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Characterization of human lymphocyte subpopulations: alloreactive cytotoxic T-lymphocyte precursor and effector cells are phenotypically distinct from Leu 2+ suppressor cells.

The OKT8/Leu 2+ human T-cell subset contains cells which perform suppressor and cytotoxic functions. We have recently produced two monoclonal antibodies (termed 2D2 and D12) which define four subpopulations of human E+ cells. Previous studies have shown that the Leu 2+ cells that suppress T-cell proliferative responses have the 2D2+D12+ phenotype. In the present studies, we have used these antibodies and fluorescence-activated cell sorter techniques to characterize the phenotype of cytotoxic T lymphocytes generated in allogeneic mixed lymphocyte cultures. These studies indicate that the cytolytic effector cells which recognize class I major histocompatibility antigens express the 2D2+D12- phenotype. The phenotype of the precursor cells for these cytotoxic T cells was similarly demonstrated to be 2D2+D12-. The subset of E+ cells with NK cytolytic activity expressed the 2D2-D12+ phenotype. These data demonstrate that the Leu 2+ precursor and effector cytotoxic T cells reactive against class I alloantigens are phenotypically distinct from the Leu 2+ cells previously shown to suppress T-cell proliferative responses.

Antibodies, Monoclonal↗

Prevalence of debrisoquine oxidation phenotypes in glaucoma patients.

The oxidation of debrisoquine, a sympatholytic antihypertensive agent, exhibits genetic polymorphism. The debrisoquine/4-OH-debrisoquine metabolic ratio (MR) separates the population to poor (PM, MR greater than 12.6) and extensive (EM, MR less than 12.6) metabolizers. 5-10% of the caucasians belong to the PM phenotype. The oxidation of many other drugs, like timolol, correlates with the debrisoquine phenotype. We determined the debrisoquine phenotype in 102 glaucoma patients. The majority of the patients was treated with ophthalmic timolol. Five patients were classified as PMs. However, two of them were on quinidine, a well known inhibitor of debrisoquine oxidation. The prevalence of debrisoquine PM phenotype in glaucoma patients was 2.9% (excluding patients on quinidine) or 4.9% (with patients on quinidine). The figures are slightly lower than the mean value reported for the normal Finnish population. However, both figures lay within the 95% confidence limits of the prevalence of PM phenotype in the normal Finnish population. The beta-blocking activity of oral timolol is increased in PMs. The significance of timolol oxidation phenotype during ocular timolol therapy warrants further investigation.

Cross-Sectional Studies↗

Phenotypic variations among enterotoxigenic O-groups of Escherichia coli from various human populations.

ETEC isolates from various sources (children from Ethiopia and some Asian countries, and Swedish tourists suffering from traveller's disease) were analysed with the aid of "biochemical fingerprinting", which is a highly discriminative, computerized method designed to subdivide E. coli isolates into different phenotypes. Isolates belonging to the most common ETEC O-groups and others which had not been typeable with available O-antisera were selected. It was found that certain phenotypes of O-groups 6 and 114 could be found in materials from several continents. Phenotypes of other O-groups were usually more restricted to certain geographic areas. Among children in Addis Abeba, 19 out of 25 isolates carrying O-antigen 78 belonged to the same phenotype. Some possible explanations for the fact that certain phenotypes of enterotoxigenic E. coli could be found over the whole world are that they might represent relatively recent developed clones, or they may represent unusually stable clones. Of the isolates that had been nontypeable with available antisera, some had lost their LT-productivity after one year of storage. These isolates proved to belong to a wide variety of phenotypes, whereas nontypeable isolates which were stable LT-producers could be clustered into distinct groups. It is suggested that the non-stable LT-producers are members of the normal E. coli flora of these children, which have occasionally picked up the enterotoxin-producing plasmids, whereas most stable LT-producers represent true ETEC clones.

Adult↗

Lipid biosynthesis in cultured arterial smooth muscle cells is related to their phenotype.

