barriers in certain layers of cell walls, such as endo- and exodermis. With respect to water permeability, changes in the suberization of roots are most relevant. Water transport or hydraulic conductivity of roots (Lpr) can be described by the composite transport model and is known to be very variable between plant species and growth conditions and root developmental states. In this review, we summarize how anatomical structures and apoplastic barriers of roots can diversely affect water transport, comparing the model plant Arabidopsis with crop plants, such as barley and rice. Results comparing the suberin amounts and water transport properties indicate that the common assumption that suberin amount negatively correlates with water and solute transport through roots may not always be true. The composition, microstructure and localization of suberin may also have a great impact on the formation of efficient barriers to water and solutes.

Interactions between growth-dependent changes in cell size, nutrient supply and cellular elemental stoichiometry of marine Synechococcus.

The factors that control elemental ratios within phytoplankton, like carbon:nitrogen:phosphorus (C:N:P), are key to biogeochemical cycles. Previous studies have identified relationships between nutrient-limited growth and elemental ratios in large eukaryotes, but little is known about these interactions in small marine phytoplankton like the globally important Cyanobacteria. To improve our understanding of these interactions in picophytoplankton, we asked how cellular elemental stoichiometry varies as a function of steady-state, N- and P-limited growth in laboratory chemostat cultures of Synechococcus WH8102. By combining empirical data and theoretical modeling, we identified a previously unrecognized factor (growth-dependent variability in cell size) that controls the relationship between nutrient-limited growth and cellular elemental stoichiometry. To predict the cellular elemental stoichiometry of phytoplankton, previous theoretical models rely on the traditional Droop model, which purports that the acquisition of a single limiting nutrient suffices to explain the relationship between a cellular nutrient quota and growth rate. Our study, however, indicates that growth-dependent changes in cell size have an important role in regulating cell nutrient quotas. This key ingredient, along with nutrient-uptake protein regulation, enables our model to predict the cellular elemental stoichiometry of Synechococcus across a range of nutrient-limited conditions. Our analysis also adds to the growth rate hypothesis, suggesting that P-rich biomolecules other than nucleic acids are important drivers of stoichiometric variability in Synechococcus. Lastly, by comparing our data with field observations, our study has important ecological relevance as it provides a framework for understanding and predicting elemental ratios in ocean regions where small phytoplankton like Synechococcus dominates.

Developmental and nutritional regulation of isoflavone secretion from soybean roots.

Isoflavones play important roles in plant-microbe interactions in rhizospheres. Soybean roots secrete daidzein and genistein to attract rhizobia. Despite the importance of isoflavones in plant-microbe interactions, little is known about the developmental and nutritional regulation of isoflavone secretion from soybean roots. In this study, soybeans were grown in hydroponic culture, and isoflavone contents in tissues, isoflavone secretion from the roots, and the expression of isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) were investigated. Isoflavone contents did not show strong growth-dependent changes, while secretion of daidzein from the roots dramatically changed, with higher secretion during vegetative stages. Coordinately, the expression of ICHG also peaked at vegetative stages. Nitrogen deficiency resulted in 8- and 15-fold increases in secretion of daidzein and genistein, respectively, with no induction of ICHG. Taken together, these results suggest that large amounts of isoflavones were secreted during vegetative stages via the hydrolysis of (malonyl)glucosides with ICHG.

Biomechanical properties of the tomato (Solanum lycopersicum) fruit cuticle during development are modulated by changes in the relative amounts of its components

