Modulation of rabbit keratocyte production of collagen, sulfated glycosaminoglycans and fibronectin by retinol and retinoic acid.

This study elucidates the biochemical response of rabbit corneal keratocytes (fibroblasts) to retinol and retinoic acid in their production of collagen, fibronectin, sulfated glycosaminoglycans, collagenase, and [3H]thymidine incorporation. The morphologic appearance of cultured keratocytes was not altered by retinoid treatment. Collagen production and [3H]thymidine incorporation demonstrated a parallel decline in response to retinoids. Collagen type was unaffected as was collagenase activity. Retinoids had minimal effect on cell layer-associated 35S-labeled glycosaminoglycans, however medium-soluble 35S-glycosaminoglycans were increased. The most dramatic effect was in fibronectin synthesis which was increased 2-3-fold. These data demonstrate that rabbit keratocytes alter their biosynthesis of extracellular matrices in response to retinoids. This may be significant in corneal pathology, since the delicate balance of these extracellular macromolecules is responsible for corneal integrity and stability.

The effects of boric acid (BA) on testicular cells in culture.

High-dose boric acid (BA) exposure produces testicular lesions in adult rats characterized by inhibited spermiation that may progress to nonrecoverable atrophy. The mechanism for the testicular toxicity of BA is unknown. To examine possible direct effects, the present study evaluated selected aspects of various testicular cell culture systems after in vitro BA exposure. Specifically, the hallmarks of the BA testicular toxicity were addressed: the mild hormone effect, the initial inhibition of spermiation, and atrophy. No effect of BA on the steroidogenic function of isolated Leydig cells was observed, supporting the contention of a CNS-mediated rather than a direct hormone effect. Since increased testicular cyclic AMP (cAMP) produces inhibited spermiation, and a role for the serine proteases plasminogen activators (PAs) in spermiation has been proposed, we evaluated both Sertoli cell cAMP accumulation in Sertoli-germ cell cocultures and the stage-specific secretion of PA activity in cultured seminiferous tubules after in vitro BA exposure, respectively. The results showed that the inhibited spermiation is not due to BA effects on either process. To address the atrophy, we evaluated BA effects in Sertoli-germ cell cocultures on 1) morphology/germ cell attachment, which might identify a target cell; 2) Sertoli cell energy metabolism, because lactate, secreted by Sertoli cells, is a preferred energy source for germ cells; and 3) DNA/RNA synthesis, because germ cells synthesize DNA/RNA and BA impairs nucleic acid synthesis in liver and may do so in testis. Despite the absence of overt morphologic changes and germ cell loss, the most sensitive in vitro endpoint was DNA synthesis of mitotic/meiotic germ cells, with energy metabolism in Sertoli or germ cells affected to a lesser extent. A re-evaluation of testis sections from rats exposed to BA revealed a decrease in the early germ cell/Sertoli cell ratio prior to atrophy. Thus, although the mechanism for the inhibited spermiation is still undefined and is the subject of future work, these combined studies revealed some changes offering a plausible explanation for the atrophy aspect of the BA testicular lesion.

Cloning and characterization of a genomic probe for malignant catarrhal fever virus.

A genomic probe specific for malignant catarrhal fever (MCF) virus was cloned by using purified viral DNA from MCF-virus strain WCll. Restriction endonuclease analysis of the purified viral DNA was used to identify the cloned viral genomic fragment. Dot blot hybridization by use of the genomic probe (pRP-5) indicated that the probe hybridized specifically with WCll-MCF virus, as well as with one other isolate of MCF-associated herpesvirus. Hybridization also was observed to a non-MCF virus strain of bovine herpesvirus.

Comparison of herpesvirus isolates from falcons, pigeons and psittacines by restriction endonuclease analysis.

Field isolates of herpesviruses recovered from falcon, pigeon, and psittacine birds were compared by restriction endonuclease (RE) analysis using four separate enzymes. Pigeon and falcon herpesviruses had strikingly similar DNA cleavage patterns, while DNA cleavage pattern of virus isolates from a double-yellow headed Amazon and an African grey parrot had different genomic patterns to both the pigeon and falcon herpesviruses. These findings support the field observations that pigeon herpesvirus causes a fatal herpesviral infection in the livers of pigeon-eating falcons.

Nucleotide sequence of a 3.5 kilobase fragment of malignant catarrhal fever virus strain WC11.

A 3.5 kilobase DNA fragment of the malignant catarrhal fever virus (MCFV), strain WC11, was mapped with a number of restriction enzymes, subcloned and sequenced. The fragment was subcloned into plasmid vector, pUC19, for direct sequencing. A complete open reading frame of 2,058 base pairs and a partial open reading frame of 630 base pairs were identified. The sequence of 3,389 nucleotides was compared to other herpesviruses. A 310 base pairs sequence in gene A was 57% homologous to a sequence in reading frame BXLF1 of Epstein-Barr virus strain B95-8.

Degradation of rRNA in Salmonella strains: a novel mechanism to regulate the concentrations of rRNA and ribosomes.

