Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1 (hOCT1/SLC22A1)

Ann. Hum. Genet . (1999), 63 , 473–482
Correction
The authors wish to add the following correction to this paper:
The genomic organization of the human organic cation transporter (hOCT1/SLC22A1) has recently been described by us to consist of 7 exons [Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1( hOCT1 / SLC22A1 ); Ann. Hum. Genet . 63 : 473–482]. A reexamination revealed 11 exons instead of 7. The mistake occurred through cDNA contamination. The corrected gene structure of the hOCT1 gene is available at EMBL under the following accession numbers:
AJ243995 (Exon 1), AJ243996 (Exon 2), AJ276051 (Exon 3), AJ276052 (Exon 4), AJ276053 (Exon 5 and 6), AJ245460 (Exon 7), AJ243998 (Exon 8), AJ243999 (Exon 9 and 10) and AJ244000 (Exon 11).     

Nucleotide sequence and chromosomal assignment of a cDNA encoding the large isoform of human glutamate decarboxylase

Glutamic acid decarboxylase (GAD) catalyses the conversion of l -glutamic acid to the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Two forms of human GAD, GAD₆₅ and GAD₆₇, are encoded by two separate genes.
A full length human GAD₆₇ cDNA has been isolated from a human frontal cortex cDNA library and the nucleotide sequence determined. The GAD₆₇ gene has been mapped to chromosome 2 using the polymerase chain reaction to amplify specifically the human sequence in rodent/human somatic cell hybrid DNA. This confirms that human GAD₆₇ is not syntenic with the smaller GAD isoform GAD₆₅ which has been assigned to chromosome 10. Production of polyclonal antiserum to a baculovirus-expressed GAD₆₇ enabled immunocytological detection of GAD in the rat brain.     

Cloning and chromosomal mapping of human cytochrome b5 reductase (DIA1)

We have isolated a cDNA clone that codes for human cytochrome b ₅ reductase. The cDNA was used to analyse, by Southern-blot hybridization, DNA isolated from a panel of 11 independent human-rodent somatic cell hybrids. The results indicate that cytochrome b ₅ reductase is encoded by a single gene located on human chromosome 22.     

PD-L1抑制小鼠脾淋巴细胞增殖药物筛选模型的建立及应用

目的通过构建重组的小鼠pcDNA3.1(+)-PD-L1质粒,并在真核细胞中进行高表达,建立PD-L1抑制小鼠脾淋巴细胞增殖药物筛选模型用于免疫调节药物的筛选。方法以RT-PCR方法扩增得到小鼠的PD-L1全长片段;以pcDNA3.1(+)真核表达质粒为载体,构建得到重组质粒pcDNA3.1(+)-PD-L1;脂质体转染到小鼠成纤维细胞(L929),通过G418筛选得到高表达PD-L1的稳定细胞株并经免疫荧光鉴定;用丝裂霉素处理过的PD-L1高表达细胞与小鼠脾淋巴细胞共培养;成功建立PD-L1抑制小鼠脾淋巴细胞增殖的模型之后向其中加入不同种类、不同浓度的天然产物提取物,并设复孔。结果通过Eco RI/Xho I双酶切及测序证实构建的重组质粒pcDNA3.1(+)-PD-L1正确。RT-PCR和免疫荧光方法鉴定重组质粒在L929细胞中高效表达,应用流式细胞术成功建立PD-L1抑制小鼠脾淋巴细胞增殖的模型,并筛选出阻断PD-L1活性的有效药物。结论获得了稳定高表达PD-L1的细胞,并且用于免疫调节的药物筛选,为免疫调节药物的筛选奠定了实验基础。     

The organic cation transporter 3 (OCT3) as molecular target of psychotropic drugs: transport characteristics and acute regulation of cloned murine OCT3

