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PubMed2004

Somatropin and its variants: structural characterization and methods of analysis.

Human Growth hormone (hGH, somatotrophin) is a 22 kDa, 191 amino-acid single chain protein produced by somatroph cells of the anterior pituitary gland. It is the major physiological regulator of growth, and deficiencies in growth hormone levels have long been recognized as the underlying cause of growth disorders (dwarfism). The ability of exogenous hGH to restore normal rates of growth in both human and animal models of growth retardation has long been recognized and the use of hGH in therapy goes back several decades. Initial preparations were prepared by extraction and purification from cadaveric pituitary tissue, and since 1984, hGH has been prepared by recombinant Deoxyribosenucleic acid (rDNA) technology. As is usually the case with "biologicals", characterization of the drug substance depended on a combination of physico-chemical and biological methods, and the hGH molecule became well characterized fairly early in its life as a drug. Indeed, by 1980 the major degradation forms and structural variants of the hGH molecule had been described and reviewed. Little satisfactory progress had been made in refining biological assays for hGH, and, although in vitro assays were described, potency-defining assays remained dependant on the whole body growth response in rats, and were both invasive and imprecise. In the early 1990's a series of collaborative studies on analysis of recombinant hGH (somatropin) established that available bioassays were much less selective that physico-chemical methods in detecting and quantifying structural degradation, and 1994 saw an international consensus to replace the bioassays with physico-chemical analytical methods for the routine batch release of somatropin. During the last decade in most markets somatropin has, unusually for a protein, been subject to batch release and control dependent entirely on physico-chemical analysis, without the routine use of any form of bioassay. During that time there has been a continuous development and refinement of methods, and the identification of a range of structural variants and degradation products of the molecule. The present review sets out to summarise the current knowledge on physico-chemical analytical methods for somatropin, and the structural variants that have been identified and characterized. A survey of available biological analytical methods is beyond the scope of this review, as is consideration of the earlier pituitary preparations and the recombinant 192 amino-acid methionyl form of the molecule (somatrem), although it is likely that many of the methods and variants described would be equally applicable to somatrem.

Chromatography, High Pressure Liquid↗
PubMed1991

Hb Mizuho or alpha 2 beta (2)68(E12)Leu----Pro in a Caucasian boy with high levels of Hb F; identification by sequencing of amplified DNA.

Hb Mizuho, an unstable beta chain variant with a Leu----Pro substitution at position beta 68, was observed in a young Caucasian boy from Kentucky. Identification was made by sequence analyses of amplified DNA and by hybridization of amplified DNA with specific probes. The patient had high Hb F levels up to the present age of nearly 5 years; this high Hb F might in part be responsible for his condition which was considerably milder than that seen in the two patients described in earlier reports. The increased gamma chain synthesis may be due to special characteristics of the beta-globin gene cistron which are comparable to those observed in sickle cell anemia patients with a relatively mild disease.

Amino Acid Sequence↗
PubMed2002

A dual reporter screening system identifies the amino acid at position 82 in Flp site-specific recombinase as a determinant for target specificity.

We have developed a dual reporter screen in Escherichia coli for identifying variants of the Flp site-specific recombinase that have acquired reactivity at an altered target site (mFRT). In one reporter, the lacZalpha gene segment is flanked by mFRTs in direct orientation. In the other, the red fluorescence protein (RFP) gene is flanked by the native FRTs. Hence, the color of a colony on an X-gal indicator plate indicates the recombination potential of the variant Flp protein expressed in it: blue if no recombination or only FRT recombination occurs, red if only mFRT recombination occurs and white if both FRT and mFRT recombinations occur. The scheme was validated by identification and in vivo characterization of Flp variants that show either relaxed specificity (active on FRT and mFRT) or moderately shifted specificity toward mFRT. We find that alteration of Lys-82 to Met, Thr, Arg or His enables the corresponding Flp variants to recombine FRT sites as well as altered FRT sites containing a substitution of G-C by C-G at position 1 of the Flp binding element (mFRT11). In contrast, wild-type Flp has no detectable activity on mFRT11. When Lys-82 is replaced by Tyr, the resulting Flp variant shows a small but reproducible preference for mFRT11 over FRT. However, this preference for mFRT11 is nearly lost when Tyr-82 is substituted by Phe.

