The impact of infrared radiation, solar radiation, and burial exposure on the efficacy of forensic immunoassay testing for blood, semen, and saliva.

Determining the biological source of a stain can be important information for both investigators and the judiciary in criminal cases. Immunochromatographic assays are commonly used in forensic science for the identification of human biological material. It has previously been demonstrated that various environmental, thermal and chemical insults can affect the efficacy of ABAcard® HemaTrace® in the detection of human blood. In this study, the efficacy of three tests - ABAcard® HemaTrace®, ABAcard® p30, and RSID™-Saliva - was determined for the detection of blood, semen, and saliva respectively, after the fluids had been exposed to adverse environmental conditions. Each biological fluid was deposited on cotton swatches and exposed to infrared (IR) light using a 100 W heat lamp emitting IR light between 620 and 750 nm and heat of 32° for 24, 36 and 48 h. Cotton swatches bearing biological fluids were also buried in outdoor soil for 3, 4 and 5 weeks. To test common forensic scenarios where biological material may be exposed to solar light, samples were placed on a car bonnet and left for 24, 36 and 48 h. ABAcard® HemaTrace® was able to detect haemoglobin in blood that had been exposed to IR and solar light up to 48 h. False negative ABAcard® HemaTrace® results were obtained from 60 % of blood samples buried for 3 and 4 weeks, and 80 % of blood samples buried for 5 weeks. ABAcard® p30 was able to detect p30 in semen that had been exposed to IR and solar light up to 48 h, except for one false negative after 48 h of IR exposure. False negative ABAcard® p30 results were obtained from all semen samples buried for 3, 4 and 5 weeks. RSID™-Saliva was able to detect α-amylase in saliva in all instances, with no false negative results observed. The findings from this study highlight the need to consider the context in which human blood, semen and saliva are found when reporting on negative immunoassay results.

Proteolipid protein 2 mRNA is expressed in the rabbit embryo during gastrulation.

Differential display technology applied to rabbit blastocysts identified an mRNA that encodes a motif similar to that of the proteolipid protein PLP2/A4 of man, mouse and sheep. The open reading frame (456bp) has 88% amino acid identity to human PLP2/A4. The gene is maximally expressed at the beginning of gastrulation: in situ hybridizations exhibited a sickle-shaped area of labelling at the posterior pole of day 7 post-coitum embryos, which appeared at day 6.5 and decreased in size up to day 8. Weaker labelling was found in the extraembryonic mesoderm, in the anterior part of the primitive streak and in the trophoblast. Time and site of gene expression coincide with emerging morphogenetic activities at the posterior pole of the embryo at the beginning of gastrulation.

Purification of uteroglobin using monospecific antibodies coupled to divinylsulphone-activated agarose.

As a model for the isolation of a labile or trace protein, the purification of uteroglobin (UGL) by immunoaffinity chromatography is described. Antibody was isolated from sheep antiserum by immunoprecipitation, and coupled to divinylsulphone-activated agarose (Mini Leak). For the immunoabsorption stage rabbit uterine mucosal scrapings were defatted and incubated directly with the immunosorbent. After washing and desorption, the UGL preparation contained relatively few high molecular weight impurities and these were removed by gel chromatography. Purification was monitored at each step by two-dimensional SDS polyacrylamide gel electrophoresis and immunoelectrophoresis. Furthermore, affinity-purified UGL was tritiated with N-succinimidyl[2,3-3H]propionate and assayed by fluorography. In order to determine absolute UGL concentrations a competitive ELISA was developed.

MADS-box genes are involved in floral development and evolution.

MADS-box genes encode transcription factors in all eukaryotic organisms thus far studied. Plant MADS-box proteins contain a DNA-binding (M), an intervening (I), a Keratin-like (K) and a C-terminal C-domain, thus plant MADS-box proteins are of the MIKC type. In higher plants most of the well-characterized genes are involved in floral development. They control the transition from vegetative to generative growth and determine inflorescence meristem identity. They specify floral organ identity as outlined in the ABC model of floral development. Moreover, in Antirrhinum majus the MADS-box gene products DEF/GLO and PLE control cell proliferation in the developing flower bud. In this species the DEF/GLO and the SQUA proteins form a ternary complex which determines the overall "Bauplan" of the flower. Phylogenetic reconstructions of MADS-box sequences obtained from ferns, gymnosperms and higher eudicots reveal that, although ferns possess already MIKC type genes, these are not orthologous to the well characterized MADS-box genes from gymnosperms or angiosperms. Putative orthologs of floral homeotic B- and C-function genes have been identified in different gymnosperms suggesting that these genes evolved some 300-400 million years ago. Both gymnosperms and angiosperms also contain a hitherto unknown sister clade of the B-genes, which we termed Bsister. A novel hypothesis will be described suggesting that B and Bsister might be involved in sex determination of male and female reproductive organs, respectively.

