Selective Growth Conditions Research Articles

Induction of c-fos expression following stimulation of pulmonary neuroendocrine cell proliferation by alterations in CO2/O-2 concentration

Pulmonary neuroendocrine (PNE) cells were harvested and cultivated from peripheral lung tissue of 15 day old fetal hamsters under selective growth conditions, including a 10% CO2 atmosphere. Following 24 h of serum starvation under 10% CO2, PNE cells placed in a 5% CO2 atmosphere failed to proliferate over the next 24 h, while cells kept at 10% CO2 showed a 2.7-fold increase in cell number. Addition of nicotine to the cell culture medium resulted in an additional concentration-dependent increase in cell number under a 10% CO2 atmosphere, while nicotine did not stimulate the proliferation of cells maintained at 5% CO2. The nicotinic receptor antagonist hexamethonium completely blocked the stimulatory effect of nicotine on cell growth. As a first step towards determining the molecular mechanisms regulating the mitogenic stimulation of PNE cells by alterations in CO2/O-2 and nicotine, cells that had been serum starved under a 10% CO2 atmosphere for 24 h were removed from the incubator and either left untreated or treated with 1 mu M nicotine under ambient air. The cells were then returned to either a 5% or 10% CO2 atmosphere. Removal of the cells from a 10% CO2 environment and subsequent reintroduction to a high CO2 concentration resulted in a 3- to 4-fold increase in c-Sos RNA expression within 15-30 min upon return to the cell culture incubator. c-fos RNA levels their decreased back to control values by 1 h. Reintroduction into a 5% CO2 atmosphere, which failed to stimulate cell growth in the proliferation assay, caused only a 2-fold increase in the levels of c-fos transcripts. The CO2-mediated increases in c-Sos transcripts were observed both in the presence and absence of nicotine, suggesting that the effects of nicotine were mediated through a fos-independent pathway. Neither the alterations in CO2/O-2 concentration or treatment with nicotine altered the levels of c-myc or c-jun gene transcripts. Nicotine thus acts indirectly but synergistically with high CO2 concentrations to stimulate PNE cell proliferation.

Selectable marker recycling in the chloroplast

The bacterial geneaadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of theaadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking theaadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which theaadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas theaadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse theaadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.

Expression of hepatocyte growth factor and c-met mRNAs during rat chemically induced hepatocarcinogenesis

The receptor for hepatocyte growth factor (HGF), a potent hepatocyte mitogen, is the product of the protooncogene c-met. In order to cast light on their significance for hepatocarcinogenesis, levels of both HGF and c-met mRNA were evaluated in rat livers during development of 2-acetylaminofluorene (2-AAF)-selected preneoplastic nodules and carcinomas following diethylnitrosamine (DEN) initiation. Rats were given a single i.p. injection of 200 mg/kg body wt DEN and, starting 2 weeks later, were administered 0.015% 2-AAF in the diet for up to 6 weeks. All rats were subjected to partial hepatectomy (PH) at week 3. Additional animals undergoing the DEN, 2-AAF and PH regimen were sacrificed at week 40 to allow evaluation of carcinomas. Oval cell proliferation, glutathione S-transferase placental form (GST-P)-positive preneoplastic lesion development and HGF and c-met mRNA levels were sequentially analyzed after PH. Numerous oval cells were observed 1 week after PH, but were remarkably reduced 2 weeks thereafter. The areas of GST-P-positive foci and nodules rapidly increased with time not only during 2-AAF feeding, but also to the same degree for at least 2 weeks after cessation of carcinogenic insult. Dot blot analysis showed HGF transcripts to be elevated after PH and during the selective growth conditions of 2-AAF feeding, dropping after cessation of carcinogenic insult. In the c-met transcript case transient increases were observed after PH, followed by a decrease. c-met over-expression in nodular livers did not correlate with the presence of 2-AAF or lesion development. In most hepatocellular carcinoma samples expression of both HGF and c-met mRNAs was below levels in non-neoplastic regions. These data suggest that HGF and c-met are directly involved in a paracrine growth pathway controlling proliferation in normal hepatocytes and oval cells, but not in preneoplastic and neoplastic cells.

