Variation In Number Of Cells Research Articles

α2β1 Integrin Regulates Lineage Commitment in Multipotent Human Colorectal Cancer Cells*

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the β1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to β1 integrin. Function-blocking antibodies to α2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in α2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of α2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type α2 integrin or a non-signaling chimeric α2 integrin. Overexpression of wild-type α2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric α2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor α2β1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.

Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

BackgroundIn women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro.MethodsTumors from 38 dogs were sliced and cultured with or without doxorubicin 1 μM for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples ≥ 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors.ResultsCulture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction ≥ 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed.ConclusionThree-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.

Miniaturization and HTS of a FRET-Based Membrane Potential Assay for Kir Channel Inhibitors

The K(ir) family of potassium-selective ion channels is characterized by their inward (anomalous) rectifying current-voltage relationship. K(ir) channels are widely expressed in mammalian cells and through their role in regulation of the cell membrane potential have been implicated in diverse physiological functions. To enable the identification of novel K(ir) channel inhibitors, a fluorescence resonance energy transfer (FRET)-based membrane potential assay was developed using a Chinese hamster ovary cell line stably expressing a human K(ir) channel. The FRET-based assay incorporates the use of two dyes {N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidylethanolamine (CC2-DMPE) and bis(1,3-diethylthiobarbiturate)trimethine oxonol [DiSBAC(2)(3)]} to track changes in membrane potential, thus enabling all of the advantages of ratiometric readout: reduced inaccuracies arising from well-to-well variation in cell number, dye loading, signal intensities, and plate inconsistencies. The assay was miniaturized to a 1,536-well microtiter plate format and read on a fluorometric imaging plate reader (FLIPR(Tetra), Molecular Devices, Sunnyvale, CA). The assay was automated and utilized to perform a primary high-throughput screening campaign to identify novel inhibitors of the K(ir) channel.

Stochastic Analysis of Multistate Systems

The treatment necessary to remove all cells for a periodic cell cycle specific regimen is analyzed using stochastic equations of a multistate model with state transition governed by residence time distributions. While the objective is to remove all cells, exactly when this occurs is uncertain due to the random timing of state transitions. Previous work has shown that removal of all cells requires great certainty that the number of cells is small; an expected population that is six standard deviations less than one cell is an indicator when cure is nearly likely. In developing the necessary stochastic framework, the expression for the standard deviation for multistate processes is derived and a simple two-state example is presented that illustrates the potential coupling between subpopulations. Finally, the methodology is applied to cell cycle specific chemotherapy and the number of cycles required to nearly be assured that zero cells remain is calculated. This method has computational advantages over Monte Carlo simulations when the initial number of cells is not small and only the variation in the total number of cells is of interest.

Altered lymphocyte homeostasis after oral prion infection in mouse

Transmissible spongiform encephalopaties (TSEs) or prion diseases develop as central nervous system (CNS) disorders characterized by extremely long incubation periods. Although TSEs do not go along with inflammatory infiltrates and/or antibody production against the prion protein (PrP Sc), the immune system plays an important role in pathogenesis as long as different lymphoid organs (Peyer's patches, lymph nodes and spleen) may facilitate the accumulation and further spread of prions after peripheral exposure. In this work we investigated the changes in lymphoid and dendritic cell (DC) populations as well as the implications of different cytokines during disease progression after experimental oral inoculation of prions in a transgenic mouse model. At different days post-inoculation (dpi), T and B lymphocytes and DC populations from lymphoid organs, blood and brain were analyzed by flow cytometry and immunohistochemistry. Besides time related variations in lymphoid cell numbers due to the aging of the animals significant changes related with the infection were found in mesenteric lymph nodes, peripheral blood leukocytes (PBLs) as well as in spleen, affecting the CD4/CD8 ratio. In contrast, little or no variation was detected in Peyer's Patches or in thymus either associated with aging or the infection status. At individual time points significant differences between infected and control mice were seen in the CD8, CD4 and DC populations, with less evidence of differences in the B cell compartment. Finally, a pro-inflammatory phenotype occurred at early times in the spleen, where the levels of lymphotoxin-β mRNA were found augmented with respect to controls. Altogether, these results suggest that normal regulation of lymphocyte populations becomes altered along the progression of a prion infection.

