Negative Control Experiments Research Articles

E4F1, a Novel Estrogen-Responsive Gene in Possible Atheroprotection, Revealed by Microarray Analysis

Estrogen has been postulated to be involved in inhibition of vascular smooth muscle cell (VSMC) proliferation mainly via estrogen receptor (ER), but the detailed mechanism has remained primarily unknown. Therefore, in this study, microarray analysis was used in two types of cultured human VSMCs: one positive for ER alpha, and the other for ER beta, which were treated by estrogens to detect the estrogen-responsive genes. We also used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate mRNA levels of selective target gene (TG) in these cells. We further studied whether the TG product was involved in inhibition of proliferation using small interfering RNA (siRNA) of the TG transfection. We subsequently used quantitative RT-PCR and in situ hybridization analysis to evaluate the expression of these gene products in human aorta. E4F1, a possible inducer of cell growth arrest, was markedly increased only in ER alpha-positive VSMCs by estrogens in both microarray and RT-PCR analyses. Blocking of E4F1 using siRNA suppressed estrogenic inhibition of ER alpha-positive VSMC proliferation. E4F1 mRNA was abundant in premenopausal female aorta with mild atherosclerotic changes. E4F1 is therefore considered one of the estrogen-responsive genes involving ER alpha-mediated inhibition of VSMC proliferation and may play an important role in estrogen-related atheroprotection of human aorta.

Chiroptical rhythmicity, part 3: implications on the activity of the functional cell-surface receptor model

The effect of ligand-induced rhythmical structural changes of the artificial receptor 1 on its capacity to mediate ion transport across lipid bilayer membranes was studied. The changes of the intravesicular pH of egg yolk phosphatidylcholine-small unilamellar vesicles (EYPC-SUVs) after application of a transmembrane proton gradient were measured by double-channel fluorescence kinetics in the presence of 1 at various concentrations of the extravesicular ligand l-His. Comparison with negative control experiments using d-His indicated that increasing concentrations of l-His affect the activity of 1 in a presumably rhythmical manner. Compared to the extent of structural rhythmicity, the functional rhythmicity of 1 appeared, however, less significant. The need for refined assay systems to fully delineate the importance of rhythmical activity of 1 with respect to biological rhythmicity is briefly discussed.

Pellicle precursor protein crosslinking characterization of an adduct between acidic proline-rich protein (PRP-1) and statherin generated by transglutaminase.

Recent work with oral transglutaminase indicated that this enzyme, derived from oral epithelial cells, crosslinked pellicle precursor proteins which may be important in the formation of the acquired enamel pellicle. The purpose of this study was to investigate whether purified acidic PRP-1 can form crosslinks with statherin, and whether such a crosslink is derived from a transglutaminase-catalyzed reaction between glutaminyl and lysyl side-chains, leading to a covalent bond formation. Enzymatic reaction products were analyzed by SDS-PAGE and reverse-phase HPLC. The SDS electrophoretogram revealed a protein band with an apparent molecular weight of 32 kDa, which is consistent with the combined apparent molecular weight of acidic PRP-1 (24 kDa) and statherin (8 kDa). A reaction product isolated by HPLC was characterized by amino acid analysis, which showed a stoichiometry consistent with being an adduct composed of one molecule of acidic PRP-1 and one molecule of statherin. In negative control experiments, it could be shown that this adduct was not detected when the lysines of both substrates were modified by reductive methylation prior to the enzymatic reaction. In addition, amino acid analysis and mass spectrometry confirmed the presence of a gamma-glutamyl-epsilon-lysine dipeptide after enzymatic hydrolysis and the absence of this dipeptide after acid hydrolysis. Analysis of the data obtained indicates that oral transglutaminase is capable of crosslinking acidic PRP-1 and statherin in vitro. In addition, this finding exemplifies the potential of post-secretory processing of salivary proteins, which may represent an additional mechanism to generate new protein species.

