The assessment of cellular proliferation by immunohistochemistry: A review of currently available methods and their applications

Summary Immunohistochemical methods using antibodies to cell cycle-related antigens may be used as a means of assessing various aspects of proliferation in tissue, and have the important advantage of preserving the spatial orientation of proliferating cells in histological sections. Currently, the most widely available antibodies for this purpose are antibodies to bromodeoxyuridine (BrdU), Ki67 and antibodies to proliferating cell nuclear antigen (PCNA). BrdU is a thymidine analogue incorporated during the S phase of the cell cycle, which can be introduced by in vivo administration or by in vitro incubation, and monoclonal antibodies are available to display its localization. Ki67 demonstrates a nuclear antigen expressed in all phases of the cell cycle, except G₀ and early G₁, but can only be applied to frozen tissue. PCNA is a nuclear antigen which is essential for DNA synthesis, two commercially available antibodies to PCNA work in paraffin-embedded tissue, but may have different staining characteristics under different conditions of fixation. The main advantages and disadvantages of these different techniques are discussed, together with their main applications to date.     

Serum-free medium for fermentor cultures of hybridomas

Hybridomas lend themselves particularly well to large scale cultivation techniques since they grow as single cells in suspension without requiring attachment to a substrate. Furthermore, many cell strains have been adapted to grow in serum-free (SF) media to a similar cell density and antibody production as in serum containing media. This review will concern itself mainly with the cultivation of hybridomas in SF-media in bioreactors of various types with the ultimate goal of producing large quantities of monoclonal antibodies (mAb).     

Are there seasonal variations of trace element concentrations (Cd,Pb, Zn) in wood of Fagus trees in Germany?

Concentrations of Cd, Pb and Zn were determined in stem wood of beech trees (Fagus sylvatica L.) from 3 sites in northern Germany. Distinct radial distribution patterns of the elements were observed in the xylem. Concentrations of Cd and Pb increased from the youngest, outermost annual rings towards the center of the stem. With Zn intermediate concentrations were observed in the sapwood and higher levels at the center of the stem.Temporal and spatial stability of such distribution patterns in the trunks was investigated. Wood samples taken from the same individual tree in different months of the year were analysed. Marked seasonal variations of mineral concentrations were observed. Also the shape of the distribution patterns of the elements varied with the season. Such variations were larger than those observed with samples taken simultaneously from different sides of the trunk. Furthermore, Pb concentrations in the stem showed variations with height above ground.The results indicate, that radial distribution patterns of Cd, Pb and Zn in xylem rings of beech are not stable. Biomonitoring trace element pollution levels by analysis of beech wood is, thus, questionable. To obtain a reliable historical record of pollution from tree rings, the distribution patterns should be stable over a long period of time. This basic requirement of the dendroanalytical method does not hold for the examined beech. Still, with other tree species and under more favourable conditions the dendroanalytical biomonitoring method may prove valuable.Presented as symposium paper at the V International Congress of Ecology, Yokohama, August 23–30, 1990.     

Ecology Institute prizes 1991 in the field of limnetic ecology

Ecology Institute prizes 1991 in the field of limnetic ecology     

Distribution of sensory neurones of the pudendal nerve in the dorsal root ganglia and their projection to the spinal cord

Summary The morphology and distribution of the sensory neurones of the pudendal nerve within the spinal ganglia of rats were investigated by use of horseradish peroxidase (HRP). The labelling was visualized in diaminobenzidine (DAB) or tetramethyl-benzidine (TMB)-stained sections. Injection of HRP directly into the pudendal nerve labelled perikarya predominantly in the sixth lumbar DRG (L6). Following injection of HRP into the scrotal skin, however, additional cells were labelled in L5 and SI. Labelling was invariably unilateral. Approximately equal numbers of small (<30 m) and large neurones (>40 m) were labelled following subcutaneous injections although injections into the nerve marked twice as many small cells as large cells. This suggests that, in the rat, most of the small-diameter fibres within the pudendal nerve ascend through L6. Although a cluster of neurones was observed in one experiment, the remaining 25 experiments did not reveal any somatotopic arrangement since the labelled perikarya were distributed evenly throughout the ganglion. Similar numbers of retrogradely labelled neurones (somatopetal transport of the tracer) were observed in both DAB- and TMB-stained sections, although TMB allowed the demonstration of anterograde (somatofugal) HRP transport by terminal labelling in the superficial laminae of the lumbar spinal cord, extending into laminae II–IV.Partially supported by grants from the DFG to HWK (Ko 758/1)     