Amino Acid Substitution↗
PubMed2003

Allele-specific Hras mutations and genetic alterations at tumor susceptibility loci in skin carcinomas from interspecific hybrid mice.

We have investigated the effects of germ-line variants that influence skin tumor susceptibility loci on the patterns of somatic genetic alterations in mouse skin cancers. Using a two-stage skin carcinogenesis model, we previously identified at least 13 skin tumor susceptibility (Skts) loci in a large interspecific F1 backcross [(NIH/Ola x M. spretus) x NIH/Ola] study. In this report, we describe the analysis of allele-specific alterations at these loci in skin tumors from the same backcross animals. The mouse Hras gene, located close to Skts2 on chromosome 7, had specific activating mutations in the Mus musculus allele in 23 of 26 carcinomas. In all cases, tumors with Hras mutations also showed specific imbalance of chromosome 7 markers that favored the chromosome carrying the mutant allele. Allele-specific quantitative microsatellite analysis was also carried out, using DNA from 62 carcinomas from (NIH/Ola x M. spretus) x NIH/Ola mice. Frequent allelic imbalance was detected at five additional tumor-susceptibility loci on chromosomes 4, 6, 7, 9, and 16 (Skts7, Skts12, Skts1, Skts6, and Skts9, respectively). At all except Skts7, we found loss of the allele inherited from the resistant strain or amplification of the allele from the susceptible strain. We conclude that polymorphisms in some low-penetrance tumor modifier genes are reflected in the pattern of somatic alterations in tumors. Analysis of such allele-specific changes in tumors may facilitate the identification of functional germ-line variants that control tumor susceptibility.

Alleles↗
PubMed2005

Characterisation of the canine parvovirus type 2 variants using minor groove binder probe technology.

Two minor groove binder (MGB) probe assays were developed for rapid identification and quantitation of the canine parvovirus type 2 (CPV-2) variants in the faecal samples of dogs. The MGB probe assays using type-specific probes labeled with different fluorophores (FAM and VIC) were able to detect the single nucleotide polymorphisms existing between types 2a/2b and 2b/2c. Both the MGB probe assays were found to be highly specific, sensitive and reproducible, ensuring a precise quantitation of the CPV-2 DNA, as confirmed by comparing the obtained viral DNA loads to those calculated using a real-time TaqMan-based PCR assay established previously. A total of 414 faecal specimens collected from diarrhoeic dogs and tested positive to CPV-2 were analysed by the MGB probe assays and by conventional methods (haemagglutination inhibition with MAbs, PCR-RFLP and sequence analysis) for prediction of the CPV-2 antigen specificity, with an overall agreement of 100%. These results make the established MGB probe assays an attractive tool for direct and rapid characterisation of type 2a, 2b and 2c CPVs.

Amino Acid Substitution↗
PubMed1972

Numerical diagnostic key for the identification of Enterobacteriaceae.

A numerical diagnostic key for enteric organisms is described which permits the identification of typical strains and of biochemical variants with high accuracy. Unknown strains are inoculated into a basic set of five media which permit the testing of eight biochemical reactions. The positive reactions are assigned points, and the score of a strain is added up, after which the identification of the strain is obtained from a table. In many instances, the final identification is obtained with this set of biochemical tests; and, in other instances, a small number of additional tests are required to distinguish between organisms giving the same score in the basic set of biochemical tests. The key permits an accurate, rapid, and economical differentiation of the typical and the more common atypical biotypes of enteric organisms in the clinical laboratory.

Culture Media↗
PubMed1997

Molecular analyses of HIV-1 group O and HIV-2 variants from Africa.

Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Polymorphism screening of PIK4CA: possible candidate gene for chromosome 22q11-linked psychiatric disorders.

