In vivo regulation of MAP kinases in Ratus norvegicus renal papilla by water loading and restriction

In cultured renal cells, hypertonicity activates multiple mitogen-activated protein kinases (MAPKs) and enhances the expression of heat shock proteins (HSPs). In rats, 24 h water restriction increased mean urinary osmolality (Uosm) from 2, 179+/-153 mOsm/kg to 2,944+/-294 mOsm/kg (P < 0.001) and was associated with significant (P < 0.05) increases in the papillary activity of c-Jun NH2-terminal protein kinase (JNK) by 22%, extracellular signal-regulated protein kinase (ERK) by 49%, and p38 MAPK by 15%. Conversely, 24 h of water-loading (Uosm 473+/-33 mOsm/kg) caused suppression of JNK activity by 43% (P < 0.001), ERK by 39% (P < 0.05), and p38 MAPK by 26% (P < 0.05). No such modulation was observed in the isotonic cortex. c-Jun phosphorylation was decreased in papilla from water-loaded rats by 45% versus controls. Expression of Hsp 110, inducible Hsp 70, and Hsp 25 was greater in the hyperosmotic papilla than the isosmotic cortex but was not affected by the animal's hydration state. In cultured inner medullary collecting duct cells, HSP expression was maximal at 500 mOsm/kg, while activation of JNK continued to increase. We conclude that under basal conditions of hydration, these HSPs are maximally expressed in the hypertonic inner medulla, while the activation of all three members of the MAPK family approaches but is not maximal.     

MAP kinases and vascular smooth muscle function

Integrins are a subclass of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. Integrins influence transendothelial migration of lymphocytes and monocytes and are suitable targets for experimental immunotherapy. They are critically involved in the pathogenesis of autoimmune neuritis and abnormally expressed in human neuropathies. Also, the role of integrins in myelination, neurite outgrowth, and nerve regeneration suggests that they could be involved in the recovery phase of immune-mediated neuropathies. We investigated by immunohistochemistry the expression of a number of integrin subunits during the course of experimental autoimmune neuritis (EAN). Results were compared with the human immune neuropathy Guillain-Barre syndrome (GBS) and extended in vitro. Inflammation and demyelination in both EAN and GBS induced the down-regulation of beta4 integrin in Schwann cells (SCs), whereas loss of alpha2 was noted only in EAN. When axonal loss was present, SCs displayed alpha5 integrin, in both EAN and GBS. In vitro, basal lamina and inflammatory cytokines modulated the expression of beta4 in SCs, but they did not influence alpha2 and alpha5 expression. Finally, integrins were differentially expressed in blood vessels during EAN. In conclusion, the spatiotemporal changes in integrin expression may be used to characterize, stage, and better understand the pathogenesis and evolution of inflammation during GBS and EAN. This may help to establish useful, novel therapy for immune-mediated neuropathies.     

Differences in the regulation of microtubule dynamics by microtubule-associated proteins MAP1B and MAP2

The regulation of microtubule dynamics in vitro by microtubule-associated proteins (MAPs) was examined, using purified porcine MAP1B and MAP2. MAP1B has a significantly smaller effect on the observed critical concentration for microtubule assembly than MAP2. Assembly is faster in the presence of either MAP, and the resulting microtubules are shorter, indicating that nucleation is substantially promoted by the MAPs. Both MAPs stabilise the microtubule lattice as observed from podophyllotoxin-induced disassembly, but the effect of MAP1B is weaker than the effect of MAP2. At steady-state of assembly MAP1B still allows microtubule dynamic instability to occur as inferred from microtubule length changes. The comparison of the effects of MAP1B and MAP2 indicates that the reduction of the observed critical concentration is attributable to the reduction of the depolymerisation rate and correlates with the extent of suppression of dynamic instability. Numerical simulations illustrate that microtubule dynamics are strongly influenced by relatively small changes in the strength of a limited subset of subunit interactions in the lattice. The observed characteristic differences between the MAPs may be important for the regulation of distinct populations of microtubules which coexist in the same cell, where differences in stability and dynamics may be essential for their different spatial roles as, for example, in developing neurons.     

