New crown motif of an HIV-1 V3 loop sequence from a Ugandan AIDS patient.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
J Eberle
Publications and source records attributed to J Eberle.
Resistance of HIV type 1 to proteinase inhibitor Ro 31-8959.
During replication of human immunodeficiency virus type 1 (HIV-1), proteolytic cleavage of Gag and Gag-Pol precursor proteins into different functional protein subunits is catalyzed by the viral proteinase, and this enzyme is the target of the antiviral proteinase inhibitor, Ro 31-8959. We investigated in vitro which HIV mutants with reduced sensitivity to Ro 31-8959 emerged during proteinase inhibition treatment; from three different HIV-1 strains, comparable progeny virus resistant to proteinase inhibitor were found, whereas the same experimental protocol detected no resistant HIV-2 mutants. Molecular analysis of the mutations underlying resistance revealed a multistep mechanism in which an amino acid exchange was common to all resistant isolates, and in all experiments preceded further exchanges at position 90 (leucine to methionine) and/or at position 54 (isoleucine to valine). For wild-type strains the 90% inhibitory concentrations of Ro 31-8959 were close to 20 nM, whereas HIV-1 mutants with all 3 amino acid exchanges had more than 50-fold increased 90% inhibitory concentrations (above 1000 nM). The primary event (Gly-48 to valine) occurs at the hinge of the flaps of the proteinase, thus hampering entry of the inhibitor to the active center and suggesting steric hindrance. Detailed knowledge of this stereotypic process could open inhibitor design, thus preventing conceivable escape of resistant virus on proteinase inhibitor action.
Amino Acid Sequence↗
Reduced capacity of antibodies from patients infected with human immunodeficiency virus type 1 (HIV-1) group O to neutralize primary isolates of HIV-1 group M viruses.
Neutralizing antibody patterns in sera of persons infected with human immunodeficiency virus type 1 (HIV-1) groups M and O to their homologous and heterologous primary isolates were determined in a peripheral blood mononuclear cell-based neutralization assay and correlated with their ability to bind to V3 loop synthetic peptides. Most HIV-1 group M sera (9/16) neutralized HIV-1 group O viruses, whereas fewer group O sera (3/13) only weakly neutralized HIV-1 group M viruses. Group M sera neutralizing HIV-1 group O viruses neutralized other HIV-1 group M viruses with titers of 1:10-1:1280. V3 loop binding capacity of sera did not reflect their neutralizing capacity of the homologous isolate. Despite the reduced neutralizing capacity of group O-infected patients' sera to group M viruses, some group M-infected patients' sera neutralized both HIV-1 group M and O isolates, suggesting that they share some conserved neutralizing epitopes.
Acquired Immunodeficiency Syndrome↗
Renal cell carcinoma extending into the vena cava: surgical approach, technique and results.
OBJECTIVE: To describe the technique and results of a thoraco-abdominal approach to removing the caval thombi in patients with renal cell carcinoma extending into the vena cava. PATIENTS AND METHODS: Between 1970 and 1990 35 patients presenting with renal cell carcinoma extending into the vena cava were treated at the Department of Urology, Innsbruck. Twenty-three of these patients underwent radical tumour nephrectomy including cavotomy and thrombectomy or caval resection. A transabdominal approach had been used in this department for radical tumour nephrectomy including cavotomy and thrombectomy or caval resection until 1987. Since 1988, a thoraco-abdominal approach has been employed. In group I patients the approach was via the seventh intercostal space, whereas in group II and III patients the thoraco-abdominal incision was made through the fifth intercostal space. In the present study the anatomy of the thoraco-abdominal approach is described. RESULTS: Tumour staging and grading yielded stage T3b in 15 patients (grade I, 1; grade II, 6; grade III, 8); another eight patients with stage T3b were found to have metastatic disease (N1, 6; N2, 2; M1, 3). On the basis of the extension of the caval thrombus the patients were classified as follows: group I, 16; group II, 3; group III, 4. In T3b N0 M0 patients the 5-year-survival rate was 62.5%, while in patients with positive lymph nodes the mean survival rate was 15.5 months. CONCLUSION: Our results suggest that the thoracoa-abdominal approach is the method of choice for the safe removal of renal cell carcinomas associated with caval thombi. If resection of the caval tumour is complete, prognosis is dependent on known factors, such as tumour invasion, nodal involvement and distant metastases rather than the extension of the tumour thrombus. An aggressive approach is not warranted in patients with nodal involvement and/or distant metastases, as it does not improve survival.
