[A family with developmental glaucoma and microcornea due to novel ADAMTS18 gene mutations].

This case report presents a family with developmental glaucoma accompanied by microcornea resulting from novel mutations in the ADAMTS18 gene. The index case involves a 5-year-old twin brother, who, during a routine examination, exhibited elevated intraocular pressure persisting for over a month. The peak intraocular pressure reached approximately 25 mmHg (1 mmHg=0.133 kPa) in both eyes, with a corneal diameter of less than 10 mm. Ocular examination revealed an enlarged cup-to-disc ratio, and optical coherence tomography (OCT) demonstrated thinning of the retinal nerve fiber layer and ganglion cell layer. Ultrasound biomicroscopy combined with gonioscopy indicated partial angle closure and abnormal anterior chamber angle development. The ocular manifestations in the twin brother were consistent with those observed in the twin sister. The clinical diagnosis was bilateral developmental glaucoma with microcornea. Genetic sequencing identified two novel compound heterozygous mutations in the ADAMTS18 gene in the twins: Mutation 1 (M1) involving the variant site 1 (c.3436C>T:p.R1146W) and Mutation 2 (M2) involving the variant site 2 (c.1454T>G:p.F485C). Ocular examinations of four additional family members were normal. Genetic testing revealed that the twins' father and sister carried M1, while the index case's mother and brother carried M2. This report underscores a unique association between ADAMTS18 gene mutations and developmental glaucoma with microcornea within a familial context, emphasizing the importance of genetic screening for early diagnosis and targeted management strategies.

Cold Spray: Over 30 Years of Development Toward a Hot Future.

Cold Spray (CS) is a deposition process, part of the thermal spray family. In this method, powder particles are accelerated at supersonic speed within a nozzle; impacts against a substrate material triggers a complex process, ultimately leading to consolidation and bonding. CS, in its modern form, has been around for approximately 30 years and has undergone through exciting and unprecedented developmental steps. In this article, we have summarized the key inventions and sub-inventions which pioneered the innovation aspect to the process that is known today, and the key breakthroughs related to the processing of materials CS is currently mastering. CS has not followed a liner path since its invention, but an evolution more similar to a hype cycle: high initial growth of expectations, followed by a decrease in interest and a renewed thrust pushed by a number of demonstrated industrial applications. The process interest is expected to continue (gently) to grow, alongside with further development of equipment and feedstock materials specific for CS processing. A number of current applications have been identified the areas that the process is likely to be the most disruptive in the medium-long term future have been laid down.

[A cross-sectional study on metabolic syndrome and its abnormal components in cancer patients].

From December 2017 to December 2018, 3 509 subjects who had regular physical examination in Health Management Center, Tianjin Medical University General Hospital were enrolled in our study, including 399 cancer patients, 1 555 chronic disease patients, and 1 555 healthy control, respectively. The mean age was (55.87±11.98) years, and 31.38% were men. The prevalence of MS among chronic disease group (42.44%) was higher than that of cancer group (34.59%) and healthy control group (18.65%) (P<0.001). Compared with healthy control group, the OR (95%CI) values of MS risk in cancer group and chronic disease group were 2.13 (1.61-2.83) and 2.85 (2.23-3.66), respectively; the OR (95%CI) values of MS risk were 3.56 (2.04-6.21) and 2.77 (1.46-5.25) in breast cancer and thyroid cancer, respectively.

[Lamina cribrosa defect and progress of glaucoma].

Lamina cribrosa (LC) is considered as the original site of glaucomatous damage of axons of retinal ganglion cells, and therefore understanding the morphological changes in the LC will help to uncover the pathogenesis of glaucoma. Previous studies have indicated that the progress of glaucomatous optic neuropathy may be associated with the LC defects. Based on imaging by swept source optical coherence tomography B-Scan of the optic discs of patients with glaucoma, for the first time the spontaneous local LC defects have been found to balance the gradient between intraocular and cerebrospinal fluid pressures, which in turn can slow down the progress of glaucomatous optic neuropathy. This article provides the direct evidence supporting the role of intraocular and cerebrospinal fluid pressure gradient in the pathogenesis of glaucoma. This finding will increase our understanding of the mechanisms underlying glaucoma and help to develop novel strategies for its treatment and prognosis analysis. (Chin J Ophthalmol, 2020, 56: 17-20).