The Prognostic Importance of T and NK-Cells in a Consecutive Series of Newly Diagnosed Patients with Chronic Lymphocytic Leukemia.

Background: Patients with CLL have a variable clinical course. Current novel prognostic indicators (ZAP-70, CD38, IgVH gene mutation status, FISH) are able to identify early-stage CLL patients at high risk of rapid disease progression. These prognostic factors focus on characteristics of the malignant B cell clone and are not currently changeable factors. Identification of modifiable characteristics of the host unrelated to the leukemic B-cell but that relate to CLL-specific survival may provide opportunities for therapeutic intervention. Since the immune system is likely to modulate disease progression in CLL patients, we evaluated critical features of that system in relation to the size of the circulating clonal B-cell population in a CLL cohort.Methods: 186 consecutive patients with CLL who were evaluated at Mayo Clinic within 2 months of diagnosis between 2000 and 2002 were identified. The primary end-point of the study was to assess if host immunity affected time to treatment (TTT). Baseline flow cytometry analysis was available for 166 patients (88%) and was used to calculate the absolute number of T-cell and natural kill (NK)-cells present at diagnosis. The size of the T-cell/NK-cell compartment relative to the size of the malignant monoclonal B-cell (MBC) compartment was then evaluated by calculating the NK:MBC ratio and T:MBC ratio. Relationships with other prognostic parameters and with TTT were evaluated.Results: Patients exhibited substantial variation in the absolute number of T-cells and NK cells as well as T:MBC and NK:MBC ratios at the time of diagnosis. Higher T:MBC and NK:MBC ratios were observed among patients with early Rai stage and mutated IgVH genes (all P≤0.0002). As continuous variables, both the T:MBC ratio (p value=0.03) and NK:MBC ratio (p value=0.02) were associated with TTT. Since they correlated with TTT as continuous variables, thresholds to classify MBC, T:MBC ratio, and NK:MBC were identified using the method of Contal and O'Quigly. On multivariate Cox modeling including stage, CD38, absolute MBC count, T:MB ratio, and NK:MB ratio, the independent predictors of TTT were disease stage, T:MB ratio, and NK:MB ratio (Table 1).Conclusion: Measurable characteristics of the host immune system appear to relate to the rate of disease progression in patients with newly diagnosed CLL. These characteristics can be modified and continued evaluation of immunomodulatory drugs, vaccination strategies, and cellular therapies to delay/prevent disease progression are warranted. Additional studies exploring how interactions between the host immune system and the leukemic clone influence clinical outcomes are needed.

Effect of Chemical and Physical Properties of River Water in Shatt Al‐Hilla on Phytoplankton Communities

This paper aims to study the chemical and physical properties in the ecological system of Shatt Al‐Hilla in Babylon Governorate in Iraq and its effect on phytoplankton population. In this context, several limnological parameters were evaluated during the period from December 2003 through November 2004 from four sampling stations sited along Shatt Al‐Hilla. The physical parameters included: temperature, turbidity and electrical conductivity. The chemical parameters included: pH, alkalinity dissolved oxygen, total hardness and the concentrations of nitrite, nitrate, phosphate and sulphate. A total of 154 species were recorded. Ninety‐seven species of the total belong to Bacillariophyceae, 37 species belong to Chlorophyceae, 13 species to Cyanophyceae, 5 species to Chrysophyceae, and 2 species to Euglenophyceae. Bimodal variation of phytoplankton was observed. Five genus of phytoplankton were the highest number of species ( Nitzschia, Navicula, Gomphonema, Cymbella and Scendesmus). Some species was occurred continuously during study period such as, Cyclotella ocellata, Cyclotella meneghiniana, Aulacoseria distans, and Gomphonema abbreviatum. The phytoplankton communities at all sampling sites showed a clear seasonal variation in phytoplankton cell number. However, no significant correlation between total cell number of phytoplankton and nutrient concentration was observed. The study was revealed the city sewage discharge, agriculture and urban run‐off were affecting the water quality of Shatt Al‐Hilla.