Carcinoma, Renal Cell↗
Incomplete expression of the tyrosinase gene family (tyrosinase, TRP-1, and TRP-2) in human malignant melanoma cells in vitro.
Sequence analysis of two clones found repressed in melanoma cell lines in earlier studies showed 9F2 to be identical with the TRP-1 gene and 6F5 with TRP-2 containing a long untranslated 3' end. For further investigation of the expression of the tyrosinase gene family in normal and malignant melanocytic cells, a series of melanoma cell lines and of cultured melanocytes were analyzed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). The Northern blots were probed with cDNA fragments specific for TRP-1, TRP-2, and tyrosinase, for nested tyrosinase-PCR the outer primers specified a 284 bp and the nested primers a 207 bp fragment. Investigations on 14 established melanoma cell lines grown in different media compared with seven normal human melanocyte (NHM) cultures revealed that all three pigment genes were expressed in NHM, whereas pigment gene expression was found repressed in nearly all melanoma cell lines and was completely absent in 4 of 14 specimen. In particular, tyrosinase and TRP-2 genes were found always to be expressed together, and TRP-1 mRNA alone was absent in four melanoma cell lines. Negativity of cultured melanoma cells for tyrosinase mRNA was confirmed by nested RT-PCR, and gene deletion was ruled out by genomic Southern blots. The gene expression seemed independent from the type of medium used for cultivation. These findings indicate repressed or lacking expression of pigment genes in melanoma cell lines, most likely due to regulatory mechanisms, and that differences may exist between tyrosinase and TRP-2 on one hand and TRP-1 on the other. Overall, it seemed that RT-PCR for tyrosinase has limited value for identifying melanoma cells in the peripheral blood of melanoma patients; TRP-1, TRP-2, and other, additional markers may be required.
Base Sequence↗
Androgen receptor alterations in patients with disturbances in male sexual development and in prostatic carcinoma.
The androgen receptor, a ligand-activated nuclear transcription factor belonging to the large superfamily of nuclear receptors, mediates the intracellular action of androgens. It plays a central role in male sexual development and in prostatic carcinoma as a target of endocrine therapy. We have looked for androgen receptor mutations as a cause of male sexual ambiguity and as a possible reason for failure of androgen ablation therapy on prostatic carcinoma. In 5 patients of 2 families with perineoscrotal hypospadia and undescended testes, we have identified a mutation ala596-->thr in the DNA-binding domain of the androgen receptor. This mutation interferes with DNA binding of the receptor. Reactivation of this mutant receptor by binding of an antibody or by interaction with other proteins and by exchange of the amino acid thr602-->ala indicates that the dimerization step is affected. A point mutation ser703-->gly was detected in a newborn male child with perineoscrotal hypospadias. This mutation decreased receptor-hormone affinity. As a consequence its transactivation activity was dependent on the androgen concentration. Although the molecular mechanisms of these two mutations are completely different, both resulted in partial androgen insensitivity and interfered with virilization in the affected patients. A different kind of mutation was present in a tumor specimen derived from an advanced therapy-resistant prostatic carcinoma. This point mutation resulted in exchange of valine-->methionine at amino acid position 715 in the receptor protein. In contrast to the former two mutations this receptor showed a gain in function.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
[Polymerase chain reaction (PCR)].