The diagnostic accuracy of CT-guided percutaneous core needle biopsy and fine needle aspiration in pulmonary lesions: a meta-analysis.

AIM: To determine and compare the diagnostic value of computed tomography (CT)-guided percutaneous core needle biopsy (PCNB) and percutaneous fine-needle aspiration biopsy (PNAB) in pulmonary lesions. MATERIALS AND METHODS: PubMed, EMBASE, and the Web of Science were systematically searched for relevant studies that investigated the diagnostic accuracy of CT-guided PCNB and/or PNAB for pulmonary lesions up to December 2014. After study selection, data extraction, and quality assessment, the sensitivity (SEN), specificity (SPE), diagnostic odds rate (DOR), positive likelihood ratios (PLR), negative likelihood ratios (NLR), and summary receiver operating characteristic (SROC) curves were calculated using the Meta-Disc 1.4 software. RESULTS: Nineteen publications, including 21 independent studies, met the inclusion criteria. Of them, 15 studies were included in the PCNB group and six studies in the PNAB group. The pooled SEN, SPE, DOR, PLR, NLR, and SROC were 0.95, 0.99, 54.72, 0.06, 821.90, and 0.98 in the PCNB group and 0.90, 0.99, 24.71, 0.14, 210.72, and 0.98 in the PNAB group, respectively. CONCLUSION: Based on current evidence, both PCNB and PNAB can be used as diagnostic methods to distinguish benign and malignant pulmonary lesions; the difference between PCNB and PNAB regarding diagnostic accuracy of benign or malignant pulmonary lesions is not obvious.

Correlation between serum level of chemokine (C-C motif) ligand 18 and poor prognosis in breast cancer.

In this study, we investigated the correlation between serum chemokine (C-C motif) ligand 18 (CCL-18) and the prognosis as well as clinical characteristics of breast cancer. Blood samples from 207 breast cancer patients, 126 individuals with benign breast tumors, and 93 healthy women were collected. Serum CCL-18 expression was detected by enzyme-linked immunosorbent assay. Mann-Whitney's U tests were carried out to analyze the relationship between serum CCL-18 and clinicopathological variables. The Kaplan-Meier method was used to evaluate the overall survival (OS), whereas differences between groups were analyzed by log-rank tests. The COX proportional hazard regression model was used to determine the association between clinicopathological characteristics and survival. We found that serum CCL-18 was significantly higher in breast cancer patients (290.06 ± 89.52 pg/mL) as compared to that in individuals with benign tumors (170.14 ± 26.57 pg/mL) or healthy women (119.36 ± 38.77 pg/mL) (P < 0.05). Serum CCL-18 was correlated with clinical cancer stages (P = 0.007), and was associated with advanced cancer stage (P < 0.05). The 5-year OS rate of breast cancer patients with high serum CCL- 18 was 35.9% (HR = 3.908, 95%CI = 2.546-12.090, P < 0.05), which was significantly lower as compared to that for patients with low serum CCL-18 (85.5%). Based on our results, we conclude that CCL-18 is a prognostic biomarker in patients with advanced breast cancer in Chinese patients.

A novel frameshift deletion in the COL1A1 gene identified in a Chinese family with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetically heterogeneous group of disorders, characterized by abnormal bone fragility, blue sclera, deafness, joint laxity, and soft-tissue dysplasia. The purpose of this study was to elucidate the genetic or molecular basis for OI type IA in a Chinese family. We evaluated the members of a family, in which six individuals are affected with increased bone fragility and blue sclera. Results of exome sequencing revealed a novel 1-bp deletion (c.2329delG, p.A777fs) in exon 33 of the COL1A1 gene in two affected individuals, but not in a control family member without OI. The variation co-segregated with the disease in all the OI patients but not in the unaffected family members. The mutation caused a frameshift alteration after codon 777, leading to premature termination of the COL1A1 protein. Thus, our findings identified a novel frameshift deletion c.2329delG (p.A777fs) in the COL1A1 gene, which is associated with OI type IA in a Chinese family.

