Cellular components of the immune barrier in the spinal meninges and dorsal root ganglia of the normal rat: immunohistochemical (MHC class II) and electron-microscopic observations

This report deals with the distribution, morphology and specific topical relationships of bone-marrow-derived cells (free cells) in the spinal meninges and dorsal root ganglia of the normal rat. The morphology of these cells has been studied by transmission and scanning electron microscopy. Cells expressing the major histocompatibility complex (MHC) class II gene product have been recognized by immunofluorescence. At the level of the transmission electron microscope, free cells are found in all layers of the meninges. Many of them display characteristic ultrastructural features of macrophages, whereas others show a highly vacuolated cytoplasm and are endowed with many processes. These elements lack a conspicuous lysosomal system and might represent dendritic cells. Scanning electron microscopy has revealed that free cells contact the cerebrospinal fluid via abundant cytoplasmic processes that cross the cell layers of the pia mater and of the arachnoid. Cells expressing the MHC class II antigen are also found in all layers of the meninges. They are particularly abundant in the layers immediately adjacent to the subarachnoid space, in the neighbourhood of dural vessels, along the spinal roots and in the dural funnels. In addition to the meninges, strong immunoreactivity for MHC class II antigen is observed in the dorsal root ganglia. The ultrastructural and immunohistochemical findings of this study suggest the existence of a well-developed system of immunological surveillance of the subarachnoid space and of the dorsal root ganglia.     

Molecular evaluation of an Alu repeat including a polymorphic variable poly(dA) (AluVpA) in the vitamin D binding protein (DBP) gene

We investigated an Alu element at the end of intron 8 of the human vitamin D-binding protein (hDBP, group-specific component, GC) gene that shows a polymorphic poly(A) tail due to a variable number of tandem repeats (AluVpA) forming the 3 end of this member of the most abundant class of short interspersed repeated DNA element (SINES). The Alu element sequence in intron 8 of the GC gene was identical in all three common GC alleles (GC*1F, GC*1S, and GC*2) and could be classified as an Alu-Sa or Alu class-II sequence. The polymerase chain reaction was used to amplify selectively a fragment of about 200 bp containing the identified (TAAA)n repeat from genomic DNA of 188 unrelated human subjects. The size of the amplified products was determined by polyacrylamide gel electrophoresis. Four alleles (named GC-18*6, GC-I8*8, GC-I8*10, and GC-18*11) were found that differed in size by multiples of four nucleotides. The allele frequencies ranged from 0.0053 to 0.8511 and the observed heterozygosity was 26%. The stable inheritance of this polymorphic patterned poly(A) sequence was confirmed by a segregation study of a highly informative family with 19 members. Statistically significant linkage disequilibrium between the AluVpA and the GC isoelectric focusing (IEF) phenotypes was found in a sample of 188 unrelated individuals and delta values were calculated from the observed haplotype distribution.     

Involvement of Ubiquitin in Phosphoenolpyruvate Carboxylase Degradation

Western immunoblot analysis of protein extracts prepared from epidermal peels, whole leaves, and mesophyll protoplasts with ubiquitin and PEPCase antibodies indicated ubiquitinated PEPCase bands and degradation products only in crude extracts which have been obtained in the presence of the proteolysis inhibitors leupeptin and hemin. After ammonium sulfate precipitation and further purification, PEPCase forms were stable and not ubiquitinated. It is assumed, that only a certain part of PEPCase is degraded via the ubiquitin-dependent proteolysis.     

Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells

Early dissemination of malignant cells is the main cause for metastatic relapse in patients with solid tumours. By use of monoclonal antibodies (mAbs) specific for cytokeratins, disseminated individual epithelial tumour cells can now be identified in mesenchymal organs such as bone marrow. Further to characterize such cells in patients with prostate cancer, an immunocytochemical procedure was developed for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). In a first step, cells were incubated with mAb ER-PR8 against PSA and secondary gold-conjugated goat anti-mouse antibodies. In a second step, biotinylated mAb CK2 to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with the Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. The sensitivity and specificity of the technique was demonstrated on cryostat sections of hyperplastic prostatic tissue, and cytological preparations of LNCaP prostatic tumour cells. Double staining was restricted to cells derived from the secretory epithelium of the prostate. Cross-reactivity between both detection systems was excluded by several controls, including the use of unrelated antibodies of the same isotype and the staining of CK18⁺/PSA^– HT29 colon carcinoma cells. CK18⁺ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 13 patients with carcinomas of the prostate, a finding that is consistent with the relative fraction of double-positive LNCaP cells. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hypertrophy. In conclusion, the approach presented appears to be a reliable method to phenotype individual prostatic carcinoma cells.     

Kidney structure and function of obligate and facultative hibernators: the white-tailed prairie dog (Cynomys leucurus) and the black-tailed prairie dog (Cynomys ludovicianus)

The white-tailed prairie dog is an obligate hibernator that enters a heterothermic phase when maintained in the cold with low intensity light and ad libitum food and water. The black-tailed prairie dog (a facultative hibernator) will not hibernate under similar conditions. It has been suggested that the black tailed prairie dog remains active during the winter because it can conserve water more effectively due to a more efficient kidney. The present study revealed no significant differences between the species in renal morphology: relative medullary thickness, nephron heterogeneity, renal vasculature, or fornix dimensions, all of which are structures associated with the urinary concentrating mechanism. In addition, there was no difference in number of nephrons between the two species. The black-tailed prairie dog does produce a more concentrated urine when food and water deprived. However, this difference was not observed when the animals were salt loaded. The water-deprivation and salt-loading experiments suggest that the higher urine osmolality produced by the back-tailed prairie dog during fasting is a result of a higher urea load due to a greater protein catabolism and not because of a differential capacity to concentrate urine.Abbreviations C cortex - GFR glomerular filtration rate - H height - IS inner stripe - IZ inner zone of medulla - L length - OS outer stripe - PE polythylene - RMT relative medullary thickness - T _a ambient temperature - W width     

Identification of receptor binding sites by competitive peptide mapping: phages T1, T5, and phi 80 and colicin M bind to the gating loop of FhuA.

Previously we proposed a transmembrane model of the FhuA receptor protein in the outer membrane of Escherichia coli. Removal of the largest loop at the cell surface converted the FhuA transport protein into an open channel and rendered cells resistant to the FhuA-specific phages T1, T5, and phi 80 and to colicin M. In the present study we employed acetylated hexapeptide amides covering the entire surface loop to investigate binding of the phages and of colicin M. Competitive peptide mapping proved to be a powerful technique to uncover three ligand binding sites within a region of 34 amino acid residues. Hexapeptides derived from three specific regions of the surface loop inhibited infection of cells by the phages and killing by colicin M. Two of these regions were common among all four FhuA ligands. Electron microscopy of phage T5 revealed that one inhibitory peptide triggered a strong conformational change leading to the release of DNA from the phage head. These results suggest that the FhuA gating loop is the target for specific binding of phages T1, T5, and phi 80 and colicin M.