All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression <em>factor</em> 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks <em>21</em> others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.

Hepatocyte <em>growth</em> <em>factor</em> (HGF) is a potent mitogen for mature hepatocytes in vitro. The receptor for HGF has recently been characterized as the product of the proto-oncogene c-met. We have examined the possible involvement of HGF in hepatic <em>growth</em> and differentiation in the rat. The experimental systems used were acetylaminofluorene treatment combined with partial hepatectomy to induce proliferation and differentiation of oval cells in adult liver and the pre- and postnatal liver. In the acetylaminofluorene model, Northern blot analysis showed that level of HGF transcripts increased one day after partial hepatectomy, reached a peak by day 6, were maintained at that level until day 13, and then declined, reaching normal level at 20 days. The expression of c-met also increased gradually, reached a peak around 9 to 13 days after partial hepatectomy, at which time oval cell proliferation was most prominent. In the developing liver, an elevated level of HGF transcripts was found between 4 and <em>21</em> days after birth. The expression of c-met also slightly increased at the same time. In situ hybridization showed that the transcripts for HGF were localized in desmin-positive Ito cells, whereas the transcripts for c-met were strongly expressed by oval cells. We have shown earlier that Ito cells and oval cells proliferate simultaneously and exist in close proximity in the acetylaminofluorene model and that Ito cells are a primary source of <em>growth</em> <em>factors</em> such as transforming <em>growth</em> <em>factor</em>-alpha and acidic <em>fibroblast</em> <em>growth</em> <em>factors</em>. The data presented here suggest that HGF is, in combination with other <em>growth</em> <em>factors</em>, involved in the proliferation and differentiation of oval cells via a paracrine mechanism.

Because of the prominent neovascularization observed in the <em>growth</em> of brain tumors, we studied the occurrence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), a potent angiogenic <em>factor</em> in astrocytomas, the most aggressive of which often have marked vascular hyperplasia. Using immunohistochemical methods, we examined <em>21</em> examples of such tumors, 7 glioblastomas multiforme, 7 anaplastic astrocytomas, and 7 low grade astrocytomas. Using polyclonal and affinity-purified rabbit antisera to human bFGF, we detected immunoreactive bFGF in all cases of glioblastoma multiforme. bFGF was present in both endothelial cells and neoplastic astrocytes. In 4 of 7 anaplastic astrocytomas, the tumor astrocytes had bFGF immunoreactivity and, in 5 of 7 cases, endothelial cells were also immunopositive. In glioblastomas multiforme and anaplastic astrocytomas, capillaries adjacent to tumor showed bFGF immunoreactivity, whereas capillaries distant from the tumors were not immunostained. In low grade astrocytomas, astrocytic cells were weakly immunoreactive in 2 of 7 cases, and in only 1 of the 7 cases capillaries were immunostained. In each grade, reactive astroglial cells showed variable bFGF immunoreactivity. The immunostaining was not seen with the flow-through fraction obtained after affinity purification of the bFGF antiserum with pure recombinant bFGF. These results suggest a possible role for bFGF in tumor <em>growth</em> and in angiogenesis in astrocytomas.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) increases glucose uptake. It is unknown if FGF<em>21</em> serum levels are affected by exercise.

RESULTS

This was a comparative longitudinal study. Anthropometric and biochemical evaluation were carried out before and after a bout of exercise and repeated after two weeks of daily supervised exercise. The study sample was composed of 60 sedentary young healthy women. The mean age was 24±3.7 years old, and the mean BMI was <em>21</em>.4±7.0 kg/m². The anthropometric characteristics did not change after two weeks of exercise. FGF<em>21</em> levels significantly increased after two weeks of exercise (276.8 ng/l (142.8-568.6) vs. (460.8 (298.2-742.1), p<0.0001)). The delta (final-basal) log of serum FGF<em>21</em>, adjusted for BMI, showed a significant positive correlation with basal glucose (r = 0.23, p = 0.04), mean maximal heart rate (MHR) (r = 0.54, p<0.0001), mean METs (r = 0.40, p = 0.002), delta plasma epinephrine (r = 0.53, p<0.0001) and delta plasma FFAs (r = 0.35, p = 0.006). A stepwise linear regression model showed that glucose, MHR, METs, FFAs, and epinephrine, were <em>factor</em>s independently associated with the increment in FGF<em>21</em> after the exercise program (F = 4.32; r² = 0.64, p<0.0001).

CONCLUSIONS

Serum FGF<em>21</em> levels significantly increased after two weeks of physical activity. This increment correlated positively with clinical parameters related to the adrenergic and lipolytic response to exercise.

BACKGROUND

ClinicalTrials.gov NCT01512368.

