Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Osteonectin/SPARC induction by ectopic beta(3) integrin in human radial growth phase primary melanoma cells

Expression of the beta(3) integrin subunit in melanoma in situ has been found to correlate with tumor thickness, the ability to invade and metastasize, and poor prognosis. Transition from the radial growth phase (RGP) to the vertical growth phase (VGP) is a critical step in melanoma progression and survival and is distinguished by the expression of beta(3), integrin. The molecular pathways that operate in melanoma cells associated with invasion and metastasis were examined by ectopic induction of the beta(3), integrin subunit in RGP SBcl2 and WM1552C melanoma cells, which converts these cells to a VGP phenotype. We used cDNA representational difference analysis subtractive hybridization between beta(3)-Positive and -negative melanoma cells to assess gene expression profile changes accompanying RGP to VGP transition. Fourteen fragments from known genes including osteonectin (also known as SPARC and BM-40) were identified after three rounds of representational difference analysis. Induction of osteonectin was confirmed by Northern and Western blot analysis and immunohistochemistry and correlated in organotypic cultures with the beta(3)-induced progression from RGP to VGP melanoma. Expression of osteonectin was also associated with reduced adhesion to vitronectin, but not to fibronectin. Osteonectin expression was not blocked when melanoma cells were cultured with anti-alpha(v)beta(3) LM609 mAb, mitogen-activated protein kinase, or protein kinase C inhibitors, indicating that other signaling pathway(s) operate through a(v)beta(3) integrin during conversion from RGP to VGP.

Siderophore Production by Bacillus megaterium: Effect of Growth Phase and Cultural Conditions

Siderophore production by Bacillus megaterium was detected, in an iron-deficient culture medium, during the exponential growth phase, prior to the sporulation, in the presence of glucose; these results suggested that the onset of siderophore production did not require glucose depletion and was not related with the sporulation. The siderophore production by B. megaterium was affected by the carbon source used. The growth on glycerol promoted the very high siderophore production (1,182 μmol g−1 dry weight biomass); the opposite effect was observed in the presence of mannose (251 μmol g−1 dry weight biomass). The growth in the presence of fructose, galactose, glucose, lactose, maltose or sucrose, originated similar concentrations of siderophore (546–842 μmol g−1 dry weight biomass). Aeration had a positive effect on the production of siderophore. Incubation of B. megaterium under static conditions delayed and reduced the growth and the production of siderophore, compared with the incubation in stirred conditions.

Vertical growth phase and positive sentinel node in thin melanoma

Sentinel node (SN) status is the most important prognostic factor for localized melanoma. Usually, patients with Breslow thickness of less than 1.0 mm are not included in SN protocols. However, the literature presents a rate ranging from 3 to 7% of nodal recurrence in thin melanoma. Ulceration, regression and high mitotic rate have been considered to be indications for an SN biopsy. The metastatic potential of the vertical growth phase is uncertain. To correlate pathological features in thin melanoma with SN metastasis, we reviewed 358 patients submitted to SN biopsy. Seventy-seven patients with lesions of 1 mm or smaller were included in the study group. Histological evaluation of the primary tumor included thickness, Clark level, mitotic rate, ulceration, regression, and growth phase. Lymphoscintigraphy was performed on all patients. Lymphatic mapping and gamma probe detection were both used for SN biopsy. Histological examination of SN consisted of hematoxylin-eosin and immunohistochemical staining. Median follow-up was 37 months. Six patients had micrometastases. Statistical analysis by the Fisher test showed that ulceration (P = 0.019), high mitotic rate (P = 0.008) and vertical growth phase (P = 0.002) were positively correlated with micrometastases. If other studies confirm these results, more melanoma patients must be submitted to SN biopsy.

