Insulin impairs the maturation of chondrocytes in vitro

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 µM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [³H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.

Neonatal administration of citalopram delays somatic maturation in rats

We investigated the somatic maturation of neonate rats treated during the suckling period with citalopram, a selective serotonin reuptake inhibitor. Groups with 6 male neonates were randomly assigned to different treatments 24 h after birth. Each litter was suckled by one of the dams until the 21st postnatal day. Body weight, head axis and tail length were measured daily from the 1st to the 21st postnatal day. Time of ear unfolding, auditory conduit opening, incisor eruption, and eye opening was determined. Pups received 5 mg (Cit5), 10 mg (Cit10) or 20 mg/kg (Cit20) citalopram sc, or saline (0.9% NaCl, w/v, sc). Compared to saline, body weight was lower (24.04%, P < 0.01) for Cit10 from the 10th to the 21st day and for Cit20 from the 6th to the 21st day (38.19%, P < 0.01). Tail length was reduced in the Cit20 group (15.48%, P < 0.001) from the 8th to the 21st day. A reduction in mediolateral head axis (10.53%, P < 0.05) was observed from the 11th to the 21st day in Cit10 and from the 6th to the 21st day in Cit20 (13.16%, P < 0.001). A reduction in anteroposterior head axis was also observed in the Cit20 group (5.28%, P < 0.05) from the 13th to the 21stday. Conversely, this axis showed accelerated growth from the 12th to the 21stday in the Cit5 group (13.05%, P < 0.05). Auditory conduit opening was delayed in the Cit5 and Cit20 groups and incisor eruption was delayed in all citalopram groups. These findings show that citalopram injected during suckling to rats induces body alterations and suggest that the activity of the serotoninergic system participates in growth mechanisms.

Maturation of pig oocytes in vitro in a medium with pyruvate

The aim of in vitro maturation oocyte systems is to produce oocytes of comparable quality to those derived in vivo. The present study was designed to examine the surface morphological changes of the cumulus-oocyte complex (COC) and nuclear maturation in a culture system containing pyruvate. Ovaries were obtained from a slaughterhouseand transported to the laboratory within 2 h at 35-39ºC,and rinsed three times in 0.9% NaCl. The COCs were harvested from the ovaries and in vitro maturation was evaluated in San Marcos (SM) medium, a chemically defined culture system containing 22.3 mM sodium pyruvate. Oocytes were cultured in SM, SM + porcine follicular fluid (pFF) and in SM + pFF + gonadotropins (eCG and hCG) for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Oocytes matured in SM (40.9%) and SM + pFF (42.9%) showed moderate cumulus expansion, whereas oocytes matured in SM + pFF + gonadotropins (54.6%) showed high cumulus expansion. The maturation rate of cultured oocytes, measured in function of the presence of the polar corpuscle, did not differ significantly between SM (40.9 ± 3.6%) and SM + pFF (42.9 ± 3.7%). These results indicate that pig oocytes can be successfully matured in a chemically definedmedium and suggest a possible bifunctional role of pyruvate as an energy substrate and as an antioxidant protecting oocytes against the stress of the in vitro environment.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), α-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with α-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Induced maturation of hepatic progenitor cellsin vitro

Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.

Stimulated mast cells promote maturation of myocardial microvascular endothelial cell neovessels by modulating the angiopoietin-Tie-2 signaling pathway

Angiopoietin (Ang)-1 and Ang-2 interact in angiogenesis to activate the Tie-2 receptor, which may be involved in new vessel maturation and regression. Mast cells (MCs) are also involved in formation of new blood vessels and angiogenesis. The present study was designed to test whether MCs can mediate angiogenesis in myocardial microvascular endothelial cells (MMVECs). Using a rat MMVEC and MC co-culture system, we observed that Ang-1 protein levels were very low even though its mRNA levels were increased by MCs. Interestingly, MCs were able to enhance migration, proliferation, and capillary-like tube formation, which were associated with suppressed Ang-2 protein expression, but not Tie-2 expression levels. These MCs induced effects that could be reversed by either tryptase inhibitor [N-tosyl-L-lysine chloromethyl ketone (TLCK)] or chymase inhibitor (N-tosyl-L-phenylalanyl chloromethyl ketone), with TLCK showing greater effects. In conclusion, our data indicated that MCs can interrupt neovessel maturation via suppression of the Ang-2/Tie-2 signaling pathway.