In this study, growth-dependent changes in the mechanical properties of the tomato (Solanum lycopersicum) cuticle during fruit development were investigated in two cultivars with different patterns of cuticle growth and accumulation. The mechanical properties were determined in uniaxial tensile tests using strips of isolated cuticles. Changes in the functional groups of the cuticle chemical components were analysed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR). The early stages of fruit growth are characterized by an elastic cuticle, and viscoelastic behaviour only appeared at the beginning of cell enlargement. Changes in the cutin:polysaccharide ratio during development affected the strength required to achieve viscoelastic deformation. The increase in stiffness and decrease in extensibility during ripening, related to flavonoid accumulation, were accompanied by an increase in cutin depolymerization as a result of a reduction in the overall number of ester bonds. Quantitative changes in cuticle components influence the elastic/viscoelastic behaviour of the cuticle. The cutin:polysaccharide ratio modulates the stress required to permanently deform the cuticle and allow cell enlargement. Flavonoids stiffen the elastic phase and reduce permanent viscoelastic deformation. Ripening is accompanied by a chemical cleavage of cutin ester bonds. An infrared (IR) band related to phenolic accumulation can be used to monitor changes in the cutin esterification index.

The Boyd Amputation in Children

The level of amputation in the pediatric population requires appropriate planning to provide an optimal residual limb for prosthetic fitting and must include long-term strategies to accommodate future growth of the extremity. A retrospective review over a 15-year period was performed of all Boyd procedures (calcaneotibial fusion) in the pediatric limb deficiency population at a single institution. A chart review and radiographic analysis was performed to identify the indications, surgical outcomes, complications, need for additional surgical intervention, and nature of the postoperative prosthetic management. Optimal positioning of the calcaneotibial fusion and the growth-dependent changes in the morphology of the fusion site were determined by radiographic analysis. A total of 109 children (117 limbs) were identified for inclusion in the study. The average age at the time of the Boyd procedure was 2.8 years. The most common indication for the Boyd procedure was a diagnosis of postaxial limb bud deficiency, which accounted for 66% of cases. Concomitant procedures were performed in 24% of cases and included proximal tibial epiphyseodesis, tibial osteotomy, or knee fusion in the majority of cases. Additional procedures were required in 33% of cases either for treatment of complication (9%) or optimization of the residual limb (24%). For the entire cohort, the complication rate was 14%. Complications were most common when the Boyd procedure was used as a treatment strategy for congenital pseudoarthrosis of the tibia. Prosthetic management utilizing supramalleolar suspension with complete end-bearing through the residual limb was possible for the majority of cases. The Boyd procedure is an effective treatment for various conditions of the lower extremity. Concomitant or additional procedures after the initial intervention may be required for complete optimization of the residual limb. Level IV.

Changes in the major cell envelope components of Mycobacterium tuberculosis during in vitro growth.

One-third of the world's population is infected with Mycobacterium tuberculosis (M.tb), which causes tuberculosis. Mycobacterium tuberculosis cell envelope components such as glycolipids, lipoglycans and polysaccharides play important roles in bacteria-host cell interactions that dictate the host immune response. However, little is known about the changes in the amounts and types of these cell envelope components as the bacillus divides during in vitro culture. To shed light on these phenomena, we examined growth-dependent changes over time in major cell envelope components of virulent M.tb by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thin-layer chromatography, mass spectrometry, immunoblotting and flow cytometry. Our studies provide evidence that major mannosylated glycoconjugates on the M.tb cell envelope change as M.tb grows in vitro on the widely used Middlebrook 7H11 agar. In particular, our compositional analyses show that from Day 9 to 28 the amounts of mannose-containing molecules, such as mannose-capped lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, change continuously in both the cell envelope and outer cell surface. Along with these changes, mannan levels on the outer cell surface also increase significantly over time. The implications of these differences in terms of how M.tb is grown for studies performed in vitro and in vivo for assessing M.tb-host recognition and establishment of infection are discussed.