Lithium is potent non-competitive inhibitor of an enzyme involved in the metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P(2)), a critical phosphoinositide (PI) that regulates signal transduction and synaptic vesicle function. Interestingly, a number of genes involved in the regulation of PtdIns-4,5-P(2) synthesis and dephosphorylation are found in regions of the genome previously mapped in bipolar disorder (BPD) including 10p, 18q, 21q, and 22q. One is PIK4CA, a member of the phosphatidylinositol 4-kinase family that phosphorylates PtdIns at the D4 position of the inositol ring as part of the PtdIns-4,5-P(2) synthetic pathway. PIK4CA maps to 22q11 in a region believed to contain a susceptibility gene for psychiatric disorders. Screening of two functional domains of PIK4CA and the promoter region resulted in the identification of 15 different polymorphisms. Rare variants at a consensus splice donor site and the promoter region were found in a total of three patients with BPD, three with schizophrenia (SZ) and only one control. Several common non-synonymous changes and a common single nucleotide polymorphism (SNP) at position -31 in the putative promoter were identified and analyzed in patients with BPD, SZ, and controls. There was no difference in the allele distribution in mentally ill subjects and controls for two variants, R2259C and E2079Q, both located in the PIK4CA catalytic domain. There was, however, a trend toward significance in the distribution of the -31 promoter genotypes in bipolar subjects and controls. Although the results of this analysis were modest, considering the heterogeneity of BPD and SZ and the hypothesis that BPD may be caused by abnormalities in genes that regulate PI-mediated phenomena in the brain, the polymorphisms we detected in the PIK4CA gene should be analyzed in a larger data set to help determine their significance in 22q11-linked mental disorders.

1-Phosphatidylinositol 4-Kinase↗
PubMed1983

Invariance and sex-specific variations of the glomerular organization in the antennal lobes of a moth, Mamestra brassicae, and a butterfly, Pieris brassicae.

The glomerular organization of the antennal lobes was analyzed in the moth Mamestra brassicae and comparatively in the butterfly Pieris brassicae. The invariance of the lobes in number, position, and size of the glomeruli was verified quantitatively in the moth for all the glomeruli in individuals of the same sex (67 in males, 68 in females) and for 56 sex-invariant glomeruli which can be identified in all individuals whatever their sex. In the butterfly, the positional variability is greater than in the moth and hinders identification. The most conspicuous sex-variant glomeruli are two adjoining macroglomeruli in the male moth which have homologs of very small size in the female. No such dimorphism was observed in Pieris, a species in which males are attracted to female by visual stimuli and not by a sex pheromone as in Mamestra. There are other sex-variant glomeruli in Mamestra: four varying in location, three subdivided in the female, two found only in the male, and three found only in the female. Consequently, differing olfactory sensitivity related to species and sex could correlate with detectable modifications in the glomerular organization. A hypothesis on the function of glomeruli is discussed in which most of the glomeruli are viewed as "olfactory generalists" giving rise collectively to proposed "across-glomeruli patterns."

Animals↗
PubMed1997

Identification of a homolog of the C alpha 3'/hs3 enhancer and of an allelic variant of the 3'IgH/hs1,2 enhancer downstream of the human immunoglobulin alpha 1 gene.

Although four regulatory elements are known downstream of the mouse IgH alpha gene, a single enhancer homologous to hs1,2 has been thus far described downstream of each human alpha gene (Chen, C. and Birshtein, B. K., J. Immunol. 1997. 159: 1310). We characterized a 10-kb region downstream of the human alpha 1 gene. Two B cell-specific regulatory elements homologous to the murine C alpha 3'/hs3 and hs1,2,3' enhancers were found, which are duplicated downstream of alpha 2. The hs1,2 element is in inverted orientation by comparison with a recently reported alpha 1 hs1,2 element: it appears as a common allelic variant carrying an internal tandem repeat insertion and its prevalence in the human population is 60%. As in the mouse, the human hs1,2 enhancer is flanked with long inverted repeats which may have promoted inversion events through homologous recombination. Although the palindromic organization of the region is maintained in human, sequence identity with rodents focuses on core enhancer elements rather than on flanking repeats. Concerted divergence of both sides of the dyad symmetry suggests that inverted repeats are not just evolutionary remnants but rather play an architectural role in the LCR function.

Alleles↗
PubMed1998

Polymerase chain reaction-single strand conformation polymorphism or how to detect reliably and efficiently each sequence variation in many samples and many genes.