In-situ observation of GaAs selective epitaxy on GaAs (111)B substrates

Selective area epitaxy has been carried out by μ-RHEED (reflection high energy electron diffraction)/SEM (scanning electron microscopy) MBE (molecular beam epitaxy) on (111)B patterned substrates masked by SiO2 and conditions for selective growth have been determined as a function of substrate temperature and Ga beam intensity. Below the critical Ga flux, a smooth epitaxial layer grows without any deposition of polycrystals on the masked area. However, in the high temperature region (over 660°C), the epilayer grown in the window is rather rough, due to the high re-evaporation rate of Ga atoms. In the high Ga flux region, polycrystalline deposition on the mask occurs and the coverage of the masked area by poly-GaAs nuclei decreases from the region close to the window to the center of the mask. It has been found that there is a strong lateral surface flow of Ga atoms from the window to the mask region.

Selective area metalorganic chemical vapor deposition growth for hexagonal-facet lasers

In order to obtain low threshold hexagonal-facet (HF) lasers by reducing their size, it is necessary to know the selective growth conditions of a hexagonal prism on a smaller area. We describe the metalorganic chemical vapor deposition (MOCVD) growth conditions for GaAs and AlGaAs hexagonal prisms on a SiO 2 masked (111)B substrate. The crystal shape and the facet strongly depend on the size of the mask windows, on the working pressure during growth, and on the growth parameters of the substrate temperature and the partial pressure of arsine. Furthermore, we explain the mechanism of the hexagonal prism growth by using the change of growth rate between low-index facet planes. By using optimized growth conditions, a 2 μm sized HF laser structure with flat GaAs/AlGaAs heterointerface can be grown.

Production of extracellular xylanases by Penicillium janthinellum. Effect of selected growth conditions.

Xylanase production by Penicillium janthinellum using 10-100 mM of 2,2-dimethylsuccinate (DMS) buffer, in a range of pH 4.5-6.0 was studied. The enzyme activity was enhanced using oat xylan as the carbon source. Under these conditions a culture produced 1.14 mumol/min (11.4 U/mL or 84.4 U/mg) of beta-xylanase after 5 d of growth in a 10-mM buffer solution at pH 4.5. Protease was absent in the DMS buffer except when 100 mM phosphate buffer at pH 6.0 was used (4 U/mL). beta-Xylosidase was only found at a pH of 4.5 in all the buffer concentrations. At a 50 mM DMS buffer concentration at pH 4.5 beta-xylanases were induced by both oat and birch xylans, having a greater effect with oat spelt xylans. Electrophoretic analyses showed that the birchwood xylan induction exhibited different proteins profiles. No beta-xylosidase or beta-glucosidase was induced until d 5. The beta-xylanases were rapidly inactivated at 50 degrees C, however, birch xylanase appeared to be more stable than oat xylanase. Using oat xylan as an inductor, the beta-xylosidase and beta-glucosidase were 85 and 91 U/L, respectively, on d 7. The xylanase produced by induction from sugar cane bagasse hydrolyzate was used for pulp biobleaching. A 20% decrease on the Kappa value in Kraft pulp using the culture extract was obtained. These selective growth conditions led us to modulate the xylanase production for pulp delignification.

Purification and DNA-binding properties of FHLA, the transcriptional activator of the formate hydrogenlyase system from Escherichia coli.

FHLA is the transcriptional activator for the expression of the genes coding for components of the formate hydrogenlyase system of Escherichia coli. The cloned fhlA gene was overexpressed under selected growth conditions, and a purification protocol for the FHLA protein was developed. Purified FHLA in the native state is a homotetramer. It binds to the upstream regulatory sequences of the fdhF gene and of the intergenic region between the divergently transcribed hyc and hyp operons. An additional binding site of FHLA located between the hycA and hycB genes was identified. While binding to the hypA-hycA intergenic region is responsible for activation of expression of the hyc operon, the newly identified site is required for the activation of the sigma 54-dependent promoter located upstream of the hyp operon. Formate seems to have no effect on the DNA-binding properties of FHLA. The binding sites of FHLA were characterized by DNase I footprinting; sequence motifs putatively involved in the interaction with FHLA are described.

Room-temperature selective growth of dielectric films by liquid-phase deposition

In multilevel interconnection processes, because thermal stress affects device characteristics and wiring reliability, low-temperature deposition has been required for interlayer dielectrics (SiO2). This research investigated high growth rate and selective growth conditions of room-temperature interlayer dielectrics formed using the liquid-phase deposition method. The dependence of deposition rate on growth temperature and H3BO3 concentration is confirmed, and a concentration of 3.8 mol 1-1 of H2SiF6 is used to achieve a high deposition rate of 1250 AA h-1. The degree of supersaturation of silica of the immersing solution is found to define three ranges of growth conditions: the non-deposition range, selective deposition range, and conformal deposition range. A selective deposition model is proposed to clarify the mechanism of room-temperature selective SiO2 growth.

