[Study on the clinical characteristics of auditory neuropathy patients with normal pure tone average or mild hearing loss].

Objective: The purpose of this study was to analyze the clinical characteristics of auditory neuropathy (AN) patients with normal hearing or mild hearing loss. Methods: Data from Multicenter Study on Clinical Diagnosis and Intervention of Acoustic Neuropathy (registration number: ChiCTR2100050125). According to the Chinese clinical practice guideline of auditory neuropathy (version 2022), these patients divided into two groups: the normal hearing group (PTA Normal, PTAN group, the average hearing threshold<20 dB HL) and the mild hearing loss group (PTA Mild hearing loss, PTAM group, the average hearing threshold between 20-35 dBHL). The audiology characteristics, clinical features, and follow-up were analyzed. Data analysis was conducted using GraphPad Prism 8 and SPSS 20.0 software. Results: A total of 75 AN with normal hearing or mild hearing loss were included in this study. The PTAN group consisted of 19 patients (38 ears), including 12 males and 7 females. The average onset age was (16.9±4.5) years old, while the test age was (22.1±5.8) years old for PTAN group. The PTAM group consisted of 56 patients (112 ears), including 29 males and 27 females. The average onset age was (16.2±7.9) years old, while the test age was (23.9±9.0) yeas old for PTAM group. The average hearing threshold of low frequency (0.125-0.5 kHz) was significantly decreased. ABR disappeared in 86.00% (126/150) of the patients. The speech recognition rate was 71.80±22.44% in the PTAN group and 58.08±29.28% in the PTAM group.-SP/AP was 0.98±0.47 in the PTAN and 1.07±0.63 in PTAM group; 40 (53.33%) patients had tinnitus. 29 patients (58 ears) were followed up, including 10 patients (20 ears) in the PTAN group and 19 patients (38 ears) in the PTAM group. There was no significant change in hearing threshold in short-term follow-up (<3 years). With the extension of the disease duration (>3 years), the PTAN group tended to decrease at low frequency, and the PTAM group decreased at high frequency first. The hearing threshold at 0.25 kHz in the PTAN group and 4 kHz in the PTAM group decreased significantly. Conclusions: AN patients with normal hearing or mild hearing loss exhibit abnormal results in audiological examination results, including ABR, electrocochleography and speech discrimination score. A combination of audiological tests should be used to make the diagnosis of AN. With the progression of the disease, AN with normal hearing or mild hearing loss tends to decrease.

[Research on the variation in distortion product otoacoustic emissions in patients with auditory neuropathy during the natural course of the disease].

Objective: The purpose of this study was to investigate the characteristics of distortion product otoacoustic emissions (DPOAE) in patients with auditory neuropathy (AN). The factors affecting DPOAE elicitation rate of each frequency, elicitation rate of each ear and change rate of first and last diagnosis in the natural course were analyzed. Methods: The sample was obtained from the Multicenter Study on Clinical Diagnosis and Intervention of AN (registration number: ChiCTR2100050125), and the diagnostic criteria for AN were based on the Chinese Clinical Practice Guidelines of Auditory Neuropathy (version 2022). Patients with bilateral AN who underwent 2 or more DPOAE tests were screened and divided into infant groups (≤3 years old) and non-infant groups (>3 years old) according to the age of detection, and the trend of DPOAE elicitation rate of each frequency, elicitation rate of each ear and change rate in the natural course of disease were analyzed, in order to explore the relevant influencing factors. Results: A total of 165 patients (330 ears) with AN were included in the study. The overall DPOAE elicitation rate per ear was 77.0%±29.4% at the initial diagnosis and 65.1%±35.2% at the final diagnosis, with a reduction observed in the elicitation rate of 171 ears (51.82%). In the infant group, there were 49 cases (98 ears), including 28 males and 21 females, whose found age ranged from 0 to 3 years old, with a median age of 0.7 years. DPOAE elicitation rate per ear was 57.9%±35.5% in the initial diagnosis, and 32.4%±32.1% in the final diagnosis, with a reduction observed in the elicitation rate of 69 ears (70.41%). In the non-infant group, there were 116 cases (232 ears), including 59 males and 57 females, ranging in found age from 3.9 to 40 years old, with a median age of 14 years old. DPOAE elicitation rate per ear was 84.6%±23.4% in the initial diagnosis, and 78.3%±27.1% in the final diagnosis, with a reduction observed in the elicitation rate of 102 ears (43.97%). Age was found to be correlated with DPOAE changes by multicategorical unordered logistic regression analysis (B=-0.224, OR=0.799, P<0.001). Conclusions: The elicitation rate of DPOAE in AN patients decreases or even disappears with increasing disease duration; The rate of DPOAE extraction is found to be lower in infant patients with auditory neuropathy (AN) compared to non-infant AN patients. Additionally, it is observed that the decrease in DPOAE extraction rate is more pronounced in infant AN patients as the disease progressed, as compared to non-infant AN patients. DPOAE and cochlear microphonic potentials should be fully combined for accurate diagnosis, and regular follow-up should be conducted to understand the natural course of the disease and give personalized guidance and assistance.