Uterine fibroblast growth factor-2 and embryonic fibroblast growth factor receptor-1 at the beginning of gastrulation in the rabbit.

Fibroblast growth factor-2 (FGF-2) induces gastrulation of rabbit blastocysts in vitro and is present in the uterine secretion at day 6 after mating. The following study was made in order to show if changes in the uterine FGF-2 concentration or in the FGF receptor concentration of the embryonic tissues point to a regulation of this event. By the use of the ELISA technique and immunohistochemistry, FGF-2 concentration was determined in the endometrial tissue, uterine secretion and blastocyst between day 4 and day 8 of pregnancy, in the uterine secretion after induction of pseudopregnancy, in day 6 blastocysts after in vitro culture, and FGF immunoreactivity was localized in the endometrial tissue. FGF receptor-1 (FGFR-1) concentration was examined correspondingly in the blastocyst. Cross-linking experiments using 125I-FGF-2 were done to identify binding proteins in the blastocyst. In the uterine secretion, FGF-2 was constantly high up to day 6.5 but showed an increase thereafter. Similar values in pseudopregnant uterine secretions indicated that the growth factor was of uterine origin. It was probably synthesized by the uterine epithelium as shown by immunohistochemistry. Under culturing conditions, the blastocyst produced small amounts of FGF-2. In the blastocyst, FGFR-1 as well as binding of 125I-FGF-2 showed a dramatic increase from day 6.0 to day 6.5, coinciding with the onset of gastrulation. Receptor antigenicity was located in the embryonic disc at day 6.5 and day 7.0. Two binding proteins of about 200 and 130 kDa were found by cross-linking. The results indicate that a regulation of growth factor influence on embryonic differentiation is more probable via expression of the embryonic receptor than via differential release of the uterine growth factor.

Fibroblast growth factor induces primitive streak formation in rabbit pre-implantation embryos in vitro.

Culturing of rabbit pre-implantation embryos was performed in Ham's F10 medium supplemented with polyvinylpyrrolidone. Under these culture conditions, day 6 post coitum blastocysts increased their diameter within 24 h to 80% of that of day 7 blastocysts grown in vivo. Despite this substained growth, the embryonic disc remained undifferentiated with clear signs of degeneration after 24 h of culture. Basic fibroblast growth factor (bFGF) was able to overcome this developmental block. After 12 h of culture, day 6 blastocysts showed pear-shaped embryonic discs, and after 24 h, the primitive streak with Hensen's node was visible. The bFGF had no comparable effects on day 5 and day 7 blastocysts. The embryonic discs of day 5 blastocysts degenerated, even in the presence of bFGF, whereas day 7 blastocysts were able to form their primitive streak, also in the absence of bFGF. TGF beta 1 did not promote embryonic development in vitro. The data indicate that the onset of mesoderm formation in the rabbit is controlled by a growth factor of the FGF-family.

Placental lactogen-like proteins in the rabbit placenta.

Rabbit placentae and embryos at days 11 and 12 were analyzed by two-dimensional gel electrophoresis and by light microscopic histology for the presence of placental lactogen-like proteins. Immunoblotting and immunohistochemistry were performed by using a goat anti-human placental lactogen serum as well as a monoclonal mouse anti-human prolactin immunoglobulin; the results were similar. In the gel electrophoresis of placental tissue, three protein spots at pH 5.6 and 43, 39, and 35 kDa were immunostained; they were absent in the embryo. Immunoresponse was restricted to the cytotrophoblast. Immunofluorescent cells were mainly found on the proximal parts of the placental trabeculae.

Uteroglobin in the developing rabbit conceptus in vivo and in vitro.

