Pituitary Macroadenoma and Visual Impairment: Postoperative Outcome Prediction with Contrast-Enhanced FIESTA.

BACKGROUND AND PURPOSE: Contrast-enhanced FIESTA can depict anterior optic pathways in patients with large suprasellar tumors. We assessed whether the degree of kink in the optic nerve at the optic canal orifice on contrast-enhanced FIESTA correlates with the postoperative improvement of visual impairment in patients with pituitary macroadenoma. MATERIALS AND METHODS: Thirty-one patients with pituitary macroadenoma who underwent preoperative MR imaging and an operation were evaluated. We measured the optic nerve kinking angle on sagittal oblique contrast-enhanced FIESTA parallel to the optic nerve; the optic nerve kinking angle was defined as the angle between a line parallel to the planum sphenoidale and a line parallel to the intracranial optic nerve at the optic canal orifice. We used logistic regression analyses to determine whether the clinical (sex, age, and duration of symptoms) and imaging (tumor height, chiasmal compression severity, hyperintense optic nerve on T2WI, and optic nerve kinking angle) characteristics were associated with the postoperative improvement (good-versus-little improvement) of visual acuity disturbance and visual field defect. RESULTS: There were 53 impaired sides before the operation: 2 sides with visual acuity disturbance alone, 25 with visual field defect alone, and 26 with both. After the operation, good improvement was found in 17 of the 28 sides with visual acuity disturbance and in 32 of the 51 sides with visual field defects. Only the optic nerve kinking angle was significantly associated with good improvement of the visual acuity disturbance (P = .011) and visual field defect (P = .002). CONCLUSIONS: The degree of the optic nerve kinking angle was an independent predictor of postoperative improvement, indicating that irreversible damage to the optic nerve may be associated with its kinking at the optic canal orifice.

Protein tyrosine phosphatase epsilon is a negative regulator of FcepsilonRI-mediated mast cell responses.

Modulation of mast-cell activation may provide novel ways to control allergic diseases. Here, we show that protein tyrosine phosphatase epsilon (PTPepsilon; Ptpre) plays key regulatory roles during mast-cell activation mediated by the high-affinity IgE receptor (FcepsilonRI). Bone marrow-derived mast cells (BMMC) from Ptpre(-/-) mice exhibited enhanced FcepsilonRI-induced Ca(2+) mobilization and mitogen-activated protein kinase (MAPK) (JNK and p38) activation, and showed corresponding enhancement of evoked degranulation and cytokine production, but not leukotriene production. Examination of proteins linking tyrosine kinase activation and Ca(2+) mobilization revealed that the absence of PTPepsilon leads to increased phosphorylation of the linker for activation of T cells and SH2 domain-containing leucocyte phosphoproteins of 76 kDa, but not Grb2-associated binder-2 (Gab2). Because Gab2 is considered to be situated downstream of Fyn kinase, we reasoned that Fyn may not be a target of PTPepsilon. In the event, Syk but not Lyn was hyperphosphorylated in PTPepsilon-deficient BMMC. Thus, PTPepsilon most likely exerts its effects at the level of Syk, inhibiting downstream events including phosphorylation of SLP-76 and linker of activated T cells and mobilization of Ca(2+). Consistent with the in vitro data, antigen- and IgE-mediated passive systemic anaphylactic reactions were augmented in Ptpre(-/-) mice. Given that the number of mast cells is unchanged in these mice, this observation most likely reflects alterations of mast cell-autonomous signalling events. These data suggest that PTPepsilon negatively regulates FcepsilonRI-mediated signalling pathways and thus constitutes a novel target for ameliorating allergic conditions.

Phosphorylation of MAP4 affects microtubule properties and cell cycle progression.