Prognostic role of the systemic immune-inflammation index and pan-immune inflammation value for outcomes of breast cancer: a systematic review and meta-analysis.

OBJECTIVE: This review examined the literature for evidence on the prognostic ability of systemic immune-inflammation index (SII) and pan-immune inflammation value (PIV) for predicting overall survival (OS) and disease-free survival (DFS) in breast cancer patients. MATERIALS AND METHODS: PubMed, Embase, Scopus, and Web of Science were searched with Google Scholar for gray literature. All types of studies reporting the association between SII or PIV and OS or DFS of breast cancer were eligible. RESULTS: 13 studies on SII and 4 studies on PIV were included. Meta-analysis showed that a high SII was a significant predictor of OS (HR: 1.97 95% CI: 1.54, 2.52 I2=76%) and DFS (HR: 2.07 95% CI: 1.50, 2.86 I2=79%) in breast cancer patients. These results did not change on sensitivity analysis and were more or less stable on multiple subgroup analyses. Pooled analysis showed that high PIV was also a significant predictor of poor OS (HR: 2.63 95% CI: 1.46, 4.74 I2=71%) and DFS (HR: 1.64 95% CI: 1.23, 2.17 I2=0%) in breast cancer patients. CONCLUSIONS: High SII and PIV can predict poor OS and DFS in breast cancer patients. High heterogeneity and the observational nature of data are important limitations of the review. Further studies are needed specifically on PIV to increase the strength of the evidence.

[Identification of a novel mutation of SOX10 gene and analysis of the phenotype].

Objective: To explore the clinical features and pathogenic mechanisms of a special syndrome with congenital sensorineural hearing loss, albinism, heterochromia iridis, nystagmus and myelin dysplasia. Methods: Detailed medical history, systematic audiology tests, ophthalmic and neurological examinations were carried out to analyze the clinical features of the child, and further molecular genetic tests including chromosome karyotype analysis, and deafness gene screening were conducted. Results: A new de novo heterozygous mutation (c.336G>T/p.Met112Ile) was detected in the child, while both his parents were demonstrated to be wild-type and symptom free. The analysis of clinical features indicated the diagnosis of PCW syndrome. Conclusion: This study identified a new mutation of SOX10 gene, which enriched the mutation spectrum of this gene. And the analysis of clinical characteristics of this patient also expanded the phenotype of this gene. This study provided a reference for clinical diagnosis and genetic diagnosis of PCW syndrome.

[The ability of gap detection in patients with acoustic neuroma].

Objective:We aimed to provide a basis for the clinical study of acoustic neuroma through investigating the ability of temporal gap detection in acoustic neuroma patients and comparing the abilities with those in people with normal and impaired hearing. Method:Twenty-two patients with confirmed acoustic neuroma, 30 normal hearing patients and 16 patients with sensorineural hearing loss were enrolled in this study, and the interval threshold for awareness of each group was tested. Result:The mean temporal gap detection test（TGDT） threshold of the normal hearing group was （3.56±0.82） ms; the sensorineural hearing loss group's was （3.91±1.46） ms; TGDT threshold of healthy side of acoustic neuroma patients was （4.01±1.86） ms; TGDT threshold of the impaired side of acoustic neuroma patients was （9.48±9.46）ms. After statistical analysis, we found that excepting for the test of phonetically balanced maximum （PBmax） and TGDT, other results in the sensorineural hearing loss group and normal hearing group is of no statistical difference. The difference between the affected side of the acoustic neuroma group and the other groups was statistically significant （P<0.05）. There was no linear correlation between the value of TGDT threshold and PBmax （P> 0.05）. TGDT value of normal people has no significant difference among people of different genders and ears of different individuals. Conclusion:The TGDT of the healthy ear of the patients with acoustic neuroma is not affected, and there is no significant change compared with normal people. The TGDT test has a good consistency with the PBmax results. The time interval response ability of the affected ear of the acoustic neuroma is significantly weaker than that of the normal person. The combined test of PBmax and TGDT will contribute to the diagnosis of retrocochlear disease.

