Quantitative microphotometric studies of Feulgen-stained nuclei on sections of the human uterine cervix: optimization of preparation and measuring technique

The total extinction of apparently normal intermediate cell nuclei was microphotometrically measured in formalin-fixed Feulgen-stained paraffin sections of the uterine cervix. We investigated whether reproducible microphotometric results can be obtained from a histologic section expected to contain a certain amount of sectioned nuclei and stained by a standard technique. The results have shown that exact reproduction of microphotometrically measured mean total extinction values of intermediate cell nuclei can be achieved with a threshold value of 90% transmission in 10 microns sections.     

Substance P and enkephalin in transected axons of medulla and spinal cord

The accumulation of transported materials in cut axons is demonstrated by the light and electron microscopic immunocytochemical localization of substance P and enkephalin in the caudal medulla and cervical spinal cord of adult rat. Two days following unilateral knife-cuts in the caudal medulla or spinal (C2-C3) levels, substance P and enkephalin-like immunoreactivity (SPLI and ELI) are detected in lesioned axons located rostral and caudal to the transection. Rostrally, SPLI and ELI are detected in the lateral reticular region and ventrolateral fasciculus corresponding to the location of previously identified bulbospinal pathways. Caudally, previously unidentified, propriospinal pathways showing SPLI are detected in the dorsal columns and in the dorsolateral fasciculus. In contrast, ELI is found caudal to the transection only in the reticular region of the medulla. For both peptides, immunoreactivity is present throughout axons containing numerous large, dense core, and small clear vesicles. These results support the concept of both particulate and soluble modes of transport for substance P and enkephalin within axons of the central nervous system.     

Suppressor T cells arising in mice undergoing a graft-vs-host response.

We investigated the ability of mice to generate antibody-forming cells when undergoing a graft-vs-host reaction. (C57BL/6 X DBA/2)F1 mice (BDF1) injected with C57BL/6 spleen cells generated suppressor T cells which inhibit antibody synthesis by BDF1 spleen cells in vitro. These T cells arose from the donor inoculum. They differ from helper T cells in size and they act directly on antigen reactive B cells. The suppressor T cells were specifically directed against components of the H-2 region of the reciprocal parental strain (DBA/2 = H-2d) in the hybrid F1 mouse.     

Exfoliative cytology of a lymphoepithelioma-like carcinoma in a cervical smear. A case report

BACKGROUND: Lymphoepithelioma-like carcinoma of the cervix (LELC) is cytologically identical to its counterparts at other sites, such as the nasopharynx. LELC can be suspected on a cervical cytologic smear. The differential diagnosis includes nonkeratinizing squamous cell carcinoma with prominent stromal inflammation, carcinoma with intense stromal eosinophilia, glassy cell carcinoma, malignant lymphoma (especially lymphoepitheloid-Lennerts lymphoma) and metastatic Schmincke-Regaud tumor. CASE: A 55-year-old female presented with an ulcerated endophytic tumor in the cervix. Exfoliative cytology showed uniform, large tumor cells, often associated with inflammatory cells, with round or oval nuclei and one or more prominent nucleoli. The cytoplasm was finely granular to flocculent, and the nuclei were uniformly vesicular. The chromatin was peripherally marginated. The cell borders were indistinct. There was no evidence of dyskeratotic or keratinized cells, koilocytes or glandlike formations. These findings were highly suspicious for LELC and were confirmed by biopsy. Flow cytometry showed DNA aneuploidy, with a DNA index of 1.08. In situ hybridization was negative for human papillomavirus 16 and 18. CONCLUSION: LELC of the uterine cervix has cytologic features that are sufficiently characteristic for a specific cytologic diagnosis. The diagnosis, nevertheless, has to be proven by histology.     

