Nodular fasciitis involving the palm.

Nodular fasciitis (NF) is a self-limiting fibrous neoplasm that can be mistaken for a soft tissue sarcoma. It is characterised by rapid growth, slight pain and local tenderness. Although it is frequently found in the forearm, a lesion distal to the wrist is quite rare. We present two unusual cases of NF involving the palm, supported by detecting ubiquitin specific protease 6 gene rearrangement. The first patient had non-intraneural NF presenting as peripheral neuropathy affecting the digital nerve while the second patient suffered from painless, non-tender NF in the palm, which had not regressed spontaneously during the five months prior to surgery.

ϒ-secretase and LARG mediate distinct RGMa activities to control appropriate layer targeting within the optic tectum.

While a great deal of progress has been made in understanding the molecular mechanisms that regulate retino-tectal mapping, the determinants that target retinal projections to specific layers of the optic tectum remain elusive. Here we show that two independent RGMa-peptides, C- and N-RGMa, activate two distinct intracellular pathways to regulate axonal growth. C-RGMa utilizes a Leukemia-associated RhoGEF (LARG)/Rho/Rock pathway to inhibit axonal growth. N-RGMa on the other hand relies on ϒ-secretase cleavage of the intracellular portion of Neogenin to generate an intracellular domain (NeICD) that uses LIM-only protein 4 (LMO4) to block growth. In the developing tectum (E18), overexpression of C-RGMa and dominant-negative LARG (LARG-PDZ) induced overshoots in the superficial tectal layer but not in deeper tectal layers. In younger embryos (E12), C-RGMa and LARG-PDZ prevented ectopic projections toward deeper tectal layers, indicating that C-RGMa may act as a barrier to descending axons. In contrast both N-RGMa and NeICD overexpression resulted in aberrant axonal-paths, all of which suggests that it is a repulsive guidance molecule. Thus, two RGMa fragments activate distinct pathways resulting in different axonal responses. These data reveal how retinal projections are targeted to the appropriate layer in their target tissue.

Hand metastasis from a sacral chordoma.

Chordomas are rare, low grade, malignant tumours derived from the ectopic remnants of the notochord that line the axial skeleton. They are characterised by their slow growth, long disease course and propensity for local relapse. Furthermore, up to 40% of non-cranial chordomas metastasise. We describe the first reported case of a hand metastasis arising from a conventional sacral chordoma after carbon ion radiotherapy. The common occurrence of distant metastasis with chordomas makes it important to perform a systemic examination, in part because their resection might improve patient prognosis.

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells.

AIMS/HYPOTHESIS: The molecular basis of the exocytosis of secretory insulin-containing granules (SGs) during biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells remains unclear. Syntaxin (SYN)-1A and SYN-4 have been shown to mediate insulin exocytosis. The insulin-secretory function of SYN-3, which is particularly abundant in SGs, is unclear. METHODS: Mouse pancreatic islets and INS-1 cells were treated with adenovirus carrying Syn-3 (also known as Stx3) or small interfering RNA targeting Syn-3 in order to examine insulin secretion by radioimmunoassay. The localisation and distribution of insulin granules were examined by confocal and electron microscopy. Dynamic single-granule fusion events were assessed using total internal reflection fluorescence microscopy (TIRFM). RESULTS: Depletion of endogenous SYN-3 inhibited insulin release. TIRFM showed no change in the number or fusion competence of previously docked SGs but, instead, a marked reduction in the recruitment of newcomer SGs and their subsequent exocytotic fusion during biphasic GSIS. Conversely, overexpression of Syn-3 enhanced both phases of GSIS, owing to the increase in newcomer SGs and, remarkably, to increased SG-SG fusion, which was confirmed by electron microscopy. CONCLUSIONS/INTERPRETATION: In insulin secretion, SYN-3 plays a role in the mediation of newcomer SG exocytosis and SG-SG fusion that contributes to biphasic GSIS.

Mechanisms of exocytosis.

