561 research outputs found
Neural differentiation of the human neuroblastoma cell line IMR32 induces production of a thyrotropin-releasing hormone-like peptide
The human neuroblastoma cell line IMR32 produces and secretes substantial amounts of TRH-immunoreactivity (TRH-IR) as measured with radioimmunoassay (RIA) using the nonspecific antiserum 4319. It was found that synthesis of TRH-IR is dependent on neural differentiation: under serum-free conditions these cells exhibit neural characteristics as defined by morphological and biochemical standards. After culture for 2–5 days in serum-free medium cells grew large neural processes and expressed neuron-specific markers whereas glial-specific markers were absent. TRH-IR became detectable after 4–8 days serum-free conditions. Northern blot and chromatographic analysis, however, failed to detect proTRH mRNA and authentic TRH in these cells. Moreover, TRH-IR was undetectable in the RIA using TRH-specific antiserum 8880. TRH-IR produced by differentiated cells was retained on a QAE Sephadex A-25 anion-exchange column and thus negatively charged. HPLC analysis showed coelution with the synthetic peptide pGlu-Glu-ProNH2. Study of the mechanisms regulating production of this novel peptide in these cells should further elucidate the role differentation plays in the synthesis of neuropeptides
Renal Clearance of the Thyrotropin-Releasing Hormone-Like Peptide Pyroglutamyl-Glutamyl-Prolineamide in Humans
Evidence that the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in human serum may not be secreted by the pituitary gland
Recent studies have revealed that TRH-like immunoreactivity (TRH-LI) in
human serum is predominantly pGlu-Glu-ProNH2 (< EEP-NH2), a peptide
previously found in, among others tissues, the pituitary gland of various
mammalian species. In the rat pituitary, < EEP-NH2 is present in
gonadotrophs and its pituitary content is regulated by gonadal steroids
and gonadotrophin-releasing hormone (GnRH). Hence, we reasoned that <
EEP-NH2 in human serum may also arise, at least in part, from the
pituitary, and that its secretion may correlate with that of
gonadotrophins. Therefore, blood was simultaneously sampled from both
inferior petrosal sinuses, which are major sites of the venous drainage of
the pituitary gland, and a peripheral vein from seven patients with
suspected adrenocorticotrophin-secreting pituitary tumours. In addition,
in six postmenopausal and six cyclic women, peripheral vein blood was
collected at 10-min intervals for 6 h, then a standard 100 micrograms GnRH
test was performed. In the sera, TRH-LI was estimated by RIA with
antiserum 4319, which binds most tripeptides that share the N- and
C-terminal amino acids with TRH (pGlu-His-ProNH2). In addition, LH and FSH
were measured in these sera b
Effects of thyroid status and thyrostatic drugs on hepatic glucuronidation of lodothyronines and other substrates in rats - Induction of phenol UDP-glucuronyltransferase by methimazole
Glucuronidation of iodothyronines in rat liver is catalyzed by at least three UDP-glucuronyltransferases (UGTs): bilirubin UGT, phenol UGT, and androsterone UGT. Bilirubin and phenol UGT activities are regulated by thyroid hormone, but the effect of thyroid status on hepatic glucuronidation of iodothyronines is unknown. We examined the effects of hypothyroidism induced by treatment of rats with propylthiouracil (PTU) or methimazole (MMI) or by thyroidectomy as well as the effects of T4-induced hyperthyroidism on the hepatic UGT activities for T4, T3, bilirubin, p-nitrophenol (PNP), and androsterone. Bilirubin UGT activity was increased in MMI- or PTU-induced hypothyroid and thyroidectomized rats, and decreased in hyperthyroid animals. T4 and, to a lesser extent, T3 UGT activities were increased in MMI- or PTU-induced hypothyroid rats, and T4 but not T3 glucuronidation also showed a significant increase in thyroidectomized rats. T4 but not T3 UGT activity was slightly decreased in hyperthyroid rats. While PNP UGT activity was decreased in thyroidectomized rats and increased in hyperthyroid animals, it was also markedly increased by MMI and slightly increased by PTU-induced hypothyroidism. In T4-substituted rats, MMI did not affect T4, T3, bilirubin and androsterone UGT activities but again strongly induced PNP UGT activity, indicating that this represented a direct induction of PNP UGT by the drug independent of its thyrostatic action. Androsterone UGT activity was hardly affected by thyroid status. Our results suggest a modest, negative control of the hepatic glucuronidation of thyroid hormone by thyroid status, which may be mediated by changes in bilirubin UGT activity. To our knowledge, this is the first report of the marked induction of a hepatic enzyme by MMI, which is not mediated by its thyroid hormone-lowering effect