During the atherogenic process in vivo, arterial smooth muscle cells (SMC) undergo changes in their phenotype. In the present study, rat SMC from primary cultures and from subcultures before 10 and after 200 passages, showing contractile-like, synthetic and transformed phenotypes, respectively, were compared in regard to their lipid content and biosynthesis. The rationale for comparing these phenotypes rests in the similar changes in phenotype of SMC that occur in the formation and progression of atherosclerotic lesions. Phenotype changes were shown to be associated with changes in the phospholipid content of SMC. Phospholipid levels increased, but not as significantly as did cholesterol levels when passing from contractile to synthetic and transformed cells (1.23 +/- 0.18, 2.28 +/- 0.26 and 3.25 +/- 0.23 micrograms/10(6) cells, respectively). Cholesterol normalized in respect to cell protein was increased to the same extent. Lipid synthesis as judged by [14C]acetate incorporation was increased 3- to 12-fold in the synthetic and transformed cells, respectively, compared to contractile cells. After thin-layer chromatography, radioactivity was shown to be markedly increased in most of the lipid fractions, but label in the cholesterol fraction of synthetic and transformed cells was increased by 7- and 21-fold, respectively. Thus, SMC in vitro were shown to drastically increase cholesterol biosynthesis associated with phenotype changes. Such changes are known to occur in vivo and might represent a critical step in the deposition of excess cholesterol within foam cells.

Animals↗

In vivo and in vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate of Escherichia coli.

A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.

Cloning, Molecular↗

Abnormal enzyme phenotype (E1a E1f): normal response to succinylcholine.

The enzyme serum cholinesterase responsible for the hydrolysis of the muscle relaxant succinylcholine exists in the form of several variants. These may be identified in serum by using substances which inhibit their activity to different degrees. The heterozygote for the atypical and fluoride resistant enzymes (E1a E1f) is one of the phenotypes which has been reported to be sinsitive to succinylcholine. A case is described where succinylcholine given on two separate occasions did not induce apnoea in an individual phenotyped as E1a E1f by at least five methods of inhibition. This is the first reported example of such insensitivity to the drug in this phenotype. Temperature activities for the patient's serum over the range of 20 degrees C to 45 degrees C differed from that of an established E1a E1f phenotype used as a control. There was a progressive inactivation of the control serum at temperatures higher than 35 degrees C, as previously reported for this phenotype. Activity in the serum of the subject of this study did not exhibit the peak activity at 35 degrees C but continued to rise and probably reached a peak between 40 degrees C and 45 degrees C. The significance of these results in the context of current methods of phenotyping is discussed.

Adolescent↗

[Clinical value of the CA 19-9 tumor marker with special reference to the Lewis phenotype].

BACKGROUND: Because of structure and biosynthesis of CA 19-9, it was postulated that patients with the Lewis phenotype Le(a-b-) are not able to synthesize CA 19-9. But some patients with Le(a-b-) on red blood cells showed elevated levels of this tumor marker. PATIENTS AND METHOD: In 164 patients suffering from benign or malignant diseases both CA 19-9 and the Lewis phenotype were determined in sera. In addition in 51 patients red blood cells were tested for Lewis substances. RESULTS: The frequencies of the different Lewis phenotypes on red blood cells were compared with the results found in sera. The prevalence of the phenotype Le(a-b-) on erythrocytes was significantly higher than in sera. In 51 patients both determinations were performed. These results were compared additionally. The phenotype Le(a-b-) found on red blood cells agreed with the results found in sera only in 30% of the cases. A loss of Lewis substances on erythrocytes could be seen both in malignant and benign diseases. Only in patients with Lewis substances found in sera elevated levels of CA 19-9 could be seen. CONCLUSION: Considering only the Lewis phenotype in sera, it could be confirmed that patients with the genotype Le(a-b-)are not able to express elevated concentrations of CA 19-9.

Aged↗

CYP2D6 extensive, intermediate, and poor phenotypes and genotypes in a Polish population.

OBJECTIVE: This study investigated the distribution of the CYP2D6 genotypes and phenotypes in a Polish population and compared the concordance of the two methods. METHODS: Six hundred unrelated healthy individuals from southwestern Poland were studied. The CYP2D6 phenotype was analyzed in 300 individuals using sparteine as a model drug. The CYP2D6 genotype was analyzed in 300 individuals by polymerase chain reaction amplification and restriction fragment length polymorphism techniques for the CYP2D6*1, CYP2D6*3, and CYP2D6*4 alleles. Additionally, in 60 randomly selected healthy individuals both the CYP2D6 phenotype and genotype was assessed to determine accordance between the methods. RESULTS: Of 300 participants in the study 25 (8.3%) were classified as poor metabolizers, 44 (14.7%) as intermediate metabolizers, and 231 (77%) as extensive metabolizers of sparteine. The frequency of CYP2D6*1, CYP2D6*3, and CYP2D6*4 alleles among the genotyped 300 persons was 75.7%, 1.3%, and 23.0%, respectively. The frequency of CYP2D6 deficient genotypes in a Polish population (8.0%) was similar to phenotyping results. The comparison of phenotype and genotype in 60 randomly selected individuals showed a good concordance of the obtained results. CONCLUSIONS. The frequencies of poor metabolizers for CYP2D6 phenotype (8.3%) and genotype (8.0%) in a Polish population from the southwestern region are in concordance and compare well with most results of poor oxidation metabolizers in other white populations.