We have previously shown that the 23S rRNA of Salmonella strains is highly fragmented by specific enzyme cleavages. In this article, we report that 23S rRNA of Salmonella strains is rapidly degraded as the cells enter the stationary phase. More than 90% of the 23S rRNA is degraded when the cells reach the stationary phase. The rate of degradation of 23S rRNA correlated with its degree of fragmentation. This degradation is probably mediated by newly synthesized protein factor(s), since treatment with chloramphenicol or rifampin inhibits the rRNA degradation. We propose that degradation of 23S rRNA is a novel mechanism in the regulation of the bacterial 23S rRNA and ribosome concentration and that this additional regulatory mechanism provides some selective advantage to cells.

Comparison of genomes of malignant catarrhal fever-associated herpesviruses by restriction endonuclease analysis.

The restriction endonuclease DNA cleavage patterns of eight isolates of malignant catarrhal fever-associated herpesviruses were examined using the restriction endonucleases HindIII and EcoRI. The eight viruses could be assigned to two distinct groups. Virus isolates from a blue wildebeest, a sika deer and an ibex had restriction endonuclease DNA cleavage patterns that were in general similar to each other. The restriction pattern of these three viruses was distinct from the other five. Of these five, four were isolated from a greater kudu, a white tailed wildebeest, a white bearded wildebeest, and a cape hartebeest. The fifth isolate C500, was isolated from a domestic cow with malignant catarrhal fever. These five viruses had similar DNA cleavage patterns.

Genetic diversity of avian infectious bronchitis virus California variants isolated between 1988 and 2001 based on the S1 subunit of the spike glycoprotein.

Twenty-nine isolates of avian infectious bronchitis virus (IBV) recovered from commercial chicken flocks in California between 1988 and 2001 and identified as California variants by serotype and direct automated cycle sequencing of the IBV spike glycoprotein S1 subunit, were further characterized phylogenetically and by nucleotide sequence comparison. California variants were grouped according to production type of chicken, by comparison with public access sequence databases (NCBI GenBank and EMBL), or based on phylogenetic analysis. Fisher's Exact test was used to compare mutations per year, purifying and positive selection, predictive antigenicity, and a > or = 6 bp deletion between California variant groups.A high number of mutations at the nucleotide level ( p = 0.013) and a > or = 6 bp deletion in the nucleotide sequence ( p = 0.006) was significantly associated with broiler-type chickens. However, 88% of significant comparisons at the amino acid level such as purifying and positive selection were seen in layer-type chickens. A pronounced predictive antigenicity in the HVR2 region was also associated with layer-type chickens ( p = 0.001). The study indicates that IBV in California is in a phase of slow evolution with different evolutionary patterns being associated with the production type of chicken.

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers.

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.

A diagnostic method to detect alcelaphine herpesvirus-1 of malignant catarrhal fever using the polymerase chain reaction.

A sensitive diagnostic method specific for alcelaphine herpesvirus-1, causative agent of malignant catarrhal fever, has been developed. Based on the nucleotide sequence of the alcelaphine herpesvirus-1 genomic DNA, a pair of 30 nucleotide primers was selected and synthesized for detecting the virus genome using the polymerase chain reaction (PCR). The virus genome was detected in crude cell lysate using the amplification reaction.

Diversity of cleavage patterns of Salmonella 23S rRNA.

The recent discovery of the phenomenon that some prokaryotes fragment their 23S rRNA during post-transcriptional processing of precursor rRNA has been shown to be particularly prevalent among strains and species of Salmonella. Some strains of Salmonella cleaved 23S rRNA at multiple sites producing several fragments. The cleavage patterns of 23S rRNA differed among Salmonella strains and sometimes among the rRNA operons in the same strain. Fragmentation of 23S rRNA was not observed in strains of the closely related species Escherichia coli. Fragmentation of 23S rRNA occurred in Brucella and Agrobacterium but the cleavage pattern was not as diverse as that demonstrated in Salmonella. Introduction of cleavage sites into precursor 23S rRNA of Salmonella is probably a recent evolutionary event.

The cell-cell adhesion receptor Mel-CAM acts as a tumor suppressor in breast carcinoma.

Mel-CAM (MUC18 or CD146) is a cell adhesion molecule sharing sequence homology with members of the immunoglobulin gene superfamily. Mel-CAM was originally described as a marker associated with invasion and metastasis in melanoma. We determined here the distribution and biological significance of Mel-CAM in normal, benign proliferative, and neoplastic breast ductal epithelium. Using a Mel-CAM-specific monoclonal antibody, we, immunohistochemically demonstrate Mel-CAM expression in 14 of 14 (100%) normal breast epithelia and benign proliferative ductal epithelial lesions, whereas Mel-CAM expression can only be focally detected in 12 of 72 (17%) breast carcinomas. Solid-phase cell adhesion assay revealed that breast carcinoma cells in culture express the ligand for Mel-CAM. Transfection of Mel-CAM cDNA into breast carcinoma cells induces a more cohesive cell growth pattern and establishes smaller tumors in immunocompromised mice than mock transfectants. In conclusion, Mel-CAM is distributed throughout normal and benign proliferative mammary ductal epithelium, but it is frequently lost in carcinomas; it functions as a heterophilic cell-cell adhesion molecule in breast epithelium, and loss of Mel-CAM expression in breast carcinoma may be an important step for tumor progression.

