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At least 1,261 records · Page 70Linked to original sources
PubMed1989

Metastatic gestational choriocarcinoma. Unusual pathologic variant following therapy.

Two cases of metastatic gestational choriocarcinoma to the lungs have been encountered that presented unusual histologic patterns. Both lesions were solitary metastases that followed multiple courses of chemotherapy, and the patients had low serum beta-human chorionic gonadotropin (hCG) levels. Surgical excision appeared to be curative in both cases. Both neoplasms were characterized by a predominance of uniform but highly atypical mononucleate trophoblastic cells. These cells infiltrated pulmonary parenchyma, forming nests of tumor with central dense necrotic debris. Syncytiotrophoblastic cells (STB) were present but showed scant cytoplasm and little vacuolization. Hemorrhage was only focal. Immunohistochemistry revealed that some of the multinucleate STB and occasional mononucleate cells produced hCG, while human placental lactogen was focally produced in the tumors. By electron microscopy the STB were identified but lacked open lacunae lined by microvilli. Most mononucleate cells showed greater maturation evidenced by a more complex cytoplasm than is seen in typical cytotrophoblastic cells (CTB). The results suggest that these tumors are a distinctive subtype of choriocarcinoma composed largely of a form of trophoblastic cell with features intermediate between CTB and STB, yet different from the intermediate trophoblast of the placental site tumor. Identification of this morphologic variant of choriocarcinoma may have clinical utility as additional cases are studied.

Adult↗
PubMed1991

Biochemical identification of B-F and B-G allelic variants of the chicken major histocompatibility complex.

Biochemical methods were used to analyse B-F and B-G antigens of the chicken major histocompatibility complex (MHC). In a panel of 12 inbred or partially inbred chicken lines the MHC haplotypes, originally defined by serological and histogenetical methods, were compared. Using monoclonal 18-6G2, allele-specific B-G patterns were obtained by immunoblotting. Comparison of B-G12 and B-G2 revealed a shared banding pattern, but additional products were detected for B-G12. The B-F products of B2 and B12 had identical IEF patterns. The identical B-F products and partially shared B-G products might explain the serological cross-reaction between these haplotypes. In addition, the IEF pattern of B-F21 appeared similar to B-F2 and B-F12, but the partial proteolysis map showed a clear difference. Although two B-F bands could be detected per haplotype, no evidence for the expression of more than one B-F locus was found. The biochemical methods enabled a precise definition of expressed MHC products and can be a useful tool for the identification of B-alleles in other chicken lines or outbred chickens for their MHC antigens.

Alleles↗
PubMed2005

A mutational analysis of the 8-17 deoxyribozyme core.

The 8-17 deoxyribozyme is a small RNA-cleaving DNA enzyme of significant applicative interest. We measured the kinetics of over 60 variants of 8-17, mutated within the "core" region. The data were analyzed according to a conceptual framework in which deleterious substitutions can either decrease the stability of the reaction's transition state, or favor unreactive ground-state conformations. In agreement with earlier in vitro evolution studies, the most severe functional effects were observed upon mutating four conserved residues, whose role was further explored by replacing them with non-standard nucleotides. Removal or modification of individual functional groups on the A6 and G7 bases suggested that these residues are involved in a close-contact interaction and form a network of functionally important hydrogen bonds. Mutagenesis of residues C13 and G14 was less revealing, but argued strongly against a role of C13 as a general acid/base catalyst. The use of non-standard nucleotides also led to the identification of one deoxyribozyme variant that, under some ionic conditions, is substantially more active than the wild-type construct. Finally, the effects of mutations in the intramolecular "core stem" correlated only in part with changes in helical stability, suggesting that a stable stem is required but not sufficient for optimal activity.

DNA Mutational Analysis↗
PubMed1996

Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection.

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electroosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The approximately 90 fl injection volume for individual cells is 7 x 10(4) times lower than those previously reported. Adult (normal and elevated A1), sickle (heterozygous), and fetal erythrocytes were analyzed with the amounts of hemoglobins AO, A1c, S and F determined. The pH gradient for cIEF was linear (r = 0.9984), which allowed tentative identification of Hb Fac. Variants differing by as little as 0.025 pl units were resolved.

Adult↗
PubMed2006

Mutation analysis of DARPP-32 as a candidate gene for schizophrenia.