The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells

We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

Postharvest respiratory activity and changes in some chemical constituents during maturation of yellow mombin (Spondias mombin) fruit

Mature fruit from the yellow mombin (Spondias mombin) was monitored for its respiration activity. Mature green fruit from the yellow mombin was stored in closed glass chambers and the concentration of oxygen and carbon dioxide at the end of a six hour respiration period was determined. At the same interval of time, the lid of the chamber was opened for air renewal. The increase in carbon dioxide and decrease in oxygen concentration demonstrated that the fruit was climacteric. The maximum liberation of CO2 54.2 mL Kg-1 h-1 and maximum absorption of O2 49.0 mL Kg-1 h-1 occurred 186 hours after the harvest which, obviously, represented the optimum fruit quality after the senescence process started. The respiratory quotient of fruit at a climacteric maximum was 1.11 representing the oxidation of carbohydrates. Total soluble solids increased from 9.1 °Brix (initial) to 13.7 °Brix (climacteric maximum) during maturation, while the total number of acids in the fruit decreased during maturation i.e. from 1.55% initially to 1.40% at pre-climacteric, 1.0% at climacteric maximum and 0.8% in the post-climacteric stage. A similar behaviour was observed in the case of ascorbic acid. There was a continuous decrease in chlorophyll and a continuous increase in the carotenoid content of fruit during maturation.

Effect of high hydrostatic pressure on antioxidant content of 'Ataulfo' mango during postharvest maturation

The objective of this study was to evaluate the effect of pressurization on the concentration of some antioxidant compounds and the antiradical efficiency during the ripening process of 'Ataulfo' mango. The fruits at physiological maturity stage were pressurized at 15, 30, or 60 MPa for 10 or 20 min. Control fruits were not pressurized. The fruits were stored at 25 °C and changes in the concentration of ascorbic acid, total phenols, total flavonoids, total carotenoids, and antiradical efficiency were evaluated. It was demonstrated that in 'Ataulfo' mango high hydrostatic pressure treatments at 60 and 30 MPa for 20 minutes induced the synthesis of ascorbic acid during storage maybe as a consequence of physiological changes and possible structural modification of the cells, while the fruits pressurized at 15 MPa showed no effect on this parameter. On the other hand, the use of 15 MPa for 10 minutes increased the synthesis of phenols, flavonoids, carotenoids, and antiradical efficiency in 'Ataulfo' mango compared to that of the control fruit. In conclusion, this behavior seemed to be due to the low hydrostatic pressure treatments (15 Mpa), which stimulated the synthesis of antioxidants in the mango fruit and ripening was not inhibited.

Growth and maturation of pequi fruit of the Brazilian cerrado

The objective of this study was to characterize the development of pequi fruit (Caryocar brasiliense) of the Brazilian cerrado. It takes 84 days (12 weeks) for pequi to develop with the onset of flowering in September and early fruit set in January. Pequi fruit showed a simple sigmoid growth curve, and its growth was characterized based on fresh mass and longitudinal and transverse diameters. The contents of titratable acidity, soluble solids, β-carotene, and vitamin C increased during fruit growth, reaching their maximum values at the 12th week (84 days) after anthesis. Pequi is a fruit with an extremely high respiratory activity; its respiratory rate decreased during its development. Pequi fruit has been classified as a non-climacteric fruit due to the decrease of both respiration and ethylene production rates during maturation and ripening.

Effect of seed maturation stages on physical properties and antioxidant activity in flaxseed ( Linum usitatissimum L.)

Abstract The changes in flaxseed constituents at different stages of maturity are reported. The physical properties and antioxidant activity of flaxseed oil during flaxseed development have been evaluated. Continuous decrease in total polyphenol content during flaxseed development. All the results showed no significant differences between HPLC-MS and TLC for quantitative determination of phospholipids classes. The fatty acid compositions of individual phospholipids were also reported. The antioxidant activity of oilseed was assessed by means of 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging assay. The test demonstrated that the antioxidant activity of the flaxseed oil was found to be harvesting time-dependant.

