Biomedical subjects

R J Wilkins

Publications and source records attributed to R J Wilkins.

At least 19 recordsLinked to original sources

PubMed2006

Influences of buffer systems on chondrocyte growth during long-term culture in alginate.

OBJECTIVE: Chondrocyte behavior is very sensitive to culture environment such as physical and biochemical conditions. As extracellular pH (pHo) and the existence of bicarbonate could affect the chondrocyte fate, hence, the purpose of this study is to investigate the buffer system effect on chondrocyte fate during relatively long-term culture. METHODS: In order to examine whether effects seen were due to bicarbonate or to pHo, we had to devise a system which could differentiate between the two effects. Culture media buffered by N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) only and the combination of HEPES and bicarbonate were used. Bovine articular chondrocytes were cultured in alginate beads for up to 12 days. pHo was kept constant by culture of 3 beads in 2 ml culture medium. Cell density, intracellular pH (pHi) and glycosaminoglycan (GAG) were measured at day 5 and day 12. Cell morphology, distribution and viability in alginate beads were monitored over 12 days of culture. RESULTS: Compared to culture in the absence of bicarbonate, a higher proliferation rate of chondrocytes was observed in the presence of bicarbonate. pHi was more alkaline, about 0.2 pH unit, in the presence of bicarbonate than that in the absence of bicarbonate. About 50% more GAG was deposited in alginate beads when chondrocytes were cultured in the combination of HEPES and bicarbonate, compared to chondrocytes cultured in the absence of NaHCO3 at the end of 12 days of culture. CONCLUSION: The presence of bicarbonate results in more alkaline in the pHi of bovine chondrocytes after long-term culture. The combination of bicarbonate and HEPES in culture medium improves cell growth, matrix production in three-dimensional alginate beads.

Alginates↗

PubMed2006

The effect of O2 tension on pH homeostasis in equine articular chondrocytes.

OBJECTIVE: To determine the effects of varying O(2) on pH homeostasis, based on the hypothesis that the function of articular chondrocytes is best understood at realistic O(2) tensions. METHODS: Cartilage from equine metacarpophalangeal/tarsophalangeal joints was digested with collagenase to isolate chondrocytes, and then loaded with the pH-sensitive fluorophore 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein. The radioisotope(22)Na(+) was used to determine the kinetics of Na(+)/H(+) exchange (NHE) and the activity of the Na(+)/K(+) pump, and ATP levels were assessed with luciferin assays. Levels of reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescein diacetate. RESULTS: The pH homeostasis was unaffected when comparing tissue maintained at 20% O(2) (the level in water-saturated air at 37 degrees C) with that at 5% O(2) (which approximates the normal level in healthy cartilage); however, an O(2) tension of <5% caused a fall in intracellular pH (pH(i)) and slowed pH(i) recovery following acidification, an effect mediated via inhibition of NHE activity (likely through acid extrusion by NHE isoform 1). The Na(+)/K(+) pump activity and intracellular ATP concentration were unaffected by hypoxia, but the levels of ROS were reduced. Hypoxic inhibition of NHE activity and the reduction in ROS levels were reversed by treatment with H(2)O(2), Co(2+), or antimycin A. Treatment with calyculin A also prevented hypoxic inhibition of NHE activity. CONCLUSION: The ability of articular chondrocytes to carry out pH homeostasis is compromised when O(2) tensions fall below those normally experienced, via inhibition of NHE. The putative signal is a reduction in levels of ROS derived from mitochondria, acting via altered protein phosphorylation. This effect is relevant to both physiologic and pathologic states of lowered O(2), such as in chronic inflammation.

Adenosine Triphosphate↗

PubMed2006

Prolactin delays hair regrowth in mice.

Mammalian hair growth is cyclic, with hair-producing follicles alternating between active (anagen) and quiescent (telogen) phases. The timing of hair cycles is advanced in prolactin receptor (PRLR) knockout mice, suggesting that prolactin has a role in regulating follicle cycling. In this study, the relationship between profiles of circulating prolactin and the first post-natal hair growth cycle was examined in female Balb/c mice. Prolactin was found to increase at 3 weeks of age, prior to the onset of anagen 1 week later. Expression of PRLR mRNA in skin increased fourfold during early anagen. This was followed by upregulation of prolactin mRNA, also expressed in the skin. Pharmacological suppression of pituitary prolactin advanced dorsal hair growth by 3.5 days. Normal hair cycling was restored by replacement with exogenous prolactin for 3 days. Increasing the duration of prolactin treatment further retarded entry into anagen. However, prolactin treatments, which began after follicles had entered anagen at 26 days of age, did not alter the subsequent progression of the hair cycle. Skin from PRLR-deficient mice grafted onto endocrine-normal hosts underwent more rapid hair cycling than comparable wild-type grafts, with reduced duration of the telogen phase. These experiments demonstrate that prolactin regulates the timing of hair growth cycles in mice via a direct effect on the skin, rather than solely via the modulation of other endocrine factors.