Evaluation of babassu meal in feed for layer hens during the growth phase

Studies were conducted to evaluate the nutritional value and inclusion levels of babassu meal (BM) in the diet of grower layer pullets in substitution to wheat meal. Digestibility, metabolism and growth trials were conducted. Twelve cecectomized roosters were used in the digestibility assay to determine the coefficients of standardized digestibility of amino acids (CSDAA). The metabolism trial was conducted with 30 adult roosters to determine the apparent metabolizable energy corrected for nitrogen (AMEn) of BM. A growth trial was performed to determine replacement levels of wheat midds by BM diet using 360 six-week-old commercial layer pullets. BM was included at the 0, 75 and 150 g/kg of BM, during grower and development rearing phases, respectively. Feed intake, body weight gain, and feed conversion were evaluated. BM AMEn was determined as 1,474 kcal/kg, on as-fed basis. The CSDAA determined for BM were below 88% for all AA. The inclusion of BM in the feed of grower layers (7-18 week) significantly decreased feed intake (p < 0.05), but significantly improved body weight gain and feedconversion ratio (p < 0.05) at 15% inclusion level. Considering the nutritional value and performance results, BM can replace wheat midds in diets of grower layer pullets.

Photosynthetic capacity and water use efficiency in sugarcane genotypes subject to water deficit during early growth phase

The objective of this study was to compare the gas exchange, photosynthetic capacity and water potential of sugarcane genotypes cultivated under water deficit conditions imposed during the initial growth phase. Experiments were performed in a greenhouse using two sugarcane genotypes namely: HoCP93-776 (drought susceptible) and TCP02-4587 (drought tolerant). Sixty days after planting, two different water treatments were applied (i.e., with or without water deficit). At 0,30 and 60 days after the treatment, gas exchange variables were evaluated for their relationship with water use, intrinsic instantaneous water use efficiency and instantaneous carboxylation efficiency. The SPAD index, photosynthetic pigments, water potential and relative water content in the leaves were also analyzed. The genotype HoCP93-776 was more sensitive to drought treatment as indicated by the significantly lower values of SPAD index, photosynthetic pigments, water potential (Ψw) and relative water content (RWC) variables. The genotype TCP02-4587 had higher water potential, stomatal control efficiency, water use efficiency (WUE), intrinsic instantaneous water use efficiency (WUEintr), instantaneous carboxylation efficiency and photosynthetic capacity. The highest air vapor pressure deficit during the drought conditions could be due to the stomatal closing in the HoCP93-776, which contributed to its lower photosynthetic capacity.

The effects of growth phase and salinity on the hydrogen isotopic composition of alkenones produced by coastal haptophyte algae

The isotopic fractionation of hydrogen during the biosynthesis of alkenones produced by marine haptophyte algae has been shown to depend on salinity and, as such, the hydrogen isotopic composition of alkenones is emerging as a palaeosalinity proxy. The relationship between fractionation and salinity has previously only been determined during exponential growth, whilst it is not yet known in which growth phases natural haptophyte populations predominantly exist. We have therefore determined the relationship between the fractionation factor, alpha alkenones-water, and salinity for C37 alkenones produced in different growth phases of batch cultures of the major alkenone-producing coastal haptophytes Isochrysis galbana (strain CCMP 1323) and Chrysotila lamellosa (strain CCMP 1307) over a range in salinity from ca. 10 to ca. 35. alpha alkenones-water was similar in both species, ranging over 0.841-0.900 for I. galbana and 0.838-0.865 for C. lamellosa. A strong (0.85 <= R**2 <= 0.97; p < 0.0001) relationship between salinity and fractionation factor was observed in both species at all growth phases investigated. This suggests that alkenone dD has the potential to be used as a salinity proxy in coastal areas where haptophyte communities are dominated by these coastal species. However, there was a marked difference in the sensitivity of alpha alkenones-water to salinity between different growth phases: in the exponential growth phase of I. galbana, alpha alkenones-water increased by 0.0019 per salinity unit (S 1), but was less sensitive at 0.0010 S 1 and 0.0008 S 1 during the stationary and decline phases, respectively. Similarly, in C. lamellosa alpha alkenones-water increased by 0.0010 S 1 in the early stationary phase and by 0.0008 S 1 during the late stationary phase. Assuming the shift in sensitivity of alpha alkenones-water to salinity observed at the end of exponential growth in I. galbana is similar in other alkenone-producing species, the predominant growth phase of natural populations of haptophytes will affect the sensitivity of the alkenone salinity proxy. The proxy is likely to be most sensitive to salinity when alkenones are produced in a state similar to exponential growth.