The Sargasso Sea Eel Expedition 1979

The background and aims of the Sargasso Sea Expedition for exploring the reproduction of the eel (Anguilla spp.) in the North Atlantic are described. The ships used for the investigations in the Sargasso Sea and during transects across the North Atlantic were F. R. V. Anton Dohrn and R. V. Friedrich Heincke, provided by the Federal Republic of Germany. Their technical and scientific equipment and their staffs are described. The courses of the cruises are mapped and the geographical positions of the investigations as well as the different instruments employed are tabulated as a source of basic data for more specialized papers published in this issue or elsewhere. Reference is also given to relevant papers which have been published elsewhere. Important but unpublished preliminary results are mentioned. Suggestions for future research needs are made.     

Turgor pressure and water transport properties of suspension-cultured cells of Chenopodium rubrum L.

The turgor pressure and water relation parameters were determined in single photoautotrophically grown suspension cells and in individual cells of intact leaves of Chenopodium rubrum using the miniaturized pressure probe. The stationary turgor pressure in suspension-cultured cells was in the range of betwen 3 and 5 bar. From the turgor pressure relaxation process, induced either hydrostatically (by means of the pressure probe) or osmotically, the halftime of water exchange was estimated to be 20±10 s. No polarity was observed for both ex- and endosmotic water flow. The volumetric elastic modulus, , determined from measurements of turgor pressure changes, and the corresponding changes in the fractional cell volume was determined to be in the range of between 20 and 50 bar. increases with increasing turgor pressure as observed for other higher plant and algal cells. The hydraulic conductivity, Lp, is calculated to be about 0,5–2·10^–6 cm s^–1 bar^–1. Similar results were obtained for individual leaf cells of Ch. rubrum. Suspension cells immobilized in a cross-linked matrix of alginate (6 to 8% w/w) revealed the same values for the half-time of water exchange and for the hydraulic conductivity, Lp, provided that the turgor pressure relaxation process was generated hydrostatically by means of the pressure probe. Thus, it can be concluded that the unstirred layer from the immobilized matrix has no effect on the calculation of Lp from the turgor pressure relaxation process, using the water transport equation derived for a single cell surrounded by a large external volume. By analogy, this also holds true for Lp-values derived from turgor pressure changes generated by the pressure probe in a single cell within the leaf tissue. The fair similarity between the Lp-values measured in mesophyll cells in situ and mesophyll-like suspension cells suggests that the water transport relations of a cell within a leaf are not fundamentally different from those measured in a single cell.     

Non-random intrachromosomal distribution of chromatid aberrations induced by X-rays,alkylating agents and ethanol in Vicia faba

A reconstructed karyotype of Vicia faba with all chromosomes individually distinguishable was treated with triethylene melamine (TEM), cytostasan (CYT) (a new benzimidazol nitrogen mustard), mitomycin C (MI), ethanol (EA) and X-rays. The distribution within chromosomes of induced chromatid abberations was non-random for all agents. The number of segments involved in aberration clustering corresponded to the number of sites representing constitutive heterochromatin, or the regions immediately adjacent to these, as evidenced by the position of Giemsa marker bands. Which of these potential regions of aberration clustering reacted with preferential involvement in aberrations was, in part at least, dependent upon the inducing agent used. It is argued that this may be due to differences in the base composition and/or molecular conformation of heterochromatic regions. Unexpectedly, the distribution pattern of chromatid aberrations induced by mitomycin C was found to be different from those after treatment with the alkylating agents TEM and cytostasan although mitomycin C is assumed to induce aberrations via alkylation. If mitomycin C-induced aberrations are indeed due to alkylation, this indicates that different alkylating agents do not necessarily result in identical patterns of abberation clustering. The other two alkylating agents and ethanol resulted in similar patterns of preferential distribution of abberations. X-Ray induced chromatid aberrations also showed a non-random intrachromosomal distribution, but the clustering was less pronounced than after treatment with the chemical agents.     