Transcriptional regulation of aquaporin-2 water channel gene by cAMP

In patients awaiting heart transplantation, end-stage disease of a second organ may occasionally require consideration of simultaneous multiorgan transplantation. Outcome statistics in multiorgan transplant recipients are needed to define optimal utilization of scarce donor resources. Incidence of cardiac allograft rejection, actuarial recipient survival, and cardiac allograft rejection-free survival were evaluated in 82 recipients of 84 simultaneous heart and kidney transplants. Twenty-three of the 82 dual-organ recipients have died with 1, 6, 12, and 24-month actuarial survival rates of 92%, 79%, 76%, and 67%, respectively. The actuarial survival rates in the heart-kidney recipients were similar to those observed in 14,340 isolated heart recipients (United Network for Organ Sharing Scientific Registry) during the same period (92%, 86%, 83%, and 79%, respectively; P=0.20). Clinical data on all episodes of treated rejection in either organ and on immunosuppressive regimens were available on 56 patients; 48% of these patients have had no rejection in either organ, 27% experienced heart rejection alone, 14% experienced kidney rejection alone, and 11% had both heart and kidney allograft rejection. Heart allograft rejection was less common in heart-kidney recipients, as compared with isolated heart transplant recipients; 0, 1, and > or = 2 treated cardiac allograft rejection episodes occurred in 63%, 20%, and 18% of heart-kidney recipients compared with 46%, 27%, and 28% of 911 isolated heart recipients reported by Transplant Cardiologists' Research Database (P=0.02). The rejection-free survival rates at 1, 3, and 6 months were 88%, 74%, and 71% in the double-organ recipients, as compared with 66%, 44%, and 39%, respectively, in the single-organ recipients. Compared with isolated heart transplantation, combined heart-kidney transplantation does not adversely affect intermediate survival and results in a lower incidence of treated cardiac allograft rejection. The findings suggest that combined heart-kidney transplantation may be an acceptable option in a small subset of potential heart transplant recipients with severe renal dysfunction.     

Synergistic activation of SAPK1/JNK1 by two MAP kinase kinases in vitro

Mitogen-activated protein kinases (MAPKs) mediate many of the cellular effects of growth factors, cytokines and stress stimuli. Their activation requires the phosphorylation of a threonine and a tyrosine residue located in a Thr-X-Tyr motif (where X is any amino acid) [1]. This phosphorylation is catalysed by MAPK kinases (MKKs), which are all thought to be 'dual specificity' enzymes that phosphorylate both the threonine and the tyrosine residue of the Thr-X-Tyr motif [2]. Here, we report that the MAPK family member known as stress-activated protein kinase-1c (SAPK1c, also known as JNK1) [3] is activated synergistically in vitro by MKK4 ([4] [5] [6]; also called SKK1 and JNKK1) and MKK7 ([7] [8] [9]; also called SKK4 and JNKK2). We found that MKK4 had a preference for the tyrosine residue, and MKK7 for the threonine residue, within the Thr-X-Tyr motif. These observations suggest that the full activation of SAPK1c in vivo may sometimes require phosphorylation by two different MKKs, providing the potential for integrating the effects of different extracellular signals. They also raise the possibility that other MAPK family members may be activated by two or more MKKs and that some MKKs may have gone undetected because they phosphorylate the tyrosine residue only, and therefore do not induce any activation unless the threonine has first been phosphorylated by another MKK.     

Transcriptional and post-transcriptional regulation of interleukin-8

We have previously shown that vaccines expressing virus-derived cytotoxic-T-lymphocyte (CTL) epitopes as short minigenes can confer effective protection against virus challenges, and here we extend these studies to the bacterium Listeria monocytogenes. Host defense against this important human pathogen appears largely T cell mediated, and a nonamer CTL epitope from the listeriolysin O (LLO) protein has been identified in BALB/c mice. We have synthesized this nonamer as a minigene, expressed it in a recombinant vaccinia virus (VV-list), and used this to immunize mice. Memory CTLs cultured from VV-list-immunized mice specifically lyse target cells pulsed with a nonamer peptide identified at LLO amino acid residues 91 to 99. Four weeks postimmunization, mice were challenged with L. monocytogenes. By day 6 following challenge with a sublethal dose of L. monocytogenes, mice immunized with VV-list showed a approximately 2,000- to 6,000-fold reduction in bacteria CFU in the spleen and liver. At this time point, with control mice, bacterial were readily detectable by Gram stain of the liver but were undetectable in the VV-list-immunized animals. Additionally, when a normally lethal dose of bacteria was given, death was delayed in VV-list-immunized animals. This study has demonstrated that a single immunization with a recombinant vaccinia virus bearing only nine amino acids from a bacterial pathogen can induce specific CTLs able to confer partial protection against bacterial challenge.