Regulation of nitric oxide synthase 2 in rabbit corneal cells.

PURPOSE: The purpose of these studies was to investigate the role of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and transforming growth factor-beta (TGF-beta) in the regulation of inducible nitric oxide synthase (NOS2) activity in rabbit corneal cells. METHODS: Rabbit corneal epithelial, stromal, and endothelial cells were grown in culture and treated with cytokines and growth factors, alone or in combination. NOS activity was measured at times up to 72 hours after treatment by assaying the culture medium for nitrite using the Griess reaction. Cell lysates were analyzed by Western blot analysis for NOS2 protein. RNA was isolated and amplified with NOS1-, NOS2-, and NOS3-specific primers by RT-PCR. RESULTS: NOS2 expression was induced by combined cytokine treatment from nondetectable levels to abundant levels in low passage (<4) stromal cells and to low levels in corneal endothelial cells but not in corneal epithelial cells. In the absence of IFN-gamma, little or no nitrite accumulation was induced by TNF-alpha, IL-1beta, or lipopolysaccharide (LPS) treatment. The inductive effects of IFN-gamma were antagonized in a dose-dependent manner by the myxoma virus rabbit IFN-gamma receptor homolog, M-T7. rRaIFN-gamma, in combination with IL-1beta and TNF-alpha, induced the appearance of NOS2 mRNA within 24 hours but detectable nitrite did not accumulate in large amounts (>10 microM) until after 24 hours postinduction. NOS2 was identified as a 130 kDa protein on Western blot analysis using monoclonal antibody against murine NOS2. TGF-beta(1) and beta(2) inhibited the accumulation of cytokine-induced nitrite in a dose-dependent manner while not significantly reducing the steady state level of NOS2 mRNA. The activity of the induced NOS was inhibited by 1400W, a NOS2-selective inhibitor, but not 7-nitroindazole, a NOS1-selective inhibitor. CONCLUSIONS: In cultured corneal stromal cells, NOS2 expression was upregulated by IFN-gamma in combination with IL-1beta and TNF-alpha but not by any of these cytokines alone, while TGF-beta downregulated the activity. Cultures of corneal epithelial cells could not be induced to express NOS2, yet cultures of endothelial cells produced low amounts of NO in response to cytokines. The NOS1 and NOS3 isoforms were not detected in any of these corneal cells.

Tissue-specific accumulation of latency-associated transcripts in herpes virus-infected rabbits.

PURPOSE: Herpes simplex virus (HSV) DNA persists in the corneas of patients and animals with a history of herpetic keratitis. The purpose of this study was to detect viral transcripts in the corneas of latently infected rabbits with a history of herpetic keratitis to determine whether the viral DNA represents latent virus, characterized by the restricted transcription of HSV genes and accumulation of the stable latency-associated transcripts (LATs), as occurs in neurons. METHODS: Rabbits were injected in the subalveolar mucosa with HSV strain RE. After 30 days, corneas were infected by intrastromal injection of HSV. Corneal disease was evaluated, and 7 to 378 days after infection, the rabbits were killed. DNA and RNA were isolated from corneas and trigeminal ganglia and amplified by PCR using gene-specific primers. RESULTS: Herpetic keratitis developed in all rabbits. All corneas of these immune rabbits contained viral DNA as many as 120 days after infection and then the frequency decreased over the next 260 days. Overall, viral DNA was detected in all ganglia and in 57% of corneas. All latently infected ganglia but no corneas contained LATs. Transcripts of the early viral gene for thymidine kinase were detected in 23 of 30 ganglia and 10 of 17 corneas. Transcripts for the late viral glycoprotein C were not detected in either tissue. CONCLUSIONS: These data document that after HSV keratitis, viral DNA persists in corneas in the absence of stable LATs and with restricted expression of other viral genes.

Cloning and characterization of an alpha-amylase gene from Streptomyces lividans.

The alpha-amylase gene (amy) of Streptomyces lividans TK24 was cloned in an amylase deficient mutant strain S. lividans M2. The cloned gene contained an open reading frame (ORF) of 2757 nucleotides (919 amino acids) coding for a protein of 100 kDa. Sequencing of the amino terminus of the extracellular alpha-amylase protein revealed the presence of a signal peptide of 33 amino acid residues. The transcriptional initiation site was mapped by the primer extension method with T4 DNA polymerase and was found to be transcribed from an unique promoter. The alpha-amylase protein produced by S. lividans was larger than those derived from other origins. It also contained the four common conserved regions characteristic of other alpha-amylase proteins.

[Effects of solute on growth and intracellular salt concentration of Pediococcus halophilus CCRC 12884 isolated from Inyu mash].

Optimal growth conditions for Pediococcus halophilus CCRC 12884, a halophilic strain of lactic acid bacterium isolated from Inyu (black bean sauce) mash, were MRS broth with 10% NaCl and 35 degrees C of incubation temperature. The NaCl in media could not be replaced by sucrose or glycerol of same concentrations, and under certain osmolarity, growth of P. halophilus CCRC 12884 only in NaCl was better than that in NaCl plus sucrose or glycerol. To determine effects of monovalent and divalent ions on P. halophilus CCRC 12884, it was found that close relations were present among Na+, K+ and PO4(-3). The intracellular concentration of Na+ in P. halophilus CCRC 12884 was not correspondent to its extracellular concentration of NaCl, but intracellular K+ and quaternary ammonium compounds significantly increased as extracellular NaCl increased. Adding 0-3.5 mM betaine to media with different contents of NaCl would not affect the growth of this halophilic bacterium. On feeding 14C-ethanolamine to the bacterium, no intracellular 14C-betaine was synthesized.