Animals↗

PubMed2004

Effects of anti-GLUT antibodies on glucose transport into human erythrocyte ghosts.

We have studied the effects of anti-GLUT1 antibodies on the uptake of glucose into erythrocytes. Glucose transport into human erythrocyte ghosts was measured directly using 3H-2-deoxy-glucose, or indirectly by monitoring associated volume changes using light scattering. The uptake of glucose was significantly inhibited in ghosts resealed in solutions containing specific antibodies against GLUT1. Such an effect was not observed when an antibody against the oestrogen receptor, lacking specificity towards GLUT1, was employed instead. The antibodies were also without effect on the efflux of preloaded glucose from erythrocyte ghosts. The demonstration that anti-GLUT antibodies can inhibit glucose uptake is support for the hypothesis that they exaggerate the cytoplasmic barrier to glucose uptake created by endofacial segments of GLUT1.

Antibodies, Monoclonal↗

PubMed2004

Mechanisms contributing to intracellular pH homeostasis in an immortalised human chondrocyte cell line.

The maintenance of chondrocyte pH is an important parameter controlling cartilage matrix turnover rates. Previous studies have shown that, to varying degrees, chondrocytes rely on Na(+)/H(+) exchange to regulate pH. HCO(3)(-)-dependent buffering and HCO(3)(-)-dependent acid-extrusion systems seem to play relatively minor roles. This situation may reflect minimal carbonic anhydrase activity in cartilage cells. In the present study, the pH regulation of the human chondrocyte cell line, C-20/A4 has been characterised. Intracellular pH (pH(i)) was measured using the H(+)-sensitive fluoroprobe BCECF. In solutions lacking HCO(3)(-)/CO(2), pH(i) was approximately 7.5, and the recovery from intracellular acidification was predominantly mediated by a Na(+)-dependent, amiloride- and HOE 694-sensitive process. A small additional component which was sensitive to chloro-7-nitrobenz-2-oxa-1,3-diazole, an inhibitor of the V-type H(+)-ATPase, was also apparent. In solutions containing HCO(3)(-)/CO(2), pH(i) was approximately 7.2. Comparison of buffering capacity in the two conditions showed that this variable was not significantly augmented in HCO(3)(-)/CO(2)-containing media. The recovery from intracellular acidification was more rapid in the presence of HCO(3)(-)/CO(2), although under these conditions it was again largely dependent on Na(+) ions and inhibited by amiloride and HOE 694. A small component was inhibited by SITS, although this effect did not reach the level of statistical significance. These findings indicate that HCO(3)(-)-dependent processes play only a minimal role in pH regulation in C-20/A4 chondrocytes. pH regulation instead relies heavily on the Na(+)/H(+) exchanger together with a H(+)-ATPase. The absence of extrinsic (HCO(3)(-)/CO(2)) buffering is likely to reflect the low levels of carbonic anhydrase in these cells. In addition to providing fundamental information about a widely-used cell line, these findings support the contention that the unusual nature of pH regulation in chondrocytes reflects the paucity of carbonic anhydrase activity in these cells.

Acidosis↗

PubMed2004

The influence and interactions of hydrostatic and osmotic pressures on the intracellular milieu of chondrocytes.