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Base Sequence↗
Androgen signal transduction and prostatic carcinoma.
In the prostate, androgen action affects the growth of the gland, its morphology, and regulation of protein expression. Endocrine therapy of non-organ-confined tumors is based on the androgen dependence of the vast majority of prostatic carcinomas. Although initial response rates are high, this therapy is only temporarily effective. Critical molecular changes ultimately resulting in androgen independence of tumor cells are unknown at this time. The androgen signal-transduction cascade and its central element, the androgen receptor (AR), are possible targets for such changes. Immunohistological analysis using anti-AR antibodies has revealed the presence of AR in a vast majority of therapy-responsive as well as therapy-unresponsive prostatic carcinomas, indicating that loss of AR expression is not the reason for androgen independence. On the other hand, molecular-biology studies have revealed qualitative and quantitative impairment of AR expression in prostatic tumor cell lines that represent very late stages of prostatic carcinoma development. Mutant ARs were detected in the prostatic tumor cell line LNCaP and in two specimens from primary prostatic tumors. The LNCaP mutant AR as well as mutant AR715met, one of the mutant receptors detected in tumor tissue, show a gain of function as compared with the wild-type receptor. In addition to androgens, the natural activators of the AR, the LNCaP receptor is activated also by progestagenic and estrogenic steroids and by the nonsteroidal antiandrogen flutamide. AR715met is activated by adrenal androgens and progesterone in addition to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)
Androgens↗
P53 mutation and c-fos overexpression are associated with detection of the antigen VLA-2 in human melanoma cell lines.
In this study, we examined the expression of c-fos, c-myc, mutant c-Ha-ras and mutant p53 proteins in three normal human melanocyte cell lines and the following 12 melanoma cell lines: M5, Mewo, A375, Bro, Mel 2a, O-Mel II, IgR 39, SkMel-13, -19, -28 Mel-57 and NKI-4, using an immunohistochemical assay (APAAP). An effort was made to correlate oncogene expression with growth parameters, differentiation antigens (HMB-45, vla-2, k.1.2.58, HLA-DR, HLA-I), and pigmentation. All melanocyte cell lines were negative for the oncogenes examined, whereas six of the melanoma cell lines were found also positive (three for c-fos, two for c-myc, one for c-Ha-ras, and four for p53). Three melanoma cell lines expressed one oncogene and three the combination c-fos/p53. These three melanoma cell lines were positive for the "late" tumor progression marker A. 1.43 (vla-2 adhesion molecule) and negative for the differentiation marker k. 1. 2. 58. Positivity for A. 1. 43 combined with negative staining for k. 1. 2. 58 was found in six out of the 12 cell lines. The observed oncogene expression correlated neither with growth parameters nor melanin content. The present findings revealed a coexpression of mutant p53 and c-fos proteins being associated with a highly malignant phenotype in melanoma cell lines. Further studies are necessary to clarify the significance of the above findings.
Biomarkers, Tumor↗
A new subtype of human immunodeficiency virus type 1 (MVP-5180) from Cameroon.
A new subtype (MVP-5180) of human immunodeficiency virus type 1 (HIV-1) was isolated from a Cameroonian AIDS patient. MVP-5180 was grown in several human T-cell lines and the monocytic U937 line. MVP-5180 DNA could not be amplified by nested primer PCR with conventional env primers and could be only very faintly amplified with gag and pol primers. Most German, Ivoirian, and Malawian anti-HIV-1 sera reacted faintly or moderately with Env proteins in an MVP-5180 immunoblot, whereas some Cameroonian sera reacted strongly. Of HIV-1-infected Cameroonians, 8% were identified by serological methods as infected with MVP-5180; 7% were positive when MVP-5180-specific PCR env primers were used. DNA sequence analysis of MVP-5180 showed that its genetic organization was that of HIV-1, with 65% similarity to HIV-1 and 56% similarity to HIV-2 consensus sequences. The env gene of MVP-5180 had similarities to HIV-1 and HIV-2 of 53 and of 49%, respectively. V3 loop analysis identified a crown of Gly-Pro-Met-Arg by using cloned DNA and Gly-Pro-Leu-Arg by using PCR-amplified DNA, neither of which configuration has been described for other HIV strains. In an analysis of relationships, MVP-5180 occupied a position distant to all other HIV-1 strains, including the chimpanzee simian immunodeficiency virus type 1 SIVcpz and the Uganda virus U455, and closer to the HIV-1/HIV-2 divergence node. MVP-5180, together with another Cameroonian isolate, ANT-70, constitutes a group subtype O of the most divergent HIV-1 isolates yet identified. Characterization of MVP-5180 is important for understanding the natural history of the primate immunodeficiency viruses and for the development of vaccines and diagnostics.