A simple and fast method with high reliability is necessary for the identification of mutations, polymorphisms and sequence variants (MPSV) within many genes and many samples, e.g. to clarify the genetic background of individuals with multifactorial diseases. We evaluated polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis to identify MPSV in several genes, which are thought to be involved in the pathogenesis of multifactorial autoimmune diseases like multiple sclerosis. The method is based on the property, that the electrophoretic mobility of single-stranded nucleic acids depends not only on its size but also on its sequence. The target sequence was amplified, digested into fragments ranging from 50-200 bp, heat-denatured and analyzed on native gels. The analysis of 55 PCR systems, including a total of 145 fragments demonstrates, that the detection rate of MPSV depends primarily on the fragment lengths. Appropriate dilutions of samples enhances the proportion of ssDNA compared to dsDNA. Changing the gel conditions, glycerol concentrations and/or the addition of urea may increase fragment resolution in some cases. In general, the detection of MPSV is neither influenced by their location within the fragment nor by the type of substitution, i.e. transitions or transversions. The standard PCR-SSCP system described here provides high reliability and detection rates and allows the efficient analysis of many samples and many genes.

Genetic Predisposition to Disease↗
PubMed2003

Evasive maneuvers by hepatitis C virus.

The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use of HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

Comment↗
PubMed2000

Identification of a novel alternative splicing of human FGF receptor 4: soluble-form splice variant expressed in human gastrointestinal epithelial cells.

Among four closely related members of the FGF receptor family, FGFR 1, 2, and 3 have alternative splicing forms encoded by different exons for the C-terminal half of the third Ig-like domain, but FGFR 4 has no such alternative exon. Furthermore, FGFR 1, 2, and 3 have another splice variant of nontransmembrane type; however, such a variant has not been reported for FGFR 4. While searching for a novel receptor-type tyrosine kinase by RT-PCR, we identified a non-transmembrane-type receptor of FGFR 4 in human intestinal epithelial cell lines (Intestine 407 and Caco-2). Sequence analysis of this receptor revealed that exon 9 coding the single transmembrane domain was displaced by intron 9. Consequently, this variant form was 120 bp shorter than the normal form and had no transmembrane portion. Moreover, the signal sequence in exon 2 was maintained, suggesting that this splice variant is a soluble receptor. This soluble receptor was detected in human gastrointestinal epithelial cells and pancreas, and also in gastric, colon, and pancreatic cancer cell lines. Single cell RT-PCR showed that this soluble receptor was expressed simultaneously with the transmembrane-type receptor in the same cell. Western blot analysis revealed that this receptor was secreted from the transfected COS7 cells. Thus, a soluble-form splice variant of FGFR 4 was identified in human gastrointestinal epithelial cells and cancer cells. This is the first report of alternative splicing of FGFR 4.

Alternative Splicing↗
PubMed1994

Structure of the gene for the testis-specific proprotein convertase 4 and of its alternate messenger RNA isoforms.

Proprotein convertase 4 (PC4) is a mammalian secretory serine endoproteinase similar to the yeast KEX2 gene product and specifically expressed in testicular germs cells. PC4 mRNA isoforms that vary in size and 3' coding sequence have been reported (N. G. Seitah, R. Day, J. Hamelin, A. Gaspar, M. W. Collard, and M. Chrétien, 1992, Mol. Endocrinol. 6: 1559-1570). To determine the origin of these various forms, the mouse PC4 gene was cloned and its organization determined. The structural gene is approximately 9.5 kb long. It contains 15 exons and 14 introns. The exon-intron organization is very similar to that of the genes for the related convertases furin, PC1, and PC2. The upstream region carries several GGGCGG and three CCAAT but no TATAA motifs. Analysis of the 5' end of PC4 mRNA in the testis has led to the identification of two novel 5' splice variants that might encode a nonsecretory enzyme. The multiple forms of PC4 mRNA can all be explained by alternate splicing of primary transcripts of a single gene.

Alternative Splicing↗
PubMed1990

Hb D Los Angeles (D-Punjab) and Hb Presbyterian: analysis of the defect at the DNA level.

Amplification of the beta-globin gene by the polymerase chain reaction (PCR) and direct sequencing were used for a fast and reliable identification of the beta-globin variant Hb D Los Angeles and revealed the predicted G----C substitution in codon 121. The same method showed the molecular defect in Hb Presbyterian to be a C----G substitution in codon 108; this eliminates a MaeII restriction site.

Base Sequence↗
PubMed2002

Studying genotype-phenotype relationships: cardiovascular disease as an example.