The intracellular milieu of chondroctyes is regulated by an array of proteins in the cell membrane which operate as transport pathways, allowing ions and nutrients such as glucose and amino acids and metabolites such as lactate to cross the plasma membrane. Here we investigated the influence of hydrostatic pressure on intracellular calcium concentrations ([Ca(2+)](i)) in isolated bovine articular chondrocytes. We found that short applications of high hydrostatic pressures led to a significant increase in [Ca(2+)](i). The pressure-induced rise was abolished for long (240 sec) but not short (30 sec) pressure applications by removal of extracellular Ca(2+). The rise in pressure was also blocked by the inhibitors neomycin and thapsigargin confirming that pressure, by generating IP(3), led to an increase in [Ca(2+)](i) by mobilising the pool of Ca(2+) ions contained within intracellular stores. We also found that intracellular [Na(+)] was affected by a rise in osmotic pressure and further affected by application of hydrostatic pressure. The effect of hydrostatic pressure on sulphate incorporation depended strongly on extracellular osmolality. Since significant gradients in extracellular osmolality exist across intact cartilage, the results imply that responses of chondrocytes to the same pressure will vary depending on location in the joint. The results also indicate that hydrostatic pressures can affect several different transporter systems thus influencing the intracellular milieu and chondrocyte metabolism.

Animals↗

PubMed2003

Intracellular pH changes in isolated bovine articular chondrocytes during the loading and removal of cryoprotective agents.

The addition and removal of a cryoprotective agent (CPA) are necessary steps in the cryopreservation of natural or engineered tissue products. However, the introduction and removal of CPAs induces dramatic chemical changes inside tissues and cells and these could cause irreversible damage. This study examined the effect of CPA loading and removal on the intracellular pH of isolated bovine articular chondrocytes using a fluorimetric technique. Chondrocytes that had been isolated from bovine articular cartilage were loaded with the pH-sensitive fluorophore 2('),7(')-bis(carboxyethyl)-5(6)-carboxyfluorescein. After removal of the extracellular fluorophore, the intensity of fluorescence was used to measure the intracellular pH according to a pre-determined calibration curve. Changes of intracellular pH in chondrocytes were measured following their exposure to dimethyl sulfoxide (Me(2)SO) and glycerol at concentrations of 0.6, 0.9, and 1.2M and later to the isotonic or hypertonic solutions that were used to remove the CPA. The effect of the presence of NaCl on the intracellular pH during CPA removal was also examined. The temperature was maintained at 37 degrees C. Trypan blue exclusion was used to quantify cell membrane integrity after the addition and removal of CPA. It was found that when the cells were exposed to CPA, the intracellular pH decreased quickly and recovered gradually later. During CPA removal, the intracellular pH rose following exposure to isotonic Hepes-buffered medium, but the opposite was observed if the Hepes buffer solution contained no NaCl; this was ascribed to the role of NaCl in cell membrane transport. It was noted that the change in intracellular pH correlated with the cell volume excursion, which could be estimated by the Kedem-Katchalsky model, and was linked to cell survival. The resulting alteration of pH inside the cells might contribute to cell damage and loss of function after cryopreservation.

Animals↗

PubMed2003

Effects of hypotonic shock on intracellular pH in bovine articular chondrocytes.

Chondrocytes inhabit an unusual environment, in which they are repeatedly subjected to osmotic challenges as fluid is expressed from the extracellular matrix during static joint loading. In the present study, the effects of hypotonic shock on intracellular pH, pH(i), have been studied in isolated bovine articular chondrocytes using the pH-sensitive fluroprobe BCECF. Cells subjected to a 50% dilution rapidly alkalinised, by approximately 0.2 pH units, a sustained plateau being achieved within 300 s. The effect was not altered by inhibitors of pH regulators, such as amiloride, bafilomycin and SITS, but was absent when cells were subjected to hypotonic shocks in solutions in which Na(+) ions were replaced by NMDG(+). The response was found to be sensitive to Gd(3+) ions, blockers of stretch-activated cation channels. Alkalinisation was also inhibited by treatment with Zn(2+) ions, at a concentration reported to block voltage-activated H(+) channels (VAHC). Depolarisation using high K(+) solutions supplemented with valinomycin also induced intracellular alkalinisation. Measurements using a membrane potential (E(m)) fluorescent dye showed that E(m) was approximately -44 mV, but was depolarised by over 50 mV following HTS. The depolarisation was also inhibited by Na(+) substitution with NMDG(+) or treatment with Gd(3+). We conclude that in response to HTS the opening of a stretch-activated cation channel leads to Na(+) influx, which results in a membrane depolarisation. Subsequent activation of VAHC permits H(+) ion efflux along the prevailing electrochemcial gradient, leading to the alkalinisation, which we record.

Alkalies↗

PubMed2003

Homeostasis of intracellular Ca2+ in equine chondrocytes: response to hypotonic shock.