Role of Trpc channels, Stim1 and Orai1 in PGF2α-induced calcium signaling in NRK fibroblasts

Normal rat kidney (NRK) fibroblasts exhibit growth-dependent changes in electrophysiological properties and intracellular calcium dynamics. The transition from a quiescent state to a density-arrested state results in altered calcium entry characteristics. This coincides with modulation of the expression of the genes encoding the calcium channels Trpc1, Trpc6 and Orai1, and of the intracellular calcium sensor Stim1. In the present study we have used gene selective short hairpin (sh) RNAs against these various genes to investigate their role in (a) capacitative store-operated calcium entry (SOCE); (b) non-capacitative OAG-induced receptor-operated calcium entry (ROCE); and (c) prostaglandin F2α (PGF2α)-induced Ca2+-oscillations in NRK fibroblasts. Intracellular calcium measurements revealed that knockdown of the genes encoding Trpc1, Orai1 and Stim1 each caused a significant reduction of SOCE in NRK cells, whereas knockdown of the gene encoding Trpc6 reduced only the OAG-induced ROCE. Furthermore, our data show that knockdown of the genes encoding Trpc1, Orai1 and Stim1, but not Trpc6, substantially reduced the frequency (up to 60%) of PGF2α-induced Ca2+ oscillations in NRK cells. These results indicate that in NRK cells distinct calcium channels control the processes of SOCE, ROCE and PGF2α-induced Ca2+ oscillations.

Protein phosphorylation in mitochondria – A study on fermentative and respiratory growth ofSaccharomyces cerevisiae

Phosphorylation as a posttranslational protein modification is a common subject of proteomic studies, but phosphorylation in mitochondria is still poorly investigated. The study presented here applied 2-DE to characterize phosphorylation in the yeast mitochondrial proteome and identified 59 spots corresponding to 34 phosphorylated mitochondrial or mitochondria-associated proteins. Most of these proteins presented putative substrates of mitogen-activated protein and target of rapamycin kinases, cAMP-dependent protein kinase, cyclin-dependent kinases and Snf1p suggesting them as key players in the phosphorylation of mitochondrial or mitochondria-associated proteins. The dynamic behaviour of the phosphoproteome under a major metabolic change, the shift from fermentation to respiration (diauxic shift), was further studied. Eight proteins (Ald4p, Eft1p/2p, Eno1p, Eno2p, Om14p, Pda1p, Qcr2p, Sdh1p) had growth dependent changes in their phosphorylation, indicating a role of phosphorylation-dependent regulation of translation, metabolic pathways (e.g. glucose fermentation, tricarboxylic acid cycle, pyruvate dehydrogenase and its bypass) and respiratory chain.

Growth-Dependent Changes in the Contribution of Carbon Monoxide to Arteriolar Function

Background/Aims: Endothelium-dependent dilation of skeletal muscle arterioles is mediated by unknown factors in very young rats. We assessed the possible contribution of carbon monoxide (CO) to this dilation and to dilation in older animals. Methods: The effects of de-endothelialization or various pharmacological inhibitors on responses to CO or endothelium-dependent dilators were studied in gracilis muscle arterioles from rats at 3–4 weeks (‘weanlings’) and 6–7 weeks (‘juveniles’). Results: Exogenous CO constricted, rather than dilated, arterioles from both age groups. This constriction was reduced by endothelial removal or NOS inhibition in juvenile, but not weanling, arterioles. In contrast, this constriction was abolished by K⁺ channel inhibition in weanling, but not juvenile, arterioles. The heme precursor δ-aminolevulinic acid constricted juvenile arterioles but did not affect weanling arterioles. The heme oxygenase inhibitor chromium (III) mesoporphyrin IX abolished the endothelium-dependent dilation of juvenile arterioles to simvastatin, and reduced ACh- and simvastatin-induced dilations of weanling arterioles. Conclusion: These findings suggest that relatively high concentrations of exogenous CO can cause constriction by inhibiting endothelium-derived NO in juvenile arterioles and inhibiting K⁺ channels in weanling arterioles. Endogenous CO produced at lower concentrations can contribute to endothelium-dependent dilation in both age groups.