The organic cation transporter 3 (OCT3) is a widely expressed transporter for endogenous and exogenous organic cations. Of particular interest is OCT3 expression and function in the brain, where it plays a role in serotonin clearance and influences mood and behavior. Protein kinase signaling mediates rapid modulation of cerebral processes, but little is known about acute regulation of OCT3 by protein kinases. Therefore, we cloned mouse OCT3 (mOCT3) and generated a human embryonic kidney cell line stably expressing the transporter to study transport characteristics, acute regulation by protein kinases, and interaction with psychotropic drugs. Uptake measurement was performed using the fluorescent cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP⁺, 1 μM) as a substrate. The translational value of these findings was determined by comparing results obtained with cloned mouse and human OCT3. mOCT3-mediated transport is membrane potential dependent and pH independent. ASP⁺ uptake by mOCT3 and human OCT3 (hOCT3) was efficiently inhibited by 1-methyl-4-phenylpyridinium, tetrapentylammonium (TPA⁺), corticosterone, serotonin, and histamine and by the drugs ketamine, fluoxetine, and diazepam. The half maximal inhibitory concentrations of mOCT3 and hOCT3 for TPA⁺, serotonin, diazepam, and ketamine are significantly different. Diazepam is a non-transported inhibitor. Furthermore, the activities of mOCT3 and hOCT3 are acutely regulated by the p56 ^lck tyrosine kinase by decreasing their V _max. Studies with freshly isolated renal proximal tubules from mOCT1/2^?/? mice, in which mOCT3 is the only OCT present, confirmed this regulation pathway. Only the activity of hOCT3 is regulated by calmodulin. These findings suggest that even though many transport properties of mOCT3 and hOCT3 are similar, there are also species-specific aspects of OCT3 function.     

X-linked adrenoleukodystrophy in Spain. Identification of 26 novel mutations in the ABCD1 gene in 80 patients. Improvement of genetic counseling in 162 relative females

In this study, we analyzed the ABCD1 gene in 80 X-linked adrenoleukodystrophy (X-ALD) patients from 62 unrelated families. We identified 53 different mutations, of which 26 are novel and two are non-pathogenic sequence variants (L516L and 3'UTR, 2246C/G) that have been previously described. The Spanish population had significant allelic heterogeneity, in which most of the mutations were exclusive to a single family 47/53 (88.7%). Only six mutations (Y174S, G277R, FsE471, R518Q, P543L, and R554H) were found in more than one family. Mutations G277R, P543L, and R554H were the most frequent, each of them being found in three patients (5%). Intra-familiar phenotype variability was observed in most of the families, but in one, with the novel mutation R120P, only the adult mild phenotype was present (five hemizygous family members). We detected 80 heterozygous women by mutation analysis, but only 78 of them showed increased very-long-chain fatty acid levels. In conclusion, this study extends the spectrum of mutations in X-ALD and facilitates the identification of heterozygous females. Our results are also consistent with previous studies reporting the difficulty of predicting genotype-phenotype correlation.     

Construction of cloning‐friendly minigenes for mammalian expression of full‐length human NF1 isoforms

The neurofibromatosis type 1 (NF1) tumor suppressor gene is one of the most frequently mutated genes in human tumors. Research on the NF1 proteins has been partially hindered by the difficulties in cloning and propagating the full‐length coding cDNAs. We have now established a condition for propagating the natural open reading frames (ORFs) and have assembled the ORFs for human NF1 type 1 and 2 isoforms. Furthermore, we were able to eliminate the cDNA cloning toxicity by introducing a mini‐intron. These NF1 minigenes were expressed similarly to the intronless version and could be used to purify full‐length NF1 proteins. The NF1 isoforms expressed from the minigenes showed Ras‐GAP activity in vivo and in vitro, while the type 1 was more potent. Our constructs expand currently available full‐length NF1 constructs and should be valuable tools in expediting the understanding of NF1, particularly the isoform‐specific functions and regulation.     