The quantitative trait gene latexin influences the size of the hematopoietic stem cell population in mice

We mapped quantitative trait loci that accounted for the variation in hematopoietic stem cell (HSC) numbers between young adult C57BL/6 (B6) and DBA/2 (D2) mice. In reciprocal chromosome 3 congenic mice, introgressed D2 alleles increased HSC numbers owing to enhanced proliferation and self-renewal and reduced apoptosis, whereas B6 alleles had the opposite effects. Using oligonucleotide arrays, real-time PCR and protein blots, we identified latexin (Lxn), a gene whose differential transcription and expression was associated with the allelic differences. Expression was inversely correlated with the number of HSCs; therefore, ectopic expression of Lxn using a retroviral vector decreased stem cell population size. We identified clusters of SNPs upstream of the Lxn transcriptional start site, at least two of which are associated with potential binding sites for transcription factors regulating stem cells. Thus, promoter polymorphisms between the B6 and D2 alleles may affect Lxn gene expression and consequently influence the population size of hematopoietic stem cells.

Human pelvic extramural ganglion cells: a semiquantitative and immunohistochemical study

In pelvic surgery, much attention is paid to nerve bundles but not to ganglion cells. Using serial section histology of 14 postmortem-treated hemipelvis (eight males, six females; mean, 79 years old), we examined the population number, distribution, and tyrosine hydroxylase-immunoreactivity (TH-IR; suggesting sympathetic neurons) of extramural pelvic ganglion cells. All pelvic ganglion cells were uniformly sized (25-30 microm) contrasting with small intramural rectal neurons. Abundant ganglion cells (30,000-140,000 unilaterally) existed not only along the pelvic viscera except for the rectum, but also along the hypogastric nerve, pelvic splanchnic nerve, pelvic plexus, and associated branches excluding those within the mesorectum. The intrapelvic ganglion cells outside the sympathetic trunk did not form macroscopically identifiable ganglia, but made small clusters (0.1-2.0 mm of maximum diameter) or were diffusely scattered within nerve bundles. More than half of these cells appeared TH-IR positive, although the positive/negative proportion differed between nerves and specimens. Greater numbers of ganglion cells were found in dorsosuperior sites (e.g., around the seminal vesicle) rather than in ventroinferior sites (e.g., along the urethra) in males, and vice versa in females. However, in total cell numbers, interindividual variations were evident rather than intergender difference. Due to significant interindividual variations in cell number, differences are likely to exist between patients in "resistance" to surgical stresses. We hypothesized that pelvic ganglion cells are liable to be damaged due to drying along the surgical margin, hypoxia in venous bleeding, pressure from surgical retractors, extension stress with taping and excess traction and/or direct injury with electrical scalpels.

Delay differential equations and the dose-time dependence of early radiotherapy reactions

The dose-time dependence of early radiotherapy reactions impacts on the design of accelerated fractionation schedules--oral mucositis, for example, can be dose limiting for short treatments designed to avoid tumor repopulation. In this paper a framework for modeling early reaction dose-time dependence is developed. Variation of stem cell number with time after the start of a radiation schedule is modeled using a first-order delay differential equation (DDE), motivated by experimental observations linking the speed of compensatory proliferation in early reacting tissues to the degree of tissue damage. The modeling suggests that two types of early reaction radiation response are possible, stem cell numbers either monotonically approaching equilibrium plateau levels or overshooting before returning to equilibrium. Several formulas have been derived from the delay differential equation, predicting changes in isoeffective total radiation dose with schedule duration for different types of fractionation scheme. The formulas have been fitted to a wide range of published animal early reaction data, the fits all implying a degree of overshoot. Results are presented illustrating the scope of the delay differential model: most of the data are fitted well, although the model struggles with a few datasets measured for schedules with distinctive dose-time patterns. Ways of extending the current model to cope with these particular dose-time patterns are briefly discussed. The DDE approach is conceptually more complex than earlier descriptive dose-time models but potentially more powerful. It can be used to study issues not addressed by simpler models, such as the likely effects of increasing or decreasing the dose-per-day over time, or of splitting radiation courses into intense segments separated by gaps. It may also prove useful for modeling the effects of chemoirradiation.

