Reduced basal CaMKII levels in hippocampal CA1 region: possible cause of stress-induced impairment of LTP in chronically stressed rats.

Chronic psychosocial stress markedly reduces the expression of high-frequency stimulation (HFS)-evoked early long-term potentiation (LTP) in the CA1 region of the hippocampus of anesthetized rats. Immunoblotting was performed to determine changes in molecular levels of key signaling proteins that might be responsible for this inhibitory effect. Western blot analysis of the CA1 region demonstrates that chronic psychosocial stress decreases basal levels of calcium calmodulin kinase II (CaMKII), phosphorylated (P)-CaMKII, calmodulin, and protein kinase C (PKCgamma) while markedly increasing protein phosphatase 2B (calcineurin) levels. The decrease of basal levels of P-CaMKII may be triggered primarily by excessive dephosphorylation resulting from enhanced basal levels of calcineurin. The decline in the basal levels of the upstream molecules, PKCgamma and calmodulin may be a consequence of the diminished basal P-CaMKII levels. Analysis of signaling molecules in CA1 region of chronically stressed rat subjected to HFS in vivo showed only one difference compared to similarly stimulated control rats; no increase in P-CaMKII levels. Our results suggest that decreased P-CaMKII levels may be primarily responsible for the stress-induced reduction in LTP expression.

Chronic psychosocial stress decreases calcineurin in the dentate gyrus: a possible mechanism for preservation of early ltp.

Chronic psychosocial stress impairs early long-term potentiation (LTP) in the hippocampal CA1 region but not in the dentate gyrus of anesthetized rats. Analysis of putative signaling molecules involved in the expression of LTP was performed to determine the possible reason(s) for the apparent resistance of the LTP of the dentate gyrus to chronic psychosocial stress. Immunoblotting was used to determine possible changes in the basal levels of various fractions of calcium-dependent calmodulin kinase II (CaMKII), phosphorylated CaMKII (P-CaMKII), calmodulin, protein kinase C gamma (PKCgamma) and calcineurin in the dentate gyrus of chronically stressed rats. Western blot analysis revealed that chronic stress significantly decreased the levels of the total CaMKII without affecting P-CaMKII levels. No significant change was detected in the levels of the upstream effectors, calmodulin and PKCgamma. However, chronic stress produced a significant decrease in calcineurin levels. The data suggest that the dentate gyrus of chronically stressed rats may have developed a compensatory mechanism whereby calcineurin levels are reduced to maintain normal P-CaMKII levels, which may be responsible for the normal early LTP of the dentate gyrus of chronically stressed rats. The results of this work will increase understanding of why certain brain regions are more resistant to deleterious effects of conditions that deteriorate learning and memory.

Alternative imaging of the lung.

This article illustrates the roles of ultrasonography, scintigraphy, and computed tomography (CT) as alternative techniques for pulmonary imaging in small animals. The advantages and limitations of each modality, normal anatomic features, and technical considerations will be discussed. Selected applications will be examined and include pulmonary consolidation, neoplasia and other masses, atelectasis, pneumothorax, dystrophic mineralization, diffuse infiltrative disease, and pulmonary embolism. The use of ultrasound and CT-guided interventional procedures will also be briefly discussed.

Hepatic abscesses in 13 dogs: a review of the ultrasonographic findings, clinical data and therapeutic options.

Historical, physical examination, clinicopathologic, radiographic and ultrasonographic findings of 13 dogs with hepatic abscesses were reviewed. Liver abscessation was characterized by number, size, shape, echogenicity and location. Solitary lesions greater than 3 cm were more common than multiple ones. The abscesses were mainly poorly echogenic lesions, often with central cavitation. The shape of the lesion ranged from round to oval or irregular. Enhancement artifact, abdominal effusion, regional lymphadenopathy and hyperechoic perihepatic fat, were identified in several dogs. Ultrasound-guided aspiration was performed in 10 of 13 dogs, and confirmed abscessation with cytologic and microbiologic evaluation. Ultrasound-guided percutaneous drainage of abscesses was performed as an adjunct to medical management in four dogs.

Delivery of DNA-cationic liposome complexes by small-particle aerosol.

Aerosol delivery of gene therapy for treatment of lung diseases allows topical treatment of the airways with DNA concentrations not obtainable by systemic administration. We have investigated delivery of cationic liposomes complexed to plasmid DNA in a small particle aerosol. Plasmid cDNA-DMRIE/DOPE complexes were nebulized using either an Aerotech II or Puritan-Bennett 1600 (PB1600) nebulizer. Reservoir sampling showed that DNA-DMRIE/DOPE complexes were damaged to a significant degree during nebulization, such that activity of transfected gene was diminished. Of the nebulizers analyzed, DNA-DMRIE/DOPE complexes were more stable in the PB1600. The loss of effective transfection by DNA-DMRIE/DOPE, as detected by decreased reporter gene activity in A549 lung cells, was consistent with denaturation of the DMRIE/DOPE. In contrast, nebulized DNA-DOSPA/DOPE complexes retained complete ability to transfect. Adjustments to flow rate and reservoir volume of the PB1600 allowed a longer period of delivery of active DNA-DMRIE/DOPE particles. DNA-DMRIE/DOPE was radiolabeled with Technetium-99m (99mTc), nebulized, and the output captured in either an Andersen Sampler (AS) (Andersen, 1958) cascade impactor particle size analyzer or an all glass impinger. cDNA-cationic lipid complexes were detected in size ranges of 0.4-10 microns, with most particles found between 1-2 microns. Aerosol output was consistent from 0 to 5 min. These results show the feasibility of aerosol delivery of DNA-cationic lipids for the purposes of gene therapy to the lung.