Biodegradation of phenanthrene and analysis of degrading cultures in the presence of a model organo-mineral matrix and of a simulated NAPL phase

Two mixed bacterial cultures (C(B-BT) and C(I-AT)) degraded phenanthrene when it was: (i) in the presence of either hexadecane as a non aqueous phase liquid or a montmorillonite-Al(OH)x-humic acid complex as a model organo-mineral matrix; (ii) sorbed to the complex, either alone or in the presence of hexadecane. The cultures had different kinetic behaviours towards phenanthrene with or without hexadecane. The degradation of Phe alone as well as that of Phe in hexadecane ended in 8 and 15 days with C(B-BT) and C(I-AT) cultures, respectively. Hexadecane increased Phe bioavailability for C(I-AT) bacteria which degraded Phe according to first-order kinetics. The same effect was observed for C(B-BT) bacteria, but with an initial 2 days lag phase and in accordance with zero-order kinetics. The presence of hexadecane did not affect the degradation of phenanthrene sorbed and aged on the complex by C(I-AT )culture. This capability was exhibited also after experimental aging of 30 days. The dynamics of the bacterial community composition was investigated through PCR-DGGE (denaturing gradient gel electrophoresis) of 16S rRNA gene fragments. Individual bands changed their intensity during the incubation time, implying that particular microbe's relative abundance changed according to the culture conditions. Isolation of phenanthrene and/or hexadecane degraders was in accord with cultivation-independent data. Growth-dependent changes in the cell surface hydrophobicity of the two cultures and of the isolates suggested that modulation of cell surface hydrophobicity probably played an important role for an efficient phenanthrene assimilation/uptake.

Growth-dependent changes in endothelial factors regulating arteriolar tone

Previous studies from this laboratory suggest that during maturation, rapid microvascular growth is accompanied by changes in the mechanisms responsible for regulation of tissue blood flow. To further define these changes, we studied isolated gracilis muscle arterioles from weanling ( approximately 25 days) and juvenile ( approximately 44 days) Sprague-Dawley rats to test the hypothesis that endothelial mechanisms for the control of arteriolar tone are altered with growth. Responses to the endothelium-dependent dilator acetylcholine (ACh) were greater in weanling arterioles (WA) than in juvenile arterioles (JA), whereas there were no consistent differences between age groups in arteriolar responses to other endothelium-dependent agonists (A-23187, vascular endothelial growth factor, and simvastatin). Inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-l-arginine methyl ester (l-NAME) attenuated ACh-induced dilation in JA but not in WA. In JA, combined inhibition of NOS and cyclooxygenase (with indomethacin) reduced the dilator responses to ACh and simvastatin by approximately 90% and approximately 70%, respectively, but had no effect in WA. Cytochrome P450 epoxygenase inhibition [with 2-(propargyloxyphenyl) hexanoic acid] had no effect on responses to ACh or simvastatin in either age group. Inhibition of Ca(2+)-activated or ATP-dependent potassium channels (with tetraethylammonium or glibenclamide, respectively) reduced these arteriolar responses in JA but not those in WA. These findings suggest that in fully grown microvascular networks, endothelium-dependent arteriolar dilation is mediated by the combined release of endothelial nitric oxide and vasodilator prostanoids, and in part through activation of Ca(2+)-activated and ATP-dependent potassium channels. However, during earlier microvascular growth, this dilation is mediated by other factors yet to be identified. This may have significant implications for the regulation of tissue perfusion during microvascular development.

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

The small intestinal epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and differentiation. The intestinal epithelium constitutes a permeability barrier regulating the vectorial transport of ions, water, and solutes. Morphological changes during cell differentiation, as well as changes in the activity of brush-border enzymes and the expression of transport proteins, are well established. However, little is known about the arachidonic acid (AA) cascade underlying epithelial cell differentiation or its role in the development of epithelial barrier function. The main purpose of this study was to examine the activity of the high-molecular-weight phospholipases A(2) (PLA(2)) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA(2) activity, AA release, COX-2 expression, prostaglandin E(2) (PGE(2)) production, and paracellular permeability were studied in preconfluent, confluent, and differentiated Caco-2 cell cultures. Our results show that Caco-2 differentiation induces a decrease in both calcium-independent PLA(2) activity and COX-2 expression and, consequently, a decrease in AA release and PGE(2) synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE(2) to differentiated cells, at concentrations similar to those detected in nondifferentiated cultures, induces the disruption of epithelial barrier function. These results suggest that AA release by calcium-independent PLA(2), COX-2 expression, and subsequent PGE(2) release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.