Hepatic lipase gene expression is transiently induced by gonadotropic hormones in rat ovaries
Hepatic lipase (HL) gene expression was studied in rat ovaries. A transcript lacking exons 1 and 2 could be detected by reverse transcription-polymerase chain reaction (RT-PCR) in the ovaries of mature cyclic females and of immature rats treated with pregnant mare serum followed by human chorionic gonadotropin (hCG) to induce superovulation. By competitive RT-PCR the HL transcript was quantified. Low levels of HL mRNA were detected in ovaries of mature cyclic females and of immature rats. During superovulation HL mRNA was several fold higher than in mature cyclic rats and transiently increased to a maximum at 2 days after hCG treatment. Pulse-labelling of ovarian cells and ovarian slices with [35S]methionine followed by immunoprecipitation with polyclonal anti-HL IgGs showed de novo synthesis of a 47 kDa HL-related protein. Expression of the protein was transiently induced by gonadotropins with a peak at 2 days after hCG treatment. Induction of liver-type lipase activity occurred only after HL mRNA and synthesis of the HL-related protein had returned to pre-stimulatory levels. We conclude that in rat ovaries the HL gene is expressed into a variant mRNA and a 47 kDa protein. The expression of the HL gene in ovaries is inducible and precedes the expression of the mature, enzymatically active liver-type lipase
Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans
TRH-like peptides have been identified that differ from TRH
(pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like
immunoreactivity (TRH-LI) in human serum and urine by RIA with
TRH-specific antiserum 8880 or with antiserum 4319, which binds most
peptides with the structure pGlu-X-ProNH2. TRH was undetectable in serum
(< 25 pg/mL), but TRH-LI was detected with antiserum 4319 in serum of 27
normal subjects, 21 control patients, and 12 patients with carcinoid
tumors (range 17-45, 5-79, and 18-16,600 pg/mL, respectively). Because
serum was kept for at least 2 h at room temperature, which causes
degradation of TRH, pGlu-Phe-ProNH2, and pGlu-Tyr-ProNH2, serum TRH-LI is
not caused by these peptides. On high-performance liquid chromatography,
serum TRH-LI coeluted with pGlu-Glu-ProNH2 (< EEP-NH2), a peptide produced
in, among others, the prostate. Urine of normals and control patients also
contained TRH-LI (range 1.14-4.97 and 0.24-5.51 ng/mL, respectively), with
similar levels in males and females. TRH represented only 2% of urinary
TRH-LI, and anion-exchange chromatography and high-performance liquid
chromatography revealed that most TRH-LI in urine was < EEP-NH2. In
patients with carcinoid tumors, increased urinary TRH-LI levels were noted
(range 1.35-962.4 ng/mL). Urinary TRH-LI correlated positively with
urinary creatinine, and the urinary clearance rate of TRH-LI was similar
to the glomerular filtration rate. In addition, serum TRH-LI was increased
in 17 hemodialysis patients (43-373 pg/mL). This suggests that serum <
EEP-NH2 is cleared by glomerular filtration wit
Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans
TRH-like peptides have been identified that differ from TRH
(pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like
immunoreactivity (TRH-LI) in human serum and urine by RIA with
TRH-specific antiserum 8880 or with antiserum 4319, which binds most
peptides with the structure pGlu-X-ProNH2. TRH was undetectable in serum
(< 25 pg/mL), but TRH-LI was detected with antiserum 4319 in serum of 27
normal subjects, 21 control patients, and 12 patients with carcinoid
tumors (range 17-45, 5-79, and 18-16,600 pg/mL, respectively). Because
serum was kept for at least 2 h at room temperature, which causes
degradation of TRH, pGlu-Phe-ProNH2, and pGlu-Tyr-ProNH2, serum TRH-LI is
not caused by these peptides. On high-performance liquid chromatography,
serum TRH-LI coeluted with pGlu-Glu-ProNH2 (< EEP-NH2), a peptide produced
in, among others, the prostate. Urine of normals and control patients also
contained TRH-LI (range 1.14-4.97 and 0.24-5.51 ng/mL, respectively), with
similar levels in males and females. TRH represented only 2% of urinary
TRH-LI, and anion-exchange chromatography and high-performance liquid