Catecholamines and peptides secreted from dense-core vesicles (DCVs) of adrenal chromaffin cells regulate a wide variety of physiological processes. For instance, the release of noradrenaline and adrenaline plays a key role in regulating heart rate and blood pressure. Thus understanding the mechanisms of secretory processes of DCVs is crucial for understanding the basis of diseases such as hypertension. DCVs undergo several stages of secretory processing before they are exocytosed. These include docking, priming and triggering of membrane fusion/exocytosis. Molecular studies of DCV exocytosis have identified many proteins critically involved in DCV secretion. These proteins include SNARE proteins, Munc18-1, phosphatidylinositol transfer protein, type I phosphatidylinositol-4-phosphate-5-kinases, NSF, Munc13, CAPS1, synaptotagmins, RalA/RalB GTPases and exocyst proteins. In this article, I will discuss the functions of these proteins within the context of the stages (i.e. docking, priming and triggering of membrane fusion/exocytosis) in DCV secretion.

Prognosis and postoperative lymphocyte count in patients with hepatocellular carcinoma who received intraoperative allogenic blood transfusion: a retrospective study.

AIMS: The effect of perioperative blood transfusion on the survival of hepatocellular carcinoma (HCC) has not been fully investigated. To clarify the prognostic value of intraoperative allogenic blood transfusion, we conducted a comparative retrospective analysis of 224 patients with HCC who underwent hepatic resection. METHODS: We compared clinicopathologic background and survival after hepatic resection between patients who received intraoperative blood transfusion (n=101) and those who did not (n=123). RESULTS: Patients with blood transfusion had a larger tumor and more frequent vascular invasion than those without blood transfusion. The 5-year cancer-related survival rate after hepatic resection, but not the disease-free survival rate, was significantly lower in patients who underwent blood transfusion than in those who did not (38.3% vs. 66.7%, P<0.01). Multivariate analysis showed intraoperative blood transfusion (P=0.02), microscopic portal invasion (P<0.01), and preoperative serum alpha-fetoprotein elevation (P=0.03) to be independent risk factors for poor outcome after hepatic resection. The negative effect of blood transfusion on postoperative survival was observed only in patients with a tumor larger than 50mm in diameter. The absolute peripheral blood lymphocyte count on postoperative day 1 was significantly lower in patients who underwent blood transfusion (880/mm(3)) than in those who did not (1081/mm(3)) (P<0.01). CONCLUSIONS: Our data suggest that intraoperative blood transfusion results in immunosuppression in the early postoperative period, allowing for progression of residual HCC after resection. Therefore, intraoperative allogenic blood transfusion should be avoided in patients with resectable HCC, particularly in those with a large tumor.

Induced expression of c-fos in the diencephalon and pituitary gland of goats following transportation.

To identify regions of the caprine diencephalone and pituitary gland related to transportation stress, the expression of c-fos protein was examined immunohistochemically as an indicator of neural activation. Ten castrated Shiba goats (Capra hircus), five transported and five controls, were used. Transported goats were trucked for 1 h and killed by transcardiac perfusion 1 h after the end of transportation. Control goats were housed in single pens killed in the same manner and at the same time as the transported goats. The diencephalon and the pituitary gland were removed after perfusion and used for immunostaining. Plasma cortisol concentrations during and after transportation also were investigated. During transportation, plasma cortisol concentrations increased (P < 0.05) compared with those in the controls. In the diencephalon, c-fos immunoreactive cells were detected in the subcallosa, the lateral septal area, the bed nucleus of stria terminalis (BNST), the preoptic hypothalamic area (POA), the suprachiasmatic nucleus (SCN), the supraoptic nucleus, the paraventricular hypothalamic nucleus parvocellular (PVNp), the paraventricular hypothalamic nucleus magnocellular (PVNm), the arcuate nucleus (ARC), the paraventricular thalamic nucleus, and the stria medullaris in both control and transported goats. The numbers of c-fos immunoreactive cells were increased (P < 0.05) by transportation in the PVNm, the PVNp, the BNST, the POA, the ARC, and the SCN (P < 0.10). In the anterior pituitary gland, the number of c-fos immunoreactive cells in transported goats was 4 to 30 times as much as in control goats; however, there were no differences in the intermediate and posterior lobes between control and transported goats. This study has identified regions in the caprine diencephalon and pituitary gland that show transport-induced increases in c-fos immunoreactive cells. In conclusion, the PVNm, the PVNp, the BNST, the POA, the SCN in the diencephalons, and the anterior lobe of pituitary gland may be involved in the stress responses of goats to transportation.