Interaction between smoking and asbestos in human lung adenocarcinoma: role of K-ras mutations.

To investigate the role of tobacco smoking and asbestos fibers in the etiology of human lung cancer, we examined the activating point mutations in the K-ras oncogene in DNA samples from 49 patients. Mutations were found more often in tissue from adenocarcinomas (12/21) than in tissue from tumors other than nonadenocarcinomas of the lung (3/28). Among the adenocarcinoma patients, asbestos exposure was predictive of K-ras mutation (odds ratio, 4.9; 95% confidence interval, 0.7-34.3); in patients with other types of lung cancer, the relation appeared to be an inverse one, but the numbers were small. The proportion of heavy smokers (over 50 pack-years) was 60% among people with K-ras mutations and 35% among the K-ras-negative subjects, suggesting that smoking causes K-ras mutations. If mutations in K-ras genes are caused by smoking, asbestos would act as a promoting agent by conferring selective growth conditions for clonal expansion on these mutated cells. Asbestos may favor recruitment of (initiated) K-ras mutation-positive cells in the multistage process of carcinogenesis by stimulating cellular growth.

Methods of assessment of transfer of a gentamycin-resistance gene (aacC1) from genetically engineered microorganisms into agriculturally important soil bacteria

In order to assess the risk associated with the deliberate release of genetically engineered microorganisms (GEMs) into the agricultural environment, the transfer of plasmids between bacterial strains was investigated under laboratory conditions. Genetically modified Rhizobium leguminosarum and Agrobacterium tumefaciens strains carrying the gentamycin acetyltransferase resistance gene (aacC1) on various plasmids were investigated for their ability to transfer the aacC1 gene to their wild-type (w.t.) counterparts, as well as to Pseudomonas syringae. Conjugation experiments between the various strains, were carried out after the relevant characteristics and conditions for selective growth of each bacterial strain had been ascertained. After conjugations on filters had been completed, the putative transconjugants were grown in media containing antibiotics and assessed for the presence of aacC1 gene by: (a) DNA plasmid profile; (b) expression of AAC(3)-I enzyme activity; (c) colony hybridization using a 32P-labelled DNA probe complementary to the aacC1 gene. The results obtained indicate that transfer of the aacC1 gene from genetically modified strains of R. leguminosarum into a plasmid-free strain of A. tumefaciens occurred via self-transmissible plasmids. Alternatively, genetically modified A. tumefaciens bearing the aacC1 gene on plasmids acquired from R. leguminosarum strains, transferred it ineffectively to a hardly detectable frequency. No transfer of the aacC1 gene from genetically modified R. leguminosarum or A. tumefaciens strains into P. syringae has been observed. These data indicate that in the absence of the RP4 element, genetically modified A. tumefaciens is not able to efficiently transfer aacC1 into w.t. R. leguminosarum and P. syringae.

Effects of Growth Conditions and Substrate Orientation on the Properties of InSb

AbstractTwo new organometallic Sb sources, tris(dimethylamino)antimony (TDMASb), and tertiarybutyldimethylantimony (TBDMSb) are being investigated. The growth of InSb using TDMASb, or TBDMSb and TMIn was investigated over a temperature range of 350 to 475 °C using a range of V/III ratios, growth rates and pressures. Misoriented InSb substrates were also used for selected growth conditions The growth rates of lnSb using TMIn and either TDMASb or TBDMSb at temperatures <= 425 °C were proportional to both the TMIn flow rate and the temperature. The surface morphology of InSb grown using either TDMASb or TBDMSb was very rough for growth temperatures <=425 °C. This may be due to the complex decomposition mechanisms involved and the presence of methyl groups on the surface. Smoother backgrounds were generally found when using off axis substrates. The details of the defects observed on the surfaces were dependent on the type of misorientation and can be related to the atomic structure of the surface steps. Both n- and p-type InSb were grown using TBDMSb or TDMASb and TMIn with mobilities up to 68,990 and 7773 cm2/Vs, respectively, at 77 K. The mobility for lnSb using either TDMASb or TBDMSb was optimized by going to lower temperatures, pressures and V/III ratios. The opposite was true for surface morphology which improved with higher temperature, pressure, and V/III ratio. The growth of high mobility InSb with smooth surfaces at T< =425 °C was not achieved with TDMASb or TBDMSb and TMIn under the conditions investigated in this work.