A new alpha 1-antitrypsin mutation, Thr–Met 85, (PI Zbristol) associated with novel electrophoretic properties

A new AAT allele ( PI Z _bristol ) has been discovered in a woman with an obstetric history of three perinatal deaths from fulminant liver disease and no living offspring. She and her father were both PI M1Z _bristol heterozygotes. The Z_bristol protein is active as a proteinase inhibitor but appeared to be deficient in the plasma to about the same degree as the S protein in MS heterozygotes. It focuses on the basic side of Z and lacks the normal pattern of secondary isoforms associated with the commonly occurring AAT variants and migrates faster than normal on an SDS electrophoresis gel. The Z _bristol mutation was found to be a C to T transition at codon 85 changing A C G (Thr) to A T G (Met). This disrupts the N -glycosylation site starting at Asn 83 preventing glycosylation at residue 83 in the PI Z_bristol protein and explains the protein isoelectric focusing and SDS gel electrophoresis results. An analysis of haplotypes in the propositus and her father indicated that the Z _bristol mutation occurred on the common M1 ( Val 213 ) genetic background. The new mutation also led to the generation of an Nla III restriction endonuclease recognition site. Cell lines from two offspring tested for the presence of this Nla III site revealed that one had the variant and the other did not. Thus, the relationship between Z_bristol and fulminant liver disease in the offspring is unclear.     

OCT4 spliced variants are differentially expressed in human pluripotent and nonpluripotent cells

OCT4 is a master regulator of self-renewal in embryonic stem cells and can potentially encode two spliced variants, designated OCT4A and OCT4B. We have examined the expression pattern of these OCT4 isoforms in various human pluripotent and nonpluripotent cells. Our data revealed that whereas OCT4A expression is restricted to embryonic stem (ES) and embryonal carcinoma (EC) cells, OCT4B can be detected in various nonpluripotent cell types. Furthermore, we detected a novel OCT4 spliced variant, designated OCT4B1, that is expressed primarily in human ES and EC cells and is downregulated following their differentiation. We also found a significantly higher level of OCT4B1 expression in stage-specific embryonic antigen-3 (SSEA3)(+) compared with SSEA3(-) subpopulations of cultured ES cells. Taken together, our data demonstrated a distinctive expression pattern for OCT4 spliced variants in different cell types and highlight the necessity of defining the type of OCT4 when addressing the expression of this gene in different human cells.     

Human ClCa1 modulates anionic conduction of calcium-dependent chloride currents

Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca²⁺-dependent Cl⁻ currents. The present study examines the relationship between the hClCa1 protein and Ca²⁺-dependent Cl⁻ currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl⁻ currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca²⁺-dependent Cl⁻ currents and show that hClCa1 increases the amplitude of Ca²⁺-dependent Cl⁻ currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl⁻ conductance while decreasing the G _SCN−/ G _Cl− conductance ratio from ∼2–3 to ∼1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca²⁺-dependent Cl⁻ channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca²⁺-dependent Cl⁻ channels by lowering the energy barriers for ion translocation through the pore.     

奥曲肽通过G0/G1期阻滞抑制胆管癌细胞的增殖

为观察四种胆管癌细胞系（RBE、NEC、QBC939、SSP－25）是否能够表达生长抑素受体（SSTR），以及八肽生长抑素类似物奥曲肽（OCT）对胆管癌细胞的抑制作用，采用逆转录－聚合酶链反应方法检测四种细胞系2，3型SSTR的表达；四氮唑蓝法检测不同浓度OCT（10、1、0.1、0.01和0.001mg/L)在体外对胆管癌细胞增殖的影响。并应用碘化丙啶（PI）及Annexin V-PI染色后于流式细胞仪检测经OCT作用后胆管癌细胞周期和凋亡情况。结果显示四种胆管癌细胞系均可以表达SSTR2 mRNA，但未检测到SSTR3 mRNA的表达。体外10、1和0.1mg/L OCT明显抑制四种胆管癌细胞的增殖（与各自对照组相比，P＜0.05)；1mg/L OCT作用48h后流式细胞分析表现为G0／G1期细胞增加，S期和G2／M细胞减少（与各自对照组相比，P＜0.01)，但没有明显凋亡发生。从而证明OCT抑制胆管癌细胞增殖的机制主要与引起细胞周期阻滞有关，而不是促进凋亡。对于表达生长抑素受体的胆管癌，OCT有望用于临床辅助治疗。     