REASONS FOR PERFORMING STUDY: Ca2+ homeostasis in articular chondrocytes affects synthesis and degradation of the cartilage matrix, as well as other cellular functions, thereby contributing to joint integrity. Although it will be affected by mechanical loading, the sensitivity of intracellular Ca2+ concentration ([Ca2+]i) in equine articular chondrocytes to many stimuli remains unknown. HYPOTHESIS: An improved understanding of Ca2+ homeostasis in equine articular chondrocytes, and how it is altered during joint loading and pathology, will be important in understanding how joints respond to mechanical loads. METHODS: [Ca2+]i was determined using the fluorophore fura-2. We examined the effects of hypotonic shock, a perturbation experienced in vivo during mechanical loading cycles. We used inhibitors of Ca2+ transporters to ascertain the important factors in Ca2+ homeostasis. RESULTS: Under isotonic conditions, [Ca2+]i was 148 +/- 23 nmol/l, increasing by 216 +/- 66 nmol/l in response to reduction in extracellular osmolality of 50%. Resting [Ca2+]i, and the increase following hypotonic shock, were decreased by Ca2+ removal; they were both elevated when extracellular [Ca2+] ([Ca2+]o) was raised or following Na+ removal. The hypotonicity-induced rise in [Ca2+]i was inhibited by exposure of cells to gadolinium (Gd3+; 10 micromol/l), an inhibitor of mechanosensitive channels. [Ca2+]i was also elevated following treatment of cells with thapsigargin (10 micromol/l), an inhibitor of the Ca2+ pump of intracellular stores. CONCLUSIONS: A model is presented which interprets these findings in relation to Ca2+ homeostasis in equine articular chondrocytes, including the presence of mechanosensitive channels allowing Ca2+ entry, a Na+/Ca2+ exchanger for removal of intracellular Ca2+ and intracellular stores sensitive to thapsigargin. POTENTIAL RELEVANCE: A more complete understanding of Ca2+ homeostasis in equine chondrocytes may allow development of future therapeutic regimes to ameliorate joint disease.

Animals↗

PubMed2003

Comparison of grass and legume silages for milk production. 1. Production responses with different levels of concentrate.

Silages prepared from pure stands of ryegrass, alfalfa, white clover, and red clover over two successive year were offered to lactating dairy cows in two feeding experiments. Proportional mixtures of all cuts prepared in a yr were used to ensure that the forage treatments were representative of the crop. Additional treatments involved mixtures of grass silage with either white clover silage or red clover silage (50/50, on a DM basis). Silages were prepared in round bales, using a biological inoculant additive, and wilting for up to 48 h. Although the legumes were less suited to silage-making than grass, because of their higher buffering capacity and lower water-soluble carbohydrate content, all silages were well-fermented. A standard concentrate was offered at a flat-rate (8 kg/d in yr 1, and 4 or 8 kg/d in yr 2). All of the legume silages led to higher DM intake and milk yields than for the grass silage, with little effect on milk composition. Intake and production responses to legumes were similar at the two levels of concentrate feeding and with forage mixtures they were intermediate to those for the separate forages. An additional benefit of the clover silages, particularly red clover silage, was the increase in levels of polyunsaturated fatty acids, particularly alpha-linolenic acid, in milk. Legume silages also led to a lower palmitic acid percentage in milk. The efficiency of conversion of feed N into milk N declined with increasing levels of legume silage. White clover silage led to a higher N-use efficiency when the effect of N intake level is taken into account.

Animal Nutritional Physiological Phenomena↗

PubMed2003

Comparison of grass and legume silages for milk production. 2. In vivo and in sacco evaluations of rumen function.