Acquired Immunodeficiency Syndrome↗
Neurospora crassa blue light-inducible gene bli-3.
Blue light induces various physiological, morphological and biochemical reactions in the filamentous fungus Neurospora crassa. This light response is accompanied by a global change in gene expression, and several light-inducible transcripts (bli-genes) have been cloned. We isolated the genomic clone of the gene bli-3, whose mRNA we have previously shown to be induced 2 minutes after the beginning of illumination. Its DNA sequence predicted a transcriptional unit of 1050 bp encoding a novel, hydrophilic protein of 209 aminoacids. Comparison to other N. crassa genes revealed a group of inducible genes which share this promoter structure: a well conserved TATA-box, similar transcription start box (TCATCANC) and repeats of pyrimidines (CT) between these boxes. Based upon this group, we propose a consensus sequence for one possible type of inducible promoter in N. crassa.
Amino Acid Sequence↗
[Transfusion-associated HIV infection].
OBJECTIVE: Estimating the risk of HIV transmission by blood or inactivated plasma transfusion. A discussion of the methods and techniques for the diagnosis of HIV infection in blood donors. DATA SOURCES: Reports in German and English on this topic as well as own experiences of the authors. SELECTION CRITERIA: No specific selection criteria. RESULTS: Transfusion associated HIV infection may be prevented by donor selection, the very efficient anti-HIV testing, by p24-antigen testing that may detect some anti-HIV negative donations especially during the early time of seroconversion and by more recent introduced techniques like polymerase chain reaction (PCR) and signal amplification assay (SAA). PCR and SAA are under development and until now not sensitive and specific enough to contribute significantly to an earlier detection of HIV infected blood. Procedures for the inactivation of HIV in plasma or whole blood have been described. Until now use of psoralens, methylene blue or direct UV irradiation may reduce viral load but have not definitely been proven by clinical trials to be 100% efficient. CONCLUSIONS: To minimize transfusion associated HIV infection in future years reduction of total amount of transfused units, restriction to regional donor recruiting and further refinement of tests will be necessary. A 100% safety of blood transfusion for infectious agent cannot be achieved, especially considering new agents and further spread of until now geographically restricted viruses.
AIDS Serodiagnosis↗
DNA sequence of the androgen receptor in prostatic tumor cell lines and tissue specimens assessed by means of the polymerase chain reaction.
Essentially all prostatic carcinomas relapse to an androgen-independent stage during androgen ablation therapy. The underlying genetic changes are still unclear. Such changes are suspected to affect the androgen-signalling pathway as well as growth promoting and inhibiting factors. This study was undertaken to test for structural changes of the androgen receptor in prostatic tumor cell lines and primary tumors. Complementary DNA (cDNA) fragments of the androgen receptor (AR) were isolated from the cell lines LNCaP, PC-3, and DU 145, ten tissue specimens obtained by radical prostatectomy, and five fine-needle biopsies by means of the polymerase chain reaction (PCR) technique. Fragments encoding the hormone- and DNA-binding domains were analyzed by DNA sequencing. The PCR technique is highly sensitive and especially recommended for the analysis of small tissue samples, such as those obtained by fine-needle aspiration. No alterations were detected in the tissue specimens and the five fine-needle aspirates. In the three tumor cell lines that represent late stages of prostatic tumor, different findings were obtained. The androgen-independent DU 145 cells did not express androgen receptors, whereas the PC-3 cells, which are also androgen-independent, expressed very low levels of normal AR. In contrast to this, the androgen-dependent LNCaP cells expressed high levels of structurally abnormal androgen receptors. These results suggest that androgen receptor mutations are probably uncommon molecular events in the early stages of prostatic cancer. Qualitative and quantitative changes, however, seem to occur in advanced prostatic cancer.