Functional analysis of LKB1/STK11 mutants and two aberrant isoforms found in Peutz-Jeghers Syndrome patients

Peutz-Jeghers Syndrome (PJS) is thought to be caused by mutations occurring in the widely expressed serine/threonine protein kinase named LKB1/STK11. Recent work has led to the identification of four mutants (R304W, I177N, K175-D176del, L263fsX286) and two novel aberrant LKB1/STK11 cDNA isoforms (r291-464del, r485-1283del) in a group of PJS Italian patients. Three of the four mutations only change 1 or 2 amino acids in the LKB1/STK11 catalytic domain. Here we demonstrate that all six LKB1/STK11 variants analysed are completely inactive in vitro as they were unable to autophosphorylate at Thr336, the major LKB1/STK11 autophosphorylation site, and to phosphorylate the p53 tumour suppressor protein. We also show that 5 out of the 6 variants are entirely localised in the nucleus in contrast to the wild type LKB1/STK11, which is detected in both the nucleus and cytoplasm. Finally we demonstrate that all 6 LKB1/STK11 variants, in contrast to wild type LKB1/STK11, are unable to suppress the growth of melanoma G361 cells. Taken together, these results demonstrate that the LKB1 mutations investigated in this study lead to the loss of serine/threonine kinase activity and are therefore likely to be the primary cause of PJS development in the patients that they were isolated from.     

Sequence variants in the 5' flanking region of the leptin gene are associated with obesity in women

Few mutations have been found in the human leptin gene and the relationship between leptin gene sequence variation and human overweight is uncertain. To determine whether sequence variation within the leptin gene and its regulatory elements contribute to extreme obesity, we screened ∼3 kb of the 5' flanking region and the three exons in 125 unrelated extremely obese (BMI ≥ 40 kg/m²) and 86 average weight women (BMI < 27 kg/m²). Within the protein coding regions only one heterozygous silent mutation was found (codon 102; AAC/AAT). Within the 5' flanking region, six frequent sequence variants were detected ( q > 0.10), and the allele frequencies of three of these variants differed between obese and average weight Caucasian women (+19, χ²= 4.46, p = 0.035; −1823, χ²= 4.36, p = 0.037; −2548, χ²= 5.73, p = 0.017). Nine infrequent sequence variants were detected ( q < 0.05) but they did not occur more often among obese women compared with those of average-weight. For extremely obese women, three polymorphisms (+19, −188, and −633) predicted the degree of obesity. Allelic variants may influence the regulation of the leptin gene and thereby influence body weight, particularly among extremely obese women. However, given the low variability in coding regions and the high variability in the 5' flanking region, discerning the functional significance of each variant is likely to be difficult.     

Neurofibromin (NF1) genetic variant structure–function analyses using a full‐length mouse cDNA

Neurofibromatosis type 1 (NF1) is caused by pathogenic variants or mutations in the NF1 gene that encodes neurofibromin. We describe here a new approach to determining the functional consequences of NF1 genetic variants. We established a heterologous cell culture expression system using a full‐length mouse Nf1 cDNA (mNf1) and human cell lines. We demonstrate that the full‐length murine cDNA produces a > 250 kDa neurofibromin protein that is capable of modulating Ras signaling. We created mutant cDNAs representing NF1 patient variants with different clinically relevant phenotypes, and assessed their ability to produce mature neurofibromin and restore Nf1 activity in NF1^?/? cells. These cDNAs represent variants in multiple protein domains and various types of clinically relevant predicted variants. This approach will help advance research on neurofibromin structure and function, determine pathogenicity for missense variants, and allow for the development of activity assays and variant‐directed therapeutics.     