[Classification of sudden deafness prognosis based on deep learning method].

Objective:This study aimed to develop predictive models for sudden sensorineural hearing loss through deep belief network (DBN) and explore whether the model performances differ when adopting different outcome criteria. Method: 228 potential predictors involving the clinical characteristics, audio logical data, and serological parameters out of 1 220 hospitalized SSHL patients who were admitted from June 2008 to December 2015 were analyzed retrospectively. The hearing data of sudden deafness were classified into two or four categories based on Chinese criteria and Siegel criteria, which were used to develop the DBN models. The area under the receiver operating characteristic curve (ROC-AUC) and accuracy were used to compare the predictive performance of different models. Result: The DBN model developed for predicting the dichotomized outcomes had better performance than that of the fourcategory outcomes. When the iteration number reached 500 times, DBN model constructed for prediction of dichotomized outcomes based on Siegel's criteria had demonstrated the best performance with an accuracy of 76.25% and an AUC of 0.81. According to indices from first layer weights, DBN gave a rank of top 10 sensitive features for hearing outcome prediction focusing on indicators regarding coagulation, demographics and pre-treatment hearing levels independent of the outcome assessment criteria. Conclusion: DBN provides a robust outcome prediction ability in SSHL datasets with rich and complex variables, especially when utilized to predict dichotomized outcomes based on the Siegel criteria. In addition, this advanced deep learning technique can automatically extract valuable predictors, which is consistent with those that had been verified in previous studies by traditional statistical methods. This study provides further evidence for extending the use of DBN algorithm to the field of developing prediction or classification models for other otological diseases in the future..

Fulvestrant inhibits the glycolysis of prolactinoma GH3 cells by downregulating IRE1/XBP1 signaling pathway.

OBJECTIVE: We aimed to evaluate the effects of fulvestrant on the glycolysis of prolactinoma GH3 cells, and reveal the potential regulatory mechanisms. MATERIALS AND METHODS: Prolactinoma cell line GH3 was treated with different concentrations of fulvestrant (0, 0.12, 0.25, 0.5 and 1 ng/ml) for 4 h. siRNAs XBP1s and XBP1u were constructed to treat GH3 cells. The expression levels of XBP1s, XBP1u, IRE1, PKM2 and GRP78 of GH3 cells were detected by Western blot. Meanwhile, the glycolytic activity of GH3 cells, including the glucose uptake, ATP/ADP, and lactate production were detected. RESULTS: The expression levels of XBP1s and XBP1u were significantly inhibited by fulvestrant in a dose-dependent manner. The glucose uptake, ATP/ADP and lactate production of GH3 cells were significantly inhibited by fulvestrant as well as siRNA XBP1s and XBP1u (p < 0.05). Western blot analysis suggested that the expression levels of IRE1, PKM2 and GRP78 were significantly decreased in GH3 cells treated by fulvestrant as well as siRNA XBP1s and XBP1u, compared with those in normal control (p < 0.05). CONCLUSIONS: Fulvestrant could inhibit the glycolysis of GH3 cells by downregulating IRE1/XBP1 signaling pathway, and this process was closely related with the downregulation of PKM2.

Nicotinamide Facilitates Mesenchymal Stem Cell Differentiation Into Insulin-Producing Cells and Homing to Pancreas in Diabetic Mice.