Dopamine- and cAMP-regulated phosphoprotein of relative molecular mass 32kDa (DARPP-32) plays a pivotal role in the signal transduction of several neurotransmitters and neuromodulators that are implicated in the pathophysiology of a variety of neuropsychiatric disorders. A postmortem study reported a significantly reduced DARPP-32 expression in the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia, suggesting possible involvement of DARPP-32 in the pathophysiology of schizophrenia. Hence, DARPP-32 was considered as a candidate gene for schizophrenia in this study. We first systemically searched for mutations in the DARPP-32 gene in 50 Han Chinese patients with schizophrenia from Taiwan. Five molecular variants were identified, including a C-to-G substitution (g.-2036C>G) in the putative core promoter that obliterated a predictive AP-2 transcription factor binding site, a G deletion in the untranslated exon 2 (g.1238delG), a G-to-A and an A-to-G substitutions in intron 2 (IVS2+31G>A) and intron 6 (IVS6+32A>G), respectively, and a three-base pair deletion of AGA in exon 6 that resulted in deletion of a glutamate at codon 135 (E135del). Further SNP- and haplotype-based association study in 249 patients and 273 control subjects, however, did not detect association of these markers with schizophrenia. Hence, our results suggest that the reduced DARPP-32 protein in patients with schizophrenia is unlikely caused by mutations in the DARPP-32 gene itself and the DARPP-32 gene is also unlikely a major susceptibility gene for schizophrenia. Nevertheless, the identification of these molecular variants should help the study of gene regulation and structure-function relationship of DARPP-32, and the association study of DARPP-32 gene with other neuropsychiatric disorders.

Alleles↗
PubMed1997

Identification of novel human tumor cell-specific CaMK-II variants.

CaMK-II (the (type II) multifunctional Ca2+/CaM-dependent protein kinase) has been implicated in diverse neuronal and non-neuronal functions, including cell growth control. CaMKII expression was evaluated in a variety of human tumor cell lines using RT-PCR (reverse transcriptase coupled polymerase chain reaction). PCR primers which flanked the CaMK-II variable domain were used so that all possible variants of the four mammalian CaMK-II genes (alpha, beta, gamma and delta) could be identified. 8 distinct CaMK-II isozymes were identified from human mammary tumor and neuroblastoma cell cDNA, each of which represented a variant of beta, gamma or delta CaMK-II. They included 2 beta isozymes (beta e, beta 'e), 4 gamma isozymes (gamma B, gamma C, gamma G, gamma H) and 2 delta isozymes (delta C, delta E) This is the first report of human beta and delta CaMK-II sequences. A panel of human cell types was then screened for these CaMK-II isozymes. As expected, cerebral cortex predominately expressed alpha, beta and delta A CaMK-II. In contrast, tumor cells, including those of neuronal origin, expressed an entirely different spectrum of CaMK-II isozymes than adult neuronal tissue. Tumor cells of diverse tissue origin uniformly lacked alpha CaMK-II and expressed 1-2 beta isozymes, at least 3 gamma isozymes and 1-2 delta isozymes. When compared to undifferentiated fibroblasts, beta e, beta'e, gamma G and gamma H were preferentially expressed in tumor cells. CaMK-II immunoblots also indicated that neuroblastoma and mammary tumor cells express isozymes of CaMK-II not present in their non-transformed cell or tissue counterpart. The identification of these new, potential tumor-specific CaMK-II variants supports previous indications that CaMK-II plays a role in growth control. In addition, these results provide insight into both splice variant switching and variable domain structural similarities among all CaMK-II isozymes.

Alternative Splicing↗
PubMed2007

Synthesis and metabotropic glutamate receptor activity of S-oxidized variants of (-)-4-amino-2-thiabicyclo-[3.1.0]hexane-4,6-dicarboxylate: identification of potent, selective, and orally bioavailable agonists for mGlu2/3 receptors.

