Hexagonal magnetite nanoprisms: preparation, characterization and cellular uptake.

The capacity of iron oxide nanocrystals to heat tissue when subjected to an alternating magnetic field (AMF hyperthermia) is shape-selective. Although iron oxide nanostructures with numerous shapes have been synthesized to date, hexagonal Fe3O4 prisms of low toxicity remained elusive. Here, we report the use of a dual ligand system permitting feasible reaction conditions to synthesize nearly perfect hexagonal Fe3O4 nanoplatelet structures, with edge length of 45 ± 5 nm and thickness of 5 to 6 nm. Their Specific Absorption Rate (SAR) is >750 W g(Fe)-1. The Fe3O4 hexagons were coated with a dopamine-based ligand to increase dispersibility in aqueous buffers. The Fe3O4 hexagons were only minimally toxic to RAW264.7 cells, which can be utilized in cell-based cancer targeting approaches.

Transplantation of porcine umbilical cord matrix cells into the rat brain.

Immune rejection of transplanted material is a potential complication of organ donation. In response to tissue transplantation, immune rejection has two components: a host defense directed against the grafted tissue and an immune response from the grafted tissue against the host (graft vs host disease). To treat immune rejection, transplant recipients are typically put on immunosuppression therapy. Complications may arise from immune suppression or from secondary effects of immunosuppression drugs. Our preliminary work indicated that stem cells may be xenotransplanted without immunosuppression therapy. Here, we investigated the survival of pig stem cells derived from umbilical cord mucous connective tissue (UCM) after transplantation into rats. Our data demonstrate that UCM cells survive at least 6 weeks without immune suppression of the host animals after transplantation into either the brain or the periphery. In the first experiment, UCM cells were transplanted into the rat brain and recovered in that tissue 2-6 weeks posttransplantation. At 4 weeks posttransplantation, the UCM cells engrafted into the brain along the injection tract. The cells were small and roughly spherical. The transplanted cells were positively immunostained using a pig-specific antibody for neuronal filament 70 (NF70). In contrast, 6 weeks posttransplantation, about 10% of the UCM cells that were recovered had migrated away from the injection site into the region just ventral to the corpus callosum; these cells also stained positively for NF70. In our second experiment, UCM cells that were engineered to constitutively express enhanced green fluorescent protein (eGFP) were transplanted. These cells were recovered 2-4 weeks after brain transplantation. Engrafted cells expressing eGFP and positively staining for NF70 were recovered. This finding indicates a potential for gene therapy. In the third experiment, to determine whether depositing the graft into the brain protected UCM cells from immune detection/clearance, UCM cells were injected into the tail vein and/or the semitendinosis muscle in a group of animals. UCM cells were recovered from the muscle or within the kidney 3 weeks posttransplantation. In control experiments, rat brains were injected with PKH 26-labeled UCM cells that had been lysed by repeated sonic disruption. One and 2 weeks following injection, no PKH 26-labeled neurons or glia were observed. Taken together, these data indicate that UCM cells can survive xenotransplantation and that a subset of the UCM cells respond to local signals to differentiate along a neural lineage.

Incorporation of bovine bone marrow stromal cells into porcine foetal tissues after xenotransplantation.

Bovine bone marrow stromal cells (BMSCs) were injected into the liver of foetal pigs at about 40 days of gestation to test whether these cells could populate developing tissue, and if so, which ones. Approximately 40 days after injection, the foetuses were harvested and tissue sections from many areas of the body were analysed for the presence of bovine cells using two different methods. First, using PCR, bovine repetitive DNA was found to be present in DNA extracted from foetal pig tissues. Secondly, using oligonucleotide primed in situ synthesis (PRINS), the in situ presence of bovine cells was found within porcine tissue sections. PRINS-labelled cells were found within cartilage, perichondrium, connective tissue and smooth muscle. These data suggest that bovine BMSCs integrate throughout the foetal pig.

A novel method for preparing histology slides without a microtome.

A study was undertaken to determine if it might be possible to prepare tissue sections on slides without the use of paraffin embedding, microtome sectioning, or cryostat sectioning which involve equipment and training not always available to scholars or professionals wishing to examine tissue microscopically. After evaluating many different reagents, cutting instruments and solid supports, we developed a method involving application of super glue to a slide, adhering a section of tissue to it, cutting the tissue with a disposable microtome blade, staining the tissue and removing the superglue with a commercially available product. The sections are similar to those sectioned on a microtome, but do not at this time equal their quality. However, histoarchitecture is preserved and individual cell morphology is usually good. We conclude that this is a viable method for preparing histology sections without the use of a microtome or cryostat, something long thought impossible. We have dubbed the method 'RAMP' (Rapid Adhesive-Mediated Procedure).