Amino acid residues in the carboxy-terminal region of cottontail rabbit papillomavirus E6 influence spontaneous regression of cutaneous papillomas

Previous studies have identified two different strains of cottontail rabbit papillomavirus (CRPV) that differ by approximately 5% in base pair sequence and that perform quite differently when used to challenge New Zealand White (NZW) rabbit skin. One strain caused persistent lesions (progressor strain), and the other induced papillomas that spontaneously regressed (regressor strain) at high frequencies (J. Salmon, M. Nonnenmacher, S. Caze, P. Flamant, O. Croissant, G. Orth, and F. Breitburd, J. Virol. 74:10766-10777, 2000; J. Salmon, N. Ramoz, P. Cassonnet, G. Orth, and F. Breitburd, Virology 235:228-234, 1997). We generated a panel of CRPV genomes that contained chimeric and mutant progressor and regressor strain E6 genes and assessed the outcome upon infection of both outbred and EIII/JC inbred NZW rabbits. The carboxy-terminal 77-amino-acid region of the regressor CRPV strain E6, which contained 15 amino acid residues that are different from those of the equivalent region of the persistent CRPV strain E6, played a dominant role in the conversion of the persistent CRPV strain to one showing high rates of spontaneous regressions. In addition, a single amino acid change (G252E) in the E6 protein of the CRPV progressor strain led to high frequencies of spontaneous regressions in inbred rabbits. These observations imply that small changes in the amino acid sequences of papillomavirus proteins can dramatically impact the outcome of natural host immune responses to these viral infections. The data imply that intrastrain differences between separate isolates of a single papillomavirus type (such as human papillomavirus type 16) may contribute to a collective variability in host immune responses in outbred human populations.     

Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry,Confocal and Super-Resolution Microscopy

Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol P _ino [m/s] and expression/localization of SLC5A3. P _ino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), P _ino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in P _ino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm². Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.     

A humanized IKBKAP transgenic mouse models a tissue-specific human splicing defect

Familial dysautonomia (FD) is a severe hereditary sensory and autonomic neuropathy, and all patients with FD have a splice mutation in the IKBKAP gene. The FD splice mutation results in variable, tissue-specific skipping of exon 20 in IKBKAP mRNA, which leads to reduced IKAP protein levels. The development of therapies for FD will require suitable mouse models for preclinical studies. In this study, we report the generation and characterization of a mouse model carrying the complete human IKBKAP locus with the FD IVS20+6T-->C splice mutation. We show that the mutant IKBKAP transgene is misspliced in this model in a tissue-specific manner that replicates the pattern seen in FD patient tissues. Creation of this humanized mouse is the first step toward development of a complex phenotypic model of FD. These transgenic mice are an ideal model system for testing the effectiveness of therapeutic agents that target the missplicing defect. Last, these mice will permit direct studies of tissue-specific splicing and the identification of regulatory factors that play a role in complex gene expression.     

A model of dirigent proteins derived from structural and functional similarities with allene oxide cyclase and lipocalins

Dirigent proteins impart stereoselectivity on the phenoxy radical-coupling reaction, yielding optically active lignans from two molecules of coniferyl alcohol. By an unknown mechanism, they direct the coupling of two phenoxy radicals toward the formation of optically active (+)- or (-)-pinoresinol. We show here that the dirigent protein AtDIR6 from Arabidopsis thaliana is a homodimeric all-beta protein in the superfamily of calycins. Based on its homology with calycins, the structure of AtDIR6 was modeled using allene oxide cyclase as template. The structural model of AtDIR6 was supported experimentally by confirmation of a predicted disulfide bridge and by the characterization of two N-linked glycans at the solvent-exposed protein surface. The model shows AtDIR6 as an eight-stranded antiparallel β-barrel with a central hydrophobic cavity for substrate binding, suggesting that dirigent proteins evolved from hydrophobic ligand-binding proteins. The data are fully consistent with the current view of the dirigent protein mode of action, according to which each subunit of the homodimer captures one of the substrate radicals and orients them in a way that precludes undesired reaction channels, thus favoring the formation of the optically pure coupling product.     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.     

Neuropeptide Y and peptide YY neuronal and endocrine systems

An extensive system of neuropeptide Y (NPY) containing neurons has recently been identified in the central and peripheral nervous system. In addition, NPY and a structurally related peptide, peptide YY (PYY), containing endocrine cells have been identified in the periphery. The NPY system is of particular interest as the peptide coexists with catecholamines in the central and sympathetic nervous system and adrenal medulla. Evidence has been presented which indicates that NPY may play important roles in regulating autonomic function.