Bcl-2 expression by eosinophils in a patient with hypereosinophilia

The hypereosinophilic syndrome (HES) represents a heterogenous group of disorders characterized by hypereosinophilia in the blood and bone marrow, as well as eosinophilic tissue infiltration. The mechanisms of eosinophilic accumulation in HES are largely unknown. It has been recently suggested that the tissue eosinophilia observed in allergic disorders is, at least partially, caused by inhibition of eosinophil apoptosis by T cell‐, mast cell‐, and eosinophil-derived cytokines.1 Therefore we hypothesized that abnormalities in the process of eosinophil apoptosis may play a part in the etiology of HES. Members of the B-cell lymphoma-2 (Bcl-2) family of proteins are important regulators of apoptosis in many cellular systems.2 The first member of the family, Bcl-2, was originally cloned from the breakpoint of a t(14;18) translocation present in many human B-cell lymphomas. Increased production of Bcl-2 protein as a result of t(14;18) translocations contributes to neoplastic B-cell expansion by preventing B-cell death. 2 In this article we describe an unusual case of HES with blood and tissue eosinophils that expressed, in contrast to normal eosinophils, the Bcl-2 gene. Because it was impossible to induce the Bcl-2 gene by IL-5 or GM-CSF in normal eosinophils, it is unlikely that the observed eosinophil Bcl-2 expression in this subject was the result of stimulation with eosinophil hematopoietins in vivo. Moreover, overexpression of Bcl-2 was associated with delayed death of blood eosinophils in vitro, suggesting that Bcl-2 did act as an apoptosis repressor in this cellular system.

Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy

Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy

Expression of Growth Factor mRNAs by Human Tenon's Capsule Fibroblasts

By using the techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern hybridization, we demonstrated that human Tenon's capsule fibroblasts in primary cultures, express the messenger RNA (mRNA) transcripts encoding transforming growth factor-β1 (TGF-β1), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF), but not those coding for aFGF and TGF-β2. Two PCR products were obtained for TGF-β1: a major fragment of 161 base pairs that corresponded to the expected size, and a minor sequence of 400 base pairs. An antisense oligonucleotide probe specific for TGF-β1 detected only the band of 161 base pair of PCR-amplified sequence. For bFGF, PDGF-A and PDGF-B, we obtained only a single PCR product with the anticipated length of 222, 225, and 217 base pairs, respectively. Southern blotting experiments revealed that these PCR-amplified fragments were specific for the respective growth factors. The negative control experiments without template did not reveal any amplification. No product for aFGF or TGF-β2 was detected. By radioimmunoassay, TGF-β1 protein was detected at the level of 24–30 pg ml −1per 2 million Tenon's fibroblasts during a 24-hour period in acid-activated conditioned medium. These results indicate that human Tenon's capsule fibroblasts in primary cultures express TGF-β1 at the translational as well as at the transcriptional levels, and they also have the capacity to synthesize bFGF and PDGF. Considering the significant effects of bFGF, TGF-β1, and PDGF in wound healing response, these growth factors are implicated in tissue repair process after glaucoma filtration surgery that contributes to the failure of the procedure.

Mutagenic potential of nitroguanidine and nitrosoguanidine in the Drosophila melanogaster sex-linked recessive lethal assay.

Nitroguanidine (NG) and its degradation product nitrosoguanidine (NSG) were evaluated for their mutagenic potential by using Drosophila melanogaster sex-linked recessive lethal (SLRL) assay. Following 72 h of feeding exposure, NG and NSG at concentrations of 4-8 micrograms ml-1 and 15-20 mg ml-1, respectively, were not mutagenic in the test system. The frequencies of mutations for NG and the negative control were 0.188% and 0.096%, respectively. The frequencies of mutations for NSG and the negative control experiments were 0.049% and 0.05%, respectively. The positive control mutation frequencies were 15% and 17.8% for the two assays. The differences between the mutation frequencies of NG and NSG and their negative controls were not significant.

Transactivation of Ds by Ac-transposase gene fusions in tobacco.

To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A2 promoter CHI A2. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5' regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.

Occurrence of HTLV-I antibodies in Danish patients with cutaneous T-cell lymphoma.