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis.

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).

The biochemical analysis of neurotransmitters in central neurocytomas.

Central neurocytomas are rare benign intraventricular tumours composed of small round synaptophysin-positive cells, suggesting a neuronal origin of these tumour cells. Although past electron microscopic studies demonstrated synaptic vesicles in the synapse of central neurocytomas, the kinds of neurotransmitters in central neurocytomas have never been identified. In this study we analyzed neurotransmitters in an attempt to clarify the tumorigenesis of central neurocytomas. We studied frozen central neurocytoma specimens from four patients. The tissue levels of glutamate and GABA (gamma-aminobutyric acid) in the tumours were determined by gas chromatography-mass spectrometry (GC-MS) using a selected ion monitoring method. The tissue levels of acetylcholine, choline, catecholamines and metabolites of catecholamines in the tumours were measured by high-performance liquid chromatography combined with electrochemical detection. Choline was found in extremely high concentration in all central neurocytomas when compared with levels in controls. In one central neurocytoma, GABA was found in extremely high concentration compared with controls. In all central neurocytomas, glutamate was found in lower or identical concentrations compared with controls. In all central neurocytomas and controls, dopamine and catecholamine concentrations were extremely low. These results indicated that the histogenesis of central neurocytomas begins with the subependymal stem cells, which have the potential to differentiate into cholinergic or GABA neurons.

Soluble Fas ligand and soluble Fas in ocular fluid of patients with uveitis.

AIMS: To investigate the presence of soluble Fas ligand (sFasL) and soluble Fas (sFas) in ocular fluid of patients with uveitis. METHODS: Samples of aqueous humour (AH, n=17), vitreous fluid (n=9), and serum (n=60) were collected from patients with uveitis which included Behçet's disease, Vogt-Koyanagi-Harada disease, sarcoidosis, human T lymphotropic virus type 1 (HTLV-I) uveitis, sympathetic ophthalmia, HLA-B27 associated acute anterior uveitis, and ocular toxoplasmosis. The AH of patients with age related cataract without uveitis obtained during cataract surgery was used as controls (n=20). The amounts of sFasL and sFas were measured by enzyme linked immunosorbent assay. RESULTS: Significant amounts of sFasL were detected in AH of patients with age related cataract (non-uveitis group). sFasL was also detected in AH of patients with uveitis, though the amounts were slightly lower than those in the non-uveitis group. On the other hand, the levels of sFas in AH of patients with uveitis were significantly higher than those in controls. As for the disease activity, the levels of sFasL and sFas in the vitreous fluid of patients with active uveitis were significantly higher than those in inactive uveitis. sFasL in the serum of healthy donors and patients with uveitis was below detectable levels, except for patients with HTLV-I uveitis who had significant amounts of sFasL in the serum. The levels of sFas in the serum of patients with Behçet's disease, sarcoidosis, and HTLV-I uveitis were significantly higher than those of healthy donors. CONCLUSIONS: sFasL is present in the AH of non-uveitic eyes with age related cataract. Intraocular levels of sFasL and sFas are significantly increased in uveitis, particularly in active uveitis. These data suggest that intraocular sFasL and sFas may have a regulatory role in uveitis.

Immunoregulation by aqueous humor.

PURPOSE: The eye is a classic example of an immune-privileged site. To investigate the local defense system of the eye, the immunosuppressive effects of the aqueous humor on cytokine production was examined. METHODS: Using T cell clones (TCCs) established from infiltrating cells in the aqueous humor of patients with human T lymphotropic virus type 1 (HTLV-1) uveitis as target cells and the aqueous humor of patients with senile cataract obtained during cataract surgery, we examined the effects of the aqueous humor on the production of cytokines by HTLV-1-infected TCCs. RESULTS: HTLV-1-infected TCCs produced large amounts of various cytokines. The aqueous humor inhibited the production of cytokines in a dose-dependent manner. The inhibitory activity was heat labile. First protein liquid chromatography showed at least four major peaks of different molecular size, indicating that the aqueous humor contains multiple immunosuppressive factors. Transforming growth factor-beta, alpha-melanocyte-stimulating hormone, and vasoactive intestinal peptide did not suppress cytokine production. The inhibitory activity was neutralized by monoclonal antibody to Fas ligand. CONCLUSION: These data suggest that soluble Fas ligand is a candidate suppressive factor in the aqueous humor.