Two experiments were conducted to investigate the basis for higher voluntary intakes and increased alpha-linolenic acid content in milk from cows offered clover silages. Six cows with rumen and duodenal cannulae were used in a four-period changeover-design experiment. Cows received 8 kg/d of dairy concentrate and had ad libitum access to one of six silage treatments: grass, red clover, white clover, alfalfa, and 50/50 (dry matter basis) mixtures of grass with red clover or white clover. The rumen fermentability of grass, red clover, white clover, and grass/red clover silages was also evaluated in a nylon bag study. Legume silages led to increased dry matter intake and milk production in comparison with grass silage. There was no significant effect of legume silages on rumen pH and volatile fatty acid concentrations, but a significant increase in rumen ammonia concentration with the legume silages, reflecting their higher protein content. The inclusion of white clover or alfalfa silage, but not red clover silage, in diets led to an increase in molar proportions of isobutyric, iso-valeric, and n-valeric acids in comparison with diets based on grass silage. Rumen fill was significantly lower, and rumen passage rates were significantly higher for cows offered alfalfa or white clover silages. However, the markedly different particle size distribution of rumen contents with these feeds suggests very different mechanisms for the high intake characteristics: high rates of particle breakdown and passage with alfalfa, and high rates of fermentation and passage with white clover. Microbial energetic efficiency (grams microbial N per kilogram organic matter apparently digested in the rumen) was highest for cows offered alfalfa silage, intermediate for clover silage, and lowest for cows offered grass silage. These differences reflect the higher rumen outflow rates for legume silages in comparison with grass silage. However, the effect of these differences on N-use efficiency (feed to milk) was probably quite small in comparison with effects of N intake. Although the biohydrogenation of alpha-linolenic acid was still high for red clover silage (86.1% compared with 94.3% for grass silage), there was a 240% increase in the proportion of alpha-linolenic acid passing through the rumen. This explains the increased recovery of alpha-linolenic acid from feed into milk with diets based on red clover silage.

Animal Feed↗

PubMed2003

Mechanisms involved in the increase in intracellular calcium following hypotonic shock in bovine articular chondrocytes.

The extracellular osmotic environment of chondrocytes fluctuates during joint loading as fluid is expressed from and reimbibed by the extracellular matrix. Matrix synthesis by chondrocytes is modulated by joint loading, possibly mediated by variations in intracellular composition. The present study has employed the Ca2+-sensitive fluoroprobe Fura-2 to determine the effects of hypotonic shock (HTS) on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a 50% dilution, [Ca2+]i rapidly increased by approximately 250%, a sustained plateau being achieved within 300 s. The effect was inhibited by thapsigargin or by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i reflects both influx from the extracellular medium and release from intracellular stores. Inhibition of the response by neomycin implicates activation of PLC and IP3 synthesis in the mobilisation of Ca2+ from intracellular stores. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels (LVACC) or reverse mode Na+/Ca2+ exchange (NCE) but could be significantly attenuated by ruthenium red, an inhibitor of transient receptor potential vanilloid (TRPV) channels and by Gd3+, a blocker of stretch-activated cation (SAC) channels. The HTS-induced rise in [Ca2+]i was almost completely absent in cells treated with Ni2+, a non-specific inhibitor of Ca2+ entry pathways. We conclude that in response to HTS the opening of SACC and a member of TRPV channel family leads to Ca2+ influx, simultaneously with the release from intracellular stores.

Adaptation, Physiological↗

PubMed2002

Effects of high pressure on glucose transport in the human erythrocyte.

The effects of raised hydraulic pressure on D-glucose exit from human red cells at 25 degrees C were determined using light scattering measurements in a sealed pressurized spectrofluorimeter cuvette. The reduction in the rates of glucose exit with raised pressure provides an index of the activation volume, deltaV++ (delta ln k/deltaP)(T) = -deltaV++/RT. Raised pressure decreased the rate constant of glucose exit from 0.077 +/- 0.003 s(-1) to 0.050 +/- 0.002 s(-1) (n = 5, P < 0.003). The Ki for glucose binding to the external site was 2.7 +/- 0.4 mm (0.1 MPa) and was reduced to 1.45 +/- 0.15 mm (40 MPa), (P < 0.01, Student's t test). Maltose had a biphasic effect on deltaV++. At [maltose] <250 microM, deltaV++ of glucose exit increased above that with [maltose = 0 mM], at >1 mm maltose, deltaV++ was reduced below that with [maltose = 0 mM]. Pentobarbital (2 mM) decreased the deltaV++ of net glucose exit into glucose-free solution from 30 +/- 5 ml mol(-1) (control) to 2 +/- 0.5 ml mol(-1) (P < 0.01). Raised pressure had a negligible effect on L-sorbose exit. These findings suggest that stable hydrated and liganded forms of GLUT with lower affinity towards glucose permit higher glucose mobilities across the transporter and are modelled equally well with one-alternating or a two-fixed-site kinetic models.

Biological Transport↗

PubMed2002

The effect of mechanical stress on cartilage energy metabolism.