OBJECTIVE: This study aimed to investigate the effect of nicotinamide on differentiation of mesenchymal stem cells (MSCs) into insulin-producing cells (IPCs) in vivo in mice and on homing of MSCs to the pancreas after being intravenously infused. METHODS: Streptozotocin (STZ)-induced diabetic Balb/c mice received syngeneic transplantation of carboxyfluorescein succinimidyl ester (CFSE)-labeled bone marrow MSCs into the liver or tail vein. Nicotinamide was intraperitoneally injected into mice at a dose of 500 mg/kg body weight per day after STZ administration. Mice who received saline solution injection instead of nicotinamide were involved as control. RESULTS: Mice that received nicotinamide injection showed lower blood glucose, higher serum insulin, and more improved glucose tolerance compared with the control group. Immunohistochemistry analysis showed that higher levels of insulin staining and higher percentages of CFSE+/insulin+ cells were observed in the liver and pancreas sections of mice who received nicotinamide injection compared with the control group. The percentage of CFSE+/insulin+ cells was positively correlated with serum insulin level. Real-time polymerase chain reaction results showed that the implanted MSCs in mice who received nicotinamide injection exhibited higher levels of ß-cell-related gene expression than the control group. More CFSE-labeled MSCs appeared in the pancreas of mice who received nicotinamide injection compared with the control group after being intravenously infused, whereas the amount of CFSE-labeled MSCs in the liver was not affected by nicotinamide injection. CONCLUSIONS: Nicotinamide facilitates MSCs differentiating into functional IPCs in vivo in diabetic mice and promotes intravenously infused MSCs to home to the pancreas.

First Report of Ageratum yellow vein virus Causing Tobacco Leaf Curl Disease in Fujian Province, China.

A leaf curling disease was observed on 7% of tobacco plants during December 2005 in research plots in the Cangshan District of Fuzhou, Fujian, China. Tobacco plants were infested with Bemisia tabaci, suggesting begomovirus etiology. To identify possible begomoviruses, total DNA was extracted from four symptomatic leaf samples (F1, F2, F3, and F4). The degenerate primers PA and PB were used to amplify part of the intergenic region and AV2 gene of DNA-A-like molecules (3). A 500-bp DNA fragment was amplified by PCR from all four samples. The PCR products were cloned and sequenced (GenBank Accession Nos. EF531601-EF531603 and EF527823). Alignment of the 500-bp sequences for the four isolates indicated that they shared 98.5 to 99.6% nt identity, suggesting that the plants were all infected by the same virus. Overlapping primers TV-Full-F (5'-GGATCCTCTTTTGAACGAGTTTCC-3') and TV-Full-R (5'-GGATCCCACATGTTTAAAATAATAC-3') were then designed to amplify the full-length DNA-A from sample F2. The sequence was 2,754 nucleotides long (GenBank Accession No. EF527823). A comparison with other begomoviruses indicated the F2 DNA-A had the highest nucleotide sequence identity (95.7%) with Ageratum yellow vein virus (AYVV; GenBank Accession No. X74516) from Singapore. To further test whether DNAß was associated with the four viral isolates, a universal DNAß primer pair (beta 01 and beta 02) was used (4). An amplicon of approximately 1.3 kb was obtained from all samples. The DNAß molecule from F2 was then cloned and sequenced. F2 DNAß was 1,345 nucleotides long (GenBank Accession No. EF527824), sharing the highest nucleotide sequence identity with the DNAß of Tomato leaf curl virus (97.2%) from Taiwan (GenBank Accession No. AJ542495) and AYVV (88.8%) from Singapore (GenBank Accession No. AJ252072). The disease agent was transmitted to Nicotiana tabacum, N. glutinosa, Ageratum conyzoides, Oxalis corymbosa, and Phyllanthus urinaria plants by whiteflies (B. tabaci) when field infected virus isolate F2 was used as inoculum. In N. tabacum and N. glutinosa plants, yellow vein symptoms were initially observed in young leaves. However, these symptoms disappeared later during infection and vein swelling and downward leaf curling symptoms in N. tabacum and vein swelling and upward leaf curling in N. glutinosa were observed. In A. conyzoides, O. corymbosa, and P. urinaria plants, typical yellow vein symptoms were observed. The presence of the virus and DNAß in symptomatic plants was verified by PCR with primer pairs TV-Full-F/TV-Full-R and beta 01/beta 02, respectively. The above sequence and whitefly transmission results confirmed that the tobacco samples were infected by AYVV. In China, Tobacco leaf curl Yunnan virus, Tobacco curly shoot virus, and Tomato yellow leaf curl China virus were reported to be associated with tobacco leaf curl disease (1,3). To our knowledge, this is the first report of AYVV infecting tobacco in China. A. conyzoides is a widely distributed weed in south China and AYVV was reported in A. conyzoides in Hainan Island, China (2). Therefore, this virus may pose a serious threat to tobacco production in south China. References: (1) Z. Li et al. Phytopathology 95:902, 2005. (2) Q. Xiong et al. Phytopathology 97:405, 2007. (3) X. Zhou et al. Arch. Virol. 146:1599, 2001. (4) X. Zhou et al. J. Gen. Virol. 84:237, 2003.

