PubMed Health⌕ Search

Biomedical subjects

Rainer M Bohle

Publications and source records attributed to Rainer M Bohle.

23 records · Page 2Linked to original sources
PubMed2003

Decreased protamine-1 transcript levels in testes from infertile men.

Infertile men exhibit an aberrant protamine-1 (Prm1) to protamine-2 (Prm2) ratio at both the mRNA and protein level. We therefore investigated whether male infertility could be related to the amount of Prm1 and Prm2 mRNA by applying real time quantitative PCR following RNA extraction from routinely Bouin-fixed and paraffin-embedded testicular biopsies. Samples (n = 51) were normalized to the same amount and similar size of tissue sections. The threshold cycle (C(T)) representing a measure of the initial number of mRNA copies was significantly (P < 0.001) higher for Prm1, but not Prm2, and thus the amount of Prm1 mRNA was lower in men with at least qualitatively normal spermatogenesis (Prm1: 29.88 +/- 2.99; Prm2: 34.28 +/- 2.26) and impaired spermatogenesis (Prm1: 31.89 +/- 2.54; Prm2: 35.59 +/- 2.09) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (Prm1: 29.04 +/- 1.02; Prm2: 34.91 +/- 1.40). In addition, the Prm1 - Prm2 C(T) difference (deltaC(T)) was significantly (P < 0.001) decreased in these two groups. A negative correlation (r = -0.504; P < 0.001) was demonstrated between the score for efficiency of spermatogenesis and the C(T) for Prm1. These data suggest that the decreasing amount of Prm1 and, as a consequence, the aberrant Prm1:Prm2 mRNA ratio plays an important role for male infertility and may serve as a possible predictive factor for the outcome of ICSI.

Analysis of Variance↗
PubMed2003

HER-2/neu gene copy number quantified by real-time PCR: comparison of gene amplification, heterozygosity, and immunohistochemical status in breast cancer tissue.

BACKGROUND: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods. METHODS: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer. RESULTS: The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies. CONCLUSIONS: The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.

Adult↗
PubMed2002

Protamine-1 and -2 mRNA in round spermatids is associated with RNA-binding proteins.

RNA-binding proteins in round spermatids have previously been assigned to the coding sequence of Prm1- and Prm2-mRNA. To further characterize this protein-RNA interaction, prior to cDNA synthesis, microdissected cell profiles were digested with different proteases exhibiting a specific cleavage site followed by both conventional and real-time quantitative PCR. Best results were obtained with proteinase K and A followed by factor Xa protease, genenase I, and proteases V8. While enterokinase revealed PCR signals solely for Prm2, no amplification signal was obtained using chymotrypsin. These data suggest a protein segment rich in basic amino acids to be important for the binding to Prm1- and Prm2-mRNA. The fact that phenanthroline treatment instead of protease digestion also resulted in amplification signals suggests the involvement of zinc-finger-like protein-RNA interactions. Employing different primer pairs, RNA-binding proteins were shown to be localized at the 5' end of Prm1- and Prm2-mRNA. Since protein-RNA interactions are a common principle of posttranscriptional regulation of gene expression, the combination of microdissection, protease digestion, and real-time quantitative PCR provides a suitable tool for its investigation in a cell type-specific manner. Furthermore, the presence of RNA-binding proteins within the coding sequence of mRNAs demands proteinase K treatment prior to cDNA synthesis, a compelling necessity for the study of gene expression.

Cell Separation↗
PubMedDate not supplied

Connexin 33: a rodent-specific member of the gap junction protein family?

Gap junctional intercellular communication between Sertoli cells and between Sertoli cells and spermatogonia is considered to play a key role in the regulation of both proliferation and differentiation of germ cells. A member of the gap junction protein family, Connexin 33 (cx33), probably has an inhibitory effect on the formation of gap junctions and so far it is the only cx that has been exclusively found in rat and mouse testes. Thus, this connexin seems to be a special member of the cx family. Using immunohistochemistry, Western blot analysis, polymerase chain reaction, and reverse transcription (RT)-PCR (tissue homogenate and microdissected cells), we studied the possible occurrence of cx33 at the protein, the DNA, and the RNA level in human testis. Whereas immunohistochemistry using the only commercially available anti-cx33 antibody showed similar labeling to the rat within the seminiferous epithelium, we could not find any further evidence for the existence of cx33 using Western blot analysis, PCR, and RT-PCR in human testis. Based on the demonstration of the staining pattern of mitochondria in human germ cells and on preabsorption studies, we could demonstrate anti-cx33 antibody cross-reacting with mitochondrial ferritin, a protein localized in the mitochondria of human testicular spermatids. Therefore, we were not able to abide by the suspicion that cx33 is present in human testis. Additionally, it was not possible to demonstrate cx33 via PCR and immunohistochemistry in the testis of different mammals (dog, cattle, pig, horse, and marmoset monkey) with normal spermatogenesis. These data indicate that cx33 seems to be the first rodent-specific testicular cx.

Amino Acid Sequence↗