Cartilage is routinely subjected to varying mechanical stresses which are known to affect matrix turnover by a variety of pathways. Here we show that mechanical loads which suppress sulphate incorporation or protein synthesis by articular chondrocytes, also inhibit rates of oxygen uptake and of lactate production. Although the mechanisms have not been definitively identified, it has been shown that high hydrostatic pressures reduce the activity of the glucose transporter GLUT. Furthermore, fluid expression consequent on static loading changes intracellular pH and ionic strength; intracellular changes which would reduce the activity of glycolytic enzymes. Both pathways would thus lead to a fall in rates of glycolysis and a reduction in intracellular ATP, and - since ATP concentrations directly affect sulphation of proteoglycans - a rapid fall in sulphate incorporation. Our results suggest that load-induced changes in matrix synthesis in cartilage can occur by means other than changes in gene expression.

Adenosine Triphosphate↗

PubMed2001

Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease.

1. This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. 2. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl- channel. 4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl- channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. 5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. 6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells.

Calcium Signaling↗

PubMed2001

Endogenous type D retrovirus in a marsupial, the common brushtail possum (Trichosurus vulpecula).

We have sequenced and characterized an endogenous type D retrovirus, which we have named TvERV(D), from the genome of an Australian marsupial, the common brushtail possum (Trichosurus vulpecula). Intact TvERV(D) gag, pro, pol, and env open reading frames were detected in the possum genome. TvERV(D) was classified as a type D retrovirus, most closely related to those of Old World monkeys, New World monkeys, and mice, based on phylogenetic analyses and genetic organization. Approximately 30 TvERV(D) proviruses are present in the genomes of possums, as detected by Southern hybridization. However, variability in fragment patterns between possums was observed and suggests recent (or ongoing) retrotranspositional activity.

Amino Acid Sequence↗

PubMed2001

Prolactin signaling influences the timing mechanism of the hair follicle: analysis of hair growth cycles in prolactin receptor knockout mice.

Pituitary PRL regulates seasonal hair follicle growth cycles in many mammals. Here we present the first evidence implicating PRL in the nonseasonal, wave-like pelage replacement of laboratory mice. In this study we show that messenger RNA transcripts encoding the one long and two short forms of PRL receptor are present in the skin of adult and neonate mice. The receptor protein was immunolocalized to the hair follicle as well as the epidermis and sebaceous glands. Furthermore, PRL messenger RNA was detected within skin extracts, suggesting a possible autocrine/paracrine role. Analysis of the hair growth phenotype of PRL gene-disrupted mice (PRLR(-/-)) revealed a change in the timing of hair cycling events. Although no hair follicle development differences were noted in PRLR(-/-) neonates, observations of the second generation of hair growth revealed PRLR(-/-) mice molted earlier than wild types (PRLR(+/+)). The advance was greater in females (29 days) than in males (4 days), resulting in the elimination of the sexual dimorphism associated with murine hair replacement. Heterozygotes were intermediate between PRLR(-/-) and PRLR(+/+) mice in molt onset. Once initiated, the pattern and progression of the molt across the body were similar in all genotypes. Although all fiber types were present and appeared structurally normal, PRLR(-/-) mice had slightly longer and coarser hair than wild types. These findings demonstrate that PRL has an inhibitory effect on murine hair cycle events. The pituitary PRL regulation of hair follicle cycles observed in seasonally responsive mammals may be a result of pituitary PRL interacting with a local regulatory mechanism.

Animals↗

PubMed2001

STAT5b is required for GH-induced liver IGF-I gene expression.

Although the increased expression of Igf-I in liver in response to GH is well characterized, the intracellular signaling pathways that mediate this effect have not been identified. Intracellular signaling molecules belonging to the Janus kinase-signal transducer and activator of transcription 5b (JAK2-STAT5b) pathway are activated by GH and have previously been shown to be required for sexually dimorphic body growth and the expression of liver cytochrome P450 proteins known to be regulated by the gender-specific temporal patterns of pituitary GH secretion. Here, we evaluate the role of STAT5b in GH activation of Igf-I by monitoring the induction of Igf-I mRNA in livers of wild-type and Stat5b(-/-)mice stimulated with exogenous pulses of GH. GH induced the expression of liver Igf-I mRNA in hypophysectomized male wild-type, but not in hypophysectomized male Stat5b(-/-) mice, although the Stat5b(-/-) mice exhibit both normal liver GH receptor expression and strong GH induction of Cytokine-inducible SH2 protein (Cis), which is believed to contribute to the down-regulation of GH-induced liver STAT5b signaling. Thus, STAT5b plays an important and specific role in liver Igf-I gene expression.

Animals↗