Growth dependent changes in the endothelial factors regulating arteriolar tone

Previous studies from our laboratory suggest that in young rats, the rapid growth of skeletal muscle and its vascular network is accompanied by a modification of the mechanisms responsible for the integrated regulation of blood flow. Building on this work, we studied isolated gracilis muscle arterioles from weanling (22–26 days) and juvenile (50–56 days) rats to test the hypothesis that this modification includes a change in the contribution of nitric oxide to the regulation of arteriolar tone. Normalized to dilator capacity, arteriolar responses to acetylcholine (ACh), A23187, VEGF and simvastatin were not different between age groups. Treatment with L-NAME significantly attenuated the dilation to each of these agonists in juvenile, but not in weanling arterioles. In juvenile vessels, combined treatment with L-NAME and indomethacin reduced ACh and simvastatin responses by 88–90% and 68–73%, respectively. In contrast, this combined treatment had no effect on responses to either agonist in weanling arterioles. Treatment with miconazole had no effect on ACh or simvastatin responses in either group. However, combined treatment with all three pathway inhibitors abolished ACh and simvastatin responses in juvenile arterioles, but had no consistent effect in weanling arterioles. These data suggest that age-dependent differences in the control of vascular tone exist and may have significant implications for the regulation of tissue perfusion. (NIH RO1 DK64668, AHA 0330194N, NIH HL 44012)

Biodegradation of phenanthrene by Pseudomonas sp. strain PP2: novel metabolic pathway, role of biosurfactant and cell surface hydrophobicity in hydrocarbon assimilation.

Pseudomonas sp. strain PP2 isolated in our laboratory efficiently metabolizes phenanthrene at 0.3% concentration as the sole source of carbon and energy. The metabolic pathways for the degradation of phenanthrene, benzoate and p-hydroxybenzoate were elucidated by identifying metabolites, biotransformation studies, oxygen uptake by whole cells on probable metabolic intermediates, and monitoring enzyme activities in cell-free extracts. The results obtained suggest that phenanthrene degradation is initiated by double hydroxylation resulting in the formation of 3,4-dihydroxyphenanthrene. The diol was finally oxidized to 2-hydroxymuconic semialdehyde. Detection of 1-hydroxy-2-naphthoic acid, alpha-naphthol, 1,2-dihydroxy naphthalene, and salicylate in the spent medium by thin layer chromatography; the presence of 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-2,3-dioxygenase activity in the extract; O(2) uptake by cells on alpha-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate and catechol; and no O(2) uptake on o-phthalate and 3,4-dihydroxybenzoate supports the novel route of metabolism of phenanthrene via 1-hydroxy-2-naphthoic acid --> [alpha-naphthol] --> 1,2-dihydroxy naphthalene --> salicylate --> catechol. The strain degrades benzoate via catechol and cis,cis-muconic acid, and p-hydroxybenzoate via 3,4-dihydroxybenzoate and 3-carboxy- cis,cis-muconic acid. Interestingly, the culture failed to grow on naphthalene. When grown on either hydrocarbon or dextrose, the culture showed good extracellular biosurfactant production. Growth-dependent changes in the cell surface hydrophobicity, and emulsification activity experiments suggest that: (1) production of biosurfactant was constitutive and growth-associated, (2) production was higher when cells were grown on phenanthrene as compared to dextrose and benzoate, (3) hydrocarbon-grown cells were more hydrophobic and showed higher affinity towards both aromatic and aliphatic hydrocarbons compared to dextrose-grown cells, and (4) mid-log-phase cells were significantly (2-fold) more hydrophobic than stationary phase cells. Based on these results, we hypothesize that growth-associated extracellular biosurfactant production and modulation of cell surface hydrophobicity plays an important role in hydrocarbon assimilation/uptake in Pseudomonas sp. strain PP2.