Monitoring changes in nisin susceptibility of Listeria monocytogenes Scott A as an indicator of growth phase using FACS

Listeria monocytogenes has previously been shown to adapt to a wide variety of environmental niches, principally those associated with low pH, and this compromises its control in food environments. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. The present Study aimed to gain a further understanding of the physiological basis for the differential effects of one control strategy, namely the use of the lantibiotic nisin. Using propidium iodide (PI) to probe membrane integrity it was shown that L. monocytogenes Scott A was sensitive to nisin (8 ng mL(-1)) but this was growth phase dependent with stationary phase cells (OD600=1.2) being much more resistant than exponential phase cells (OD600=0.38). We demonstrate that, using a combination of techniques including fluorescence activated cell sorting (FACS), the membrane adaptations underpinning nisin resistance are triggered much earlier (OD600 < 0.5) than the onset of stationary phase. The significance of these findings in terms of mechanism and application are discussed. (c) 2005 Elsevier B.V.All rights reserved.

Impact of salinity and growth phase on alkenone distributions in coastal haptophytes

Batch cultures of Isochrysis galbana (strain CCMP 1323) and Chrysotila lamellosa (strain CCMP 1307) were grown at salinity ca. 10 to ca. 35 and the alkenone distributions determined for different growth phases. UK'37 values decreased slightly with salinity for C. lamellosa but were largely unaffected for I. galbana except during the decline phase. The values decreased with incubation time in both species. The proportion of C37:4, used as proxy for salinity, increased in both species at 0.16-0.20% per salinity unit, except during the stationary phase for I. galbana. C37:4 was much more abundant in C. lamellosa (30-44%) than in I. galbana (4-12%). Although our results suggest that salinity has a direct effect on alkenone distributions, growth phase and species composition will also have a marked impact, complicating the use of alkenone distributions as a proxy for salinity in the marine environment.

Growth phase-specific promoters of cyanobacteria for synthetic biology applicatations

No abstract available.

Global changes in gene expression observed at the transition from growth to stationary phase in Listeria monocytogenes ScottA batch culture

Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually. The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L. monocytogenes adapts to environmental change are yet to be fully elucidated. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. We have utilized a proteomic approach to investigate the response of L. monocytogenes batch cultures to the transition from exponential to stationary growth phase. Proteomic analysis showed that batch cultures of L. monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone. Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase. We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase. The significance of these findings in terms of functionality and the mechanistic picture are discussed.

Estimation of R(0) from the initial phase of an outbreak of a vector-borne infection

The magnitude of the basic reproduction ratio R(0) of an epidemic can be estimated in several ways, namely, from the final size of the epidemic, from the average age at first infection, or from the initial growth phase of the outbreak. In this paper, we discuss this last method for estimating R(0) for vector-borne infections. Implicit in these models is the assumption that there is an exponential phase of the outbreaks, which implies that in all cases R(0) > 1. We demonstrate that an outbreak is possible, even in cases where R(0) is less than one, provided that the vector-to-human component of R(0) is greater than one and that a certain number of infected vectors are introduced into the affected population. This theory is applied to two real epidemiological dengue situations in the southeastern part of Brazil, one where R(0) is less than one, and other one where R(0) is greater than one. In both cases, the model mirrors the real situations with reasonable accuracy.

Growth and differentiation on a trypanosome of the subgenus Schizotrypanum from the bat Phyllostomus hastatus

The effects of temperature, pH, osmolarity and aeration on the growth and differentiation of a trypanosome ofthe subgenus Schizotrypanum isolatedfrom the bat Phyllostomus hastatus were studied. In general, the growth characteristics ofthe flagellate were similar to those of Trypanosoma (Schizotrypanum) cruzi. However, the parasite did not growth at 33 or 37C. Increase in the osmolarity and aeration promoted growth at 33C. Significant metacyclogenesis was detected only in the growth condition where maximal growth occured (28C, pH 7.3, 380m0s/kg, in tissue cullure flasks), at the end ofthe exponential growth phase. The begining of the metacyclogenesis process was coincident with most glucose utilization and lowest pH. During metacyclogenesis both culture medium pH and osmolarity increased steadly.