Temporal changes in elemental composition in decomposing filamentous algae (Cladophora glomerata and Pilayella littoralis) determined with PIXE and PIGE.

Particle-induced X-ray emission and particle-induced gamma-ray emission spectrometry were successfully applied in a study of the elemental composition of decomposing filamentous algae. Fresh brown (Pilayella littoralis) and green (Cladophora glomerata) algal materials were placed in cages at 4m depth in a water column of 8m in the Archipelago Sea, northern Baltic Sea. Every second week decaying algae were sampled from the cages to allow measurements of changes in the elemental compositions. In the study of the elemental losses the concentrations were compensated for the mass reduction. The results show that sulphur, chlorine and partly potassium were lost during decomposition of P. littoralis and C. glomerata. Most of the other elements studied were recovered in the remaining algal mass. Special attention was paid to sorption and desorption of elements, including metal binding capacity, in the decaying algal materials. The affinity order of different cations to the two algal species was established by calculation of conditional distribution coefficients, D'(M). For instance for P. littoralis the following series of binding strength (affinity) of cations were obtained: Al>Ti>Fe >> Mn>Ni, Cu>Ba, Cr, Zn>>Rb>K, Sr>Pb>>Ca>>Na>Mg. Notably is that the binding strength of strontium was more than 10 times higher for P. littoralis than for C. glomerata. Due to their high binding capacity and good affinity and selectivity for heavy metal ions these algae have great potential as biological sorbents. Large variations in elemental content during decomposition complicate the use of algae for environmental monitoring.

Systematic search for the best gene expression markers for melanoma micrometastasis detection.

Melanoma is notorious for its high tendency to metastasize and its refractoriness to treatment thereafter. Metastasis is believed to occur mostly through the lymphatic system, and the status of sentinel lymph nodes is currently recognized as the best prognostic indicator. Unfortunately, the lymphatic metastatic process is still poorly understood and the occurrence of sentinel node metastases (micrometastases) may be underestimated. We performed genome-wide gene expression analyses of melanoma lymph node micrometastases and macrometastases, and of primary melanomas and benign naevi, to characterize the early metastatic cells molecularly and to disclose the best diagnostic markers and rational targets for therapy. Significance analysis of microarrays identified 22 over- and five under-expressed genes with > or = four-fold changes in the micrometastases. Of these genes, MLANA, TYR, MIA, ERBB3, PRAME, and SPP1 were tested as potential markers by RT-PCR and immunohistochemistry. In a prospective study of 160 patients, our graded MLANA and TYR RT-PCR analyses disclosed clinically significant metastases, as assessed by disease recurrence, better than histological and immunohistochemical examinations. These results strongly suggest the clinical implementation of quantifiable RT-PCR assays to confirm and complement the pathological examination of sentinel node metastases. Furthermore, SPP1 and PRAME proved valuable as melanoma-specific markers capable of differentiating melanoma cells from benign naevi in the sentinel lymph nodes. Importantly, these two genes may also prove to be ideal targets for drug development and therapy. Most molecular traits of the micrometastases were already present in the primary tumours, suggesting that micrometastasis to sentinel lymph nodes is a fairly non-selective process.

Elemental analyses of pine bark and wood in an environmental study.

Bark and wood samples were taken from the same individuals of Scots pine (Pinus sylvestris L.) from a polluted area close to a Cu-Ni smelter in Harjavalta and from some relatively unpolluted areas in western Finland. The samples were analysed by thick-target particle induced X-ray emission (PIXE) after preconcentration by dry ashing at 550 degrees C. The elemental contents of pine bark and wood were compared to study the impact of heavy metal pollution on pine trees. By comparison of the elemental contents in ashes of bark and wood, a normalisation was obtained. For the relatively clean areas, the ratios of the concentration in bark ash to the concentration in wood ash for different elements were close to 1. This means that the ashes of Scots Pine wood and bark have quite similar elemental composition. For the samples from the polluted area the mean concentration ratios for some heavy metals were elevated (13-28), reflecting the effect of direct atmospheric contamination. The metal contents in the ashes of pine bark and wood were also compared to recommendations for ashes to be recycled back to the forest environment. Bark from areas close to emission sources of heavy metal pollution should be considered with caution if aiming at recycling the ash. Burning of bark fuel of pine grown within 6 km of the Cu-Ni smelter is shown to generate ashes with high levels of Cu, Ni as well as Cd, As and Pb.