In human cells, MAP4, a microtubule-associated protein ubiquitously expressed in proliferating cells, has been shown to undergo in vivo phosphorylation. Two phosphorylation sites, serines 696 and 787, lie within the proline-rich region of its microtubule-binding domain. To test the hypothesis that phosphorylation at these sites influences microtubule properties or cell cycle progression, we prepared stable cell lines that inducibly express versions of MAP4 in which phosphorylation of these two serines was prevented by their replacement with alanine, lysine, or glutamate residues (AA-, KK-, or EE-MAP4). All nonphosphorylatable mutant forms of MAP4 expressed in mouse Ltk- cells were localized to MT arrays that were unremarkable in appearance. Expression of nonphosphorylatable mutants of MAP4 did not affect cell doubling time; however, expression of some mutants altered progression into or through cell division. Interactions of mutant MAP4 with MTs were examined in vitro. KK mutant MAP4 bound MTs more avidly than its wild-type counterpart, WT-MAP4. In vivo MT polymer also differed among the mutants: MTs in cells expressing the KK- and AA-MAP4 forms were more resistant to nocodazole depolymerization than those in cells expressing EE- or WT-MAP4 forms. Our results demonstrate that phosphorylation alters MAP4 properties and suggest a raison d'être for phosphorylation of the MAP4 microtubule-binding domain during cell cycle progression.

Neurofilaments of Klotho, the mutant mouse prematurely displaying symptoms resembling human aging.

We reported previously that neurofilaments (NFs) of aged rats were highly packed in the axon and contained a smaller amount of NF-M as compared with those of young rats (Uchida et al. [1999] J. Neurosci. Res. 58:337-348). We studied NFs of the mutant mouse, named Klotho, which displays prematurely symptoms resembling human aging. The transport of axonal cytoskeletal proteins, including NFs, tubulin and actin, was decreased at the leading portion of the peak of transported proteins in Klotho during the process of premature aging. The nearest neighbor inter-NF distance in Klotho axons (35-39 nm) was shorter than that of the wild-type mouse (48-49 nm), indicating the packing of NFs in Klotho. The ratio of NF-M to NF-L was slightly decreased in cytoskeletons from the spinal cords of Klotho. These changes are similar, though not identical, to those observed in aged rats, and are the first evidence of age-related changes in the neurons of Klotho.

Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains.

Tau in Alzheimer neurofibrillary tangles has been shown to be hyperphosphorylated and CDK5, GSK3, MAP kinase and SAP kinases are the candidate kinases for the phosphorylation of tau. Recently, it was reported that the conversion of p35, the activator of CDK5, to p25 was upregulated in Alzheimer's disease (AD) brains, and that p35 is cleaved to yield p25 by calpain. Here we show that p35 is rapidly cleaved to p25 in rat and human brains within a short postmortem delay and that the conversion of p35 to p25 is partially dependent on calpain activity. Immunoblot analysis of brains prepared from patients with AD or age-matched control individuals with a short postmortem delay revealed no specific increase in the levels of p25 in AD brains, whereas the levels of active form of calpain were increased in AD brains compared to the those in controls. These observations suggest that the conversion of p35 to p25 is a postmortem degradation event and may not be upregulated in AD brains.

Plasma levels of mature form of adrenomedullin in patients with haemodialysis.

BACKGROUND: Adrenomedullin (AM) is a potent vasodilator and natriuretic peptide with hypotensive effects. Immunoreactive AM in human plasma consists of the biologically active mature form, AM (1-52)-CONH2 (mAM) and the intermediate form, AM-gly-COOH (iAM). However, the different effects of mAM and iAM in patients on haemodialysis (HD) have remained unclear. METHODS: Thirty-nine patients on HD and 10 controls were included in this study. We determined plasma levels of mAM and iAM using an immunoradiometric assay that recognizes total AM (tAM) and another that is specific for only mAM. RESULTS: The plasma concentrations of mAM and iAM in patients before HD were significantly higher than those in the controls (n=10) (4.76+/-0.28 vs 1.28+/-0.22 fmol/ml, P<0.001, 25.99+/-1.47 vs 8.52+/- 0.91 fmol/ml, P<0.001 respectively). The plasma levels of mAM and iAM before HD significantly and negatively correlated with systolic blood pressure (SBP) (r=-0.46, P<0.01, and r=-0.32, P<0.05 respectively) and diastolic blood pressure (DBP) (r=-0.32, P<0.05, and r=-0.35, P<0.05 respectively). After HD, plasma mAM and iAM levels as well as SBP and DBP were significantly lower than before HD. Plasma levels of mAM and iAM correlated significantly (r=0.73, P<0.001). CONCLUSIONS: These data suggest that mAM and/or iAM are involved in blood pressure regulation in patients undergoing HD, and further work is needed to understand the precise role of adrenomedullin in this regulation.