Nutritional strategies to counter stress to the immune system in athletes, with special reference to football

Although epidemiological data indicate that athletes are at increased risk of upper respiratory tract infection during periods of heavy training and the 1 – 2 week period following endurance race events, there is very limited information on the responses to football training and match-play. For several hours after heavy exertion, components of both the innate (e.g. natural killer cell activity and neutrophil oxidative burst activity) and adaptive (e.g. T and B cell function) immune system exhibit suppressed function. Although such responses to football training and competition do not appear to be as pronounced, variations in immune cell numbers and function are reported in professional footballers over the course of a season. Attempts have been made through nutritional means (e.g. glutamine, vitamins C and E, and carbohydrate supplementation) to attenuate immune changes following intensive exercise and thus lower the risk of upper respiratory tract infection. Carbohydrate supplementation during heavy exercise has emerged as a partial countermeasure and attenuates increases in blood neutrophil counts, stress hormones, and inflammatory cytokines, but has little effect on decrements in salivary IgA output or natural killer cell function. Animal research indicates that other nutritional components such as beta-glucan, quercetin, and curcumin warrant human investigations to determine if they are effective countermeasures to exercise-induced immune dysfunction.

First-line use of enfuvirtide-containing HAART regimen with dramatic clinical and immunological improvement in three cases

First-line use of enfuvirtide-containing HAART regimen with dramatic clinical and immunological improvement in three cases

Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana

Size is an important parameter in the characterization of organ morphology and function. To understand the mechanisms that control leaf size, we previously isolated a number of Arabidopsis thaliana mutants with altered leaf size. Because leaf morphogenesis depends on determinate cell proliferation, the size of a mature leaf is controlled by variation in cell size and number. Therefore, leaf-size mutants should be classified according to the effects of the mutations on the cell number and/or size. A group of mutants represented by angustifolia3/grf-interacting factor1 and aintegumenta exhibits an intriguing cellular phenotype termed compensation: when the leaf cell number is decreased due to the mutation, the leaf cell size increases, leading to compensation in leaf area. Several lines of genetic evidence suggest that compensation is probably not a result of the uncoupling of cell division from cell growth. Rather, the evidence suggests an organ-wide mechanism that coordinates cell proliferation with cell expansion during leaf development. Our results provide a key, novel concept that explains how leaf size is controlled at the organ level.

Regulation of Fruit Growth and Fruit Size in Apple

Fruit development in apple cultivars varying in their ultimate fruit size was analyzed using cytology, flow cytometry (FCM), and semi-quantitative RT-PCR. Fruit size variation across cultivars was largely explained by variation in cell number. The cell division phase lasted for less than 30 days in all varieties, less than previously believed. A distinct overlap between the cell division and cell expansion phases was present. Analysis of the relative cell production rate (rCPR) showed a major peak about 10 days after full bloom (DAFB) after which it declined. Comparison of the rCPR across varieties suggested distinct patterns of cell production with `Gala' having a low but sustained rCPR, `Pixy Crunch' a short but high rCPR, and `Golden Delicious' having a high and sustained rCPR. FCM analysis also showed similar patterns with a peak in the proportion of dividing cells about 10 DAFB followed by a decline. To further understand regulation of cell number, four cell cycle related genes were cloned from `Gala'. Cyclin Dependent Kinase B (CDK B) and Cyclin B were found to be highly cell division phase specific in their expression. Analysis of gene expression by semi-quantitative RT-PCR indicated peak expression of these two genes at 5-10 DAFB, consistent with the peaks in rCPR and proportion of dividing cells. Comparison of gene expression across the varieties showed higher peak expression of the above genes in the larger-fruited `Golden Delicious' than in the smaller-fruited `Gala.' This study provides novel insight into the regulation of fruit development in apple and also suggests a role for the cell cycle genes in fruit size regulation.

Eutely, cell lineage, and fate within the ascidian larval nervous system: determinacy or to be determined?