Epitope tag mapping of the extracellular and cytoplasmic domains of the rat parathyroid hormone (PTH)/PTH-related peptide receptor.

The PTH/PTH-related peptide (PTHrP) receptor is predicted to span the plasma membrane seven times with an amino-terminal extracellular extension and a cytoplasmic carboxyl-terminal tail. To assess this prediction, we inserted 10- or 9-amino acid epitope tags from c-myc or hemophilus influenza hemaglutinin (HA), which are recognized by the monoclonal antibodies 9E10 and 12Ca5, respectively, in different extracellular and cytoplasmic regions of the receptor and examined the immunoreactivity of the epitopes in intact and permeabilized cells. The data show that the epitopes were well tolerated when introduced into the E2 region of the extracellular amino-terminus (E2-myc and E2-HA), in the first extracellular loop (EL1), in the second and third cytoplasmic loops (CL2c and CL3), or in the carboxyl-terminal tail (T-myc). Receptors tagged at these locations were well expressed, bound PTH with high affinity, and increased cAMP accumulation with a good efficiency. Receptors tagged in the second and third extracellular loops (EL2c and EL3c) or the first cytoplasmic loop (CL1c) bound the PTH radioligand with a low affinity, stimulated cAMP accumulation with a low efficiency, and had low expression levels. The receptors tagged on presumed extracellular regions, E2-myc, E2-HA, EL1, EL2c, and EL3c, were readily detected on the surface of intact cells with the monoclonal antibody against the epitope tag. In contrast, receptors tagged with the c-myc epitope in the cytoplasmic loops (CL1c, CL2c, and CL3) or in the carboxyl-terminal tail (T-myc) did not show any 9E10 binding in intact cells. These receptors, however, were well expressed on the cell surface, as detected by the binding of the monoclonal antibody, 12Ca5, to the HA tag that was introduced into the E2 region of these constructs. The c-myc epitopes, however, became accessible after permeabilization of the cell membrane. In conclusion, these data provide experimental evidence for the sidedness of the extracellular and cytoplasmic domains of the PTH/PTHrP receptor.

Myc activates telomerase.

Telomere maintenance has been proposed as an essential prerequisite to human tumor development. The telomerase enzyme is itself a marker for tumor cells, but the genetic alterations that activate the enzyme during neoplastic transformation have remained a mystery. Here, we show that Myc induces telomerase in both normal human mammary epithelial cells (HMECs) and normal human diploid fibroblasts. Myc increases expression of hEST2 (hTRT/TP2), the limiting subunit of telomerase, and both Myc and hEST2 can extend the life span of HMECs. The ability of Myc to activate telomerase may contribute to its ability to promote tumor formation.

Downregulation of the PTH/PTHrP receptor by vitamin D3 in the osteoblast-like ROS 17/2.8 cells.

Effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the parathyroid hormone (PTH)/PTH-related peptide (rP) receptor protein and mRNA in ROS 17/2.8 cells were studied. Treatment of ROS 17/2.8 cells with 1,25(OH)2D3 caused time- and dose-dependent suppression of PTH/PTHrP receptor number and immunoreactivity. The effects required more than 24 h incubation with 1,25(OH)2D3 and were maximal by 72 h. The cells did not recover their PTH/PTHrP receptors even after 4 days of treatment with control medium. Treatment with low concentrations of 1,25(OH)2D3 (0.1 M) dramatically decreased the PTH/PTHrP receptor mRNA levels, which were maximal after 24 h of incubation. The half-life of the PTH/PTHrP receptor transcript, 6-8 h, was similar in control and 1,25(OH)2D3-treated cells, suggesting that 1,25(OH)2D3 acts in controlling transcription of the PTH/PTHrP receptor gene but does not change the degradation rate of the PTH/PTHrP receptor transcripts. These data indicate that 1,25(OH)2D3 has a potent inhibitory effect on the expression of the PTH/PTHrP receptor protein and mRNA in ROS 17/2.8 cells.

Molecular cloning of a type A chicken corticotropin-releasing factor receptor with high affinity for urotensin I.