Thick-target PIGE analysis of plant materials preconcentrated by dry ashing.

Plant materials were dry ashed at 550 degrees C and analysed using particle-induced prompt gamma-ray emission (PIGE). The analyses were performed with an external beam of 3 MeV protons incident on the target. Seven biological certified reference materials were analysed and used for the evaluation of the method for Na, Mg, Al, P and Mn. The elemental concentration to detection limit ratios were greatly enhanced by dry ashing of the biological materials. The concentrations of the elements in ashes were clearly above the values at which reliable analyses can be made. The method was applied to samples of spruce and pine. Due to the low ash content of the wood samples, the sensitivity of the method was radically improved. The detection limits for the five elements studied in spruce wood were in the range 0.014-2.5 mug g(-1). The set-up and the beam current used enabled simultaneous particle-induced X-ray emission spectrometry (PIXE) analyses, with the sensitivity optimised for heavier trace elements.

Non-specific, rapidly generated cytotoxicity in lymphocytes induced by BCG in vitro: no evidence of enhancing effect from preceding interaction between BCG and transitional cell line cells.

PURPOSE: To study short-term events in the mechanism of action of BCG with an emphasis on the interaction between BCG and T24 cell line cells. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMNC) or/and several tumor cell lines were incubated with BCG (Oncotice) using various clinical and subclinical BCG concentrations. RESULTS: 3 h BCG incubation of PBMNC at 10(7) - 5*10(5) CFU/ml., followed by a 4 h cytotoxicity test, resulted in a significant augmentation of cytotoxicity of PBMNC against T24 cells, and the augmentation was almost significant at 10(5) CFU/ml. Overnight BCG incubation of PBMNC further augmented that cytotoxicity at all concentrations down to 10(4) CFU/ml. The minimum overall time (incubation with BCG + cytotoxicity test), where stimulation of PBMNC could be detected, was only 4 h. The BCG enhanced cytotoxicity of PBMNC could be demonstrated against all the tested cell line cells in a 4 h cytotoxicity test by using a preceding overnight BCG incubation of PBMNC, and against the majority of the cell lines by using a preceding 3 h BCG incubation of PBMNC. No convincing evidence was obtained to support the hypothesis that BCG should be first processed by T24 cells to make these cells more susceptible to cell mediated lysis by PBMNC. CONCLUSIONS: Clinical and subclinical concentrations of BCG are directly stimulatory to PBMNC, which become, in a minimum time of a few hours, more capable of killing tumor cells, without a need for preceding interaction between BCG and tumor cells.

Origin of microcells in the human sarcoma cell line HT-1080.

The aim of this study was to investigate the development of microcells in the human sarcoma cell line HT-1080 after interference with thiophosphamidum. We found that damaged interphase macrocells located at the projection of the nucleolus may form one or several microcells. The micronuclei of the microcells intensively incorporate the thymidine analogue 5-bromo-2'-deoxyuridine and strongly express argyrophilic nucleolar organiser region proteins. At an early phase of the development, the micronuclei contain fragmented DNA, but in subsequent phases, the micronuclei accumulate polymeric DNA, simultaneously with an increase in their size. After desintegration of the damaged macrocell, the microcells appear in the intercellular space. The microcells can enter mitosis and they strongly express the lung resistance protein. Electron microscopic observations suggest that coiled bodies are involved in the development of the microcells. Since the observed path of microcell formation differs from apoptotic cell fragmentation into apoptotic bodies, we propose a new term for this microcell development: sporosis. We suggest that self-renewal of the tumour stem cells is likely based on sporosis.