Use of a lactoferrin assay in the differential diagnosis of female genital tract infections and implications for the pathophysiology of bacterial vaginosis.

BACKGROUND AND OBJECTIVES: Lactoferrin has served as a marker for leukocytes (polymorphonuclear neutrophils [PMN]) in clinical specimens. GOAL: To investigate the potential of a lactoferrin latex agglutination test in the differential diagnosis of female genital infection. STUDY DESIGN: Lactoferrin was quantified in the vaginal discharge of women with genital infections. Polymorphonuclear neutrophils were added to vaginal discharge and observed over 8 hours. RESULTS: Vaginal lactoferrin titers were significantly elevated in women with trichomoniasis, candidiasis, and bacterial vaginosis (BV). Using a lactoferrin titer of > or = 1:40, the assay has a sensitivity of 79.3% and a specificity of 83.3% for the presence of trichomoniasis or bacterial vaginosis. Vaginal discharge from women with BV significantly destroyed added PMN. CONCLUSIONS: Vaginal lactoferrin determinations may provide a useful screen for inflammatory genital infections and identify individuals who require additional diagnostic evaluation. The observed absence of PMN in bacterial vaginosis may result from the destruction of PMN in vaginal discharge rather than the absence of a primary inflammatory response.

Relation of polyamine synthesis and titer to aging and senescence in oat leaves.

Polyamine biosynthesis in senescing leaves of Avena sativa L. was measured by determining the activities of arginine decarboxylase (EC 4.1.1.19), ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). Polyamine content was also estimated by thin layer chromatography and high performance liquid chromatography. Arginine decarboxylase activity decreases progressively in aging attached first leaves and in senescing excised leaves in the dark. Conversely, it increases during light exposure of excised leaves, which retards senescence. Ornithine decarboxylase activity is high and constant in the attached leaf, irrespective of age; it decreases in excised leaves kept in the dark and in the light, irrespective of senescence. S-Adenosyl-l-methionine decarboxylase shows no correlation with age or senescence. Levels of putrescine, diaminopropane, agmatine, and spermidine are high in young leaves and decline with age. The best single indicator of senescence is usually spermidine, which decreases in excised leaves incubated in the dark, but increases in such leaves with time of light exposure. Spermidine generally has a reciprocal relationship with putrescine, indicating that spermidine synthase, which converts putrescine to spermidine, may exert important physiological control. These data support the view that polyamines play an important role in the regulation of plant development.

Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves : I. Inhibition of protease activity, ethylene production, and chlorophyll loss as related to polyamine content.

Excision and dark incubation of oat (Avena sativa L., var. Victory) leaves cause a sharp increase in protease activity, which precedes Chl loss. Both these senescence processes are inhibited by exogenously applied 1,3-diaminopropane (Dap), which occurs naturally in leaf segments. The inhibition of protease activity is much greater in vivo than in vitro, suggesting inhibition of protease synthesis as well as protease action by Dap. Chl breakdown in leaves of radish and broccoli, which also senesce rapidly in the dark, is only slightly inhibited by DaP. These differences between cereal and dicotyledonous plants are correlated with the natural occurrence of Dap in cereals. In the light, Dap promotes, rather than retards, the loss of Chl in oat leaves. This resembles previously described effects of other polyamines. Addition of Mg(2+) to the medium does not antagonize this effect. In the dark, the accumulated Dap also inhibits ethylene production and decreases titer of other polyamines. Addition of Ca(2+) to the incubation medium containing Dap competitively reduces the effects of Dap. Thus, Dap, like other polyamines, seems to require an initial attachment to a membrane site shared with Ca(2+) before exerting its antisenescence action.

Effects of Exogenous 1,3-Diaminopropane and Spermidine on Senescence of Oat Leaves : II. Inhibition of Ethylene Biosynthesis and Possible Mode of Action.

The effects of the polyamines spermidine and 1,3-diaminopropane on ethylene biosynthesis and chlorophyll (Chl) loss were studied in peeled leaves of oat (Avena sativa L., var. Victory) incubated in the dark. Peeling off the epidermal cells induces an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity, resulting in an enhanced ACC and ethylene formation. Both polyamines inhibit ethylene biosynthesis from methionine by inhibiting ACC synthase activity and, more effectively, the conversion of ACC to ethylene. They also inhibit Chl loss occurring between 24 and 48 h of dark incubation; but, as shown by inhibitor experiments, inhibition of Chl loss does not result from inhibition of ethylene formation. Ethylene production and Chl loss, both associated with senescence, require membrane integrity; thus, treatments which promote deterioration of membranes inhibit both processes. Ca(2+) in the incubation medium competitively reduces the polyamine-mediated inhibition of ACC conversion and Chl loss. The data suggest that polyamines initially attach to membranes, thereby inducing changes which, in turn, lead to inhibition of ethylene biosynthesis and retardation of senescence.