Interferon regulatory factor-1 is inducible by prolactin, interleukin-2 and concanavalin A in T cells.

Interferon regulatory factor-1 (IRF-1) gene expression is rapidly upregulated in the prolactin (PRL)-activated Nb2 rat T lymphoma cell line. To further elucidate its role as a T cell activation molecule, IRF-1 gene expression in response to various T cell stimuli was examined. In Nb2 T cells, PRL induced two peaks of IRF-1 gene expression: a rapid, transient peak at 1 h and a sustained peak at 12 h. PRL subsequently induced interferon-gamma (IFN-gamma) gene expression at 3-6 h. However, the early induction of IRF-1 and IFN-gamma does not appear to be interdependent. Interleukin-2 (IL-2) also induced IRF-1 gene expression in Nb2 T cells but only one broad peak at 10 h was observed. In primary mouse splenocytes, concanavalin A induced rapid and transient expression of the IRF-1 gene; maximal expression occurred by 6 h, and then returned to basal levels by 12-15 h. These results provide additional evidence for the importance of IRF-1 in T cell activation.

Prolactin receptor gene expression in lymphoid cells.

To understand the role of pituitary prolactin (PRL) and its receptor (PRL-R) in the growth and differentiation of lymphoid cells, PRL-R gene expression was analyzed in various lymphoid tissues and in a rat T lymphoma cell line, Nb2, which requires PRL for growth. The technique of reverse transcription coupled to polymerase chain reaction (RT-PCR) was used to detect the low abundance PRL-R transcripts. Within 30 min to 1 h, PRL stimulates a rapid but transient increase in PRL-R mRNA levels in Nb2 T cells. By 4 h, PRL-R mRNA returned to near basal levels and then gradually declined to a new steady-state level by 12 h. Significant increases in receptor RNA levels were observed in the presence of protein synthesis inhibitors, which suggests that PRL-R mRNA levels are under negative regulation. PRL-R gene expression was also demonstrated in normal mouse thymocytes, splenocytes, and in several lymphoid cell lines. The expression of the PRL-R gene in stimulated lymphoid cells provides additional evidence for the role of PRL as an immunomodulatory molecule.

Interferon-regulatory factor 1 is an immediate-early gene under transcriptional regulation by prolactin in Nb2 T cells.

The pituitary peptide hormone prolactin (Prl) is a potent inducer of Nb2 T lymphoma cell proliferation. To analyze the early genetic response to the mitogenic signals of Prl, a cDNA library was constructed from Nb2 T cells stimulated for 4 h with Prl and the protein synthesis inhibitor cycloheximide. Of 26 distinct clones isolated by differential screening, one clone, designated c25, exhibited extremely rapid but transient kinetics of induction by Prl and superinduction by Prl plus cycloheximide. Run-on transcription analysis indicated that c25 gene transcription was induced greater than 20-fold within 30 to 60 min of Prl stimulation. Surprisingly, DNA sequence analysis of c25 cDNA revealed that this Prl-inducible early-response gene is the rat homolog of the mouse transcription factor interferon-regulatory factor 1 (IRF-1), sharing 91% coding sequence similarity with mouse IRF-1. At the protein level, rat IRF-1 shares 97% and 92% homology with mouse IRF-1 and human IRF-1, respectively, suggesting that this molecule has been functionally conserved throughout evolution. Our studies show that the gene for IRF-1 is an immediate-early gene in Prl-stimulated T cells, which suggests that IRF-1 is a multifunctional molecule. In addition to its role in regulating growth-inhibitory interferon genes, IRF-1 may, therefore, also play a stimulatory role in cell proliferation. The gene for IRF-1 is one of the earliest genes known to be transcriptionally regulated by Prl.

Effect of 3,3'-iminodiproprionitrile (IDPN) on corticosteroidogenesis of isolated adrenocortical cells.