Adolescent↗

Influence of donor and recipient genotypes on CYP2D6 phenotype after liver transplantation: a study of mutations CYP2D6*3 and CYP2D6*4.

OBJECTIVE: After liver transplantation (LT), genotypic differences between the recipient and the transplanted liver, medications and post-LT complications may all affect drug metabolism. We have studied the effect of two CYP2D6 mutations in the donor and the recipient on post-LT CYP2D6 phenotype. METHOD: The CYP2D6 phenotype was assessed in 48 patients before and after LT with debrisoquine or dextromethorphan. CYP2D6*3 (CYP2D6A) and CYP2D6*4 (CYP2D6B) mutations were detected in the donor and the recipient using polymerase chain reaction. RESULTS: Before LT, 40 subjects were classified as extensive metabolisers (EM) and 8 as poor metabolisers (PM); after transplantation, 41 were EMs and 7 were PMs. Genotype and phenotype were in agreement in 100% of EMs and 40% of PMs. The low percentage of agreement in PMs could not be explained by severely altered liver function. The phenotype of 13 subjects was apparently changed by LT: 6 EMs became PMs and 7 PMs became EMs. All four subjects in whom genotype changed following LT had a corresponding change in phenotype: two EM subjects became PMs and two PM subjects became EMs. CONCLUSION: The low percentage of agreement in PMs may be partly explained by mutations other than CYP2D6*3 and CYP2D6*4. Nevertheless, our study shows that the CYP2D6 genotype of the donor controls the phenotype of the recipient of a liver transplantation.

Adult↗

Novel phenotypes identified by plasma biochemical screening in the mouse.

We used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for both gene function studies and use as new animal models of human disease (Nolan et al. 2000b). One focus of the program was the development of a blood biochemistry screen. At 8-12 weeks of age, approximately 300 ml of blood was collected from F1 offspring of ENU mutagenized male mice. This yielded approximately 125 ml of plasma, used to perform a profile of 17 standard biochemical tests on an Olympus analyzer. Cohorts of F1 mice were also aged and then retested to detect late onset phenotypes. In total, 1,961 F1s were screened. Outliers were identified by running means and standard deviations. Of 70 mice showing consistent abnormalities in plasma biochemistry, 29 were entered into inheritance testing. Of these, 9 phenotypes were confirmed as inherited, 10 found not to be inherited, and 10 are still being tested. Inherited mutant phenotypes include abnormal lipid profiles (low total and HDL cholesterol, high triglycerides); abnormalities in bone and liver metabolism (low ALP, high ALP, high ALT, and AST); abnormal plasma electrolyte levels (high sodium and chloride); as well as phenotypes of interest for the study of diabetes (high glucose). The gene loci bearing the mutations are currently being mapped and further characterized. Our results have validated our biochemical screen, which is applicable to other mutagenesis projects, and we have produced a new set of mutants with defined metabolic phenotypes.

Animals↗

Epigenetics and phenotypic variation in mammals.

What causes phenotypic variation? By now it is clear that phenotype is a result of the interaction between genotype and environment, in addition to variation not readily attributable to either. Epigenetic phenomena associated with phenotypic variation at the biochemical, cellular, tissue, and organism level are now well recognized and are likely to contribute to the "intangible variation" alluded to. While it is clear that epigenetic modifications are mitotically heritable, the fidelity of this process is not well understood. Inheritance through more than one generation of meioses is even less well studied. So it remains unclear to what extent epigenetic changes contribute to phenotypic variation in natural populations. How might such evidence be obtained? What are the features of phenotypes that might suggest an epigenetic component? How much of the epigenetic component is truly independent of genetic changes? The answers to such questions must come from studies designed specifically to detect subtle, stochastically determined phenotypic variation in suitable animal models.