The hypothalamic-pituitary-adrenal axis is an essential physiological system in many species. CRF, the major neuropeptide regulating ACTH secretion, is highly conserved in its primary sequence. Evolutionary conservation of the CRF sequence suggests that the CRF receptor (CRF-R) complementary DNA and examined its properties. The avian CRF-R complementary DNA encodes a 420-amino acid protein that is 87-88% identical to those of human, rat, and mouse. Most sequence divergence occurs in the putative signal peptide and the extracellular amino-terminus of the receptor. Five additional amino acids are inserted in the amino-terminus of the cCRF-R. When expressed in COS-7 cells, the cCRF-R binds the CRF and urotensin I radioligands with high affinities. Urotensin I competes for binding to the chicken CRF-R, expressed in COS-7 cells, with an apparent affinity 20 times higher than that of CRF. Both urotensin I and sauvagine were more effective in stimulating cAMP accumulation in COS-7 cells transfected with the cCRF-R than CRF. The effects of CRF and urotensin I on inositol phosphate accumulation were also tested. Urotensin I was an effective as CRF in stimulating inositol phosphate accumulation in COS-7 cells transfected with the cCRF-R. These data suggest that the sequence of the CRF-R is highly conserved from avian to mammalian species and that, despite its high sequence homology to the type A mammalian CRF-R, the ligand binding properties of cCRF-R are similar to those of the type B CRF-R i.e. a higher affinity for urotensin I than for CRF.

The alternatively spliced type II corticotropin-releasing factor receptor, stably expressed in LLCPK-1 cells, is not well coupled to the G protein(s).

Two alternatively spliced corticotropin-releasing factor receptor (CRF-R) cDNAs, type I and type II, were recently isolated from a human cDNA library. The two cDNAs are identical except that the type II cDNA encodes an additional 29 amino acid inserted in the first putative cytoplasmic loop. Since the first cytoplasmic loop is highly conserved in all the members of the hCRF receptor family we have examined whether the presence of the 29 amino acid cassette in CRF-RII influences G protein coupling in LLCPK-1 cells stably expressing the type I and type II hCRF receptors. Whether measured in intact cells or in membrane preparations, LLCPK-1 cells stably expressing CRF-RII have a 4-5 fold lower binding affinity. Maximal CRF-stimulated cAMP accumulation in LLCPK-1 cells stably expressing CRF-RI was 10-15-fold higher than that in LLCPK-1 cells expressing CRF-RII. The EC50 for CRF-stimulated cAMP accumulation in hCRF-RI-expressing cells was in the range of 0.5 +/- 0.2 nM. In contrast, the EC50 for CRF-stimulated cAMP accumulation in hCRF-RII expressing cells was 7.7 +/- 0.2 nM. hCRF increased phosphoinositide turnover in LLCPK-1 cells stably expressing CRF-RI but not in those expressing CRF-RII; this effect required hCRF concentrations of 100 nM and higher. In membrane preparations, GTP-gamma-S inhibited hCRF binding to CRF-RI and shifted the binding Kd from 4.5 nM to 16.7 nM. Conversely, GTP-gamma-S did not influence hCRF binding to CRF-RII in broken cell membranes. Additionally, CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RI was potentiated by GTP, whereas CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RII was insensitive to GTP. These data indicate that CRF-RII is not well coupled to the G protein. Since the only difference between the CRF-RII and CRF-RI is the insert in the first putative cytoplasmic loop, these data indicate that the first cytoplasmic loop plays a crucial role in hCRF receptor coupling to the G protein.

Signaling properties of mouse and human corticotropin-releasing factor (CRF) receptors: decreased coupling efficiency of human type II CRF receptor.