Recognition of HLA-A1 by murine monoclonal antibodies

Abstract: Balb/c mice were immunized with cells from the mouse mastocytoma line P815 transfected with an HLA-A1 gene. The splenocytes of the immunized mice were fused with cells from the murine myeloma NS-1. In an initial screening, supernatants of growing cultures were tested for their binding capacity to the immunizing P815/A1⁺ cells as well as to P815/A2⁺ cells. Three out of 756 hybrids produced antibodies which bound to P815/A1⁺ cells only. They were cloned and further analyzed for their binding reactivity to reference B-lymphoblastoid cells from the Tenth International Histocompatibility Workshop. One monoclonal antibody, designated 6B11, reacted only with HLA-A1⁺ cells, while the two other antibodies, 3G3 and 7F10, appeared to detect antigenic determinants shared by HLA-A1, A3, A11, A26, and A30 (3G3) and by HLA-A1, A3, A11, A26, A28 and A30 (7F10). Flow cytometric studies on B-lymphoblastoid cell lines as well as on a series of tumor cell lines, including melanoma and colon carcinoma lines, confirmed the specificity of these antibodies. Monoclonal antibodies 7F10 and 6B11 were found to be of the IgM class and 3G3 of the IgG1 class. By complement-dependent ⁵¹Cr release experiments it was further shown that the two IgM antibodies 7F10 and 6B11 were able to lyse all cell lines of the HLA-A1 haplotype tested.     

反义周期蛋白B1基因抑制HT29细胞的增殖

用ＤＮＡ重组技术将人类ｃｙｃｌｉｎＢ１基因的全长ｃＤＮＡ，反向插入到真核表达载体ｐＸＪ４１－ｎｅｏ的ＢａｍＨ１位点，得到ｃｙｃｌｉｎＢ１基因的反义ＲＮＡ表达载体ｐＡＳ－Ｂ１。利用ＤＮＡ磷酸钙共沉淀方法，将ｐＡＳ－Ｂ１转染进ＨＴ２９细胞。在含有Ｇ４１８的培养基中，挑选出独立的细胞克隆。利用ｃｙｃｌｉｎＢ１ｃＤＮＡ作探针，Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交鉴定出一株内源性ｃｙｃｌｉｎＢ１ｍＲＮＡ受到抑制的细胞株ＨＴＢ１。比较ＨＴＢ１与对照细胞ＨＴ２９发现，ｃｙｃｌｉｎＢ１的反义ＲＮＡ明显抑制ＨＴ２９细胞的增殖，细胞内３Ｈ－ＴｄＲ参入量减少。流式细胞光度术分析，处于Ｇ１期细胞比率的升高与Ｓ期、Ｇ２＋Ｍ期细胞数量的下降都十分显著。     

Association of a polymorphism at the 5'-region of the angiotensin II type 1 receptor with hypertension

Molecular variants of individual components of the renin-angiotensin system are thought to contribute to inherited predisposition towards essential hypertension. Using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequence analysis, we identified seven polymorphisms in the 5'-flanking region of the angiotensin II type 1 receptor ( AGTR1 / AT ₁ ) gene. We conducted a case-control study in a sample from the Japanese population to determine whether polymorphic markers in the 5'-flanking region of the AT ₁ gene were associated with essential hypertension. The study compared 149 hypertensive subjects to 156 normotensive control subjects. A significantly higher frequency of the AT ₁ (−535)*T allele was observed in hypertensive subjects. Evidence was obtained that the AT ₁ (−535)*T allele showed a synergistic effect on risk of hypertension with angiotensin I converting enzyme D allele ( ACE *D).