A new semi-automated instrument to improve the fine needle aspiration procedure during breast lesion cell sampling

A large and increasing number of women in the western world will at some point during their life be investigated morphologically for some type of breast lesion. Fine Needle Aspiration (FNA) is one morphological method which is considered to be the fastest, cheapest and the most patient-friendly approach. However, the frequency of conclusive samples using this method varies and is often too low, especially when performed by unexperienced operators. In this study we have developed and tested a new semi-automated instrument (“CytoTest”) designed for FNA which is intended to improve the efficacy of the technique by increasing the percentage of conclusive samples. A total of 443 consecutive aspiration procedures on palpable breast lesions were performed to compare this new “CytoTest” equipment with the standard protocol using the same type of needles. We conclude that by increasing the extent and frequency of the reciprocatory motions used by an experienced sampling operator as well as enhancing the ejection pressure, the cellular yield can be increased almost three folded compared to the standard protocol. For cases with high amounts of non-diagnostic material (such as blood or cystic fluid) which were discarded, up to four times more sample could be obtained. Furthermore, the frequency of sparse samples under 1 mg was halved with use of the “CytoTest”.     

ADP-ribosylation in isolated nuclei of Physarum polycephalum.

ADP-ribosylation of histones and non-histone nuclear proteins was studied in isolated nuclei during the naturally synchronous cell cycle of Physarum polycephalum. Aside from ADP-ribosyltransferase (ADPRT) itself, histones and high mobility group-like proteins are the main acceptors for ADP-ribose. The majority of these ADP-ribose residues is NH2OH-labile. ADP-ribosylation of the nuclear proteins is periodic during the cell cycle with maximum incorporation in early to mid G2-phase. In activity gels two enzyme forms with Mr of 115,000 and 75,000 can be identified. Both enzyme forms are present at a constant ratio of 3:1 during the cell cycle. The higher molecular mass form cannot be converted in vitro to the low molecular mass form, excluding an artificial degradation during isolation of nuclei. The ADPRT forms were purified and separated by h.p.l.c. The low molecular mass form is inhibited by different ADPRT inhibitors to a stronger extent and is the main acceptor for auto-ADP-ribosylation. The high molecular mass form is only moderately auto-ADP-ribosylated.     

Two dimensional single-strand conformation polymorphism analysis: a useful tool for the detection of mutations in long DNA fragments.

A new two-dimensional gel system for the analysis of single strand conformational polymorphisms has been developed to identify point mutations, deletions and insertions in long DNA fragments (e.g. 2.7 kb) generated by the polymerase chain reaction. In this procedure, such DNA fragments are first restricted with frequent-cutter enzymes. The resulting small fragments are then separated in the first dimension according to their size by electrophoresis under denaturing conditions; these single stranded DNA fragments are subsequently fractionated in the second dimension by electrophoresis on a non denaturing slab gel based on their fold-back conformation which is completely sequence-dependent. The method was tested on three previously characterized pH 4.5 resistant mutants of HRV14 and was then used to determine changes in three further mutants.     

Mathematical modelling of primary production in Green Bay (Lake Michigan,USA), a phosphorus-and light-limited system

Application of mathematical models in the design and evaluation of lake restoration programmes must include due consideration of three basic concepts of model development; 1) that the model framework is appropriately matched to the intended management use, 2) that selection of the proper degree of model complexity is fundamental to the achievement of model credibility and 3) that field and laboratory studies must be designed and interpreted with the aid of the model to insure development of a comprehensive, integrated tool.These concepts are demonstrated for the case of lake restoration efforts in Green Bay (Lake Michigan, USA). Striking gradients in water quality (transparency, algal standing crop, hypolimnetic oxygen depletion) and trophic state occur along the major axis of the bay in response to phosphorus loaded from the Fox River. A simple model for gross primary production is developed to permit calculation of the relative importance of internal carbon production to the total organic carbon budget of the bay. Primary production varies from high rates over a limited photic depth in the turbid, phosphorus-rich waters of the eutrophic portions of the bay to low rates over an extensive photic depth in the transparent, phosphoruspoor reaches of the oligotrophic regions. Internal production accounts for approximately 90% of the total organic carbon loaded to the system over the summer growing season. Water quality management strategies must address the stimulation of primary production by phosphorus loaded from the Fox River in any attempt to lower the standing crop of nuisance algae, improve water clarity, and reduce rates of hypolimnetic oxygen depletion in Green Bay.     

Evidence that Escherichia coli virus T1 induces a DNA methyltransferase

DNA of Escherichia coli virus T1 is resistant to MboI cleavage and appears to be heavily methylated. Analysis of methylation by the isoschizomeric restriction enzymes Sau3AI and DpnI revealed that recognition sites for E. coli DNA adenine methylase (dam methylase) are methylated. The same methylation pattern was found for virus T1 DNA grown on an E. coli dam host, indicating a T1-specific DNA methyltransferase.     

DNA repair dependent NAD+ metabolism is impaired in cells from patients with Fanconi's anemia

In vitro cultivated fibroblasts derived either from patients with Fanconi's anemia (FA) or from healthy probands were analyzed for their DNA repair-dependent NAD+ metabolism. No difference in NAD+ pools was found. NAD+ consumption after cell damage by u.v. irradiation was, however, significantly reduced in FA cells. Several FA cell lines had a lowered ability to transfer ADP-ribose to acid-precipitable material. Additionally, a decreased activity of NAD: protein ADP-ribosyltransferase was found for three FA cell lines. Our data indicate, that FA is accompanied by a defective NAD+ metabolism during DNA repair.     

Partition of protein and DNA during cytokinesis in human breast cancer cell lines.

Three human breast cancer cell lines (HTB-126, MDA-231, and HTB-122) with DNA index (DI) values between 1.26 and 1.72 were analysed together with a diploid mouse embryonal carcinoma cell line (PCC3) by a TV-video time-lapse technique (pedigree analysis). Cytochemical parameters (DNA and proteins) were studied in individual cells in a rapid scanning microspectrophotometer. Post-mitotic sister cell pairs were analysed after Feulgen-naphthol-yellow staining. The DI values of the cell lines were selected to reflect various well-known clinical ploidy entities differing in malignancy potentials. A mitotic disturbance of the partition of DNA and protein to daughter cells was found in particular in MDA-231 closest to the triploid DNA modal value (DI = 1.37). Duration of mitosis was considerably longer in the near triploid line compared to the other lines. The MDA-231 line was also least sensitive to suboptimal growth conditions. This report calls attention to a possible causality between mitotic error and intraclonal genotype and cell mass heterogeneity.     

Repair rate in human fibroblasts measured by thymine dimer excorporation

Summary The UV photoproduct, thymine dimer ( ), is excorporated with a remarkably low rate from the DNA of human fibroblasts grown in cell culture. An UV dose of 18 J/m² creates 0.045% (related to thymine). Within the first two days of repair logarithmically growing and quiescent fibroblasts exhibit the same repair rates; thereafter, the proportion of is lower in growing cells due to recovery of DNA replication. Only about 50% of the lesions are excised within 24 h. In quiescent cells, 13% of the thymine dimers originally present can be detected as late as a week after UV-irradiation. Two distinct first-order rate constants indicate that approximately half of the dimers are less accessible to repair. Repair measured by the nucleoid decondensation technique corresponds to the faster repair rate, whereas the slow repair rate cannot be detected by this method. Saturation of repair is found beyond 27 J/m². The remarkably slow rate of excision indicates that thymine dimers are not lethal lesions in human fibroblasts.