BACKGROUND

Cold exposure stimulates <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) secretion in animals, enhancing the cold-induced thermogenesis (CIT) response through browning of white adipose tissue. In humans, the effects of cold exposure on circulating FGF<em>21</em> levels are unknown.

OBJECTIVE

Our objective was to evaluate the effects of mild cold exposure on circulating FGF<em>21</em> and its relationship with CIT and lipolysis in humans.

METHODS

We conducted a randomized, single-blind, crossover intervention study at the National Institutes of Health Clinical Center.

METHODS

Participants were healthy adults.

METHODS

Subjects were exposed to a 12-h exposure to 24 or 19 C in a whole-room indirect calorimeter.

METHODS

Energy expenditure, plasma FGF <em>21</em>, nonesterified fatty acid, and adipose tissue microdialysis glycerol concentrations were evaluated.

RESULTS

At 24 C, plasma FGF<em>21</em> exhibited a diurnal rhythm, peaking at 0800 h [110 (59-178) pg/ml], and progressively dropped to a nadir at 1700 h [41 (<em>21</em>-71) pg/ml, P < 0.0001] before rising at 1900 h [60 (11-81) pg/ml, P < 0.0001]. Exposure at 19 C lessened the diurnal reduction of FGF<em>21</em> observed at 24 C from 0800-1700 h and augmented overall FGF<em>21</em> levels by 37 ± 45% (P = 0.01). The change in area under the curve plasma FGF<em>21</em> between 19 and 24 C correlated positively with the change in area under the curve adipose microdialysate glycerol (R(2) = 0.35, P = 0.04) but not with nonesterified fatty acid. Cold-induced increase in FGF<em>21</em> predicted greater rise in energy expenditure during cold exposure (β = 0.66, P = 0.027), independent of age, gender, fat mass, and lean mass.

CONCLUSIONS

Mild cold exposure increased circulating FGF<em>21</em> levels, predicting greater lipolysis and CIT. A small reduction in environmental temperature is sufficient to modulate FGF<em>21</em> diurnal rhythm in humans, which may mediate cold-induced metabolic changes similar to those in animals.

Scleroderma (systemic sclerosis, SSc) is a complex autoimmune disease caused by progressive fibrotic replacement of normal tissue architecture, a progressive and ultimately fatal process that currently has no cure. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in SSc is unclear. In comparison with the normal skin tissues, miRNAs were aberrantly expressed in limited cutaneous scleroderma and diffuse cutaneous scleroderma skin tissues. We also identified miRNAs whose expressions were correlated with SSc fibrosis: miR-<em>21</em>, miR-31, miR-146, miR-503, miR-145, and miR-29b were predicted to be involved. This study further confirmed that miR-<em>21</em> was increased whereas miR-145 and miR-29b were decreased both in the skin tissues and <em>fibroblasts</em>. As predicted target genes, SMAD7, SAMD3, and COL1A1 were regulated by these miRNAs. After stimulation with transforming <em>growth</em> <em>factor</em> β, the expression of miR-<em>21</em> was increased and that of SMAD7 mRNA was decreased. MiR-145 was upregulated whereas the mRNA level of SMAD3 was downregulated. The downregulation of miR-29b was correlated with the upregulation of COL1A1 mRNA. MiRNAs might play an important role in the pathogenesis of SSc and suggest a potential therapy.

Circulating levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a metabolic regulator of glucose, lipid, and energy homeostasis, are elevated in obese diabetic subjects, raising questions about potential FGF<em>21</em> resistance. Here we report tissue expression changes in FGF<em>21</em> and its receptor components, and we describe the target-organ and whole-body responses to FGF<em>21</em> in ob/ob and diet-induced obese (DIO) mice. Plasma FGF<em>21</em> concentrations were elevated 8- and 16-fold in DIO and ob/ob mice, respectively, paralleling a dramatic increase in hepatic FGF<em>21</em> mRNA expression. Concurrently, expression levels of βKlotho, FGF receptor (FGFR)-1c, and FGFR2c were markedly down-regulated in the white adipose tissues (WAT) of ob/ob and DIO mice. However, dose-response curves of recombinant human FGF<em>21</em> (rhFGF<em>21</em>) stimulation of ERK phosphorylation in the liver and WAT were not right shifted in disease models, although the magnitude of induction in ERK phosphorylation was partially attenuated in DIO mice. Whole-body metabolic responses were preserved in ob/ob and DIO mice, with disease models being more sensitive and responsive than lean mice to the glucose-lowering and weight-loss effects of rhFGF<em>21</em>. Endogenous FGF<em>21</em> levels, although elevated in diseased mice, were below the half-maximal effective concentrations of rhFGF<em>21</em>, suggesting a state of relative deficiency. Hepatic and WAT FGF<em>21</em> mRNA expression levels declined after rhFGF<em>21</em> treatment in the absence of the increased expression levels of βKlotho and FGFR. We conclude that overt FGF<em>21</em> resistance was not evident in the disease models, and increased hepatic FGF<em>21</em> expression as a result of local metabolic changes is likely a major cause of elevated circulating FGF<em>21</em> levels.