Selective growth of ultra-low resistance GaAs/InGaAs for high performance InGaAs FETs

To minimize the source resistance of a doped-channel InGaAs heterostructure FET grown on a GaAs substrate, GaAs, InGaAs and InAs selective growth conditions are studied by metalorganic vapor phase epitaxy (MOVPE). It is found that the lowest growth temperatures with complete selectivity for InGaAs (indium composition less than 0.2) and InAs are 540 and 400°C, respectively. The contact resistance at the regrown interface measured with the transmission line model (TLM), is minimized to 5 × 10 -9 ω cm 2 when the Si-doped GaAs is regrown using a side contact structure. However, the contact resistance increases as the In composition in the regrown InGaAs increases. This might be due to the strain or dislocations caused by lattice mismatching between GaAs and InGaAs.

Selective Epitaxial Growth of Si and Si1-xGex Films by Ultrahigh-Vacuum Chemical Vapor Deposition Using Si2H6 and GeH4

Aiming at the precise profile control of Si1-xGex, selective epitaxial growth (SEG) conditions and Si1-xGex growth were investigated by ultrahigh-vacuum chemical vapor deposition (UHV-CVD) using Si2H6 and GeH4. As long as the total amount of Si2H6 did not exceed the critical amount, Si- and Si1-xGex-SEG were both achieved independent of source gas flow rate and substrate temperature. The Ge fraction x of Si1-xGex could be decided by the flow rate ratio between Si2H6 and GeH4, independent of substrate temperature. Fast gas flow switching realized the formation of a Si(120 Å)/Si1-xGex(69 Å) strained layer superlattice at 587°C.

Culture of melanocytes obtained from normal and vitiligo subjects.

The development of human melanocyte culture in vitro from normal adult skin and uninvolved skin of vitiligo patients is essential to investigate the mechanism of depigmentation in vitiligo and other pigmentary dermatoses. By using selective growth and long-term maintenance conditions, we selectively cultured melanocytes derived from normal foreskins and arm skins, and uninvolved foreskins and arm skins of vitiligo patients. The melanocytes of the arm skins were successfully cultured from the roofs of suction blisters. Melanocyte Growth Media (MGM) consisting of MCDB-153 formulation with basic fibroblast growth factor (bFGF), bovine pituitary extract (BPE), insulin, hydrocortisone, phorbol 12-myristate 13-acetate (PMA) and 10% human AB serum was sufficient to grow the melanocytes from normal and vitiligo donors. Melanocytes from uninvolved skin of vitiligo donors showed no different morphologic features, initial seeding capacity and population doubling time compared with those from normal skin. Melanocytes from both cell types grew without any lag period for more than 6 months (6-11 passages). Melanocytes obtained from foreskins had higher initial seeding capacity and shorter population doubling time than those obtained from arm skins using suction-blistered roofs. Our results suggest that the culture method using suction blisters may be a simple and easy way to obtain melanocytes. In addition, vitiligo melanocytes can be successfully cultured with appropriate growth conditions and may show no defective growth patterns. This culture system will be applied to investigate the basic pathophysiology of vitiligo and other various pigmentary dermatoses.

Limiting conditions of Si selective epitaxial growth in Si2H6 gas-source molecular beam epitaxy

The limiting conditions are presented of selective epitaxial growth (SEG) on a SiO2-patterned Si (001) substrate for Si gas-source molecular-beam epitaxy by use of 100% Si2H6. In the initial stage of growth, epitaxial Si was selectively grown on a Si surface in the wide temperature range of 500–850 °C. On the other hand, polycrystalline Si nucleation on a SiO2 surface was intimately related to the total volume of supply gas. At a substrate temperature of 700 °C and a Si2H6 flow rate of 60 sccm, a SEG layer could be deposited at a rate as high as 645 Å/min.