Adaptor Proteins, Signal Transducing↗

Multiplex inheritance of component phenotypes in a murine model of lupus.

We analyzed the linkage of GN and a wide spectrum of serological phenotypes associated with systemic lupus erythematosus in a (NZM2410 x C57BL/6)F2 cross. Some phenotypes, such as glomerulonephritis (GN) and anti-chromatin IgG antibody production, were more penetrant in females, but others, such as anti-dsDNA antibody production, did not show a gender bias. These results suggest that gender bias affects only a subset of SLE-component phenotypes, and that NZM2410 can be used to dissect the genetic basis of this phenomenon. Genome scanning linked six chromosomal intervals with the expression of one or more component phenotypes. These loci included two Sle loci previously identified in an (NZM2410 x B6)F1 x NZM2410 backcross, loci identified by others in the NZB/W model. Our analysis also suggested two new intervals on chromosomes (Chrs.) 10 and 11. Detailed analysis of the segregation of different phenotypes within these intervals suggests that they encompass more than one susceptibility locus. This clustering has been a common finding in several murine polygenic traits. Each of NZM2410 susceptibility loci can be aligned with a specific genetic pathways contributing to SLE pathogenesis on the basis of the spectrum of component phenotypes expressed.

Animals↗

Changes in osteopontin mRNA expression during phenotypic transition of rabbit arterial smooth muscle cells.

Transition from a contractile to a synthetic phenotype appears to be an early key event during the development of intimal thickening after arterial wall injury. We examined the expression of osteopontin mRNA, proliferation, and phenotypic properties of smooth muscle cells (SMCs) in rabbit neointima after balloon denudation and in primary culture. A strong osteopontin mRNA signal was detected in the thickened intima 1 week after balloon denudation and in the surface layer of the intima 2 weeks after balloon denudation. Ki-67 immunohistochemistry showed that osteopontin mRNA expression increased when SMCs entered the proliferating phase in the intima. Rabbit arterial SMCs on type I collagen after 1 day of primary culture with growth factors, as well as freshly isolated cells, were in the G0 phase (contractile phenotype) and did not express osteopontin mRNA. After 3 days of culture, most cells entered the G1B phase (synthetic phenotype) and expressed osteopontin mRNA. In the absence of growth factors, most cells transferred to the G1A phase (intermediate phenotype) after 3 and 7 days, but did not express osteopontin mRNA. Our findings indicate that the osteopontin gene provides a marker that can be used to distinguish the phenotypic properties of vascular SMCs.

Animals↗

A comparative analysis of phenotype expression in human osteoblasts from heterotopic ossification and normal bone.

BACKGROUND AND AIMS: Heterotopic ossification (HO) is a pathological bone formation process in which ectopic bone is formed in soft tissue. The formation of bone depends on the expression of the osteoblast phenotype. Earlier studies have shown conflicting results on the expression of phenotype markers of cells originating from HO and normal bone. The hypothesis of the present study is that cells from HO show an altered expression of osteoblast-specific phenotype markers compared to normal osteoblasts. The aims of the study were to further characterize the expression of osteoblast phenotypemarkers and to provide a comparison with other study results. PATIENTS AND METHODS: Using an in vitro technique, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and immunohistochemistry, we compared the phenotype gene expression (type I collagen, alkaline phosphatase, Cbfa-1, osteocalcin) of osteoblasts from resected HO and normal bone (iliac crest). RESULTS: Cells from HO expressed the osteoblast phenotype (type I collagen, alkaline phosphatase) but were characterized by a depleted osteocalcin expression. The expression of Cbfa-1 (osteocalcin transcription gene) showed a large variety in our study. Preoperative radiotherapy had no effect on phenotype expression in cells from HO. CONCLUSION: Our results provide a characterization of cells originating from HO and support the thesis of an impaired osteoblast differentiation underlying the formation of HO. The transcription axis from Cbfa-1 to osteocalcin could be involved in the pathogenesis of HO.

Adult↗

Structural variation in the Waxy gene and differentiation in foxtail millet [Setaria italica (L.) P. Beauv.]: implications for multiple origins of the waxy phenotype.