Uteroglobin (UGL) was measured in day-4 to day-10 rabbit conceptuses by a competitive ELISA. Levels in blastocyst fluid, tissues, coverings and in the early fetus were determined separately. The total amount of UGL increased from 18.4 ng to 6.8 micrograms per conceptus. The UGL content of individual day-6 blastocysts was studied in vitro. Culturing was carried out up to 60 h in Ham's F10 medium with polyvinylpyrrolidone as macromolecular component, with and without progesterone, and with progesterone plus estradiol. UGL was determined in the blastocyst fluids, tissues with coverings and in the culture media. After labelling with [35S]-methionine, protein patterns of total blastocysts and of culture media were analysed by two-dimensional gel electrophoresis and fluorography. The morphology of cultured blastocysts was examined by electron microscopy. During 60 h of culture, the blastocysts expanded in diameter by 84%, and released 19% of their initial UGL content into the medium, independent of the hormonal substitution. Neither de novo synthesis, nor degradation of UGL was found: the protein remained unlabelled in fluorography, and its total quantity was not significantly different from that of non-cultured controls. Trophoblast, endoderm and embryoblast cells showed well preserved cell organelles and intercellular junctions, while the morphological differentiation of the germ layer was inhibited.

Ultrastructure, protein synthesis and secretion of day-6 rabbit blastocysts cultured in a chemically defined, protein-free medium.

Day-6 rabbit blastocysts were cultured in Ham's F10 medium supplemented with polyvinylpyrrolidone as a macromolecular component, for 4 to 12 h. The integrity of the blastocyst cells was demonstrated by electron microscopy. Expansion and biosynthesis of proteins and of DNA were studied after culturing in the presence of 35S-methionine and 3H-thymidine. Polyvinylpyrrolidone did not interfere with the subsequent protein analysis, which was performed by two dimensional gel electrophoresis followed by silver staining and fluorography. More than 600 labelled proteins were found in the blastocyst tissue, many of them were also present in the blastocyst fluid and in the blastocyst coverings. Several proteins seemed to be produced for incorporation into the blastocyst coverings; others, only detected in the culture medium, might have been synthesized for secretion into the environment.

Influence of vitamin A deficiency on the excretion of uromucoid and other substances in the urine of rats.

Male Fü-albino rats were weaned at the age of four weeks and maintained on a vitamin A-deficient diet. When they were 14-18 and 21-26 weeks old, the concentration of uromucoid, calcium and other substances possibly important for the pathogenesis of urinary calculi were determined. Reduced uromucoid excretion with hypercalciuria and reduced phosphate levels were observed. Subsequent examination of the kidneys did not demonstrate the presence of nephrocalcinosis or lithiasis. The relation between vitamin A, the synthesis of uromucoid and AMPS and calcium metabolism in the renal tubules is discussed.

[Immune histological representation of uromucoid in the kidney of man and rat (author's transl)]. / Immunhistologische Darstellung von Uromukoid in der Niere des Menschen und der Ratte

Uromucoid of the rat shows cross reactions with antihumanuromucoid serum. Localisation of this protein in the kidney of rat in comparison to the kidney of man was carried through by immune histological technique with a fluorescein isothiocyanate (FITC) conjugated antihumanuromucoid globuline. Results show that in rat and man synthesis site of Uromucoid are the distale tubules.

Estimated organ donor potential in German maximum care hospitals.

BACKGROUND: The decreasing number of organ donors in Germany remains a major issue in transplantation medicine. The aim of this study was to estimate the organ donor potential at German maximum care hospitals. METHODS: To critically review potential in comparison with organ donation rates in 2010. We separated Maximum care hospitals into university institutions (A-level) and centers with a neurosurgical unit (B-level) based upon the size of hospital as indicated by the total number of beds. To estimate the number of possible organ donors, we adopted the American model previously published by Sheehy et al: the potential was 0.015 organ donors/bed/year for hospitals with more than 350 beds. RESULTS: In 2010 overall in Germany there were 1296 organ donations resulting in 4205 transplanted organs. University hospitals realized 397 organ donations namely 0.008 organ donors/bed/year (57% of calculated organ donor potential), whereas B-level hospitals accounted for 555 of organ donors with a rate of 0.007 organ donors/bed/year (48% of calculated organ donor potential). Large variations in realizing organ donations occurred among single hospitals. CONCLUSION: Our results indicated a substantial potential to increase organ donation rates in German maximum care hospitals. These hospitals (n = 145) are responsible for 73% (non-maximum care hospital n = 1195) of the absolute organ donor pool. Further studies are needed to evaluate possibilities to address the organ shortage particularly with regard to donor detection in intensive care units and also the refusal rate by families.