chromatography revealed that most TRH-LI in urine was < EEP-NH2. In
patients with carcinoid tumors, increased urinary TRH-LI levels were noted
(range 1.35-962.4 ng/mL). Urinary TRH-LI correlated positively with
urinary creatinine, and the urinary clearance rate of TRH-LI was similar
to the glomerular filtration rate. In addition, serum TRH-LI was increased
in 17 hemodialysis patients (43-373 pg/mL). This suggests that serum <
EEP-NH2 is cleared by glomerular filtration with little tubular
resorption. The possible role of the prostate as a source of urinary
TRH-LI was evaluated in 11 men with prostate cancer, showing a 25%
decrease in urinary TRH-LI excretion after prostatectomy (0.19 +/- 0.02
vs. 0.15 +/- 0.01 ng/mumol creatinine, mean +/- SEM). However, TRH-LI was
similar in spontaneously voided urine and in urine obtained through a
nephrostomy cannula from 16 patients with unilateral urinary tract
obstruction (0.15 +/- 0.01 vs. 0.14 +/- 0.01 ng/mumol creatinine). These
data indicate that: 1) TRH-LI in human serum represents largely < EEP-NH2,
which is cleared by renal excretion; 2) part of urinary < EEP-NH2 is
derived from prostatic secretion into the blood and not directly into
urine; and 3) urinary < EEP-NH2 can be used as marker for carcinoid
tumors
The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense
Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense
Real-time and Multichannel Measurement of Contractility of hiPSC-Derived 3D Skeletal Muscle using Fiber Optics-Based Sensing
As the field of cardiac and skeletal muscle tissue engineering expands, so does the need for accurate and reliable systems to generate in vitro 3D tissues and analyze their functional properties. In this study, the Cuore is introduced, a system that integrates sensors based on optical fibers and uses the principle of light interferometry to detect the contraction of 3D Tissue Engineered Skeletal Muscles (3D-TESMs). The technology employed in the Cuore allows for reproducible and multichannel force measurements down to a nano-Newtons resolution while maintaining sterility and permitting continuous non-invasive recording within and outside standard tissue culture incubators. Thanks to the integrated electrodes for electrical pulse stimulation (EPS), 3D-TESMs generated from three independent hiPSC-derived myogenic progenitors (MPs) lines are stimulated and the contractility is recorded over the course of a week. Through the modulation of different EPS parameters, the optimal combination to induce the 3D-TESMs in producing fully fused tetani without causing damage is determined. Furthermore, 3D-TESMs from different lines exhibit characteristic signatures of spontaneous contractility and response to caffeine, verapamil, and the β-agonist clenbuterol. The ease of use, high sensitivity, and the integrated electrodes and sensors make the Cuore an ideal technology to investigate the biology of contractile tissues and their response to drugs.</p
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A dynamic supramolecular polyurethane network whose mechanical properties are kinetically controlled
We report the synthesis and characterization of a kinetically controlled, thermoreversible supramolecular polyurethane whose mechanical properties depend unusually strongly on the processing history. Materials were prepared by solution casting, quenching and annealing of quenched material, allowing pronounced micro-structural evolution, which leads to rapid increases in modulus as determined by rheological analysis. Tensile tests showed that the quenched material is soft, weak and ductile (shear modulus ~ 5 MPa, elongation ~ 250 %), but after annealing, at 70 °C for one hour, it becomes stiffer, stronger and more brittle (~ 20 MPa, ~ 20 %). FTIR and NMR spectroscopic analysis, coupled with MDSC and SAXS, were performed to investigate the network’s dynamic structural changes. SAXS results suggest the presence of a lamellar structure in the sample when solution cast at high temperature, or annealed. This ordering is unique when compared to structurally-related supramolecular bisurethane and bisurea polymers, and may be the cause of the observed path dependence. These mechanical properties, which can be switched repeatedly by simple thermal treatments, coupled with its adhesion properties as determined from peel and tack tests, make it an excellent candidate as a recyclable material for adhesives and coatings
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