Peritonitis increases MMP-9 activity in peritoneal effluent from CAPD patients.

Patients undergoing long-term continuous ambulatory peritoneal dialysis (CAPD) sometimes experience ultrafiltration failure. Mesothelial basement membrane thickening and the accumulation of submesothelial fibrotic tissue are common features of the diseased peritoneum. Peritonitis can lead to ultrafiltration failure, but the precise mechanism is not clear. The key enzymes in extracellular matrix (ECM) remodeling, namely matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), are produced by human peritoneal mesothelial cells. Using peritoneal effluent from 13 CAPD patients with peritonitis and 7 noninfected CAPD control individuals, we examined MMP and TIMP activities by gelatin and reverse zymography. Latent and activated types of MMP-2 and -9, and TIMP-1 and -2 were identified in peritoneal effluent (from all CAPD patients). Levels of latent and activated type MMP-9, as well as of TIMP-1 activities were higher at the onset of peritonitis than either during the recovery phase of peritonitis and/ or in control individuals. Activated MMP-9 activity positively correlated with leukocyte numbers and IL-6 levels in peritoneal effluent. Activities of MMP-2 and TIMP-2 in peritoneal effluent did not change between the onset of peritonitis and recovery. We concluded that increased MMP-9 and TIMP-1 levels might be associated with peritoneal ECM remodeling during peritonitis.

Increased plasma levels of mature adrenomedullin in chronic glomerulonephritis.

Adrenomedullin (AM) is a potent vasodilative and natriuretic peptide that is processed from its precursor as the intermediate form, AM-glycine-COOH (iAM). Subsequently, iAM is converted to the biologically active mature form, AM(1-52)-CONH(2) (mAM), by enzymatic amidation. Using immunoradiometric assays that recognize total AM (tAM) and only mAM, we determined the plasma and urinary levels of mAM and iAM in patients with chronic glomerulonephritis (CGN). The plasma mAM concentration was significantly higher in the patients than in the controls (1.8 +/- 0.1 vs. 1.3 +/- 0.1 fmol/ml, p < 0.01), whereas the plasma iAM concentration of the CGN patients did not significantly differ from that of the controls (9.4 +/- 0.5 vs. 8.9 +/- 0.5 fmol/ml). Levels of urinary mAM excretion in the patients did not statistically differ from those of the controls (1. 6 +/- 0.4 vs. 2.0 +/- 0.3 fmol/mg creatinine), whereas urinary iAM excretion was significantly lower in the CGN patients (3.7 +/- 0.7 vs. 5.6 +/- 0.8 fmol/mg creatinine, p < 0.05). Urinary excretion levels of mAM significantly correlated with those of sodium (r = 0. 47, p < 0.05), whereas those of iAM did not. In conclusion, the plasma ratio of mAM to iAM is augmented in CGN patients, and mAM appears to be involved in the regulation of sodium. Therefore, determination of the mAM in addition to the tAM concentration is essential in CGN patients.