The neurotoxic agent, 3,3'-iminodiproprionitrile (IDPN), is a disrupter of neurofilament- and intermediate filament-organelle association. In the present study, the effect of IDPN on corticosteroidogenesis was investigated using isolated rat (having few intermediate filaments) and domestic fowl (having abundant intermediate filaments) adrenocortical cells. Cells were incubated with or without steroidogenic agents and precursors and with or without various concentrations of IDPN for 2 hr. IDPN had similar inhibitory potencies (as indicated by the half-maximal inhibitor concentrations (ID50 values] with both rat and domestic fowl cells despite their grossly different intermediate filament content. However, the average ID50 values of IDPN varied with the different steroidogenic agents and precursors used. The average IDPN ID50 values for maximal ACTH- and 8-bromo-cyclic AMP (8-Br-cAMP)-induced corticosterone production were equivalent (49.7 and 45.7 mM, respectively). However, the IDPN ID50 values for maximal ACTH-induced cAMP production, maximal 25-hydroxycholesterol- and pregnenolone-supported corticosterone production, and maximal ACTH- and 8-Br-cAMP-induced protein synthesis varied from 3.7 to 5.4 times the average ID50 values for maximal ACTH- and 8-Br-cAMP-induced corticosterone production. Thus, the inhibitory action of IDPN was not closely linked to the inhibition of ACTH-transmembrane signaling via cAMP, protein synthesis, and steroidogenic enzyme activity. The data suggest that IDPN inhibited corticosteroidogenesis at at a step after cAMP but before cholesterol side-chain cleavage and that the inhibition was not dependent on the presence of intermediate filaments.

Potentiation of interferon's antiviral activity by the mutually synergistic interaction of MuIFN-alpha/beta and MuIFN-gamma.

The interaction of the interferons (IFNs) that cooperate to potentiate the antiviral action of IFN was studied. Serial dilutions of MuIFN-gamma and MuIFN-alpha/beta were employed separately and in combination to block virus replication in one-step, virus yield reduction experiments. To calculate the potentiation of IFN activity, protection levels obtained for each combination of MuIFN-gamma and MuIFN-alpha/beta were compared with those obtained for the separate IFNs. Potentiation levels increased with increasing concentrations of each of the IFNs in a dose-dependent manner, suggesting that potentiation of IFN's antiviral activity was the result of the mutually synergistic interaction of the IFNs. Three challenge viruses were employed: Mengo virus (positive-strand RNA virus), vesicular stomatitis virus (negative-strand RNA virus), and vaccinia virus (DNA virus). Identical results were observed with the three different viruses, suggesting that mutual synergism was a basic feature of the potentiation of IFN's antiviral activity by combined preparations of MuIFN-gamma and MuIFN-alpha/beta.

Requirement for IFN gamma in potentiation of interferon's antiviral and anticellular activities: identity of mouse and human systems.

Potentiation was originally demonstrated as a nonadditive, synergistic enhancement of interferon (IFN) activity in the mouse system for mixed preparations containing MuIFN-alpha/beta and MuIFN-gamma. Potentiation of the antiviral and anticellular activities has now been studied for mouse and human systems, and in both systems IFN-alpha and IFN-beta interacted synergistically with IFN-gamma, but not with each other. Further, the antiviral and anticellular activities of IFN-alpha and IFN-beta were potentiated equally by IFN-gamma. Potentiation was demonstrated for HuIFNs with specific activities of 10(7) U/mg of protein and higher. Naturally produced and recombinant HuIFN-alpha s had the same relative abilities to be potentiated by HuIFN-gamma. It was concluded that IFN-gamma with either IFN-alpha or IFN-beta was essential for potentiation, that potentiation of anticellular and antiviral actions occurred in similar manners, and that a close correlation existed between potentiation in mouse and human systems. These results suggest that potentiation was caused by the interaction of two dissimilar IFN types (immune versus virus-type) and that potentiation studies in the mouse may be directly relevant for humans.

Interferon potentiation occurs at a pretranscriptive stage.

Combination of interferon-gamma (IFN-gamma) with a mixture of IFN-alpha and IFN-beta (IFN-alpha/beta) produce a potentiated antiviral state. The onset of induction of the potentiated antiviral state was observed to be more rapid than the onset of induction of either IFN-gamma or IFN-alpha/beta separately. The onset of viral resistance to combined IFN-gamma and IFN-alpha/beta (25 U/ml each) began after 2 h, compared with 4 h for IFN-alpha/beta (25 U/ml) alone and 8 h for IFN-gamma (25 U/ml) alone. By adding actinomycin D at various times after interferon treatment, this more rapid onset of induction of the potentiated antiviral state was shown to be the result of a more rapid onset of cellular transcription. To determine whether increased concentrations of IFN-alpha/beta acted through a similar mechanism, we performed kinetic experiments with several concentrations of IFN-alpha/beta and with combined preparations of IFN-gamma and IFN-alpha/beta. The findings showed that treatment of cells with the combined interferons and with a high concentration of IFN-alpha/beta resulted in more rapid onset of cellular transcription and more rapid development of the antiviral state. Thus, the cellular response to potentiation appeared to involve perception by the cell of the combined interferons as a higher-than-expected effective concentration of interferon, resulting in more rapid onset of cellular transcription of mRNA for antiviral proteins.

Potentiation of interferon action.

Potentiation of interferon action was described as a non-additive, synergistic enhancement of interferon activity by combined preparations of immune and virus-type interferons. The potentiated antiviral activity was demonstrated for a variety of virus types as well as for the human and mouse in vitro systems. Potentiation by combined immune and fibroblast interferons was also demonstrated for the antitumor and direct anticellular effects of interferon. Use of partially purified immune and fibroblast interferons demonstrated that potentiation appeared to be a property of the interferon molecules themselves.