Incidence of green soybean seeds as a function of environmental stresses during seed maturation

Hot and dry weather conditions during soybean [Glycine max (L.) Merrill] seed maturation can cause forced maturation of the seed, resulting in the production of high levels of green seed, which may be detrimental to seed germination. These stressful conditions were imposed on soybean plants during seed maturation to investigate the production of green seeds and seed quality. Plants of the CD 206 cultivar were grown in a greenhouse until the R5.5 growing stage and then transferred to phytotrons at R6 and R7.2 for stress induction. Plants were subjected to two temperature regimes, high (28ºC to 36ºC) and normal (19ºC to 26ºC), and four soil water availability conditions, control (adequate water supply), 30% gravimetric moisture (GM), 20% GM and no water supply. Seed were harvested at R9. Green seed percentages and 100-seed weights from the lower, middle and upper thirds of each plant were determined. Seed quality was assessed by germination, tetrazolium (viability and vigor) and electrical conductivity tests. Occurrence of green seed varied from 9% to 86%, depending on the severity of the stresses imposed. High temperature, coupled with no water supply at R6, resulted in a pronounced occurrence of green seeds. There was no difference in the percentage of green seeds among the plant segments. Seed quality was negatively affected by the incidence of green seeds. A procedure for screening soybean genotypes in a phytotron for their tolerance and/or susceptibility to the production of green seeds was developed.

Storage of 'Swingle' citrumelo seeds in different maturation stages subjected to fungicide treatment

The establishment of citrus orchards with rootstocks from seed with a low physiological quality has been a recurring problem. Low quality seeds directly affect both the final stand and the time required for seedling production. The irregular maturation of fruits, seed recalcitrance, and the high incidence of fungi, make long term storage difficult, even at low temperatures. This study evaluated the storage potential and the use of fungicide treatments on 'Swingle' citrumelo seeds extracted from fruits collected at two maturation stages, green or ripe. The seeds were subjected to a thermal treatment, treated with Derosal, Thiram or Tecto+Captan fungicides, packaged in impermeable polyethylene bags and stored in a cold chamber for nine months. Every three months, the physiological and sanitary qualities of the seeds were evaluated from germination and sanitary tests and also from enzyme profiles. Seeds from green fruits deteriorated less than those from mature fruits; deterioration increased up to nine months of storage; treatment with the Tecto+Captan mixture gave effective pathogen control and maintained seed quality during storage. The germination of the green and ripe seeds is satisfactory (70%) after three months storage.

Physiological quality of coffee seeds produced under different levels of solar radiation and maturation stages

In Brazil, although the coffee plantations are predominantly grown under full sunlight, the use of agroforestry systems can lead to socioeconomic advantages, thus providing a favorable environment to the crop by promoting its sustainability as well as environmental preservation. However, there is a lack of information on physiological quality of the coffee seeds produced under different levels of solar radiation. Within this context, the objective of this study was to evaluate the influence of different levels of solar radiation and maturation stages on the physiological quality of coffee (Coffea arabica L.) seeds, cv. Acaiá Cerrado MG-1474. Three levels of solar radiation (plants grown under full sunlight; under plastic screens of 35% shading; and under plastic screens of 50% shading) and three maturation stages (cherry, greenish-yellow and green) were assessed. Physiological quality of seeds was assessed by using germination test, first count of germination, abnormal seedlings, dead seeds, and seedlings with open cotyledonary leaves. Electrophoretic analysis of isoenzymes catalase, esterase, superoxide dismutase and peroxidase was also performed. With the evolution of development the coffee seeds presents increases on physiological quality, and at its beginning the seeds show improvements on quality with the reduction of solar radiation.

A comparison of cognitive processes and components of neurological maturation in 3-and-11 year-old children

The relationship between the child's cogni tive development and neurological maturation has been of theoretical interest for many year s. Due to diff iculties such as the lack of sophisticated techniques for measur ing neurolog ical changes and a paucity of normative data, few studies exist that have attempted to correlate the two factors. Recent theory on intellectual development has proposed that neurological maturation may be a factor in the increase of short-term memory storage space. Improved technology has allowed reliable recordings of neurolog ical maturation.. In an attempt to correlate cogni tive development and neurological maturation, this study tested 3-and II-year old children. Fine motor and gross motor short-term memory tests were used to index cogni tive development. Somatosensory evoked potentials elici ted by median nerve stimulation were used to measure the time required for the sensation to pass along the nerve to specific points on the somatosensory pathway. Times were recorded for N14, N20, and P22 interpeak latencies. Maturation of the central nervous system (brain and spinal cord) and the peripheral nervous system (outside the brain and spinal cord) was indi~ated by the recorded times. Signif icant developmental di fferences occurred between 3-and ll-year-olds in memory levels, per ipheral conduction velocity and central conduction times. Linear regression analyses showed that as age increased, memory levels increased and central conduction times decreased. Between the ll-year-old groups, there were no significant differences in central or peripheral nervous system maturation between subjects who achieved a 12 plus score on the digit span test of the WISC-R and those who scored 7 or lower on the same test. Levels achieved on the experimental gross and fine motor short-term memory tests differed significantly within the ll-year-old group.