The larval central nervous system (CNS) of the ascidian Ciona intestinalis (L., 1767) arises from an embryonic neural plate and contains sufficiently few cells, about 330, to enable definitive counts. On the basis of such counts, there is evidence both for cell constancy (eutely) in the larval CNS and for small variations in the overall numbers of cells and among defined cell types within this total. However, evidence for the range of such deviations and the existence of a true phenotypic wild type are lacking. The record of cell lineage, i.e., the mitotic ancestry of each cell, and the fates of some of these cells have recently received increased documentation in both the genus Ciona and Halocynthia roretzi (von Drasche, 1884). Relatively few generations of cells, between 10 and 14, form the entire CNS in C. intestinalis, and cell death does not occur prior to larval hatching. The tiny complement of larval CNS cells can therefore be seen as the product of a small fixed number of determinate cleavages, and variations in cell number as the product of minor deviations in this mitotic ancestry. Within these lineage records, some cell fates have already been identified, but knowledge of most is lacking because the cells lack markers or other identifying features. Nevertheless, this tiny nervous system offers the prospect that all its cells can one day be identified, and their developmental histories and larval functions analyzed, cell by cell.

Analysis of the Tomato Fruit Growth Response to Temperature and Plant Fruit Load in Relation to Cell Division, Cell Expansion and DNA Endoreduplication

To better understand the regulation of fruit growth in response to environmental factors, the effects of temperature and plant fruit load on cell number, cell size and DNA endoreduplication were analysed. Plants were grown at 20/20 degrees C, 25/25 degrees C and 25/20 degrees C day/night temperatures, and inflorescences were pruned to two ('2F') or five ('5F') flowers. Despite a lower fruit growth rate at 20/20 degrees C, temperature did not affect final fruit size because of the compensation between cell number and size. The higher cell number at 20/20 degrees C (9.0 x 10(6) against 7.9 x 10(6) at 25/25 degrees C and 7.7 x 10(6) at 25/20 degrees C) resulted from an extended period of cell division, and the smaller cell size was due to a shorter period of expansion rather than a lower expansion rate. By contrast, the lower fruit growth rate and size of 5F fruits compared with 2F fruits resulted from the slow down of cell expansion, whereas the number of cells was hardly affected in the proximal fruit. However, within the inflorescence the decreasing gradient of fruit size from proximal to distal fruits was due to a decrease in cell number with similar cell size. Fruit size variations within each treatment were always positively correlated to variations in cell number, but not in cell size. Negative correlations between cell size and cell number suggested that cells of tomato pericarp can be seen as a population of competing sinks. Mean ploidy was slightly delayed and reduced in 5F fruits compared with 2F fruits. It was highest at 25/25 degrees C and lowest at 25/20 degrees C. Treatments did not affect ploidy and cell size in similar ways, but within each treatment, positive correlations existed between mean ploidy and cell size, though significant only in the 2F-25/20 treatment.

Genetic control of NKT cell numbers.

NKT cells play a critical role in shaping the character and strength of a wide range of immune responses, including those against pathogens, tumours, allografts and autologous tissues. Because numbers of NKT cells affect clinical outcomes in a wide range of disease models, and this characteristic demonstrates allelic variation, the mapping of the locations and identification of the coding sequences of these genes has become a matter of significant importance. Here, we review the results to date that examine the effects of targeted deletion of a number of candidate genes, as well as the congenic and genetic linkage analyses that have attempted to localize allelic loci that affect NKT cell numbers. Although a number of candidate genes have been examined, there is no evidence that any of these contribute to variation in NKT cell numbers in natural populations. Two of the most important genetic regions controlling NKT cell numbers are Nkt1 on chromosome 1, which may contribute to lupus susceptibility, and Nkt2 on chromosome 2, which appears to contribute to diabetes susceptibility. Of great interest is a third locus on chromosome 18, identified in a novel congenic line, which can confer an absolute deficiency in this important immunoregulatory lymphocyte population.