BACKGROUND

Murine <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> is a nutritionally regulated hormone secreted by the liver principally in response to peroxisome proliferator-activated receptor-alpha (PPAR alpha) activation, which plays a critical role in regulating metabolism during ketosis. FGF<em>21</em> is also a PPAR gamma target gene in mouse adipose tissue. Little information is available on FGF<em>21</em> functions in humans.

OBJECTIVE

The aim of the study was to measure plasma FGF<em>21</em> during fasting, ketogenic diet, and PPAR agonist treatment in humans.

METHODS

We conducted a prospective study involving three patient groups at two university hospitals.

METHODS

Eight healthy male volunteers underwent a 48-h period of starvation followed by 24-h refeeding (group 1); seven obese individuals were allocated to a low-carbohydrate diet for 3 months (group 2); and three groups of healthy, overweight or obese male volunteers received treatment with a PPAR alpha (20 microg/d GW590735) (n=6), PPAR delta (10 mg/d GW501516) (n=6), or PPAR gamma agonist (rosiglitazone) (n=10) for 2 wk (group 3).

METHODS

Fasting plasma FGF<em>21</em> and serum 3-hydroxybutyrate were measured.

RESULTS

There was no significant variation in human plasma FGF<em>21</em> during fasting and refeeding. A 3-month ketogenic diet was associated with a 42% decline in plasma FGF<em>21</em> levels. Circulating FGF<em>21</em> increased significantly in response to treatment with PPAR alpha (39%) and PPAR delta (32%), but not PPAR gamma agonists.

CONCLUSIONS

FGF<em>21</em> does not play a major role in regulating the fasting response or ketosis in man. However, plasma FGF<em>21</em> is elevated in response to pharmacological activation of PPAR alpha and PPAR delta and may contribute to the beneficial metabolic effects observed in response to pharmacotherapy with these compounds.

Tubulointerstitial fibrosis correlates closely with renal function, although the mechanism regulating tubulointerstitial fibrogenesis remains poorly understood. Since platelet-derived <em>growth</em> <em>factor</em> (PDGF) is a <em>growth</em> <em>factor</em> for <em>fibroblasts</em>, we examined the effect of daily (for 7 days) PDGF-AA or PDGF-BB administration on renal tubulointerstitial architecture in rats. PDGF-AA administration at a dose of 5 mg/kg did not affect the renal tubulointerstitium. By comparison, PDGF-BB induced dose-dependent renal tubulointerstitial cell proliferation and fibrosis. At 5 mg/kg, PDGF-BB increased BrdU labeling in tubulointerstitial <em>fibroblasts</em> at 24 hours (19-fold), which peaked at 72 hours (23-fold) with bromodeoxyuridine uptake returning to control values by 7 days. Tubulointerstitial proliferation was associated with the differentiation of these cells into myo<em>fibroblasts</em> as evidenced by alpha-smooth muscle actin expression beginning on day 3. The expression of alpha-smooth muscle actin peaked on day 5 and had markedly declined by day <em>21</em>. In addition, apoptotic cells were identified within the tubulointerstitium on day 3 and progressively increased through day 7, suggesting that the disappearance of myo<em>fibroblasts</em> may have occurred through apoptosis. These changes were accompanied by increased expression of alpha 1 (III) collagen mRNA and interstitial accumulation of type III collagen within the renal cortex. By morphometric analysis, an approximately twofold increase in collagen III immunolabeling within the interstitial compartment was evident at 24 hours and peaked on days 5 to 7 (approximately fourfold). These data suggest that PDGF-BB may be an important mediator of tubulointerstitial hyperplasia and fibrosis.

OBJECTIVE

To assess the role of hypoxia in causing the death and deconstruction of photoreceptors in detached retinas and the effectiveness of supplemental oxygen in limiting such damage.

METHODS

Retinal detachment was induced surgically in the right eye of each of 10 cats. The cats were allowed to survive surgery for 3 days. Two were kept for these 3 days in normoxia (room air, <em>21</em>% oxygen) and eight in hyperoxia (70% oxygen). The retinas were examined for cell death by use of labels for normal and fragmenting DNA, with antibodies and a cone sheath-specific lectin to demonstrate the status of their inner and outer segments, the synaptic structures of the outer plexiform layer, and the distribution of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and with in situ hybridization to demonstrate bFGF mRNA.