A novel FBN1 heterozygous mutation identified in a Chinese family with autosomal dominant Marfan syndrome.

The purpose of this study was to identify the clinical features and mutations in the fibrillin-1 gene (FBN1) in a large Chinese family with autosomal dominant Marfan syndrome (MFS). Seventeen members from a Chinese family of 4 generations were included in the study. All members underwent complete ophthalmic examination. Molecular genetic analysis was performed on all subjects. All exons of FBN1 were amplified by polymerase chain reaction, sequenced, and the sequences were compared with a reference database. Variations were evaluated in family members as well as 100 normal controls. Changes in structure and function of the protein induced by amino acid variation were predicted by bioinformatic analysis. Ectopia lentis, dolichostenomelia, arachnodactyly, and tall stature were present in all patients diagnosed with MFS. The novel heterozygous missense mutation c.2243 T>G (p.C781W) in exon 19 of FBN1 was identified in all 5 patients, but not in other family members or 100 normal controls. This mutation caused an amino acid substitution of cysteine to tryptophan at position 781 (p.C781W) of the FBN1 protein. This mutation occurred in a highly conserved region and may cause structural and functional changes in the protein according to our bioinformatic analysis. Our results suggest that the novel mutation C781W of FBN1 is responsible for the pathogenesis of MFS in this pedigree.

A novel mutation of PAX6 identified in a Chinese twin family with congenital aniridia complicated with nystagmus.

Genetic variations within the paired box gene 6 (PAX6) gene are associated with congenital aniridia. To detect the genetic defects in a Chinese twin family with congenital aniridia and nystagmus, exons of PAX6 were amplified by polymerase chain reaction (PCR), sequenced and compared with a reference database. Six members from the family of three generations were included in the study. The twins' father presented with congenital aniridia, nystagmus and cataract at birth, while the twins presented with congenital aniridia and nystagmus. A novel mutation c.888 insA in exon 10 of PAX6 was identified in all affected individuals. This study suggests that the novel mutation c.888 insA is likely responsible for the pathogenesis of the congenital aniridia and nystagmus in this pedigree. To the best of our knowledge, this is the first report of this mutation in PAX6 gene in pedigree with aniridia. Furthermore, no PAX6 gene defect was reported in twins with congenital aniridia.

A novel NF1 frame-shift mutation (c.702_703delGT) in a Chinese family with neurofibromatosis type 1.

This study aimed to characterize the clinical features of a Chinese pedigree with neurofibromatosis type 1 (NF1) and to identify mutations in the NF1 gene. In this three-generation family containing 8 members, 5 had been diagnosed with NF1 and the others were asymptomatic. All members of the family underwent complete medical examinations. Molecular genetic analyses were performed on all subjects included in the study. All exons of NF1 were amplified by polymerase chain reaction, sequenced, and compared with a reference database. Possible changes in function of the protein induced by amino acid variants were predicted by bioinformatic analysis. In this family, the 5 patients presented different clinical phenotypes, but all manifested typical café-au-lait macules. One novel frame-shift mutation, c.702_703delGT, in exon 7 of NF1 was identified in all affected family members, but not in the unaffected family members or in 102 normal controls. This mutation generates a premature stop codon at amino acid position 720. Additionally, a synonymous mutation c.702 G>A was found in 3 family members, including 2 affected and 1 normal individuals. In conclusion, our study suggests that a novel c.702_703delGT frame-shift mutation in NF1 is likely to be responsible for the pathogenesis of NF1 in this family. To the best of our knowledge, it is the first time that a c.702_703delGT mutation has been identified in a family with neurofibromatosis type 1.

[Therapeutic effectiveness of modified endoscopic retrograde appendicitis therapy for acute appendicitis of different severities in children].