Cardiovascular disease is a multifactorial disorder resulting from the complex interaction of a plethora of both environmental and genetic factors. The "candidate gene" approach aims at identifying the genes contributing to cardiovascular disease assessing their entire polymorphic spectrum and the consequences of their combination in appropriate large association studies. In view of the sequence data available for the entire human genome, candidate sequences will more easily be traced and located in their chromosomal context. More powerful sequencing devices are available and will hopefully give reliable and reproducible data not only on the nucleotide sequence diversity in different populations throughout candidate regions of the human genome. The choice and the assessment of disease-related or intermediate phenotypes, especially in clinical settings, will be again more crucial in the future when the knowledge of gene sequence variation significantly increases. Identification of relevant genes and genetic variants involved in the different pathophysiological steps leading to cardiovascular disease may considerably improve our understanding of the mechanisms of the disease course. This may help to identify high-risk individuals and groups or subgroups in whom specific therapeutic interventions are indicated or necessary, leading to an individually adapted clinical management.

Cardiovascular Diseases↗
PubMed2001

A short-run new analytical ultracentrifugal micromethod for determining low-density lipoprotein sub-fractions using Schlieren refractometry.

We have developed a new analytical ultracentrifugal micromethod for the determination of serum low-density lipoprotein (LDL) subclasses directly from ultracentrifugal Schlieren scans. We have used special software for the analysis of this type of single-spin density-gradient ultracentrifugation. The flotation of LDL patterns was obtained by underlayering a physiological salt solution with serum or isolated lipoprotein fractions raised to a density of 1.3 g/mL in the spinning ultracentrifugation capillary band-forming cell. The repeated analysis of Schlieren curves of the same sample from 10 to 100 microL in the 60-100 min full-speed interval time resulted in quite reproducible results. We obtained quantitative results by measuring the Schlieren areas between the sample curves and the reference baseline curve by using computerised numerical and graphic techniques. The decomposition of the integrated curve was carried out using a nonlinear regression program followed by deconvolution algorithm analysis in order to determine the parameters of the composing Gaussian subclasses. The LDL particle concentrations were calculated from the area under the integral of the Gaussian curve using a calibration data constant. The flotation range of the LDL Schlieren curves in the cell was identified with serum from which LDL had been removed by means of precipitation reagents and with centrifugation of isolated LDL aliquots. With this technique, we measured the concentration of LDL and analysed its polydispersity without the need for preceding sequential isolation of the LDL. On the basis of the Schlieren curves, the LDL samples were either physically paucidisperse, having a symmetrical peak within a narrow density range, or were polydisperse, showing an asymmetrical pattern distributed over a broader density region. The described method proved to be useful for a clear and immediate visual presentation of the concentration values of the LDL and for the identification of the heterogeneity of LDL variants without the need for the preparative isolation of that density class.

Adult↗
PubMed2006

Genetic determinants of bone mass do not relate with breast cancer risk in US white and African-American women.

INTRODUCTION: The association between high bone mass and increased breast cancer risk has been established. Identification of polymorphisms and the resultant variant receptors suggests the possibility of differential effects on hormone responsive genes when complexed with the hormones. Both estrogen receptor-alpha (ER) and vitamin D receptor (VDR) polymorphisms have been associated with bone density. Thus, we examined these polymorphisms for association with increased breast cancer risk among US African-American and white women. METHODS: A case-control study was conducted to measure ER and VDR polymorphisms and radial bone mineral density (BMD) in African-American and white women, and to examine the association between polymorphisms, bone density and breast cancer risk. Genotypes and bone density were obtained from 412 women (220 cases and 192 controls, with equal distribution between the two ethnic groups). RESULTS: We found no evidence for an association between either the ER or VDR genotypes and breast cancer risk. Also, there was no difference in the risk of breast cancer by genotypes after adjusting for ethnicity. The addition of age, sex and ethnicity-specific BMD (Z-scores) did not significantly change the odds ratio for breast cancer. CONCLUSIONS: Our data suggest that the polymorphisms investigated had no effect on risk of breast cancer in this population. Thus, we found no evidence to support our hypothesis that breast cancer cases and controls would have a different distribution of ER and VDR genotypes. Furthermore, the polymorphisms were not associated with differences in bone mass and its relationship with breast cancer risk.

Adult↗