RESULTS

Retinal detachment without oxygen supplementation caused the death of some photoreceptors; the loss of cytochrome oxidase from the inner segments and the collapse of the outer segments of surviving photoreceptors; the loss of synaptophysin profiles from the outer plexiform layer; and the loss of bFGF protein from retinal neurons and neuroglia but not from retinal vessels. Oxygen supplementation (hyperoxia) during detachment mitigated all these changes, reducing photoreceptor death, maintaining the specialized structures of surviving photoreceptors, and stabilizing the bFGF within the retina.

CONCLUSIONS

In experimental retinal detachment, hypoxia caused by the separation of outer retina from its normal source of nutrients is a factor in inducing the death and deconstruction of photoreceptors as well as in the loss of bFGF from the detached retina. Hyperoxia offered to human patients between diagnosis of retinal detachment and surgery may enhance the function of the reattached retina.

The gene and protein structure of the mouse UBF (mUBF), a transcription <em>factor</em> for mouse ribosomal RNA gene, have been determined by cDNA and genomic clones. The unique mUBF gene consists of <em>21</em> exons spanning over 13 kb. Two mRNAs coding for mUBF1 and mUBF2 having 765 a.a. and 728 a.a., respectively, are produced by an alternative splicing of exon 8. It specifies 37 amino acids constituting a part of the regions homologous to high mobility group proteins (HMG box 2). A human UBF (hUBF) cDNA obtained by polymerase chain reaction also indicates the presence of two kinds of mRNAs, the shorter form lacking the same region as mUBF2. Comparison of the cDNAs from hUBF and mUBF revealed an unusual conservation of nucleotide sequence in the 3'-terminal non-coding region. We examined the relative amounts of expression of mUBF1 and mUBF2. The eight tissues studied contained both molecular species, although mUBF2 was the predominant form of UBF. The mRNA of mUBF1 was expressed one half of the mUBF2 in quiescent mouse <em>fibroblasts</em> but reached the same amount in <em>growing</em> state.

Disruption of skeletal muscle homeostasis by substitution with fibrotic tissue constitutes the principal cause of death in Duchenne muscular dystrophy (DMD) patients, yet the implicated fibrogenic mechanisms remain poorly understood. This study identifies the extracellular PAI-1/urokinase-type plasminogen activator (uPA) balance as an important regulator of microribonucleic acid (miR)-<em>21</em> biogenesis, controlling age-associated muscle fibrosis and dystrophy progression. Genetic loss of PAI-1 in mdx dystrophic mice anticipated muscle fibrosis through these sequential mechanisms: the alteration of collagen metabolism by uPA-mediated proteolytic processing of transforming <em>growth</em> <em>factor</em> (TGF)-β in muscle <em>fibroblasts</em> and the activation of miR-<em>21</em> expression, which inhibited phosphatase and tensin homologue and enhanced AKT signaling, thus endowing TGF-β with a remarkable cell proliferation-promoting potential. Age-associated fibrogenesis and muscle deterioration in mdx mice, as well as exacerbated dystrophy in young PAI-1(-/-) mdx mice, could be reversed by miR-<em>21</em> or uPA-selective interference, whereas forced miR-<em>21</em> overexpression aggravated disease severity. The PAI-1-miR-<em>21</em> fibrogenic axis also appeared dysregulated in muscle of DMD patients, providing a basis for effectively targeting fibrosis and muscular dystrophies in currently untreatable individuals.

Transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1) induces stromal <em>fibroblast</em>-to-myo<em>fibroblast</em> transdifferentiation in the tumor-stroma interactive microenvironment via modulation of multiple phenotypic and functional genes, which plays a critical role in tumor progression. Up to now, the involvement of micro-RNAs (miRNAs) and their roles in TGF-β1-induced myo<em>fibroblast</em> differentiation in tumor-stroma interaction are unclear. Using quantitative real-time RT-PCR, we demonstrated that the expression of micro-RNA-<em>21</em> (miR-<em>21</em>) was upregulated in activated <em>fibroblasts</em> after treatment with TGF-β1 or conditioned medium from cancer cells. To determine the potential roles of miR-<em>21</em> in TGF-β1-mediated gene regulation during myo<em>fibroblast</em> conversion, we showed that miR-<em>21</em> expression was downregulated by miR-<em>21</em> inhibitor and upregulated by miR-<em>21</em> mimic. Interestingly, downregulation of miR-<em>21</em> with the inhibitor effectively inhibited TGF-β1-induced myo<em>fibroblast</em> differentiation while upregulation of miR-<em>21</em> with a mimic significantly promoted myo<em>fibroblast</em> differentiation. We further demonstrated that MiR-<em>21</em> directly targeted and downregulated programmed cell death 4 (PDCD4) gene, which in turn acted as a negative regulator of several phenotypic and functional genes of myo<em>fibroblasts</em>. Taken together, these results suggested that miR-<em>21</em> participated in TGF-β1-induced myo<em>fibroblast</em> transdifferentiation in cancer stroma by targeting PDCD4.