Physical assignment of two porcine microsatellites to the short arm of chromosome 1 by modified DISC-PCR.

Direct in situ single copy PCR (DISC-PCR) was modified to increase signal detection efficiency and physically assign two porcine microsatellites (MS), Sw552 and Sw137, to chromosome 1p at Flpter 0.1-0.2 and Flpter 0.01-0.2, respectively. These are the first two MS localized to chromosome 1p by an in situ method. The modifications of the DISC-PCR procedure included use of buffered formalin and pepsin as pre-reaction treatments, and tetramethylbenzidine (TMB) for signal detection. The detection efficiency of the modified method is twofold higher than that of the original technique.

Detection of proviral DNA of bovine immunodeficiency virus in bovine tissues by polymerase chain reaction (PCR) and PCR in situ hybridization.

In this study, experiments were designed to investigate the distribution of bovine immunodeficiency virus (BIV) proviral DNA in the tissues and cells of infected calves by solution-phase polymerase chain reaction (SP-PCR) and PCR in situ hybridization (PCR-ISH). Total DNA samples extracted from tissues of 10 BIV-infected and 5 uninfected calves were amplified by SP-PCR with the primers directed to the BIV conserved pol gene segment. The identity of the SP-PCR product was confirmed by Southern hybridization with a BIV pol gene cDNA probe. SP-PCR results demonstrated that BIV proviral DNA was present predominantly in neural tissues and some lymphoid tissues in BIV-infected calves. It also was detected frequently in other tissues including lung, heart, esophagus, and pancreas. Further investigation on cell location of BIV proviral DNA was performed by in situ amplification of DNA on formalin-fixed tissue sections. The amplified DNA was subjected to in situ hybridization with an internal biotinylated probe and detected with streptavidin-gold followed by silver enhancement. Specific BIV proviral DNA signals were observed in neurons, microglial cells, lymphocytes, septal macrophages, smooth muscle cells, and endothelial cells. On the basis of these results, we conclude that BIV replicates in a variety of bovine tissues in vivo and has a broad cell tropism.

Closely related genes encode developmental and tissue isoforms of porcine cytochrome P450 aromatase.

We examined the chromosomal basis for the synthesis of tissue (ovary, endometrium/placenta, and peri-implantation blastocyst) isoforms of cytochrome P450 aromatase in the pig. DNA fragments derived from three distinct porcine aromatase chromosomal genes were cloned and characterized. The porcine type III aromatase gene encoding the blastocyst aromatase isoform was found to consist of nine coding exons and two mutually exclusive, 5' untranslated exons (designated E1A and E1B), collectively spanning 30 kb or more. The porcine type II aromatase gene, encoding the endometrial/placental aromatase isoform, was identified by cloning of a genomic DNA fragment spanning the corresponding exons 7, 8, and 9. The DNA inserts of two other phage clones encompassed exons 2, 3, and 4 of a third chromosomal gene (type I) encoding the ovarian aromatase isoform. All intron-exon junctions in these genomic fragments were found to be identical in relative positions to those of the single-copy human aromatase gene. Comparisons of cDNA and genomic sequences indicated that nucleotide sequence variation was not uniform across the corresponding exons of these genes and that the corresponding intronic sequences were conserved. The type II and type III aromatase genes were localized to the same regional location (q16-17) on swine chromosome 1, which is homologous to the human chromosome 15 region (q21.1) in which the human aromatase gene resides. Results demonstrate that the three aromatase genes characterized in the present study appear to be similar in their overall structural organization and most likely are clustered, which could have resulted from at least two independent gene duplication events. The presence of multiple aromatase genes constitutes a newly described mechanism by which aromatase enzyme biosynthesis and functional activity can be regulated in a tissue and temporal fashion and serves to highlight further the complexity of aromatase gene expression in mammals. Moreover, the presence of a unique aromatase gene that is highly expressed in pig blastocysts may constitute a paradigm for other mammals (e.g., equids, rabbit, hamster) whose peri-implantation blastocysts are estrogenic.

Physical assignments of 68 porcine cosmid and lambda clones containing polymorphic microsatellites.

Two lambda phage and 66 cosmids containing informative porcine microsatellites were assigned to 17 of 18 porcine autosomes and the X Chromosome (Chr) by fluorescence in situ hybridization (FISH). These assignments provide additional physically anchored markers to integrate the porcine physical and genetic maps.

Physical mapping of SOD1 to bovine chromosome 1.

