Fibroepithelial polyps of the vagina in bitches: a histological and immunohistochemical study.

The histological and immunohistochemical features of 13 cases of suspected vaginal fibroepithelial polyps are reported. The characteristic microscopical features of these lesions were an abundant oedematous or fibrous stroma containing spindle-shaped and stellate cells and the presence of variable inflammation and haemorrhage. There was often a superficial layer of compressed tissue, but the stroma in the peripheral areas of the masses was generally more loosely arranged than in central areas. The connective tissue cells expressed vimentin and desmin, but did not express smooth muscle actin or calponin. Individual cases had additional changes including granulomatous inflammation, epithelial dysplasia suggestive of papillomavirus infection and a lesion resembling phyllodes tumour in women.

Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent [E1(-), E2b(-)] adenoviral vectors.

The systemic delivery of [E1(-)] adenoviral (Ad) vectors encoding a transgene results in efficient viral uptake and abundant transgene expression in the liver. However, [E1(-)]Ad vector persistence is transient due to cytotoxic T lymphocyte (CTL)-mediated loss of the Ad-infected cells. Our laboratory has previously demonstrated that additional modifications to the [E1(-)]Ad vector genome, by deletion of the Ad E2b genes, significantly decreased virus-genome-derived gene expression and simultaneously improved the long-term performance of the resultant [E1(-), E2b(-)]Ad vector. In this study, we confirmed that [E1(-), E2b(-)]Ad vector genomes could persist equally well in C57Bl/6 or Balb/c mouse hepatocytes. Despite vector genome persistence, we observed a strain-dependent variability in the duration of CMV enhancer/promoter-driven transgene expression in the liver. While Balb/c mice rapidly shut down [E1(-), E2b(-)]Ad-derived transgene expression, C57Bl/6 mice allowed for prolonged transgene expression. This occurred even when both strains were crossed into a severe combined immune-deficient background, demonstrating that host adaptive immune responses are not responsible for the phenomenon. Furthermore, differential methylation of the CMV enhancer/promoter was also not demonstrated in either strain of mouse, eliminating this mechanism as causative. Thus, alternative mechanisms for this phenomenon are discussed.

An allelic series of mutations in the kit ligand gene of mice. I. Identification of point mutations in seven ethylnitrosourea-induced Kitl(Steel) alleles.

An allelic series of mutations is an extremely valuable genetic resource for understanding gene function. Here we describe eight mutant alleles at the Steel (Sl) locus of mice that were induced with N-ethyl-N-nitrosourea (ENU). The product of the Sl locus is Kit ligand (or Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is a member of the helical cytokine superfamily and is the ligand for the Kit receptor tyrosine kinase. Seven of the eight ENU-induced Kitl(Sl) alleles, of which five cause missense mutations, one causes a nonsense mutation and exon skipping, and one affects a splice site, were found to contain point mutations in Kitl. Interestingly, each of the five missense mutations affects residues that are within, or very near, conserved alpha-helical domains of Kitl. These ENU-induced mutants should provide important information on structural requirements for function of Kitl and other helical cytokines.

An allelic series of mutations in the Kit ligand gene of mice. II. Effects of ethylnitrosourea-induced Kitl point mutations on survival and peripheral blood cells of Kitl(Steel) mice.

The ligand for the Kit receptor tyrosine kinase is Kit ligand (Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is encoded at the Steel (Sl) locus of mice. Previous studies revealed that Kitl(Sl) mutations have semidominant effects; mild pigmentation defects and macrocytic, hypoplastic anemia occur in heterozygous mice, and more severe pigmentation defects and anemia occur in homozygotes. Lethality also occurs in mice homozygous for severe Kitl(Sl) mutations. We describe the effects of seven new N-ethyl-N-nitrosourea (ENU)-induced Kitl(Sl) mutations and two previously characterized severe Kitl(Sl) mutations on pigmentation, peripheral blood cells, and mouse survival. Mice heterozygous for each of the nine mutations had reduced coat pigmentation and macrocytosis of peripheral blood. In the case of some of these mutations, however, red blood cell (RBC) counts, hemoglobin concentrations, and hematocrits were normal in heterozygotes, even though homozygotes exhibited severely reduced RBC counts and lethality. In homozygous mice, the extent of anemia generally correlates with effects on viability for most Kitl(Sl) mutations; i.e., most mutations that cause lethality also cause a more severe anemia than that of mutations that allow viability. Interestingly, lethality and anemia were not directly correlated in the case of one Kitl(Sl) mutation.

