Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.     

Microbiological characteristics of Pacific shrimp (Pandalus jordani).

Microorganisms associated with Pacific shrimp (Pandalus jordani) were isolated and identified. Those on the iced raw shrimp, which yielded an average count of 1.6 x 10(6), were predominantly Moraxella, Pseudomonas, Acinetobacter, Arthrobacter, and Flavobacterium-Cytophaga spp. The blanching and peeling reduced the microbial level to 3.3 x 10(4) and also selectively eliminated Moraxella spp. The microbial flora changed after each processing sequence, and the heat sensitivity and growth characteristics of the representative microbial groups suggested that the presence of Arthrobacter and Acinetobacter spp. in peeled shrimp may indicate inadequate cleaning of raw shrimp or a shorter blanching time. The presence of Moraxella and Flavobacterium-Cytophaga spp. would indicate the degree of secondary contamination, and the presence of Pseudomonas spp. would indicate the shelf-age of the processed shrimp.     

Effect of cerebral electro-therapy on neurohormone excretion in weather-sensitive patients

Use of a cerebral electro-therapy (CET) device, called PENTA, for weather-sensitive patients is described here. It applies 6–45 V D.C. monopolar triangular shaped pulses of 0.75 ms duration, allowing an input of 0–600 mA mean current via 2 frontal and 1 indifferent occipital electrode. To avoid adaptation it is programmed to two alternating frequencies of 20 and 65 Hz. Its successful application to 15 cases of disturbed neuro-endocrine regulation in weather sensitivity is reported. It resulted in cures for 3–12 months, depending on the type of weather sensitivity treated. The mechanism of CET treatment may be due to a bio-feedback on the limbicsystem, involving hypothalamic and pituitarian hormones. The normalising effects described here were followed up by neurohormone urinalysis of 17-KS, 17-OH, adrenaline, noradrenaline, serotonin, 5-HIAA, histamine and thyroxine.—This method proved itself as a valuable and objective tool for psychosomatic complaints due to weather sensitivity.     

Translesion synthesis of 7,8-dihydro-8-oxo-2'-deoxyguanosine by DNA polymerase eta in vivo

7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) is one of the most common DNA lesions induced by oxidative stress. This lesion can be bypassed by DNA polymerase eta (Pol η) using in vitro translesion synthesis (TLS) reactions. However, the role that Pol η plays in vivo contributing to 8-oxo-dG mutagenesis remains unclear. To clarify the role of Pol η in 8-oxo-dG mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector (pSP189) which replicates in mammalian cells. The pSP189 plasmid was treated with methylene blue plus light (MBL), which produces predominantly 8-oxo-dG in DNA, and was then replicated in GM637 cells in presence of siRNA that knocks down the expression of Pol η, or in XP-V cells, which lack functional Pol η. The mutant frequencies were increased in the Pol η siRNA knockdown cells and in XP-V cells relative to control, meaning that Pol η plays an important role in preventing 8-oxo-dG mutagenesis. In the same system, knockdown of OGG1 also led to an increase in mutagenesis. Neither the type of mutations nor their distribution along the supF gene were significantly different between control and target specific siRNA-transfected cells (or XP-V cells) and were predominantly G to T transversions. These results show that Pol η has an important role in error-free 8-oxo-dG lesion bypass and avoidance of oxidative stress-induced mutagenesis in vivo.     

Criteria for the multiple use of isolated perfused hearts in electrophysiological and metabolic experiments.

Both ethical and economic restrictions limit the availability of porcine hearts for in vitro perfusion experiments. Therefore, we tested the feasibility of multiple use of in vitro perfused working hearts for electrophysiological and metabolic investigations. Pig hearts (n=7) rejected for originally planned haemodynamic measurements because of exclusion criteria were perfused in a four-chamber working heart mode. All hearts were kept in steady-state conditions on a low haemodynamic level over 2 h during 75-channel ECG recordings and NADH fluorescence measurements before and after norepinephrine (NE) was administered. QRS and QT interval durations were in a range comparable to in vitro studies and, like QRS and T amplitudes, were found to be sensitive markers of the changing condition of the isolated heart preparation, as myocardial oedema leads to prolonged QRS and QT intervals and declining ECG voltage amplitudes. A change in NADH fluorescence following NE administration was observed in the first 150 min of perfusion, but not later. Considering a time frame of 120 min, multiple use of isolated perfused porcine hearts with low-level haemodynamics may allow a broad spectrum of investigations and could therefore represent a possibility of overcoming the restricted availability of porcine hearts.     

Reliability and performance-dependent variations of muscle function variables during isometric knee extension.

Despite the common use of standardised methods analysing neuromuscular function during knee extension, there is a lack of test-retest reliability studies. Furthermore, for most of the investigated variables it is unknown which changes of values indicate an enhancement of performance. The aim of the present study was to investigate performance-dependent variations of muscle functions during isometric contraction of knee extensors and to examine test-retest reliability of their measurement methods. For test-retest reliability sports students completed three test sessions. Highly skilled athletes, sports students and untrained subjects were investigated to determine the performance-dependent variations. The following variables were analysed: maximal voluntary contraction (MVC), voluntary activation (VA), absolute muscle reaction time (AR), muscle endurance (ME), and EMG frequency analysis (MF) of m. vastus lateralis (VL), m. vastus medialis (VM) and m. rectus femoris (RF). RESULTS TEST-RETEST-RELIABILITY: A high reliability between session 1 vs. 2 and session 2 vs. 3 was shown for MVC (ICC=0.92 and .97), VA (0.92/0.95) and ME (0.87/0.95). ICC in AR (0.23) was low between the first and second session and moderate between the second and third session (0.74). MF of VL, VM and RF showed low ICC between sessions. PERFORMANCE DEPENDENT VARIATIONS: Significant differences in nearly all variables (except VA) were found between trained (athletes and sports students) and untrained subjects.     

Mutation spectra induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of human XP-A fibroblasts

1-Nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide are oxidative metabolites that are responsible for the mutagenicity of 1-nitropyrene. In this study, the mutation spectra induced by oxidative metabolites in human cells were determined using a shuttle vector assay. The mutation frequencies induced by 1-nitropyrene 9,10-oxide were 2-3 times higher than those induced by 1-nitropyrene 4,5-oxide. The base substitutions induced by 1-nitropyrene 4,5-oxide were G --> A transitions, G --> C transversions, and G --> T transversions. In the case of 1-nitropyrene 9,10-oxide, G --> A transitions, G --> T transversions, A --> G transitions and G --> C transversions were observed. Most base substitution mutations induced by oxidative metabolites occurred at the guanine sites in the supF gene. These sequence-specific hot spots were commonly identified as 5'-GA sequences for both metabolites. On the other hand, the sequence-specific hot spots at the adenine sites were identified as 5'-CAC sequences for 1-nitropyrene 9,10-oxide. These results suggest that the oxidative metabolites of 1-nitropyrene induce sequence-specific DNA mutations at the guanine and adenine sites at high frequency.