An innovative model for international undergraduate education.

Educational exchange programs have a long academic history. Through such programs, students expand their knowledge of cultures in their home country and the larger world. Typically, students learn about a particular substantive area, such as politics, economics, health care, or language. There is also a loftier goal: to encourage understanding and respect for the people of other countries and share experiences with family and friends at home. In a biography of William J. Fulbright, the developer of the preeminent exchange program that bears his name, the senator is described as hoping that in a democratic country like the United States, "the knowledge and understanding of other cultures would inevitably trickle down through the educational system to those who were not privileged to travel abroad" (1, p. 131).

Expression and characterization of a B cell growth promoting polypeptide derived from the 12 kDa B cell growth factor gene (BCGF 1).

The expression and partial purification of recombinant 12 kDa B cell growth factor are reported. The polypeptide was derived from the genomic sequence of the gene (BCGF 1) which is here shown to be a single copy gene that localizes to human chromosome 16. When expressed as a glutathione S-transferase fusion protein in E. coli, the protein appears as a 38 kDa polypeptide in Western blot analysis using a peptide antibody. The purified fusion protein stimulates the proliferation of activated human B cells in a dose-dependent manner, and the active site resides within the 104 carboxy-terminal amino acids. The availability of biologically active recombinant 12 kDa B cell growth factor will enable its evaluation in B cell growth regulation, and provides a new means of in vitro culturing of human B lymphocytes.

Determination of equilibrium constants of alkaline earth metal ion chelates with Dowex A-1 chelating resin.

A complexation chemistry model is applied to chelating ion-exchange systems and a method is presented for the determination of equilibrium constants for metal ion chelates with these resins. Protonation constants for the iminodiacetic based chelating resin Dowex A-1 were determined from potentiometric pH-data. Equilibrium constants were determined for 1:1 beryllium, magnesium, calcium, strontium and barium chelates with the resin in a wide pH range by measuring the concentrations of respective metal ions in the aqueous phase with direct current plasma atomic emission spectrometry (DCP-AES). A batch technique was used for the equilibrium experiments. At pH below 7 protonated 1:1 species were also found to be formed with the resin. From the obtained equilibrium constants, theoretical distribution coefficients were calculated as function of pH for respective metal ion resin system.

Colorimetric solid-phase minisequencing assay illustrated by detection of alpha 1-antitrypsin Z mutation.

In solid-phase minisequencing, a defined point mutation is detected in microtiter plate-immobilized DNA by a single nucleotide primer extension reaction. We have here developed the method into a colorimetric assay and applied it to the detection of the Z mutation of the alpha 1-antitrypsin gene. We used novel nucleoside triphosphates modified with dinitrophenyl (DNP) hapten, permitting detection by anti-DNP-alkaline phosphatase conjugate, with p-nitrophenyl phosphate as substrate. The Z mutation is detected in two reactions: DNP-labeled dCTP is incorporated when the template is normal, DNP-dUTP when the Z mutation is present. Both modified nucleotides were incorporated with high specificity and with an efficiency similar to that of unmodified nucleotides. The test results are measured by spectrophotometry, yielding quantitative absorbance values. Calculation of the ratio of C to U signal permitted unambiguous distinction of normal homozygous, ZZ homozygous, and ZM heterozygous genotypes. The colorimetric minisequencing assay is rapid, standardized, and automatable, and thus provides an accurate and simple alternative for the analysis of known point mutations.

A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E.