Ser787 in the proline-rich region of human MAP4 is a critical phosphorylation site that reduces its activity to promote tubulin polymerization.

p34cdc2 kinase-phosphorylation sites in the microtubule (MT)-binding region of MAP4 were determined by peptide sequence of phosphorylated MTB3, a fragment containing the carboxy-terminal half of human MAP4. In addition to two phosphopeptides containing Ser696 and Ser787 which were previously indicated to be in vivo phosphorylation sites, two novel phosphopeptides, containing Thr892 or Thr901 and Thr917 as possible phosphorylation sites, were isolated, though only in in vitro phosphorylation. The role of phosphorylation at Ser696 and Ser787, which were differently phosphorylated during the cell cycle (Ookata et al., (1997). Biochemistry, 36: 15873-15883), was investigated in MT-polymerization, using MAP4 Ser to Glu mutants, which mimic phosphorylation at each site. Mutation of Ser787 to Glu strikingly reduced the MAP4's MT-polymerization activity, while Glu-mutation at Ser696 did not. These results suggest that Ser787 could be the critical phosphorylation site causing MTs to be dynamic at mitosis.

Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25.

Cyclin-dependent kinase 5 (CDK5) is a unique CDK, the activity of which can be detected in postmitotic neurons. To date, CDK5 purified from mammalian brains has always been associated with a truncated form of the 35-kDa major brain specific activator (p35, also known as nck5a) of CDK5, known as p25. In this study, we report that p35 can be cleaved to p25 both in vitro and in vivo by calpain. In a rat brain extract, p35 was cleaved to p25 by incubation with Ca(2+). This cleavage was inhibited by a calpain inhibitor peptide derived from calpastatin and was ablated by separating the p35.CDK5 from calpain by centrifugation. The p35 recovered in the pellet after centrifugation could then be cleaved to p25 by purified calpain. Cleavage of p35 was also induced in primary cultured neurons by treatment with a Ca(2+) ionophore and Ca(2+) and inhibited by calpain inhibitor I. The cleavage changed the solubility of the CDK5 active complex from the particulate fraction to the soluble fraction but did not affect the histone H1 kinase activity. Increased cleavage was detected in cultured neurons undergoing cell death, suggesting a role of the cleavage in neuronal cell death.

Brain-derived neurotrophic factor-induced phosphorylation of neurofilament-H subunit in primary cultures of embryo rat cortical neurons.

Phosphorylation of the neurofilament-H subunit (NF-H) was investigated in rat embryonic brain neurons in culture. A portion of the NF-H was phosphorylated in vivo at embryonic day 17 when brain neurons were prepared. When the neurons were isolated and cultured, the NF proteins disappeared once and then reappeared over the next several days in the following order: (1) NF-L/NF-M, (2) dephosphorylated NF-H and (3) phosphorylated NF-H. Phosphorylation of NF-H began around 4 days after cell plating, at about the time of synapse formation. Treatments that appeared to modulate the timing of synapse formation also affected the timing of NF-H phosphorylation: (1) earlier phosphorylation was observed at higher neuronal cell density, (2) earlier phosphorylation was observed in neurons cultured on a coating substrate that promotes rapid neurite extension and (3) phosphorylation was suppressed when neurite extension was inhibited by brefeldin A. Three possible synapse formation-induced events, excitation, cell-cell contact through adhesion proteins and elevated concentrations of neurotrophic factors, were examined for their possible involvement in generating the signal for NF-H phosphorylation. Neither excitation nor cell contact enhanced NF-H phosphorylation. Neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) stimulated phosphorylation of NF-H. The BDNF-stimulated phosphorylation was inhibited by an anti-BDNF antibody and K252a, an inhibitor of BDNF receptor TrkB tyrosine kinase. Among known NF-H kinases of cyclin-dependent kinase 5 (CDK5), external signal-regulated protein kinase (ERK) and stress-activated protein kinase (SAPK), CDK5 and SAPK showed an increase in kinase activity or an active form with a time course similar to NF-H phosphorylation in control culture. On the other hand, BDNF stimulated the kinase activity of CDK5 and induced appearance of an active form of ERK transiently. These results suggest a possibility that synapse formation induces NF-H phosphorylation, at least in part, through activation of CDK5 by BDNF.

Increased plasma levels of mature form of adrenomedullin in patients with chronic renal failure.