The origin and evolution of the waxy type of foxtail millet [Setaria italica (L.) P. Beauv] were studied by analyzing structural variation in the Waxy gene. Initially, the Waxy gene was amplified by RT-PCR, RACE and genomic PCR from a non-waxy strain to determine the structure of the wild-type gene. Secondly, we screened by PCR for polymorphisms at the Waxy locus in 79 strains with various waxy phenotypes. We then carried out genomic Southern analysis on 67 strains and identified seven RFLP classes which were designated as types I-VII. RFLP type was correlated with phenotype, such that types I and II corresponded to non-waxy, types III and VI to low-amylose, and types IV, V and VII to waxy phenotypes. The differences between RFLP types could be attributed to insertions in the Waxy gene. Types II and VI were caused by the insertion of a Tourist element into intron 1 and a SINE-like sequence into intron 12, respectively. Types III, IV, V and VII were characterized by the insertion of large sequences into the Waxy gene that may alter the expression of the gene. Thus, multiple, independent insertions in the Waxy gene appear to have caused the loss-of-function waxy phenotypes. Furthermore, the geographical distributions of the three RFLP types associated with the waxy phenotype (types IV, V and VII) were distinct, with type IV being found mainly in Taiwan and Japan, type V in Korea, and type VII in Myanmar. These results indicate a polyphyletic origin for the waxy phenotype in landraces of foxtail millet.

Base Sequence↗

Genetic regions that interact with loss- and gain-of-function phenotypes of deltex implicate novel genes in Drosophila Notch signaling.

The Notch signaling pathway is an evolutionarily conserved mechanism that regulates many cell fate decisions. The deltex (dx) gene encodes an E3-ubiquitin ligase that binds to the intracellular domain of the Notch protein and regulates Notch signaling in a positive manner. However, it is still not clear how Dx does this. We generated a transgenic line, GMR-dx, which overexpresses dx in the developing Drosophila eye disc. The GMR-dx line showed a rough-eye phenotype, specific transformation of a photoreceptor cell (R3 to R4), and a rotation defect in the ommatidia. This phenotype was suppressed in combination with a dx loss-of-function mutant, indicating that it was due to a dx gain-of-function. We previously reported that overexpression of Dx results in the stabilization of Notch in late endosomes. Here, we found that three motifs in Dx, a region that binds to Notch, a proline-rich motif and a RING-H2 finger, were required for this stabilization, although the relative activity of these variants in this assay did not always correspond to the severity of the rough-eye phenotype. In an attempt to identify novel genes of the Notch pathway, we tested a large collection of chromosomal deficiencies for the ability to modify the eye phenotypes of the GMR-dx line. Twelve genomic segments that enhanced the rough-eye phenotype of GMR-dx were identified. To evaluate the specificity of these interactions, we then determined whether the deletions also interacted with the wing phenotypes associated with a loss-of-function mutation of dx, dx24. Analyses based on whole-genome information allowed us to conclude that we have identified two novel loci that probably include uncharacterized genes involved in Dx-mediated Notch signaling.

Amino Acid Motifs↗

Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe.

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomzyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana(r)-6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana(r)-6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity.

Amino Acid Sequence↗

Genotype, enzyme activity, glutathione level, and clinical phenotype in patients with glutathione synthetase deficiency.

Glutathione synthetase (GS) deficiency is a rare autosomal recessive disorder. The clinical phenotype varies widely, and nearly 30 different mutations in the GSS gene have been identified. In the present study, genotype, enzyme activity, metabolite levels and clinical phenotype were evaluated in 41 patients from 33 families. From some of the patients, data on glutathione (GSH) levels and gamma-glutamylcysteine levels in cultured fibroblasts were also available. Twenty-seven different mutations were found: 14 missense, 9 splice, 2 deletions, 1 insertion and 1 nonsense mutation. Twenty-three patients were homozygous and 18 were compound heterozygous. The moderate and severe clinical phenotypes could not be distinguished based on enzyme activity, GSH or gamma-glutamylcysteine levels in cultured fibroblasts. However, in fibroblasts, the residual GS activity was correlated with the GSH level. All mutations causing frameshifts, premature stop codons or aberrant splicing were associated with moderate or severe clinical phenotypes including haemolytic anaemia, 5-oxoprolinuria, and (in several forms) neurodevelopmental signs. The data indicate that additional genetic or environmental factors modify at least the moderate and severe phenotypes and that the clinical classification given to the patients may be influenced by variation in follow-up. The type of mutation involved can, to some extent, predict a mild versus a more severe phenotype.

Cells, Cultured↗