Characterisation of microbial community composition of a Siberian tundra soil by fluorescence in situ hybridisation

The bacterial community composition of the active layer (0–45 cm) of a permafrost-affected tundra soil was analysed by fluorescence in situ hybridisation (FISH). Arctic tundra soils contain large amounts of organic carbon, accumulated in thick soil layers and are known as a major sink of atmospheric CO 2. These soils are totally frozen throughout the year and only a thin active layer is unfrozen and shows biological activity during the short summer. To improve the understanding of how the carbon fluxes in the active layer are controlled, detailed analysis of composition, functionality and interaction of soil microorganisms was done. The FISH analyses of the active layer showed large variations in absolute cell numbers and in the composition of the active microbial community between the different horizons, which is caused by the different environmental conditions (e.g., soil temperature, amount of organic matter, aeration) in this vertically structured ecosystem. Universal protein stain 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF) showed an exponential decrease of total cell counts from the top to the bottom of the active layer (2.3 × 10 9–1.2 × 10 8 cells per gram dry soil). Using FISH, up to 59% of the DTAF-detected cells could be detected in the surface horizon, and up to 84% of these FISH-detected cells could be affiliated to a known phylogenetic group. The amount of FISH-detectable cells decreased with increasing depth and so did the diversity of ascertained phylogenetic groups.

Growth hormone increases Stat5 and Stat1 expression in lactating goat mammary gland: a specific effect compared to milking frequency

In ruminants, both milking frequency and exogenous GH treatment affect milk production. In a previous report, we showed that the modulation of milk yield due to variations in milking frequency and GH treatment was associated with variations in mammary cell numbers. The aim of this study was to clarify the different mechanisms governing the effects of GH treatment and milking frequency on signal transducer and activator of transcription (Stat) expression and activation, and on the expression of genes involved in mammary cell differentiation. Six Saanen goats in late lactation were milked once daily from one half-udder and thrice daily from the other half-udder for 23 days. At the same time, the goats were divided into two groups: GH-treated versus control group. After slaughter of the goats, soluble mammary proteins and RNA were extracted from half-udder samples. Levels of Stat5, Stat3 and Stat1 proteins and the Stat activation by phosphorylation were analysed by Western blot. The amounts of Stat5 protein and mRNA were significantly elevated by GH treatment in all half-udders (milked once or thrice daily). Positive Stat5 immunoreactivity was principally localised in the nuclei of epithelial cells, with heterogeneous intensity between cells. No significant changes in Stat5 protein phosphorylation levels were observed. Furthermore, GH significantly increased Stat1 protein levels, without modifying the level of Stat1 tyrosine phosphorylation, and tended to reduce the abundance of Stat3 protein. In contrast, milking frequency failed to modify Stat gene expression, protein level and phosphorylation. Using Northern blot, we showed that levels of kappa casein and prolactin receptor mRNA were not affected by the treatments. These observations suggest that GH probably acts specifically on mammary cells by regulating the expression of Stat1, 3 and 5. In contrast, milking frequency does not act through this regulatory pathway.

Differences and Yearly Variation in Number and Size of Mesocarp Cells in Sweet Cherry (Prunus avium L.) Cultivars and Related Species

To characterize factors determining the varietal difference of the fruit size in sweet cherries, we studied the relationships among fruit weight, number of mesocarp cells and radial cell lengths in 56 cultivars and selections of sweet cherries and related species. In addition, the effect of flower thinning on the number of mesocarp cells and the dependence of cell number on young fruit weight were analyzed. A high correlation coefficient was obtained between fruit thickness and mesocarp thickness versus fruit weights. All parameters measured showed large varietal differences. The correlation coefficient between mesocarp cell number and length to the differences in fruit weight varied yearly from 0.611 to 0.706, and from 0.488 to 0.599, respectively. The contribution of the cell number to fruit weight was larger than that of cell length in every year tested. The yearly correlation coefficients were 0.869-0.931 for the fruit weight, 0.826- 0.852 for the number of mesocarp cells, and 0.482-0.674 for the radial cell length. Fruit weights of the wild species were considerably lighter than those of the commercial cultivars. The numbers of mesocarp cells and cell lengths of the former were about half those of the latter. From these results, we conclude that both the mesocarp cell numbers and their lengths affect the varietal differences in the fruit weight. However, the cell number has a larger contribution and is a more stable parameter. Young fruit size and flower thinning had almost no effect on mesocarp cell number. Therefore, we conclude that cell number determination in mesocarp is a useful method for estimating varietal differences in fruit weight of sweet cherry.