BACKGROUND: Adrenomedullin (AM), a novel vasodilator peptide, is produced by C-terminal amidation reaction of AM-glycine. AM-glycine, an intermediate form of AM (iAM), is processed from pro AM. AM circulating in the human blood stream was found to consist of an amidated mature form (mAM) and iAM. Biological activity is exerted only by mAM. METHODS: To investigate the pathophysiological role of mAM in renal disease, we measured plasma concentrations of mAM as well as total AM (tAM), representing both mAM and iAM, in patients with various renal diseases. In addition, plasma ANP level was measured in all patients. RESULTS: The concentrations of plasma mAM in renal failure with dialysis (2.1 +/- 0.2 fmol/ml, mean +/- SEM) and without dialysis (1.2 +/- 0.2) were significantly (p < 0.05) higher than those in control group (0.5 +/- 0.1). However, the plasma ANP level was increased only in renal failure patients with dialysis. Plasma mAM levels were significantly correlated positively with serum creatinine levels and negatively with hematocrit. No significant difference was noted in the ratio of mAM/tAM between renal failure patients and healthy subjects. CONCLUSION: These results suggest that plasma mAM is increased in renal failure in relation to deterioration of renal function, while the amidation process of AM seems to be unaffected in patients with renal failure.

Neurofilaments of aged rats: the strengthened interneurofilament interaction and the reduced amount of NF-M.

Amyotrophic lateral sclerosis is an age-related neurological disease, characterized by neurofilament (NF) accumulation in primary axons followed by degeneration of motor neurons. To elucidate age-related factors that might lead to pathological NF accumulation, NFs were compared between young and aged rats. Electron microscopic examination of sciatic nerve axons revealed that NFs were more than twice as densely packed in aged rat axons (542 +/- 180 NFs/mm2) as in young adult rat axons (211 +/- 73 NFs/mm2). The NFs isolated from aged rats also appeared to be more aggregated than those from young rats. Phosphorylation at the head or tail domains was studied as a possible candidate affecting NF organization. Western blotting with phosphorylation-dependent antibodies showed higher phosphorylation of NF-H in the tail domains of aged rat spinal cord NFs, but dephosphorylation did not diminish the differences in aggregation between aged and young rat NFs. On the other hand, when NFs were phosphorylated by A-kinase on their head domains, the extent of phosphorylation in NF-M of aged rat NFs was only one-third of young rat NFs. We found that aged rat NFs contained only 60% of the NF-M of young rat NFs in molar ratio compared to NF-L. These results raise a possibility that the decreased amount of NF-M induces the aggregates of isolated NFs and the higher packing density of NF in aged rat axons.

[Exercise-induced acute renal failure associated with renal vasoconstriction].

Exercise-induced acute renal failure without rhabdomyolysis is not a rare condition. We experienced 6 cases (5 men and a woman) during last the 8 years. All cases complained of severe loin pain and nausea after mild to moderate exercises (for example, a track race in an athletic meeting). The elevation of serum and urinary myoglobin was undetected. In 4 of 5 patients with abdominal CT, renal patchy vasoconstriction (wedge-shaped low-density lesion) was observed. This was diagnosed as exercise-induced acute renal failure with loin pain (serum creatinine levels: 1.7-8.6 mg/dl). The renal function in 5 of the 6 cases normalized in about three weeks by fluid replacement therapy and hemodialysis support, which one patient received for 3 days. One patient required a long time for improvement of renal function and renal insufficiency persisted (serum creatinine 1.8 mg/dl). In 2 patients, the concentration of serum uric acid became very low after the recovery of renal function. These two patients were diagnosed as an isolated hyperuricosuric hypouricemia. More than half of the 6 patients had previously experienced the same episodes (loin pain and nausea) after exercise. Exercise-induced acute renal failure, probably due to renal patchy vasoconstriction, seems to be not a rare disease. The etiology of renal patchy vasoconstriction after exercises remains to be elucidated. The occurrence of acute renal failure must be taken into consideration when the youngster, especially with renal hypouricemia, complains of severe loin pain and nausea after exercise such as a track race.