Objective: To investigate the effects of modified endoscopic retrograde appendicitis therapy (mERAT) on the treatment of children with different severities of acute appendicitis. Methods: This study was a case-control study. A total of 586 children with acute appendicitis, who were admitted to the Pediatric Department of Second Affiliated Hospital of Air Force Medical University between January 2019 and November 2023, were selected as the research subjects. According to the severity of the disease, the patients were divided into simple appendicitis group, suppurative appendicitis group and perforated appendicitis group. The baseline data, hospitalization treatment and costs, outcomes, and recurrence in each group were analyzed, and the difference in the effectiveness of mERAT between the groups were compared by Kruskal-Wallis H test and χ2 test. Results: Among 586 children, there were 338 males and 248 females. The age at onset was 7.0 (4.6, 9.4) years. There were 475 cases of simple appendicitis, 78 cases of suppurative appendicitis, and 33 cases of perforated appendicitis. There were no significant differences in age and gender among the three groups (F=0.59, χ2=3.31, both P>0.05). However, there were statistically significant differences in body temperature, white blood cell counts, neutrophil percentage, lymphocyte percentage, nausea or vomiting, right lower abdominal pain, umbilical pain, right lower abdominal tenderness, and right lower abdominal rebound pain (H=7.56, 161.52, 169.11, and 169.61, χ2=12.05, 13.82, 12.05, 7.74, 20.35, and 94.61, all P<0.05). Also, the treatment time, postoperative hospital stay, total hospital stay, and cost showed statistically significant differences (H=4.70, 33.66, 34.99, 30.37, all P<0.05). There was no significant difference in the initial treatment success rate (98.1% (466/475) vs. 98.7% (77/78) vs. 90.9% (30/33), P=0.057). During the 30 (23, 36) months of follow-up, the recurrence rate was 7.9% (35/433) in the simple appendicitis group, 20.8% (15/72) in the suppurative appendicitis group, and 30.0% (9/30) in the perforated appendicitis group, with a statistically significant difference (χ2=23.56, P<0.001). Among the children with recurrent appendicitis, 15 cases still chose mERAT, of them 11 cases (31.2%) had simple appendicitis, 2 cases (2/15) had suppurative appendicitis, and 2 cases (2/9) had perforated appendicitis.The latest time to recurrence in the 3 groups was 32, 35 and 10 months, respectively. Conclusion: Treatment with mERAT has a good effect in pediatric simple appendicitis, but has a higher recurrence rate despite a better initial treatment success rate in suppurative appendicitis and perforated appendicitis.

Long noncoding RNA PVT1-214 enhances gastric cancer progression by upregulating TrkC expression in competitively sponging way.

The article "Long noncoding RNA PVT1-214 enhances gastric cancer progression by upregulating TrkC expression in competitively sponging way, by S. Zhao, N.-F. Fan, X.-H. Chen, C.-H. Zhuo, C.-W. Xu, R.-B. Lin, published in Eur Rev Med Pharmacol Sci 2019; 23(10): 4173-4184-DOI: 10.26355/eurrev_201905_17920-PMID: 31173288" has been withdrawn from the authors. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/17920.

[Emerging of Japanese encephalitis virus and Getah virus from specimen of mosquitoes in Inner Mongolia Autonomous Region].

Objective: To investigate the types and distribution of blood-sucking insects and arboviruses in Inner Mongolia autonomous region, and provide basic data for the prevention of arbovirus transmitted disease. Methods: Blood-sucking insects were collected by lamp trapping method in nature. Mosquito samples were classified according to morphologic characteristics and then stored at liquid nitrogen. Viruses were isolated in cell culture and characterized, using molecular biological methods. Results: A total of 24 240 mosquitoes and 17 110 aphids were collected from 2 sites of 5 counties (Flags) in Inner Mongolia in 2014 and during 2017-2018. Among them, Japanese encephalitis virus gene was detected in Culex pipiens pallens, and 4 virus strains isolates which could be stably passaged. The isolates were identified as Getah virus and densonucleosis virus by molecular biology identification. Phylogenetic analysis on the E2 gene of the Getah virus (NMDK1813-1) showed that it belonged to the same evolutionary branch of the Gansu isolates (GS10-2) and having six common amino acid variation sites. Conclusions: The emergence of Japanese encephalitis virus and Getah virus from specimen of mosquitoes in Inner Mongolia indicated the new challenges on the prevention and control of arbovirus and related diseases. The results pf this study provided basic data for the prevention and control stretagies of arbovirus transmitted diseases in Inner Mongolia.