Polymorphisms of 5,10-methylenetetralydrofolate reductase (MTHFR), fruit and vegetable intake, and the risk of stomach cancer

Stomach cancer is a serious public health problem in China. 5,10-Methylenetetralydrofolate reductase (MTHFR) may be involved in both DNA methylation and DNA synthesis. Folate deficiency is associated with cancer risk that may be modulated by a genetic variation in the MTHFR gene in folate metabolism. The main goal of this study was to evaluate the association between polymorphisms of the MTHFR gene and the risk of stomach cancer. This study also explored the modification effects of fruit and vegetable intake (one of the main constituents is folate) on the risk of this disease. A population-based case-control study was conducted in Taixing, China, consisting of 206 newly diagnosed cases with primary stomach cancer and 415 healthy population controls. Polymorphisms of MTHFR C677T and A1298C were assayed by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) techniques. The data were analysed using the logistic regression model. No obvious association between the MTHFR A1298C polymorphism and the risk of stomach cancer was observed in this study. The frequencies of 677 C/C, C/T, and T/T were 34.5, 50.9, and 14.6%, respectively, in controls. The frequency of the MTHFR 677 wild homozygotic genotype was 25.8% in cases, which was lower than that in controls (34.5%). The adjusted odds ratio (OR) for the MTHFR 677 any T genotype was 2.05 (95% confidence interval (CI), 1.26-3.34) when compared with the C/C genotype. In the low fruit and vegetable intake group an increasing trend was observed with the T allele exposure, p=0.0056. The adjusted ORs were 1.68 (95% CI = 0.86-3.29) for the C/T genotype and 3.58 (95% CI = 1.46-8.75) for the T/T genotype, respectively. The MTHFR 677 any T genotype was associated with an increased risk of primary stomach cancer among the Chinese population. Folate deficiency might modify the MTHFR gene polymorphism and influence the risk of stomach cancer.     

Selection of Osteoprogenitors from the Jaw Periosteum by a Specific Animal-Free Culture Medium

The goal of our research work is to establish mesenchymal osteoprogenitors derived from human jaw periosteum for tissue engineering applications in oral and maxillofacial surgery. For future autologous and/or allogeneic transplantations, some issues must be addressed. On the one hand, animal-free culture conditions have yet to be established. On the other hand, attempts should be undertaken to shorten the in vitro culturing process efficiently. The aim of the present study is to compare and analyze the phenotype of osteoprogenitors from the jaw periosteum under normal FCS-containing and animal-free culture conditions. Therefore, we analyzed the proliferation rates of MesenCult-XF medium (MC-) in comparison to DMEM-cultured JPCs. Whereas jaw periosteal cells (JPCs) show relatively slow proliferation rates and a fibroblastoid shape under DMEM culture conditions, MC-cultured JPCs diminished their cell size significantly and proliferated rapidly. By live-monitoring measurements of adhesion and proliferation, we made an interesting observation: whereas the proliferation of the MSCA-1⁺ subpopulation and the unseparated cell fraction were favored by the animal-free culture medium, the proliferation of the MSCA-1^- subpopulation seemed to be repressed under these conditions. The alkaline phosphatase expression pattern showed similar results under both culture conditions. Comparison of the mineralization capacity revealed an earlier formation of calcium-phosphate precipitates under MC culture conditions; however, the mineralization capacity of the DMEM-cultured cells seemed to be higher. We conclude that the tested animal-free medium is suitable for the in vitro expansion and even for the specific selection of osteoprogenitor cells derived from the jaw periosteum.