(-)-4-Amino-2-thiabicyclo-[3.1.0]hexane-4,6-dicarboxylate (LY389795, (-)-3) is a highly potent and selective agonist of metabotropic glutamate receptors 2 (mGlu2) and 3 (mGlu3). As part of our ongoing research program, we have prepared S-oxidized variants of (-)-3, compounds (-)-10, (+)-11 (LY404040), and (-)-12 (LY404039). Each of these chiral heterobicyclic amino acids displaced specific binding of the mGlu2/3 receptor antagonist 3H-2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid (3H-LY341495) from membranes expressing recombinant human mGlu2 or mGlu3 and acted as potent agonists in cells expressing these receptor subtypes. Docking of the most potent of these derivatives, (+)-11, to mGlu2 revealed the possibility of an additional H-bond interaction between the sulfoxide oxygen of (+)-11 with tyrosine residue Y236. Pharmacokinetic analysis of mGlu active enantiomers (+)-11 and (-)-12 in rats showed each to be well absorbed following oral administration. Consistent with their mGlu2/3 agonist potency and pharmacokinetic properties, both (+)-11 and (-)-12 blocked phencyclidine-evoked ambulations in a dose-dependent manner, indicating their potential as nonclassical antipsychotic agents.

Administration, Oral↗
PubMed2001

Identification and characterization by high-performance liquid chromatography/electrospray ionization mass spectrometry of a new variant hemoglobin, Mataro [beta134(H12) Val > Ala.

This work illustrates the practical use of combined microbore reversed-phase high-performance liquid chromatography (RP-HPLC) with electrospray ionization mass spectrometry (ESI-MS) in protein identification. The approach consisted of the detection of the abnormal beta-globin chain by ESI-MS analysis of mixtures of intact globins, which simultaneously provided their molecular masses. Separation of the polypeptide globin chains was carried out using microbore C4 RP-HPLC on-line with ESI-MS. Direct peptide-mapping ESI-MS without previous chromatographic separation was performed in order to identify tryptic peptides from whole blood. For the sequence confirmation of the abnormal peptide containing the mutation point, C18 RP-HPLC tryptic separation of the globin mixture on-line with collision-induced dissociation (CID) fragmentation was done. The y series ions allowed the identification of the hemoglobin (Hb) variant as [beta134(H12) Val > Ala]. This new Hb was named Hb Mataró, after the city where it was detected.

Alanine↗
PubMed2001

t(8;21;8)(p23;q22;q22): a new variant form of t(8;21) translocation in acute myeloblastic leukemia with maturation.

The complex variants of t(8;21) involving chromosomes 8 and 21 as well as a variable chromosome account for 1.1-5% of acute myeloid leukemia (AML) patients. This paper reports a case of AML-M2 with t(8;21;8) translocation for the first time. The patient was a female, aged 47 years. Her myelogram was compatible with AML-M2. Chromosome study using R-banding technique revealed a karyotype 46, XX, t(8;8)(p23;q22). Dual-color FISH assay with two probes P1 164(green signal) and YAC 225B8 (red signal) both of which closely located on the 8q showed that one yellow signal consisting of a green signal and a red signal and one red signal appeared on the long and the short arm of the same der(8) chromosome, respectively, further confirming this translocation occurred between both homologous chromosomes 8. RT-PCR analysis detected the AML1/ETO fusion transcript in our patient, thus indicating that this chromosomal aberration was, in fact, a complex three-way rearrangement t(8;21;8)(p23;q22;q22). In conclusion, combining conventional karyotype, FISH or RT-PCR analyses is a rational strategy for identification of the complex variants of t(8;21) translocation.

Bone Marrow Cells↗
PubMed2003

Frequent E-cadherin gene inactivation by loss of heterozygosity in pleomorphic lobular carcinoma of the breast.

Pleomorphic lobular carcinoma of the breast is a variant of infiltrating lobular carcinoma that has poor prognosis. The pleomorphic appearance of this variant hinders its correct identification and differentiation from ductal carcinoma. The analysis of E-cadherin glycoprotein expression is a powerful tool for distinguishing lobular from ductal carcinomas, because complete loss of E-cadherin expression occurs in most infiltrating lobular tumors and lobular carcinomas in situ, but not in ductal tumors. In the present study, we have evaluated E-cadherin expression by immunohistochemistry in a series of 29 pleomorphic lobular breast carcinomas, including 7 cases with an in situ component. Complete loss of E-cadherin expression was observed in all the cases (29/29, 100%), in invasive and in situ components. To understand better the mechanisms underlying E-cadherin inactivation in this tumor type, the frequency of loss of heterozygosity at the E-cadherin gene locus (16q22.1) was analyzed. All informative tumors (27/27, 100%) showed loss of heterozygosity, thus implying a strong association between loss of E-cadherin expression and loss of heterozygosity at 16q22.1. Moreover, loss of heterozygosity was detected in all in situ components analyzed. These results imply that in terms of E-cadherin inactivation, pleomorphic lobular tumors are identical to classic infiltrating lobular carcinomas and distinct from ductal tumors, and therefore they should be considered a variant of lobular carcinoma of the breast, despite their aggressive behavior.