Recent extensive studies have revealed that molecular hydrogen (H(2)) has great potential for improving oxidative stress-related diseases by inhaling H(2) gas, injecting saline with dissolved H(2), or drinking water with dissolved H(2) (H(2)-water); however, little is known about the dynamic movement of H(2) in a body. First, we show that hepatic glycogen accumulates H(2) after oral administration of H(2)-water, explaining why consumption of even a small amount of H(2) over a short span time efficiently improves various disease models. This finding was supported by an in vitro experiment in which glycogen solution maintained H(2). Next, we examined the benefit of ad libitum drinking H(2)-water to type 2 diabetes using db/db obesity model mice lacking the functional leptin receptor. Drinking H(2)-water reduced hepatic oxidative stress, and significantly alleviated fatty liver in db/db mice as well as high fat-diet-induced fatty liver in wild-type mice. Long-term drinking H(2)-water significantly controlled fat and body weights, despite no increase in consumption of diet and water. Moreover, drinking H(2)-water decreased levels of plasma glucose, insulin, and triglyceride, the effect of which on hyperglycemia was similar to diet restriction. To examine how drinking H(2)-water improves obesity and metabolic parameters at the molecular level, we examined gene-expression profiles, and found enhanced expression of a hepatic hormone, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), which functions to enhance fatty acid and glucose expenditure. Indeed, H(2) stimulated energy metabolism as measured by oxygen consumption. The present results suggest the potential benefit of H(2) in improving obesity, diabetes, and metabolic syndrome.

BACKGROUND

Gastric cancer (GC) is a leading cause of cancer deaths worldwide. Since the approval of trastuzumab, targeted therapies are emerging as promising treatment options for the disease. This study aimed to explore the molecular segmentation of several known therapeutics targets, human epidermal growth factor receptor 2 (HER2), MET and fibroblast growth factor receptor 2 (FGFR2), within GC using clinically approved or investigational kits and scoring criteria. Knowledge of how these markers are segmented in the same cohort of GC patients could improve future clinical trial designs.

METHODS

Using immunohistochemistry (IHC) and FISH methods, overexpression and amplification of HER2, FGFR2 and MET were profiled in a cohort of Chinese GC samples. The correlations between anti-tumour sensitivity and the molecular segments of HER2, MET and FGFR2 alterations were further tested in a panel of GC cell lines and the patient-derived GC xenograft (PDGCX) model using the targeted inhibitors.

RESULTS

Of 172 GC patients, positivity for HER2, MET and FGFR2 alternations was found in 23 (13.4%), 21 (12.2%) and 9 (5.2%) patients, respectively. Positivity for MET was found in 3 of 23 HER2-positive GC patients. Co-positivity for FGFR2 and MET was found in 1 GC patient, and amplification of the two genes was found in different tumour cells. Our study in a panel of GC cell lines showed that in most cell lines, amplification or high expression of a particular molecular marker was mutually exclusive and in vitro sensitivity to the targeted agents lapatinib, PD173074 and crizotinib was only observed in cell lines with the corresponding high expression of the drugs' target protein. SGC031, an MET-positive PDGCX mouse model, responded to crizotinib but not to lapatinib or PD173074.

CONCLUSIONS

Human epidermal growth factor receptor 2, MET and FGFR2 oncogenic driver alterations (gene amplification and overexpression) occur in three largely distinct molecular segments in GC. A significant proportion of HER2-negative patients may potentially benefit from MET- or FGFR2-targeted therapies.