CRF is the primary neuroregulator of the function of the hypothalamic-pituitary-adrenal axis. We have recently cloned a mouse CRF receptor (mCRF-R) complementary DNA (cDNA) from an AtT-20 cell cDNA library by polymerase chain reaction. To compare the functions of mCRF-R to those of the human type I and type II CRF receptors (hCRF-RI and hCRF-RII), cDNAs were cloned into the expression vector pcDNA1 and transfected into COS-7 cells. CRF binding and CRF-stimulated cAMP accumulation as well as phosphoinositide hydrolysis were measured. Scatchard analysis of the binding of 125I-labeled [Tyr0]r/hCRF ([125I]CRF) to COS-7 cells expressing mCRF-R and hCRF-RI cDNAs revealed the same apparent Kd (9 nM). In contrast, the apparent binding Kd for hCRF-RII was 20 nM CRF. Maximal stimulatory concentrations (1 microM) of rat/human CRF-(1-41) (r/hCRF) increased cAMP accumulation in COS-7 cells transfected with mCRF-R, hCRF-RI, and hCRF-RII cDNA plasmid (10 micrograms each) from basal values of 8-19 pmol/10(5) cells.15 min to 84 +/- 10, 87 +/- 16, and 45 +/- 16 pmol/10(5) cells.15 min, respectively. The EC50 values of r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing mCRF-R and hCRF-RI cDNAs were similar at 0.4 +/- 0.2 and 0.7 +/- 0.2 nM, respectively. Conversely, the EC50 of r/hCRF-stimulated cAMP accumulation in hCRF-RII-transfected COS-7 cells was 47.5 +/- 18.9 nM. As the level of expression of hCRF-RII was lower than that of hCRF-RI, we compared r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing low and high levels of hCRF-RI. The EC50 for r/hCRF-stimulated cAMP accumulation in COS-7 cells transfected with hCRF-RI did not change when receptor expression was varied by a factor of 1- to 8.4-fold. In contrast, the EC50 for r/hCRF-stimulated cAMP accumulation mediated by hCRF-RII was at least 100-fold higher than that mediated by the hCRF-RI in COS-7 cells, which suggests poor coupling between hCRF-RII and adenylate cyclase. Inositol phosphate (IP) levels were also determined in mCRF-R, hCRF-RI, and hCRF-RII cDNA-transfected COS-7 cells stimulated with increasing concentrations of r/hCRF. r/hCRF-stimulated IPs accumulation was dose dependent in COS-7 cells expressing mCRF-R and hCRF-RI using 100 and 1000 nM r/hCRF. Concentrations of 10 (or less) nM r/hCRF had no effect on IP generation. hCRF-RII did not mediate stimulation of IP even at 1000 nM r/hCRF.(ABSTRACT TRUNCATED AT 400 WORDS)

Truncation of the carboxyl-terminal region of the rat parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of adenylyl cyclase but not phospholipase C.

The functional role of the rat parathyroid hormone(PTH)/PTH-related peptide (PTHrP) receptor's carboxyl-terminal region was characterized by comparing the binding and signaling properties of receptors that have 78 and 111 amino acid deletions (R513 and R480, respectively), with those of the 591-amino acid wild-type (WT) receptor. R480 and R513 have 4- and 1.5-fold lower apparent Kd values for rat PTH-(1-34) (rPTH), compared with the WT receptor (WT, 1.81 +/- 0.19 nM; R513, 1.24 +/- 0.12 nM; R480, 0.48 +/- 0.05 nM, mean +/- S.E.). PTH (100 nM)-stimulated cAMP accumulation and polyphosphoinositide hydrolysis both correlated positively with receptor expression. However, whereas PTH-stimulated polyphosphoinositide hydrolysis was indistinguishable among WT and either truncated mutant at comparable levels of expressed receptors, maximal PTH-stimulated cAMP accumulation was 4-6- and 2-3-fold higher in cells expressing R480 and R513, respectively. Furthermore, pretreatment of COS-7 cells with 100 ng/ml of pertussis toxin (PTX) enhanced PTH-stimulated cAMP accumulation in cells expressing the WT receptor, but failed to do so in cells expressing either R480 or R513. Thus, sequences in the PTH/PTHrP receptor's carboxyl-terminal tail lower the affinity of the WT receptor for agonist; directly interact with, or indirectly facilitate the interaction of the receptor with a PTX-sensitive G protein that inhibits adenylyl cyclase; and decrease the efficacy with which the receptor interacts with Gs.

Effect of porphyrins on the hematopoietic recovery of mice treated with gamma-radiation.