Aged↗
PubMed2003

Echocardiographic monitoring of left ventricular regional motion during hyperventilation and intravenous infusion of trometamol (tris) for detection of variant angina.

BACKGROUND: In spite of constant progress and development of new diagnostic tests, the detection of variant angina, which occurs in approximately 30% of patients with ischaemic heart disease (IHD), remains challenging. AIM: To assess the sensitivity and specificity of echocardiographically monitored transient abnormalities of regional wall motion of left ventricle (LV) during hyperventilation after intravenous infusion of trometamol (TRIS-buffer) for the detection of coronary artery spasm. METHODS: The study group consisted of 72 patients (14 women and 58 men, aged from 32 to 69 years) with IHD. A control group was composed of 20 healthy men. Patients with IHD were divided into two groups. Group I consisted of 46 patients with Prinzmetal's angina whereas group II was composed of 26 patients with exertional angina and a history of myocardial infarction. Two-dimensional echocardiographic monitoring of LV contractility was carried out during hyperventilation after an intravenous infusion of 100 ml of trometamol. RESULTS: Transient abnormalities of regional LV wall motion during infusion of trometamol and hyperventilation occurred in 91% of patients from group I and in 8% of patients from group II (p<0.00001). Electrocardiographic ST-segment changes during hyperventilation-trometamol test were observed in 63% of patients from group I and in 23% of patients from group II (p<0.0001). No transient regional dyssynergy of LV nor ST-segment changes during hyperventilation-trometamol test in the control group were observed. The sensitivity and specificity of regional LV wall motion during hyperventilation-trometamol test in the identification of patients with variant angina were 91% and 92%, respectively, and the sensitivity and specificity of ST-segment changes - 63% and 76%, respectively. CONCLUSIONS: Echocardiographic monitoring of regional LV wall motion during hyperventilation after intravenous infusion of trometamol is a sensitive and specific test for detection of variant angina.

Adult↗
PubMed1999

Splice variants of the nuclear dot-associated Sp100 protein contain homologies to HMG-1 and a human nuclear phosphoprotein-box motif.

Sp100 and PML are interferon-inducible proteins associated with a new class of nuclear domains (known as nuclear dots or PML bodies) which play a role in tumorigenesis, virus infections, and autoimmunity. While PML is extensively alternatively spliced, only two splice variants are known for Sp100. Here we describe the identification and characterization of several Sp100 splice variant proteins and support their existence by elucidation of the 3'-end of the Sp100 gene. Some of the splice variants contain a domain of significant sequence similarity with two previously described highly related interferon-inducible nuclear phosphoproteins as well as to suppressin and DEAF-1, which altogether define a novel protein motif, termed HNPP-box. One class of splice variants contains an almost complete and highly conserved copy of the DNA-binding high mobility group 1 protein sequence and thus represent novel HMG-box proteins. When expressed transiently, both major classes of Sp100 splice variant proteins localize in part to nuclear dots/PML bodies and in addition to different nuclear domains. Furthermore, PML was occasionally redistributed. These data indicate that alternatively spliced Sp100 proteins are expressed, differ in part in localization from Sp100, and might bind to chromatin via the HMG domain.

Alternative Splicing↗
PubMed2006

Vitamin D-binding protein (DBP) gene polymorphism is associated with Graves' disease and the vitamin D status in a Polish population study.