10 of 68 CTCL (cutaneous T-cell lymphoma) patients without features of ATLL had antibodies against HTLV-I (human T-cell leukaemia virus, type I). The titre of antibody in these positive patients was generally much lower than that seen in cases of ATLL (adult T-cell leukaemia/lymphoma); geometric mean of 80 for CTCL vs. 8000 for Caribbean ATLL. The presence of HTLV-I antibody was unrelated to clinical remission, relapse, or stages of the disease, and some positives were detected in the earliest phases of mycosis fungoides. Among controls and normal donors between the ages of 40 and 65, only 1 of 36 and 3 of 113, respectively, had low titre antibodies to HTLV-I in their sera. Only 5 of 354 Danish normal donors of all ages had antibody, which was identical to the rate in over 2000 US normal donors. In negative control experiments, these antibodies were unreactive with bovine leukaemia virus. These data suggest that HTLV-I or a related retrovirus crossreactive with HTLV-I occurs in a low percentage of the Danish population and patients with CTCL have such antibodies at an increased rate, but less than the rate seen for ATLL (greater than 90%).

In vivo nitrosation of amidopyrine in humans: use of ‘ethanol effect’ for biological monitoring of N-nitrosodimethylamine in urine

Under normal conditions a possible N-nitrosodimethylamine formation in vivo cannot directly be monitored in urine due to high metabolic conversion rate (greater than 99.9%). Own experiments showed an increased excretion rate (up to 2.4%) if ethanol was administered simultaneously. This model was used for monitoring experiments with respect to in vivo formation of N-nitrosodimethylamine. Amidopyrine, as a compound which is easily nitrosated, was administered (single oral dose of 500 mg) to volunteers. Under the influence of 20-30 g ethanol it was possible to detect N-nitrosodimethylamine in urine. From negative control experiments it must be concluded that this appearance of N-nitrosodimethylamine derives from in vivo nitrosation of the drug. The amount excreted in urine varied between 0.5 and 10 micrograms N-nitrosodimethylamine within 8 h and seemed to be influenced by salivary nitrite concentrations which ranged from 5 to 220 p.p.m. NO-2. In comparison with earlier excretion studies in humans it can be assumed that only 1-2% of the originally formed nitrosamine was found in urine. To our knowledge this is the first time that in vivo formation of N-nitrosodimethylamine was directly shown to occur in humans.

FACTORS INVOLVED IN THE PRODUCTION OF IMMUNITY WITH PNEUMOCOCCUS VACCINE

1. Pneumococcus vaccine was administered to 29 patients with pneumonia to determine whether a state of immunity could be induced during the course of the disease. Twenty patients received an intravenous injection of pneumococcus vaccine or pneumococcus filtrate. Nine pneumonia patients received an intradermal injection of vaccine. Eight patients with miscellaneous disease received an intravenous or intradermal injection of pneumococcus vaccine. 2. Of 23 tests in which the serum of the patient was studied for the appearance of protective substance after intravenous injection of heterologous pneumococcus vaccine, 20 or 87 per cent showed a positive response within 6 days after the administration of the antigen. The average day of onset was 4.4 days after injection. 3. Of 9 tests of the same character following the intravenous injection of pneumococcus filtrate, 8 or 89 per cent showed a positive response. The average day of onset of protective substance was 5.6 days after injection. 4. The appearance of specific protective substance following heterologous injection of pneumococcus vaccine appeared to be due to the introduction of the vaccine and not to the natural course of the disease, as was shown by negative control experiments. 5. Of 24 tests with intradermal injection of vaccine, 10 or 42 per cent developed slight protective substance of irregular degree 4.5 days after injection. 6. No immediate reactions were observed following the intravenous or intradermal injection of pneumococcus vaccine. One chill occurred after injection of pneumococcus filtrate. Of 20 cases with intravenous injection of pneumococcus vaccine or filtrate, 2 died of their disease, one a case in which homologous vaccine was used and one in which heterologous vaccine was administered. 7. Conclusions concerning the therapeutic value of the introduction of pneumococcus vaccine in pneumonia must await further investigation. These studies demonstrate that specific protective substances generally appear 4 to 5 days after intravenous injection of pneumococcus vaccine during the course of lobar or bronchopneumonia.