[Isolation and identification of Arbovirus in Hainan province, 2017-2018].

Objective: To understand the types and distribution of Arboviruses in Hainan province. Methods: Blood-sucking insects were collected in Hainan province from 2017 to 2018. After laboratory treatment, BHK-21 cells and C6/36 cells were inoculated with grinding supernatant of all blood-sucking insects to isolate all of involving virus. Arbovirus genes in blood-sucking insects were detected in parallel by RT-PCR method. Results: A total of 15 062 mosquitoes were classified into four genera (Culex, Armigeres, Aedes, Anopheles) and 11 360 midges were collected. Culex tritaeniorhynchus was in the majority and accounted for 92.88% (13 990/15 062) of all the mosquitoes collected. Four strains of virus isolates were notified by tissue culture method. Three strains of viruses belonged to Japanese encephalitis virus (JEV), with the other one as Getah virus (GETV). Five pools of JEV gene amplification were positive, from Culex tritaeniorhynchus. Results from the phylogenetic analysis showed that they belonged to genotype JEV-Ⅰ. The minimum infection rate of JEV was 0.57‰ (8/13 990). A total of 5 pools of Akabane virus (AKV) gene amplification were positive. The minimum infection rate of AKV was 0.44‰ (5/11 360). Based on the S gene and M gene sequences of the virus, data from the phylogenetic analysis showed that the five AKV strains carried by midges in Hainan province were in a separate evolutionary branch and with formed unique geographical distribution. Conclusions: JEV and GETV had been isolated again from the mosquito specimens in this survey, since the 1980s. AKV was detected from the midge specimens in Hainan province. These results showed the needs of strengthening the programs on detection and monitor of JEV, GETV and AKV that were related to animal and human diseases in order to reduce the risks of related diseases in this area.

Long noncoding RNA PVT1-214 enhances gastric cancer progression by upregulating TrkC expression in competitively sponging way.

OBJECTIVE: Long noncoding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) is aberrantly expressed and involved in the promotion of various cancers. However, the vital epigenetic function of PVT1-214, a transcript isoform of PVT1, in gastric cancer (GC) remains unknown. We aimed to investigate the dysregulation and detailed mechanism underlying the involvement of lncRNA PVT1-214 in GC. PATIENTS AND METHODS: The expression of PVT1-214 in GC tissues and cell lines was detected by qRT-PCR. The relationship between increased PVT1-214 levels and the advanced clinicopathological features of tumor tissues was analyzed using a Chi-square test. The influence of PVT1-214 on the survival rate of GC cell lines was evaluated by the log-rank test. Cell lines were used to explore the carcinogenic effects of PVT1-214 in vitro and in vivo, and specific tests included cell apoptosis determined by flow cytometry, cell proliferation assayed by Cell Counting Kit-8 (CCK-8) and colony formation, and the use of these cells for mice xenograft models. Direct complementary binding was predicted by bioinformatics and verified by dual luciferase reporter assay, RNA transfection, quantitative polymerase chain reaction (qPCR), and Western blotting. Spearman's correlation coefficient was adopted to evaluate the correlation between miR-128 and PVT1-214 levels. RESULTS: PVT1-214 expression in GC tissues and cell lines is markedly elevated. In GC patients, high expression of PVT1-214 is associated with late tumor stage, increased tumor size, and poor survival. PVT1-214 silencing represses cell proliferation and enhances apoptosis of GC cells both in vivo and in vitro. Additionally, PVT1-214 functions as a competing endogenous RNA (ceRNA) by binding to miR-128. Inhibition of miR-128 releases Tropomyosin receptor kinase C (TrkC) from the complementary binding complex, subsequently increasing the protein level of TrkC in GC cells. CONCLUSIONS: PVT1-214-induced miR-128 repression regulates TrkC to further the progression of GC, indicating that this process will provide a promising therapeutic target in GC.

Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells.