OBJECTIVE: Vitamin D-binding protein (DBP) genetic variants have an influence on vitamin D status and, therefore, they may contribute to the development of autoimmune diseases. In this case-control study, we investigated the association of DBP gene polymorphisms with susceptibility to Graves' disease (GD) in a Polish population. Furthermore, we analyzed the distribution of DBP genotypes in GD patients divided according to the clinical (gender, age of onset, ophthalmopathy, family history, smoking habits) and genetic parameters (CTLA4 49G and HLA-DRB1*03 alleles), as well as the vitamin D serum levels. METHODS: 332 polish patients with GD and 185 healthy controls were genotyped for the DBP gene single nucleotide polymorphisms (SNPs) at codon 420 ACG --> AAG (Thr --> Lys) and at codon 416 GAT --> GAG (Asp --> Glu) by the PCR-RFLP method. The variable (TAAA)N repeat polymorphism in the intron 8 was analyzed in 332 patients and 164 controls by the PCR amplification followed by the PAGE. In addition, 25(OH)D3 serum levels were measured in 110 patients. RESULTS: In patients with GD, the frequency of the Lys allele (34.2% vs. 25.7%, p = 0.005; OR = 1.50; 95% CI: 1.13-1.99) at codon 420 was significantly higher compared to controls. The distribution of codon 420 genotypes also differed significantly (p = 0.01), with the frequency of the Lys/Lys homozygotes (9.3% vs. 5.9%; OR = 1.63; 95% CI: 0.80-3.32) being higher in GD. The distribution of codon 416 alleles and genotypes did not differ in both studied groups (p = 0.59 and p = 0.81, respectively). Analysis of the intron 8 (TAAA)N repeat polymorphism led to the identification of a novel variant in the Polish population, described as 7 repeats, but no association between the intron 8 alleles and GD was observed. The 420 Lys allele was associated with lower 25(OH)D3 serum levels (p = 0.01). No correlation between the DBP genotypes and other susceptibility alleles or the GD clinical phenotype was observed. CONCLUSIONS: (i) The DBP gene Lys allele at codon 420 confers susceptibility to GD in the Polish population, (ii) the codon 416 alleles and intron 8 (TAAA)N variants are not associated with susceptibility to and clinical phenotype of GD, and (iii) the codon 420 Lys allele correlates with lower 25(OH)D3 serum concentration.

Adolescent↗
PubMed2004

Comparison of methods for the visual identification of prevalent vertebral fracture in osteoporosis.

The identification of vertebral fracture in osteoporosis is based mainly on the identification of abnormal variation in vertebral shape, but this can be misleading in the presence of a non-fracture deformity or normal variant of vertebral shape. Qualitative identification of vertebral fracture (Qual) is influenced by the subjectivity of the approach, and although more objective, the semiquantitative method (SQ) can be difficult to apply. In addition, there has been little independent evaluation of SQ in relation to other approaches. We aimed to evaluate a new algorithm-based approach for the qualitative identification of vertebral fracture (ABQ) and to compare it with SQ and Qual. Two radiologists reported spinal radiographs for 372 postmenopausal women using Qual (reader 1), and SQ and ABQ (reader 2). Non-fracture deformities and normal variants were also reported using Qual and ABQ. The prevalence of vertebral fracture by subjects was higher for SQ (24%) than for Qual (11%) and ABQ (7%). Agreement was poor between SQ and the other methods, and moderate between Qual and ABQ. Twenty-two women with vertebral fracture were agreed by all three methods, similar to the total identified by ABQ (25 women). Seventeen women diagnosed with fracture by Qual, had non-fracture deformity or normal variant (but no fracture) according to ABQ. Of the women with SQ fractures, 53% and 70% were identified negative for fracture but positive for non-fracture deformity or normal variant by ABQ and Qual. The main sources of discrepancy between SQ and the other methods were Scheuermann's disease, normal variation, and degenerative change accompanied by short anterior vertebral height. For all methods, bone mineral density (BMD) and BMD Z-scores were lower in women with vertebral fractures than in those with no fractures. Bone mineral density and BMD Z-scores were also lower at the lumbar spine and total body in women with vertebral fractures according to Qual and ABQ than they were for SQ, and were lower in women with SQ fractures agreed by Qual and ABQ, compared with those diagnosed negative for fracture by Qual and ABQ (p<0.01). We conclude that poor agreement between methods arises mainly from difficulties in differentiating true fracture from non-fracture deformity. Our new approach attempts to address this problem but requires further testing in a larger study population.

Aged↗
PubMed2000

Optimization of a simple and rapid single-strand conformation analysis for detection of mutations in the PROS1 gene: identification of seven novel mutations and three novel, apparently neutral, variants.