Base Sequence↗
Characterization of two point mutations in the androgen receptor gene of patients with perineoscrotal hypospadia.
Perineoscrotal hypospadia is a major sign of sexual ambiguity due to inadequate androgen action in genetic and gonadal males. In patients showing these symptoms we have detected two androgen receptor gene mutations. In consequence we characterized the properties of the mutant receptors with respect to hormone-binding, transactivation and DNA-binding. An amino acid substitution alanine-596-->threonine in the D-box of the androgen receptor was detected in 3 and 2 brothers, respectively. This mutant receptor, AR-thr596, bound ligand in a normal fashion. It showed a promoter-dependent defect of transactivation and was unable to induce transcription of a promoter containing one androgen responsive element but showed almost wild-type transactivation of a promoter containing two closely spaced androgen-responsive elements. The complex promoter of the human prostate-specific antigen gene was induced with intermediate efficiency. In electrophoretic mobility shift assays AR-thr596 was unable to form a complex with oligonucleotides containing 1 or 2 androgen responsive elements, however its DNA-binding activity was restored by an anti-androgen receptor antibody in the presence of ligand. A point mutation which caused substitution of serine-703 in the hormone-binding domain with glycine was detected in a new-born male with ambiguous genitalia. This mutant receptor, AR-gly703, showed a reduced ligand affinity. The total amount of specific androgen binding sites in genital fibroblasts of the patient was reduced. Transactivation activity of AR-gly703 was dependent on hormone concentration. It was inactive at low levels of androgens but was fully activated in the presence of high androgen concentrations. The nature of the promoter had no effect on transactivation properties of this mutant androgen receptor. Its DNA-binding activity in gel shift experiments was normal.
Base Sequence↗
Posterior hypospadias: long-term followup after reconstructive surgery in the male direction.
Posterior hypospadias with a scrotal or perineal meatus results from a defect in step 3 of male sexual differentiation. The different etiological factors underlying this condition result in a broad spectrum of presentations ranging from the isolated form to complex ambiguity of the external genitalia, such as male pseudohermaphroditism. Between 1952 and 1988 a total of 92 patients with posterior hypospadias underwent a 2-stage reconstruction at our department. A retrospective study was performed with the aim of evaluating the long-term results in these patients. Our special interest focused on the functional and cosmetic results, exocrine and endocrine functions, as well as the sexual lives of the patients. While satisfactory results were obtained in two-thirds of the 42 male patients available for long-term followup, there were 13 patients who at followup still presented with complex sexual ambiguity. In 6 of these patients androgen receptor defects were detected by means of biochemical as well as molecular-biological investigations. Our data emphasize the importance of androgen metabolism for male sexual development and underline the necessity of careful evaluation in these children.
Adolescent↗
[Diagnosis and staging of blunt kidney trauma. A comparison of urinalysis, i.v. urography, sonography and computed tomography].