The present study investigated the expression and regulation of GnRH and GnRH receptor (GnRHR) messenger RNAs (mRNAs) in human granulosa-luteal cells using reverse transcription-polymerase chain reaction (RT-PCR). Granulosa-luteal cells were aspirated from preovulatory follicles obtained from women undergoing in vitro fertilization. Two sets of primers derived from human hypothalamic GnRHR complementary DNA (cDNA) were used to amplify cDNAs from granulosa-luteal cells. PCR products corresponding to the expected sizes of GnRH were obtained from granulosa-luteal cells as well as the brain, but not from skeletal muscle cDNA. The authenticity of the PCR products was confirmed by Southern blot hybridization with internal oligonucleotide probes and by subsequent cloning and sequencing. Similarly, using four sets of primers specific for the human pituitary GnRHR cDNA, PCR products with the expected sizes were detected from both brain and granulosa-luteal cells, but not from skeletal muscle. PCR products were subsequently confirmed by Southern blot hybridization using an internal oligonucleotide probe or a cDNA probe which was obtained from screening a human pituitary cDNA library. Cloning and sequencing of the PCR product in the 3'-untranslated region revealed identical sequence with the reported human pituitary GnRHR cDNA sequence. RNA samples obtained from cells immediately after dissociation or after 2, 5, and 8 days of culture were analyzed by RT-PCR, and in all cases, both GnRH and GnRHR mRNA were detected. To investigate how gene expression of GnRH and GnRHR is regulated, we examined the effect of GnRH and hCG on GnRH and GnRHR mRNA levels in cultured human granulosa-luteal cells. Treatment with different concentrations of GnRH induced biphasic responses. Both GnRH and GnRHR mRNA were significantly increased by 1 nM, but slightly decreased by 1 microM GnRH; 1 nM GnRH also significantly inhibited progesterone production, whereas higher doses had no effect. Treatment with hCG (1 IU/ml) decreased GnRHR mRNA levels without altering the expression of the GnRH gene. These results demonstrate for the first time that 1) both GnRH and GnRHR mRNAs are expressed in human granulosa-luteal cells; 2) GnRH mRNA levels are autoregulated by GnRH; and 3) GnRHR gene expression is up-regulated by GnRH, but down-regulated by hCG. These findings provide strong evidence that GnRH is an autocrine regulator in the human ovary.

Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase. Asymmetry in p66 subunits of the p66/p66 homodimer.

A recombinant p66 form of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) can be obtained [(1991) Biotechnol. Appl. Biochem. 14, 69-81] from crude Escherichia coli extracts by immobilized metal affinity chromatography (IMAC). We have analyzed the p66 HIV-1 RT, isolated in the presence of 0.3 M imidazole, by gel permeation HPLC on Superose 12. The results show that it contains two major distinct p66 forms (24.1 min and 28.3 min peaks) which are distinguishable from the purified homodimeric (p66/p66) HIV-1 RT (22.2 min peak). Protein peak 1 (24.1 min) is converted to a 22.3 min peak upon storage for 20 h at 4 degrees C. Under identical conditions, the isolated peak 2 (28.3 min) appeared as a conformationally heterogeneous mixture elaborated by peaks at 22.3 min and 25.9 min. The protein species thus obtained were active in the RNA-dependent DNA polymerase and RNase H activity assays and produced heterodimeric HIV-1 RT upon incubation with the HIV-1 protease. When the IMAC-purified, imidazole-free homodimeric (p66/p66) form of the enzyme was incubated with 0.3 M imidazole for 16 h at 4 degrees C, protein peaks at 28.3 min (peak A) and 30.5 min (peak B) were isolated by gel permeation HPLC. While both of these p66-containing species were stable and displayed identical RNA-dependent DNA polymerase activities, the protein in peak B was only 50% active in RNase H function compared with the protein from peak A. These imidazole-mediated dissociation studies support the hypothesis of partial unfolding of one of the RNase H domains of the p66/p66 homodimer, suggesting that the p66 subunits are asymmetric in the native enzyme.

Simultaneous mutations at Tyr-181 and Tyr-188 in HIV-1 reverse transcriptase prevents inhibition of RNA-dependent DNA polymerase activity by the bisheteroarylpiperazine (BHAP) U-90152s.