Renal cell carcinoma in an L-shaped kidney.

A 64-year-old man with a chief complaint of an asymptomatic abdominal mass was diagnosed as having a renal cell carcinoma in his L-shaped kidney. He was successfully treated with partial nephrectomy following selective embolization of the feeder artery. It is thought to be the first reported case of renal cell carcinoma occurring in an L-shaped kidney.

Evolutionary characterization of the six internal genes of H5N1 human influenza A virus.

The entire nucleotide sequences of all six internal genes of six human H5N1 influenza A viruses isolated in Hong Kong in 1997 were analysed in detail from a phylogenetic point of view and compared with the evolutionary patterns of the haemagglutinin and neuraminidase genes. Despite being isolated within a single year in the same geographical location, human H5N1 viruses were characterized by a variety of amino acid substitutions in the ribonucleoprotein complex [PB2, PB1, PA and nucleoprotein (NP)] as well as the matrix (M) proteins 1 and 2 and nonstructural (NS) proteins 1 and 2. The presence of previously reported amino acid sequences specific for human strains was confirmed in the PB2, PA, NP and M2 proteins. Nucleotide and amino acid sequence identities of the six internal genes of H5N1 viruses examined here were separated into at least two variant groups. In agreement with the above result, phylogenetic trees of the six internal genes of human H5N1 viruses were generally composed of two minor clades. Additionally, variable dendrogram topologies suggested that reassortment among viruses contributed further to the genetic variability of these viruses. As a result, it became clear that human H5N1 viruses are characterized by divergent gene constellations, suggesting the possible occurrence of genetic reassortment between viruses of the two evolutionary lineages.

The RIM/NIM family of neuronal C2 domain proteins. Interactions with Rab3 and a new class of Src homology 3 domain proteins.

RIM1 is a putative effector protein for Rab3s, synaptic GTP-binding proteins. RIM1 is localized close to the active zone at the synapse, where it interacts in a GTP-dependent manner with Rab3 located on synaptic vesicles. We now describe a second RIM protein, called RIM2, that is highly homologous to RIM1 and also expressed primarily in brain. Like RIM1, RIM2 contains an N-terminal zinc finger domain that binds to Rab3 as a function of GTP, a central PDZ domain, and two C-terminal C(2) domains that are separated by long alternatively spliced sequences. Unexpectedly, the 3'-end of the RIM2 gene produces an independent mRNA that encodes a smaller protein referred as NIM2. NIM2 is composed of a unique N-terminal sequence followed by the C-terminal part of RIM2. Data bank searches identified a third RIM/NIM-related gene, which encodes a NIM isoform referred to as NIM3; no RIM transcript from this gene was detected. To test if NIMs, like RIMs, may function in secretion, we investigated the effect of NIM3 on calcium-triggered exocytosis in PC12 cells. NIM3 induced a dramatic increase in calcium-evoked exocytosis (50%), with no significant effect on base-line release, suggesting that NIMs, like RIMs, regulate exocytosis The combination of conserved and variable sequences in RIMs and NIMs indicates that the individual domains of these proteins provide binding sites for interacting molecules during exocytosis, as shown for the zinc finger domain of RIM, which binds to GTP-bound Rab3s. To search for additional interacting proteins for RIMs, we employed yeast two-hybrid screens with the C-terminal half of RIM1. Two members of a new family of homologous brain proteins, referred to as RIM-binding proteins (RIM-BPs), were identified. RIM-BPs bind to RIM in yeast two-hybrid and GST pull-down assays, suggesting a specific interaction. In RIMs, the binding site for RIM-BPs consists of a conserved proline-rich sequence between the two C(2) domains, N-terminal to the beginning of NIMs. RIM-BPs are composed of multiple domains, including three fibronectin type III-domains and three Src homology 3 domains, of which the second Src homology 3 domain binds to RIMs. With the RIM-BPs, we have identified a partner for RIMs that may bind to RIMs at the synapse in addition to Rab3.