In a retrospective study of 1230 patients with blunt abdominal or flank trauma, the results of microscopic urinalysis, urography, sonography, and computed tomography in the diagnosis and staging of renal injuries were compared. Haematuria was present in 98% of renal lacerations and in all pedicle injuries, whereas among 1038 patients without haematuria only one small laceration was found. The degree of haematuria showed no correlation with the severity of renal injury. 92 renal injuries were diagnosed radiologically, including 58 lacerations, 31 contusions, and three pedicle injuries. Computed tomography was performed in 39 lacerations and six contusions, and allowed correct diagnosis and staging in all cases. Urography established the correct diagnosis in 72%, and sonography in 85%. Sensitivity in the diagnosis of clinically relevant injuries (lacerations and pedicle injuries) was 98% (specificity: 99%), when both modalities were combined. Based on the results of our study, we suggest the use of sonography as a screening modality in blunt abdominal or flank trauma, and additional urography in case of haematuria. Computed tomography is the method of choice for exact staging of suspected renal injury.
Adolescent↗
Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone.
Structural changes of the androgen receptor (AR) may contribute to the development of resistance to endocrine therapy in prostatic carcinoma. We have isolated AR cDNA fragments from seven tumor specimens derived from patients with advanced metastatic prostatic tumors. In one specimen obtained from a patient who failed to respond to endocrine and cytotoxic therapy we have detected a point mutation in the hormone-binding domain of the receptor. This AR mutation is a guanine-to-adenine transition at nucleotide 2671 that leads to substitution of methionine for the wild type valine at position 715. It is a somatic mutation because it was not present in the AR genomic DNA fragments isolated from prostatic and testicular tissues of the same patient. The mutant AR was recreated in an expression vector and transiently expressed in COS-7 and CV-1 cells. Hormone-binding assays revealed that the mutant receptor does not differ from the wild type receptor in its ability to bind androgen. The dissociation constant for the synthetic androgen mibolerone was 3 nM for both receptors. There was also no significant difference in binding of other steroids and nonsteroidal antiandrogens as revealed by competition binding assays. However, transfection experiments to determine the trans-activation potential of the mutant receptor produced differences in the action of this receptor compared to the wild type receptor. Dihydrotestosterone and the synthetic androgens methyltrienolone (R1881) and mibolerone were equally proficient in conferring trans-activation activity to both the mutant and wild type receptors. Adrenal androgens such as dehydroepiandrosterone and androstenedione, as well as progesterone mediated a higher trans-activation through the mutant than through the wild type receptor. These data demonstrate that the exchange of a single valine into methionine at position 715 in the AR promoters trans-activation not only by testicular but also by adrenal androgens and progesterone. This pattern of ligand-dependent trans-activation may have significance in the process controlling the progression of prostatic carcinoma.
Adrenal Glands↗
[Proliferative activity and p53 expression in transitional cell carcinoma of the urinary bladder].
Mutations of the p53 gene are important mechanisms in malignant transformation and are associated with dysregulation of normal cell growth. In the present study the expression of mutated p53-protein and proliferating cell nuclear antigen (PCNA) was investigated in a series of 31 human transitional cell carcinomas (TCC) by immunohistochemistry (IHC). The number of PCNA-positive cells and the pattern of expression was distinct in normal urothelium being confined to the basal cell layer. In dysplastic urothelium and in Carcinoma in situ (CIS) PCNA-immunoreactive nuclei were irregularly distributed throughout all layers. In tumor cell complexes the pattern of PCNA-immunoreactivity was different in papillary and primary infiltrating TCCs. Densitometric quantification of the intensity of the PCNA-reactivity using image analysis revealed an increase from normal to dysplastic urothelium and from dysplastic urothelium to invasive tumors. 21/31 (68%) of the tumors and tumor-associated CIS showed overexpression of p53 varying in percentage, pattern and reaction intensity. The percentage of PCNA-positive cells was higher in tumors overexpressing p53. Double IHC showed colocalization of both molecules in a significant proportion of tumor cells suggesting a link of p53 overexpression and the abnormal proliferative activity. The present results show that p53 over-expression is found in a significant percentage of TCCs and indicate a close association with a defective growth regulation resulting in increased PCNA-levels and enhanced cellular proliferation.
Analysis of Variance↗