The ability of prostate-specific antigen (PSA) to predict tumor volume and stage in patients with prostate cancer would be improved if <em>factors</em> regulating its production and clearance were better defined. A thorough understanding of the pharmacokinetics (regulation of production, metabolism, and excretion) of PSA has been precluded, however, by the absence of an in vivo animal model. The purposes of this study are to develop a murine model for evaluating PSA pharmacokinetics in vivo and to assess <em>factors</em> that influence PSA production in vitro. The human prostate cancer cell line, LNCaP, was chosen because it is androgen sensitive and PSA positive. Although LNCaP cells are usually nontumorigenic when inoculated s.c. in athymic mice, coinoculation of 1 x 10(6) LNCaP cells with 1 x 10(6) human bone <em>fibroblasts</em> reliably produces PSA-secreting carcinomas. This LNCaP model provides accurate correlation between tumor volume and serum PSA levels (r = 0.94) and demonstrates that tumor volume and androgens are codeterminants of circulating PSA levels. Following castration, serum PSA levels decrease rapidly up to 8-fold and increase up to 20-fold following androgen supplementation, without detectable castration-induced tumor cell death or concomitant changes in tumor volume. Serum PSA levels increase 0.24 ng/ml/mm3 of tumor, which is approximately 5-fold less than that estimated for humans. Most likely this reduced PSA index (PSA:tumor volume ratio) results from a 7-fold faster clearance of PSA in athymic mice than in humans; other than this shorter half-life, PSA elimination in the murine model appears similar to that in humans, with both following first-order kinetics characteristic of a two-compartment model. Interestingly, following prolonged <em>growth</em> (greater than <em>21</em> days) in castrate hosts, LNCaP tumors are capable of adapting to an androgen-deprived environment whereby LNCaP tumors regain the ability to secrete PSA in amounts similar to the precastrate state. In LNCaP cells, androgens increase PSA mRNA levels 4-fold in vivo and in vitro. PSA mRNA expression is also altered by various <em>growth</em> <em>factors</em>. Changes in PSA production induced by androgens and <em>growth</em> <em>factors</em> do not always parallel changes in LNCaP cell <em>growth</em> rate induced by these <em>factors</em>, suggesting that PSA production occurs independently of cell <em>growth</em> rate and may be influenced by various interrelated <em>factors</em>, including hormonal and stromal milieu. Observations from this murine model suggest that androgens and tumor volume are independent determinants of serum PSA levels and imply that decreases in circulating PSA following antiandrogen therapy may not always reflect a corresponding reduction in tumor volume.

Lipocortin I is a high affinity substrate for the epidermal <em>growth</em> <em>factor</em> receptor/kinase that can be purified by reversible Ca2+-dependent association with cellular particulate fractions. Purified human lipocortin I was shown by ultraviolet spectroscopy to undergo conformational changes in response to high concentrations of Ca2+ (30-700 microM). Equilibrium dialysis of lipocortin I alone against 45Ca2+ showed barely detectable binding at 5-60 microM Ca2+. However, in the presence of phosphatidylserine, binding was significantly enhanced, and Scatchard analysis of the binding data showed that lipocortin I contained four Ca2+-binding sites with an apparent Kd of 75 microM. Lipocortin I associated with phosphatidylserine vesicles and F-actin in a Ca2+-dependent manner with half-maximal association occurring at 22 and 180 microM Ca2+, respectively. Neither phosphatidylcholine vesicles nor intact cultured <em>fibroblasts</em> bound detectable amounts of lipocortin I. Phosphorylation of lipocortin I by the epidermal <em>growth</em> <em>factor</em>-stimulated kinase in A431 membranes required Ca2+ (half-maximum = 28 microM) in the presence of Mg2+, but phosphorylation was not Ca2+-dependent in the presence of Mn2+. The difference in Ca2+ requirement for phosphorylation probably is a reflection of the fact that Mn2+, but not Mg2+, promoted the association of lipocortin I with phospholipid. Although the physiological role of lipocortin I phosphorylation has not yet been determined, it was observed that phosphorylation of Tyr-<em>21</em> reduced by 5-fold the amount of Ca2+ required for half-maximal association of the protein with phosphatidylserine vesicles.

The placental-derived <em>growth</em> <em>factor</em> (PIGF) is a dimeric glycoprotein showing a high degree of sequence similarity to the vascular endothelial <em>growth</em> <em>factor</em>. Alternative splicing of the PIGF primary transcript gives rise to two forms, named PIGF-1 and PIGF-2, which differ only in the insertion of a highly basic <em>21</em>-amino acid stretch at the carboxyl end. The presence of the PIGF mRNA in thyroid, placenta, lung, and goiter has indicated the tissues where this <em>factor</em> functions. However, the role of PIGF in vascular development has not yet been clearly established. In the present study, we described the purification of PIGF-1 from overexpressing eukaryotic cells and then measured the angiogenic activity of the purified PIGF-1 in vivo in the rabbit cornea and the chick chorioallantoic membrane assays. In both in vivo assays, PIGF-1 induced a strong neovascularization process that was blocked by affinity-purified anti-PIGF-1 antibody. In the avascular cornea, PIGF-1 induced angiogenesis in a dose-dependent manner and seemed to be at least as effective (if not more effective) than vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> under the same conditions and at the same concentration. PIGF-1 was shown to induce cell <em>growth</em> and migration of endothelial cells from bovine coronary postcapillary venules and from human umbilical veins. In these two in vitro assays, PIGF-1 seemed to have a comparable effect to that of vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> on the cultured microvascular endothelium (eg, capillary venule endothelial cells). In summary, this is the first study to demonstrate that PIGF-1 can induce angiogenesis in vivo and stimulate the migration and proliferation of endothelial cells in vitro.