Anticoagulant protein S (PS) deficiency is a known risk factor for thrombophilia. The structure and high allelic heterogeneity of the PS gene (PROS1), together with the presence of a 97% homologous pseudogene, complicates PROS1 analysis. We have optimized a simple, fast, and non-isotopic Single-Strand Conformation Analysis (SSCA or SSCP) method for PROS1 mutation detection. This is accomplished through the analysis of the single-stranded and heteroduplex DNA fragments corresponding to 15 PCR segments that include part of the 5'-upstream region and the 15 PROS1 exons with their intron boundaries. To standardize the method, 13 known PROS1 mutations or allele variants in 10 different fragments were analyzed under different electrophoretic conditions. The results indicated that, using a combination of two different electrophoretic settings, all the allele variants could be detected as a single-strand band shift and/or by the presence of a heteroduplex. This method was used to analyze the PROS1 gene in 31 propositi with different types of PS deficiency and thrombosis. Ten different cosegregating mutations, seven of which are novel (143C->G, L-27H, G96X, M599T, P626L, 1418delA, and 1877delT), were identified in the five families suffering from type I or quantitative PS deficiency and in four of the nine families with coexistence of type I and type III phenotypes. No clearly co-segregating PROS1 mutations were identified in any of the 17 type III propositi analyzed, although eight of them were heterozygotes for the uncommon P460 allele of the S/P460 variant. Furthermore, five apparently neutral allelic variants, three of which are novel (-296C->T, 182G->C and T57S), were identified in a normal control, two type I/III and two type III PS-deficient pedigrees.

Alleles↗
PubMed1997

Characterization of MSH2 and MLH1 mutations in Italian families with hereditary nonpolyposis colorectal cancer.

Mismatch repair genes MSH2 and MLH1 are considered to be the two major genes that are responsible for hereditary nonpolyposis colorectal cancer (HNPCC). Germline heterozygous inactivating mutations of MSH2 and MLH1 have been identified previously in a substantial fraction of individuals who are predisposed genetically to colorectal carcinoma (CRC) and other tumors of the HNPCC spectrum. With the aim of determining the relevance of these two genes in the Italian population, we submitted to mutational analysis a set of 17 HNPCC families, all of which fulfilled the "Amsterdam criteria." A combination of different techniques, including reverse transcription-polymerase chain reaction (RT-PCR) of long fragments and single-strand conformation polymorphism (SSCP) on cDNA and genomic DNA, allowed the identification of ten molecular variants, seven of which are predicted to inactivate mismatch repair function. The mutated predisposing gene was MSH2 in two families and MLH1 in five other families. All of the mutations were characterized by DNA sequencing and appeared to involve different molecular mechanisms, such as short in-frame and out-of-frame deletions, splicing errors, and nonsense mutations. This study also demonstrates that, in the Italian population, a considerable fraction of HNPCC families (at least 41%) is linked to MSH2 and MLH1 mutations.

Adaptor Proteins, Signal Transducing↗
PubMed1998

From "magic bullet" to "specially engineered shotgun loads": the new genetics and the need for individualized pharmacotherapy.

Steady progress in the identification of human pharmacogenetic variants and new discoveries of disease susceptibility genes makes the old notion of one disease/one drug untenable. Advances in the ability to rapidly identify these variants, when coupled with appropriate drug delivery systems, should revolutionize pharmacotherapy.

Genetic Predisposition to Disease↗
PubMedDate not supplied

Characterization of periplasmic Escherichia coli protein expression at high cell densities.

We have used two-dimensional electrophoresis (2-DE) to analyze changes in protein expression profiles during a microbial cultivation process on an industrial scale. An Escherichia coli strain W31 10 containing the gene for recombinant human growth hormone production was used. Samples were taken at time intervals ranging from fast to slow growth rate (late growth phase at high cell density/starvation) and 2-DE analysis combined with image analysis using the PDQuest software showed significant alterations in expression levels of a number of proteins. Twenty-four protein spots were identified using a combination of matching with SWISS-2DPAGE E. coli map, N-terminal sequence analysis and mass spectrometry matrix-assisted laser desorption/ionization (MALDI). Two of the most abundant proteins expressed at late growth phase (pI 5.4/28 kDa and pI 5.5/28 kDa) were subjected to N-terminal sequence analysis after electrotransfer of the proteins from a preparative 2-DE gel to polyvinylidene difluoride (PVDF) membrane. Sequence tags of five amino acids in combination with approximate pI and Mr identified both proteins as deoxyribose phosphate aldolase (gene name deoC). In addition, both spots were subjected to tryptic in-gel digestion and analyzed using MALDI. Peptide mass fingerprints from both spots showed similar MALDI spectra and 10 of 10 tryptic fragments confirmed the identity as deoC. The identification of the acidic variant of deoC on 2-DE gels and the observation of this variant as induced during late growth phase is novel.

Acrylic Resins↗