We describe a new technique by which single base changes in human genes can be conveniently detected. In this method the DNA fragment of interest is first amplified using the polymerase chain reaction with an oligonucleotide primer biotinylated at its 5'-end. The amplified 5'-biotinylated DNA is immobilized on an avidin matrix and rendered single-stranded. The variable nucleotide in the immobilized DNA is identified by a one-step primer extension reaction directed by a detection step primer, which anneals to the DNA immediately upstream of the site of variation. In this reaction a single labeled nucleoside triphosphate complementary to the nucleotide at the variable site is incorporated. The method is highly sensitive, allowing the use of nucleoside triphosphates labeled with radioisotopes of low specific activity (3H) as well as nonradioactive markers (digoxigenin). The procedure consists of few and simple operations and is thus applicable to the analysis of large numbers of samples. Here we applied it to the analysis of the three-allelic polymorphism of the human apolipoprotein E gene. We were able to correctly identify all possible combinations of the three apo E alleles.

Affinity-based collection of amplified viral DNA: application to the detection of human immunodeficiency virus type 1, human cytomegalovirus and human papillomavirus type 16.

We have devised a sensitive and convenient hybridization technique by combining the polymerase chain reaction (PCR) with affinity-based hybrid collection. In this method 5'-biotinylated primers are used to introduce biotin residues into the DNA fragments during the amplification. The amplified DNA fragments are detected by liquid hybridization using a 32P- or 35S-labelled oligonucleotide as probe. For measurement the hybrids are collected on polystyrene microparticles or onto microtitre wells taking advantage of the biotinavidin interaction. The method is highly sensitive allowing the detection of 30 molecules of DNA. It involves few and simple operations, and is thus suitable for routine diagnostics. The applicability of the method to the detection of HIV-1 DNA from blood, HCMV DNA from urine and HPV-16 DNA from cervical scrapes was evaluated.

Determination of stability constants for alkaline-earth and alkali metal ion complexes of glycine by spectrophotometry.

The complex equilibria between alkaline-earth and alkali metal ions with glycine were studied by a spectrophotometric method. The following stability constant (concentration) values valid at 25 degrees and ionic strength 1.0M were found: K(HL) = 10(9.57), K(LiL) = 10(-1.2), K(BaL) = 10(-0.40), K(SrL) = 10(0.14), K(CaL) = 10(0.55), K(MgL) = 10(1.17).

Determination of total iron in standard rocks by spectrophotometric titration with EDTA.

A method for the determination of total iron in silicate rocks is described. It is based on spectrophotometric titration of iron(III) with EDTA after decomposition in a PTFE bomb. No prior separation of interfering elements is needed. The method was tested by analysis of the U.S. Geological Survey reference rocks G-2, AGV-1, GSP-1, BCR-1 and PCC-1. The same sample solutions were also analysed by atomic-absorption spectrophotometry. The agreement with published and recommended values was good.

Standardization of EDTA by spectrophotometric titration, with metallic copper as standard.

The standardization of EDTA against electrolytically pure copper is described. The copper is dissolved and titrated spectrophotometrically with EDTA at pH 5.0 without an indicator. A relative standard deviation of 0.009% was achieved.

The formation of 2-hydroxy-1,3-diaminopropane-N,N,N',N'-tetra-acetic acid silver and mercury(III) chelates.

The stability constants of mononuclear (1:1) and binuclear (2:1) chelates of the Ag-HDPTA and Hg(II)-HDPTA systems (HDPTA = 2-hydroxy-1,3-diaminopropane-N,N,N',N'-tetra-acetic acid) were evaluated from pM and pH data, using a method proposed earlier by Ringbom and the author. The determinations yielded the following stability constants (concentration): The use of the reagent for the analytical determination of silver and mercury(II) ions is also discussed.

Potentiometric and spectrophotometric determination of the protonation constant of hexamethylenetetramine.

The protonation constant of hexamethylenetetramine (urotropine) was determined by a potentiometric and a spectrophotometric method. The calculations gave log K(HL) (concentration constants): 4.89 at mu = 0.1 and 5.05 at mu = 0.5. The temperature was 25 degrees and potassium chloride was used to adjust the ionic strength.