Specificity of Ca2+-dependent protein interactions mediated by the C2A domains of synaptotagmins.

Synaptotagmins represent a family of neuronal proteins thought to function in membrane traffic. The best characterized synaptotagmin, synaptotagmin I, is essential for fast Ca2+-dependent synaptic vesicle exocytosis, indicating a role in the Ca2+ triggering of membrane fusion. Synaptotagmins contain two C2 domains, the C2A and C2B domains, which bind Ca2+ and may mediate their functions by binding to specific targets. For synaptotagmin I, several putative targets have been identified, including the SNARE proteins syntaxin and SNAP-25. However, it is unclear which of the many binding proteins are physiologically relevant. Furthermore, more than 10 highly homologous synaptotagmins are expressed in brain, but it is unknown if they execute similar binding reactions. To address these questions, we have performed a systematic, unbiased study of proteins which bind to the C2A domains of synaptotagmins I-VII. Although the various C2A domains exhibit similar binding activities for phospholipids and syntaxin, we found that they differ greatly in their protein binding patterns. Surprisingly, none of the previously characterized binding proteins for synaptotagmin I are among the major interacting proteins identified. Instead, several proteins that were not known to interact with synaptotagmin I were bound tightly and stoichiometrically, most prominently the NSF homologue VCP, which is thought to be involved in membrane fusion, and an unknown protein of 40 kDa. Point mutations in the Ca2+ binding loops of the C2A domain revealed that the interactions of these proteins with synaptotagmin I were highly specific. Furthermore, a synaptotagmin I/VCP complex could be immunoprecipitated from brain homogenates in a Ca2+-dependent manner, and GST-VCP fusion proteins efficiently captured synaptotagmin I from brain. However, when we investigated the tissue distribution of VCP, we found that, different from synaptic proteins, VCP was not enriched in brain and exhibited no developmental increase paralleling synaptogenesis. Moreover, binding of VCP, which is an ATPase, to synaptotagmin I was inhibited by both ATP and ADP, indicating that the native, nucleotide-occupied state of VCP does not bind to synaptotagmin. Together our findings suggest that the C2A-domains of different synaptotagmins, despite their homology, exhibit a high degree of specificity in their protein interactions. This is direct evidence for diverse roles of the various synaptotagmins in brain, consistent with their differential subcellular localizations. Furthermore, our results indicate that traditional approaches, such as affinity chromatography and immunoprecipitations, are useful tools to evaluate the overall spectrum of binding activity for a protein but are not sufficient to estimate physiological relevance.

Clinical features of human T-lymphotropic virus type 1 uveitis: a long-term follow-up.

To investigate the clinical manifestations of human T-lymphotropic virus type-1 uveitis (HU), 112 HU patients who were followed up periodically for more than one year were retrospectively analyzed with respect to their ophthalmological and systemic complications. The gender ratio (female/male ratio) of the HU patients was 2.0 and the initial complications were foggy vision in 34.5%, ocular floaters in 33.3%, and blurred vision in 15.5%. As for the ocular symptoms, the majority (78.6%) of patients were classified as intermediate uveitis with vitreous inflammation. Recurrence of uveitis episodes was seen in one half of the patients (51.8%); 12 patients had more than six uveitis episodes. The interval of uveitis episodes varied from two weeks to 10 years. Nearly one half of the patients (43.8%) had ocular complications: e.g., cataract in 22 patients, persistent vitreous opacities in 17 patients, and glaucoma in 16 patients. Although the visual prognosis was essentially good, 11 patients had poor visual prognosis (<0.1). The causes of poor vision in these patients were cataract, cystoid macular edema, epiretinal membrane, and optic nerve atrophy. Of the 112 HU patients, two developed HTLV-I-associated myelopathy (TSP/HAM) after the onset of HU, while none developed adult T-cell leukemia. Sixteen HU patients had a previous history of Graves' disease and a past history of methimazole therapy, while Graves' disease was found in another HU patient only after HU onset and methimazole was not administered before the onset of HU. The present data of long-term follow-up indicate that (1) HU causes various ocular complications and its visual prognosis can be poor, (2) TSP/HAM can be induced even after the onset of HU, and (3) methimazole is not a risk factor of HU after Graves' disease.