Superoxide dismutase 1 (SOD1) was mapped to cattle chromosome 1q12 --> q14 by in situ methods. Both traditional in situ hybridization using tritium and a new technique, direct in-situ single copy PCR (DISC-PCR), were used in two separate laboratories. Both human and bovine SOD1 clones were tritium labeled for radioactive in situ hybridization. A primer pair based on the bovine SOD1 gene (Barendse et al., 1994b) was used for the DISC-PCR procedure. The map location of SOD1 is close to collagen 6A1. SOD1 is a potentially important type 1 anchor locus in the region where the gene for horns in cattle was recently mapped (Georges et al., 1993; Schmutz et al., 1995).

Directed integration of the physical and genetic linkage maps of swine chromosome 7 reveals that the SLA spans the centromere.

The first integrated physical and genetic linkage map encompassing the entire swine chromosome 7 (SSC7) reveals that the porcine MHC (SLA) spans the centromere. A SLA class II antigen gene lies on the q arm, whereas class I and III genes lie on the p arm, suggesting that the presence of a centromere within the SLA does not preclude a functional complex. The SLA appears smaller than other mammalian MHC, as the genetic distance across two class I, three class II, and three class III SLA gene markers is only 1.1 cM. There are significant variations in recombination rates as a function of position along the chromosome, and the SLA lies in the region with the lowest rate. Furthermore, the directed integration approach used in this study was more efficient than previous efforts that emphasized the screening of large insert libraries for random microsatellites.

Use of direct in situ single-copy (DISC) PCR to physically map five porcine microsatellites.

Direct in situ single-copy polymerase chain reaction (DISC-PCR) was used to assign and orient a linkage group to pig chromosome 1. Five microsatellites were analyzed, and all five were successfully localized using this procedure. Physical data were used to orient the linkage group with respect to the centromere and estimate the amount of coverage of chromosome 1. There was excellent concordance between the physical and linkage maps. The linear order of the microsatellites was identical, and relative distances were similar. All markers were located on the long arm of chromosome 1. Coverage was estimated at about 32%. Thus, DISC-PCR rapidly and easily assigned and ordered microsatellite markers for which large genomic clones do not exist.

Reduced numbers and intense anti-ubiquitin immunostaining of bovine motor neurons affected with spinal muscular atrophy.

Motor neurons in the spinal cord affected with bovine spinal muscular atrophy (SMA) were investigated immunohistochemically using antibodies against bovine ubiquitin. Anti-ubiquitin immunostained many chromatolytic and swollen degenerating motor neurons in the ventral horn of the SMA-affected spinal cord. The most severely swollen cells showed a lightly-stained center and a strongly-stained periphery after immunostaining. However, there were many dark, shrunken neurons in a more advanced stage that showed a completely negative reaction when immunostained. Motor neuron counts differed significantly between SMA-affected and normal animals at the lumbar intumescence and at the fourth lumbar neuromere, but not at the brachial intumescence or the second cervical level.

Gross, microscopic and ultrastructural lesions of protoporphyria in Limousin calves.

The gross, microscopic and ultrastructural lesions associated with the genetic disease, protoporphyria, in Limousin cattle were studied in detail. The clinical signs and lesions were most severe in young animals. In the liver, the lesions consisted of portal fibroplasia, bile ductule hyperplasia, parenchymal cell swelling, and pigment accumulation. Maltese cross-like crystals were evident under polarized light. Ultrastructurally, there were large secondary lysosomes comprised of electron-dense granules associated with lipid droplets in hepatocytes. Phagocytic cells in the dermis also contained large heterogeneous secondary lysosomes.

A combination histochemical stain for equine muscle.

The purpose of this study was to find a combination histochemical staining technique for the evaluation of equine skeletal muscle that is reliable and effective, while offering a substantial reduction in the labor and cost involved with currently used individual histochemical methods. Several combinations under varying conditions of pH were studied. The most uniform results were obtained using an acid preincubation step at an optimal pH of 4.2 followed by reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) and the remainder of the acid-ATPase procedure.

Rectovaginal constriction in Jersey cattle: genetics and breed dynamics.

Data from farmer-owned herds and from experimental matings supported monofactorial recessive inheritance of rectovaginal constriction in US Jersey cattle. Kempthorne's population genetics model of a recessive trait involving only male selection was extended to include mutation and converted to selection of females only. Computer analyses with that model estimate slow decline in the frequency of the gene for rectovaginal constriction. Practical dynamics of the disorder in a breed registering 50,000 females and 2,000 males annually are given for current conditions and after 500 generations of selection.