The neuronatin gene resides in a "micro-imprinted" domain on human chromosome 20q11.2.

A small fraction of the genome contains genes that are imprinted and thus expressed exclusively from one parental allele. We report here that the human neuronatin gene (NNAT) on chromosome 20q11.2 is imprinted and transcribed specifically from the paternal allele. The region containing NNAT has multiple CpG islands, and methylation analysis showed that a 1.8-kb CpG island in its promoter region exhibits differential methylation in all tissues examined. This finding is consistent with the island acting as a component of the NNAT imprint control domain. NNAT lies within the singular 8.5-kb intron of the gene encoding bladder cancer-associated protein (BLCAP), which, as we demonstrate, is not imprinted. This study provides the first example, to our knowledge, in humans of an imprinted gene contained within the genomic structure of a nonimprinted gene. Thus, NNAT is in an imprinted "microdomain," making this locus uniquely suited for the investigation of mechanisms of localized imprint regulation.

Adenovirus vectors with the 100K gene deleted and their potential for multiple gene therapy applications.

The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K-]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1-,100K-]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K-]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K-]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1-,100K-]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K-]Ad vectors suggest that they may have utility in a variety of gene therapy applications.

Dopamine D1 autoreceptor function: possible expression in developing rat prefrontal cortex and striatum.

Synthesis-modulating dopamine (DA) autoreceptor function was studied in vivo using gamma-butyrolactone (GBL) to block propagation along DA axons. DA synthesis was measured by the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) after inhibition of aromatic L-amino acid decarboxylase. GBL treatment markedly increased DOPA accumulation in both the striatum and prefrontal cortex of developing rats. The selective DA partial D1 agonist SKF-38393 inhibited this GBL-induced rise in DA synthesis in both the striatum and prefrontal cortex of 15- and 22-day-old rats, but not in adults. The effects of SKF-38393 in developing rats were mimicked by the non-catechol D1 partial agonist CY-208-243, and were blocked by the D1 antagonist SCH-23390, suggesting receptor mediation. The mixed D2/D3 agonist quinpirole attenuated DA synthesis in striatum of both two-week-old and adult rats, but failed to inhibit the GBL-induced increase in DA synthesis in the developing prefrontal cortex. These findings suggest that synthesis-modulating D1-like receptor function may emerge transiently in the developing mammalian forebrain. In the adult striatum these functions appear to be subsumed by D2-like receptors, whereas all synthesis-modulating DA receptor function in prefrontal cortex appears to be essentially lost with maturation.

Morphological assessment of neuronal aggregates in the striatum of the rat.

Regional variations in cell-packing density, culminating in the formation of cell clusters, is now a recognized morphological characteristic of the striatum that has been correlated, in some instances, with either regional histochemical variations or the distribution pattern of afferent fiber systems, or both. Within these cluster regions a further level of organization exists, in the form of discrete neuronal aggregates. The light microscopic morphology of these neurons and the nature of their intercellular contacts at the electron microscope level form the focus of this report. The neurons composing such aggregates are characterized by contiguous soma-somatic or soma-dendritic contact with extended regions of junctionlike symmetrical and consistent contacts where the distance between the cytoplasmic membranes of apposing neurons narrows to as close as 7 nm. Coated vesicles close to the contact areas are common. Three-dimensional computer reconstructions of serial 1 micron sections through aggregates in either the caudatoputamen or nucleus accumbens reveal "chains" of contiguous cells that frequently involve as many as 60 neurons. These contiguous cell aggregates are discrete entities within the larger clusters or islands. It is postulated that the cellular aggregates may represent the fundamental level of striatal organization and may be local modules for intrinsic information processing, modifying extrinsic data processed through the biochemical compartmentalization of the striatum imparted by striosomes, neuropeptides, and dopaminergic, thalamic and cortical afferents.

Dietary recall method comparison for hospitalized elderly subjects.

A study was made to compare accuracy of the 24-hour recall, including both number of items recalled and amounts of food eaten, for elderly subjects using three methods: I, unaided oral recall, II, printed questionnaire, and III, interview with actual-size color pictures of foods. Sixty hospitalized patients, age 65 to 94, were interviewed. Factors studied for influence on scores were age, sex, educational level, obesity, place of residence, and food preparer. Results showed no significant difference in scores owing to method used, except that 30 subjects above the median age of 76 scored higher on number of items recalled and amounts of food eaten when using the printed questionnaire. None of the demographic factors studied made a significant difference in scores.