Porphyrins are a group of organic compounds involved in a wide spectrum of fundamental biological processes. Non-metallic, naturally occurring and synthetic porphyrin derivatives may produce cytotoxic effects in malignant or normal tissues exposed to visible light. Supra-clinical doses of the photosensitizing porphyrin, Photofrin are hematostimulatory when administered to normal and immunosuppressed inbred mice. To determine if a non-photosensitizing metalloporphyrin has similar hematostimulatory activity, we have synthesized iron (III) hematoporphyrin chloride (FeHp) and administered it to sub-lethally irradiated mice. FeHp (10 mg/kg) given 1 and 4 days or 1, 4 and 7 days following sub-lethal (7 Gy) whole body irradiation significantly increased spleen colony forming units of progenitor cells of the granulocyte-macrophage lineage (CFU-GM) 14 days post-irradiation, relative to irradiated controls. In addition, total splenocyte numbers were significantly increased 17 days post-irradiation in mice that had received FeHp 1 and 4 days post-irradiation. When FeHp was given 24 hours prior to irradiation and again 48 hours or 48 and 96 hours post-irradiation, significant increases in splenic CFU-GM and spleen cell numbers, relative to control mice, were observed 15 days post-irradiation. A non-metallic photosensitizing monomeric fraction of Photofrin, deuteroporphyrin IX, 2,4 (4,2) hydroxyethyl vinyl (HVD) was compared to Photofrin for its ability to influence the hematopoietic recovery of irradiated mice. Only Photofrin but not HVD given in 3 doses (10 mg/kg) 1, 4 and 7 days following irradiation (4.8 Gy) significantly enhanced the recovery of spleen cellularity and splenic CFU-GM. In addition, Photofrin significantly increased bone marrow CFU-GM 7 and 10 days following the sub-lethal dose of gamma-radiation. The mechanism by which certain porphyrins augment hematopoiesis in the mouse is unknown. However, the identification of FeHp as a non-photosensitizing monomeric porphyrin with hematostimulatory activity in vivo, indicates that further study of metalloporphyrins is warranted and may reveal their clinical potential within the context of therapeutically-induced immunosuppression.

Parathyroid hormone (PTH) stimulates adrenocorticotropin release in AtT-20 cells stably expressing a common receptor for PTH and PTH-related peptide.

Complementary DNA encoding a rat bone PTH/PTHrP receptor was stably expressed in the murine corticotroph cell line, AtT-20. Several clones, expressing variable numbers of PTH/PTHrP receptors, were developed. In contrast to the relatively low binding affinity (apparent Kd = 15 nM) observed in COS-7 cells transiently expressing the PTH/PTHrP receptor, all AtT-20 stable transfectants bound [Nle8,18,Tyr34]bPTH(1-34)NH2 (NlePTH) with an affinity that was indistinguishable from that observed in ROS 17/2.8 cells expressing native PTH/PTHrP receptors. Additionally, NlePTH dramatically increased cAMP accumulation and ACTH release in AtT-20 cells expressing the PTH/PTHrP receptor with an ED50 of 0.6 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The high binding affinity and the high efficacy of NlePTH in stimulating cAMP accumulation and ACTH release indicate that the PTH/PTHrP receptor is efficiently coupled to the intracellular signalling system responsible for stimulation of ACTH release in AtT-20 cells. No additivity of cAMP accumulation or of ACTH release was observed when these cells were treated with maximally active concentrations of both NlePTH and CRF. This suggests that the receptors for both of these hormones share the same intracellular effectors, and that intracellular signaling in AtT-20 cells is not compartmentalized. Additionally, the ability of NlePTH to stimulate ACTH release in AtT-20 cells, a function that is normally performed by CRF, demonstrates promiscuity between activated receptors and distal biological functions.

[The difference of erythropoietin concentration in various disease].

The serum concentration of erythropoietin in 79 cases with various blood diseases, uremia, chronic obstructive pulmonary disease etc was determined. At comparable degrees of anemia, patients with myelodysplastic syndrome and aplastic anemia had the highest levels of erythropoietin in our study. The high level of erythropoietin titer in patients with aplastic anemia should be taken as the nom for renal synthesis and release of this hormone. The erythropoietin level in patients with uremic anemia was lower than the level in patients with anemia of other causes but still higher than that of the normal controls. Patients suffering from polycystic kidney disease with or without uremia had a high level of erythropoietin due to local hypoxia of remnant kidney tissue resulting from the pressure of cystic formation. Different methods are used to determine the erythropoietin level, which varies with the stage and etiology of the diseases. There are other stimulating or inhibitory factors of erythropoiesis when the assay is processed. Transfusion and administration of certain drugs also influence the growth of erythroid cells, thus the serum titers of erythropoietin differed markedly between patients at comparable hemoglobin concentration.