ALS2/alsin is a member of guanine nucleotide exchange <em>factors</em> for the small GTPase Rab5 (Rab5GEFs), which act as modulators in endocytic pathway. Loss-of-function mutations in human ALS2 account for a number of juvenile recessive motor neuron diseases (MNDs). However, the normal physiological role of ALS2 in vivo and the molecular mechanisms underlying motor dysfunction are still unknown. To address these issues, we have generated mice homozygous for disruption of the Als2 gene. The Als2-null mice observed through <em>21</em> months of age demonstrated no obvious developmental, reproductive or motor abnormalities. However, immunohistochemical and electrophysiological analyses identified an age-dependent, slowly progressive loss of cerebellar Purkinje cells and disturbance of spinal motor neurons associated with astrocytosis and microglial cell activation, indicating a subclinical dysfunction of motor system in Als2-null mice. Further, quantitative epidermal <em>growth</em> <em>factor</em> (EGF)-uptake analysis identified significantly smaller-sized EGF-positive endosomes in Als2-null <em>fibroblasts</em>, suggesting an alteration of endosome/vesicle trafficking in the cells. Collectively, while loss of ALS2 does not produce a severe disease phenotype in mice, these Als2-null animals should provide a useful model with which to understand the interplay between endosomal dynamics and the long-term viability of large neurons such as Purkinje cells and spinal motor neurons.

BACKGROUND

YKL-40 is secreted by macrophages and neutrophils and is a growth factor for vascular endothelial cells and fibroblasts. Elevated serum concentrations of YKL-40 are found in patients with diseases characterized by inflammation or ongoing fibrosis. The aim of this study was to seek association between serum YKL-40 in patients with ulcerative colitis (UC) and Crohn disease (CD) and clinical disease activity.

METHODS

One-hundred-and-sixty-four patients with UC and 173 patients with CD were studied. The Simple Clinical Colitis Activity Index (SCCAI) and the Harvey-Bradshaw (H-B) score were used to assess disease activity. Serum YKL-40 (determined by ELISA) was related to C-reactive protein (CRP) and disease activity.

RESULTS

In patients with UC, the median serum YKL-40 rose with increasing disease activity, and patients with severe active disease had higher serum YKL-40 (median 59 microg/L (95% CI: 26-258 microg/L), P < 0.001) than patients with inactive UC (33 microg/L (19-163)) and age-matched controls (43 microg/L (20-124)). Patients with severe active CD had higher serum YKL-40 (59 microg/L (21-654), P < 0.001) than age-matched controls, but not higher than inactive CD patients (43 microg/L (17-306)). Serum YKL-40 was elevated in 41% of the patients with severe UC, in 10% with inactive UC, in 46% with severe CD and in 30% with inactive CD. Serum YKL-40 correlated with SCCAI in UC patients but not with H-B score in CD patients. In both patient groups, low correlations were found between serum YKL-40 and CRP, albumin and leucocytes.

CONCLUSIONS

Serum YKL-40 is elevated in patients with active IBD and may be complementary to inflammatory markers and clinical characteristics in the assessment of disease activity.

OBJECTIVE

Fibroblast growth factor receptor 3 (FGFR3) mutations have been associated with achondroplastic syndromes and urinary bladder carcinomas. Here we describe changes in FGFR3 mRNA and protein expression in transitional carcinomas and determine the effect of monoclonal antibodies against FGFR3 in RT-112 cell line proliferation.

METHODS

We used microarray tools to evaluate FGFR3 mRNA expression in 22 urinary bladder carcinomas at different stages (noninvasive pTa, lamina propria invasive pT1, and muscular invasive pT2) and 7 nonneoplastic tissue controls. FGFR3 protein expression was evaluated by Western blotting in 15 different carcinomas and 3 nonneoplastic controls. Two hundred thirty-seven urinary bladder and renal pelvis carcinomas and 21 negative controls were tested on tissue microarrays by immunohistochemistry. The effect on cell proliferation in the RT-112 bladder cancer cell line of monoclonal antibodies against FGFR3 was also evaluated.

RESULTS

Overexpression of FGFR3 mRNA was found in pTa and pT1 stage carcinomas (fold change >8) and in pT2 carcinomas (fold change >4). Nonneoplastic urinary bladder samples do not express FGFR3 protein. However, 83% of pTa, 100% of pT1, and 50% of pT2 carcinomas expressed FGFR3 as determined by Western blotting. By immunohistochemistry, FGFR3 was positive in 71.4% of pTa, 72% of pT1, and 49.2% of pT2 cases as well as 61.5% of upper urinary tract carcinomas. Proliferation of the RT-112 cell line was inhibited with monoclonal antibodies against FGFR3.

CONCLUSIONS

FGFR3 seems to play an important role in transitional cell carcinoma development. Our results suggest that FGFR3 antagonists could be developed as possible therapeutics for treatment of urinary tract carcinoma.