The replacement of either Tyr-181 or Tyr-188 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) by the corresponding HIV-2 RT amino acids Ile-181 or Leu-188 is known to result in active mutant enzymes (Y181I; Y188L) with virtual loss of sensitivity towards three structural classes of nonnucleoside RT inhibitors; L-697,661, nevirapine, and TIBO R82913. The bisheteroarylpiperazine (BHAP) U-90152S, a highly specific inhibitor (IC50, 0.29 +/- 0.01 microM) of HIV-1 RT, inhibited the recombinant Y181I and Y188L HIV-1 RT mutants with IC50 values of 3.6 +/- 0.15 microM and 0.71 +/- 0.02 microM, respectively. Construction and in vitro analysis of double mutants Y181I/Y188L and Y181C/Y188L of HIV-1 RT showed > 150-fold resistance to U-90152S. An HIV-2 RT mutant containing amino acids 176-190 from HIV-1 RT acquired full sensitivity to U-90152S (IC50, 0.26 +/- 0.01 microM). It is concluded that simultaneous mutations at Tyr-181 and Tyr-188 of HIV-1 RT promotes resistance to U-90152S.

Mechanism of resistance to U-90152S and sensitization to L-697,661 by a proline to leucine change at residue 236 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase.

Bisheteroarylpiperazines (BHAPs) are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). BHAP-resistant HIV-1 is sensitized to other classes of nonnucleoside RT inhibitors and this has been primarily attributed to a proline-to-leucine substitution at amino acid 236 (P236L) of HIV-1 RT. To understand the basis for the in vitro sensitization-resistance phenomenon, single base pair mutations at amino acid P236 in HIV-1 RT were introduced to obtain P236L, P236T, P236H, P236R, and P236A HIV-1 RT mutants. Active HIV-1 RT mutants H235W, D237T, and H235W/D237T/T240K, containing substitutions from HIV-2 RT, were also cloned, expressed, and purified. Three BHAPs (U-88204E, U-87201E, and U-90125S) and the pyridinone L-697,661 were selected to quantitatively assess the effects of these amino acid substitutions on sensitization to L-697,661 and resistance to the BHAPs. The HIV-1 RT mutants bearing single (H235W; D237T) or multiple (H235W/D237T/T240K) HIV-2 RT substitutions around the conserved P236 conferred little resistance or sensitization to these RT inhibitors. The inhibition profiles of the P236 HIV-1 RT mutants demonstrated a direct correlation between sensitization to L-697,661 and resistance to the BHAPs. These results suggest alterations in the shape of the binding pocket as the mechanism by which the P236L mutation confers resistance to the BHAPs and sensitization to L-697,661.

The human gonadotropin-releasing hormone receptor gene: complete structure including multiple promoters, transcription initiation sites, and polyadenylation signals.

The interaction of gonadotropin-releasing hormone and its receptor is a critical event in the endocrine regulation of reproduction. We have recently cloned the gene encoding for the human gonadotropin-releasing hormone receptor (hGnRHR). Partial sequence analysis revealed a structural organization consisting of three exons and two introns. Exon II contains only 219 bp and the remainder of the approximately 5 kb transcript is distributed between exons I and III. The complete coding region for the hGnRHR represented only 987 bp leaving an extensive 5' and 3' non-translated region and potentially additional exons unaccounted for. This report provides the complete sequence of exon I and III and demonstrates that further exons are unlikely to be contained within this gene. Sequencing of the 5' end of the gene revealed the presence of five consensus TATA sequences distributed within a 700 nucleotide region. Primer extension analysis detected multiple transcription initiation sites associated with this cluster of TATA sequences. Transcription of this region up to the most 5' initiation site was demonstrated by the reverse transcription-polymerase chain reaction (RT-PCR) method. The 5' non-translated region stretches between 703 and 1393 bp, depending on which initiation site is used. Several consensus cis-acting regulatory sequences were identified within the 5' end. These include, among others, sites for PEA-3, AP-1, and Pit-1. In addition, cAMP response element (CRE)-like and glucocorticoid/progesterone response element (GRE/PRE)-like sequences were found.(ABSTRACT TRUNCATED AT 250 WORDS)