Limitations of selective epitaxial growth conditions in gas-source MBE using Si 2H 6

The limiting conditions of selective epitaxial growth (SEG) on SiO 2 patterned Si(001) substrate were studied for Si gas-source molecular beam epitaxy (MBE) by use of 100% Si 2H 6. In the initial stage of growth, epitaxial Si was selectively grown on a Si surface in the temperature range of 500 to 850°C. On the other hand, polycrystalline Si nucleation on a SiO 2 surface was intimately related to the impinging density of the Si 2H 6 molecules on SiO 2, so that SEG was limited by the supply gas volume. Under optimum SEG conditions such as substrate temperature of 700°C and Si 2H 6 flow rate of 60 SCCM, a SEG layer could be deposited at a rate as high as 645 Å/min.

7570. Conditions for uniform selective epitaxial growth: C I Drowley and M L Hammond,Solid St Technol,33, 1990, 135–141

A recombinant fusion protein combining the mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p), both from Saccharomyces cerevisiae, has been genetically elaborated with the aim of increasing the polar surface area of the carrier to facilitate its crystallization. The gene encoding the his-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2. The chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, which demonstrated its transport activity. The kinetic exchange properties of Anc2-Cyc1(His6)p and the wild type his-tagged carrier Anc2(His6)p were very similar. However, Anc2-Cyc1(His6)p restored cell growth less efficiently than Anc2(His6)p which correlates with the lower amount found in mitochondria. Purification of Anc2-Cyc1(His6)p in complex with carboxyatractyloside (CATR), a high affinity inhibitor of ADP/ATP transport, was achieved by combining ion-exchange chromatography and ion-metal affinity chromatography in the presence of LAPAO, an aminoxide detergent. As characterized by absorption in the visible range, heme was found to be present in isolated Anc2-Cyc1(His6)p, giving the protein a red color. Large-scale purification of Anc2-Cyc1(His6)p-CATR complex opens up novel possibilities for the use of crystallographic approaches to the yeast ADP/ATP carrier.

B cell progenitors have different growth requirements before and after immunoglobulin heavy chain commitment.

Using the clonable pre-B cell assay, we have identified B cell progenitors that are not yet committed to the production of a particular H chain allele. These cells represent approximately 10-20% of clonable pre-B cells found in 15-d fetal liver. The clonable pre-B cell assay provides an environment adequate for the expansion and differentiation of these cells into mature, Ig-secreting, cells. Using the same methodology, we have also identified progenitors that are uncommitted to the production of a particular L chain isotype. Moreover, investigating the growth requirements for clonable pre-B cells has led to the discovery of selective growth conditions that distinguish cells before and after commitment to H chain allotype.

Low-temperature (250 °C) selective epitaxy of GaAs films and p-n junction by laser-assisted metalorganic chemical vapor deposition

Low-temperature selective epitaxial growth of device quality GaAs has been achieved by laser-assisted chemical vapor deposition (LCVD). GaAs substrates thermally biased to temperatures in the range 250–500 °C were irradiated by an Ar ion laser to induce localized deposition of GaAs. Carefully selected growth conditions resulted in growth rates as low as a monolayer per second at 250 °C. This is the lowest substrate temperature for epitaxial GaAs with optical and structural quality comparable to those achieved in conventionally metalorganic chemical vapor deposition grown GaAs. Also reported is the first p-n junction by LCVD technique using zinc as the p-type dopant. This new low-temperature selective deposition process can lead to maskless fabrication of multicomponent devices on the same wafer.

Low temperature selective epitaxy of III–V compounds by laser assisted chemical vapor deposition

Laser Assisted Chemical Vapor Deposition (LCVD) is a new approach for low temperature selective epitaxy of III-V compounds with great potential for maskless multicomponent device integration on the same wafer. GaAs substrates were thermally biased to temperatures as low as 250°C and exposed simultaneously to fluxes of trimethylgallium (TMG) and arsine (AsH3). Ar ion laser scanning of the substrate, at carefully selected growth conditions, resulted in epitaxial selective deposition at rates as low as a few Å/s per scan. The LCVD growth rates as a function of deposition temperatures are reported for different laser powers and seem to follow an Arrhenius type relation. Activation energies in the range 10–13 kcal/mol were estimated for growth temperatures in the range 250–400°C. X-ray diffraction topography is demonstrated as a powerful nondestructive tool for structural characterization. We have utilized this technique to optimize the LCVD growth conditions in order to achieve device quality films that are free from lattice distortion. Low temperature photoluminescence shows the presence of impurity peaks that can be related to carbon.