OBJECTIVE

To investigate whether circulating levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), which previously has been shown to be elevated in obesity, could predict the development of type 2 diabetes in a 5.4-year, population-based, prospective study.

METHODS

Baseline plasma FGF<em>21</em> levels were measured using an enzyme-linked immunosorbent assay in 1,900 subjects from the Hong Kong Cardiovascular Risk Factor Prevalence Study (CRISPS). The prospective association of FGF<em>21</em> with diabetes development over 5.4 years was analyzed using multiple logistic regression.

RESULTS

At baseline, plasma levels of FGF<em>21</em> increased progressively with worsening dysglycemia from normal glucose tolerance, through prediabetes, to diabetes (global trend, P < 0.001). Of 1,292 subjects without diabetes at baseline, a high baseline FGF<em>21</em> level was a strong independent predictor for diabetes development (odds ratio 1.792; P < 0.01), together with waist circumference and fasting plasma glucose levels.

CONCLUSIONS

Plasma FGF<em>21</em> levels were significantly increased in subjects with prediabetes and diabetes and predicted the development of diabetes in humans.

BACKGROUND

Expression and activity of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> hormone-like protein are associated with development of several metabolic disorders. This study was designed to investigate whether serum FGF<em>21</em> level was also associated with the metabolic syndrome-related cardiovascular disease, atherosclerosis, and its clinical features in a Chinese cohort.

METHODS

Two-hundred-and-fifty-three subjects visiting the Cardiology Department (Sixth People's Hospital affiliated to Shanghai JiaoTong University) were examined by coronary arteriography (to diagnose coronary artery disease (CAD)) and hepatic ultrasonography (to diagnose non-alcoholic fatty liver disease (NAFLD)). Serum FGF<em>21</em> level was measured by enzyme-linked immunosorbent assay and analyzed for correlation to subject and clinical characteristics. The independent <em>factor</em>s of CAD were determined by multivariate logistic regression analysis.

RESULTS

Subjects with NAFLD showed significantly higher serum FGF<em>21</em> than those without NAFLD (388.0 pg/mL (253.0-655.4) vs. 273.3 pg/mL (164.9-383.7), P < 0.01). Subjects with CAD showed significantly higher serum FGF<em>21</em>, regardless of NAFLD diagnosis (P < 0.05). Serum FGF<em>21</em> level significantly elevated with the increasing number of metabolic disorders (P for trend < 0.01). After adjustment of age, sex, and BMI, FGF<em>21</em> was positively correlated with total cholesterol (P < 0.05) and triglyceride (P < 0.01). FGF<em>21</em> was identified as an independent <em>factor</em> of CAD (odds ratio = 2.984, 95% confidence interval: 1.014-8.786, P < 0.05).

CONCLUSIONS

Increased level of serum FGF<em>21</em> is associated with NAFLD, metabolic disorders and CAD.

Extracellular matrix (ECM) components significantly influence the <em>growth</em> characteristics of cardiomyocytes, development of spontaneous contractile activity and morphologic differentiation. The present study involves characterization of ES cell-derived cardiomyocytes cultured on different ECMs. We hypothesized that cardiogel, which is a naturally occurring ECM containing a complex mixture of laminin, fibronectin may better support ES cell-derived cardiomyocytes maturation in terms of chronotropic characteristics and subcellular structure development. Spontaneously differentiated cardiomyocytes from 7-day embryoid bodies (EBs) derived from mouse ES cells line Royan B1 (C57BL/6) were cultured on commercial ECM (matrigel), a <em>fibroblast</em>-derived ECM (cardiogel), and control (without ECM) for 7 + <em>21</em> days. The <em>growth</em> characteristics of cardiomyocytes were assessed by immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR), positive and negative chronotropic drugs and transmission electron microscopy (TEM). The spontaneously beating cells expressed markers characteristic of cardiomyocytes including alpha-actinin, desmin, troponin-I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin-43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic <em>factor</em> (ANF) and myosin light chain isoform-2V (MLC-2V). In addition, spontaneous beating of cardiomyocytes on both ECMs was enhanced by treatment of cells with isopernaline, while reduced more on matrigel, by carbacol when compared with control and cardiogel. However, the change in beating was similar in all groups upon treatment with phenylephrine and/or Bay-K. Ultrastructure analysis showed myofibrillar bundle organization and exhibited intercalated discs, T-tubules, Z-, A-, I-, H- and M-bands and the showed earlier maturation of the cardiomyocytes on cardiogel. We conclude that substructurally matured cardiomyocytes can be produced from ES cells and the cardiac <em>fibroblast</em>-derived ECM (cardiogel) supported earlier maturation of ES-derived cardiomyocytes, in terms of chronotropic characteristics and subcellular structure development.