Lens epithelium-derived growth factor: effects on growth and survival of lens epithelial cells, keratinocytes, and fibroblasts.

We isolated a clone encoding a protein from a human lens epithelial cell (LEC) cDNA library with antibody (Ab) from a cataract patient and named it "lens epithelium-derived growth factor" (LEDGF). LEDGF is found to be identical to p75, a coactivator of both transcription (1) and pre-mRNA splicing (2). In serum-free medium LEDGF stimulated growth of LECs, cos7 cells, skin fibroblasts, and keratinocytes, and prolonged cell survival. Without LEDGF, the aforementioned cells did not survive. Also in serum-free medium, Ab to LEDGF neutralizing LEDGF blocked cell growth and caused cell death. Thus, LEDGF, a regulatory factor, may play an important role for growth and survival of a wide range of cell types.

CIS3 and JAB have different regulatory roles in interleukin-6 mediated differentiation and STAT3 activation in M1 leukemia cells.

We have reported JAK-signaling modulators, CIS1 (cytokine-inducible SH2 protein-1), CIS3 and JAB (JAK2 binding protein), which are structurally related. In M1 myeloid leukemia cells, CIS3 was induced by neither interleukin 6 (IL6) nor interferon gamma (IFNgamma), while JAB was induced strongly by IFNgamma and slightly by IL6 and leukemia inhibitory factor (ILF). Forced expression of CIS3 and JAB in M1 cells prevented IL6- or LIF-induced growth arrest and differentiation, even when their expression levels were comparable to endogenous ones in several cell lines such as HEL, UT-7, IFNgamma-treated M1, and CTLL2 cells. Pretreatment of parental M1 cells with IFNgamma but not IFNbeta resulted in suppression of LIF-induced STAT3 activation and differentiation, further supporting that physiological level of JAB is sufficient to inhibit LIF-signaling. However, unlike JAB, CIS3 did not inhibit IFNgamma-induced growth arrest, suggesting a difference in cytokine specificity between CIS3 and JAB. CIS3 inhibited STAT3 activation with slower kinetics than JAB and allowed rapid c-fos induction and partial FcgammaRI expression in response to IL6. In 293 cells, CIS3 as well as JAB bound to JAK2 tyrosine kinase domain (JH1), and inhibited its kinase activity, however, the effect of CIS3 on tyrosine kinase activity was weaker than that of JAB, indicating that CIS3 possesses lower affinity to JAK kinases than JAB. These findings suggest that CIS3 is a weaker inhibitor than JAB against JAK signaling, and JAB and CIS3 possess different regulatory roles in cytokine signaling.

A Janus kinase inhibitor, JAB, is an interferon-gamma-inducible gene and confers resistance to interferons.

It has been shown that interferons (IFNs) exert their signals through receptor-associated Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). However, molecular mechanism of regulation of IFN signaling has not been fully understood. We have reported novel cytokine-inducible SH2 protein (CIS) and JAK binding protein (JAB) family genes that can potentially modulate cytokine signaling. Here we report that JAB is strongly induced by IFN-gamma but not by IFN-beta in mouse myeloid leukemia M1 cells and NIH-3T3 fibroblasts. NIH-3T3 cells ectopically expressing JAB but not CIS3 lost responsiveness to the antiviral effect of IFN-beta and IFN-gamma. M1 leukemic cells stably expressing JAB were also resistant to IFN-gamma and IFN-beta-induced growth arrest. In both NIH-3T3 and M1 transformants expressing JAB, IFN-gamma did not induce tyrosine phosphorylation and DNA binding activity of STAT1. Moreover, IFN-gamma-induced activation of JAK1 and JAK2 and IFN-beta-induced JAK1 and Tyk2 activation were inhibited in NIH-3T3 JAB transformants. These results suggest that JAB inhibits IFN signaling by blocking JAK activity. We also found that IFN-resistant clones derived from LoVo cells and Daudi cells expressed high levels of JAB without stimulation. In IFN-resistant Daudi cells, IFN-induced STAT1 and JAK phosphorylation was partially reduced. Therefore, overexpression of JAB could be, at least in part, a mechanism of IFN resistance.

Morphologic characteristics of N-methyl-N-nitrosourea-induced retinal degeneration in C57BL mice.

Morphologic characteristics of retinal degeneration induced by a single systemic administration of N-methyl-N-nitrosourea (MNU) in mice was investigated. The aim was to characterize the MNU-induced retinal lesions in mice and compare them with human retinitis pigmentosa. A dose of 60 mg/kg body weight MNU, injected intraperitoneally into male and female C57BL mice, evoked progressive retinal degeneration in all treated mice, while control mice remained normal. An early change was photoreceptor apoptosis followed by infiltration of macrophages and swelling of the pigment epithelial cells with phagosomal inclusions for apoptotic photoreceptor cell removal. Loss of the majority of photoreceptor cells occurred within a week. Then, Feulgen-positive corpuscles, indicative of an aggregation of degenerative photoreceptor elements, vitread the outer limiting membrane were surrounded by Müller cell processes, and the duplication of the pigment epithelial cells sclerad the outer limiting membrane were seen 2 and 3 weeks after the treatment. Finally, the Feulgen-positive corpuscles disappeared and Müller cell processes were in direct contact with the continuous lining of the single layer of pigment epithelial cells. As in retinitis pigmentosa in humans, the primary event was loss of photoreceptor cells by apoptosis, but the migration of the pigment epithelial cells within the retina was not seen in the present model.

['Ischemic tolerance' in ischemia-reperfusion injury in the optic nerve in rats].

Brief ischemia caused by high intraocular pressure induced tolerance to subsequent ischemia-reperfusion injury in rats. Male Wistar rats were subjected to 15 minutes of ischemia. This ischemic injury did not show distinct axonal damage in the optic nerve in electron microscopy. 1, 2, 4, 7, and 14 days after the first 15 min ischemia, the rats were subjected to a second ischemia for 45 minutes (ischemic tolerance). After 1 week, the rats were perfusion fixed and the optic nerves were processed for light and electron microscopy. Samples of the axonal density in the central optic nerve 2 mm behind the lamina cribrosa were observed and counted an electron micrographs. In axonal morpometric findings, 2 days and more after brief ischemia, the damage was lessened more than after 45 minutes ischemia (control) and the difference was significant. This 'ischemic tolerance' induced by brief ischemia might be considered the same stress as brain ischemia-reperfusion injury.

Antinuclear antibodies in children with chronic nonspecific complaints.

Children who are chronically complaining nonspecific symptoms such as headache, fatigue, abdominal pain, and low grade fever are commonly seen in daily pediatric outpatient clinics. Some of them are unable to go to school and are diagnosed as school refusal by physicians or educational staff. On the other hand, there are children who do not fulfill any criteria of collagen diseases and whose anti-nuclear antibodies (ANA) are found to be positive. Some of these children have chronic nonspecific complaints. We prospectively studied the prevalence of ANA in children who visited a pediatric outpatient clinic because of chronic nonspecific complaints. Surprisingly, 74 out of 140 symptomatic children (52.4%) were positive for ANA, while only 5 out of 82 healthy control children (6.1%) were positive (p < 0.0001). 39 of 74 ANA positive patients (52.1%) have low ANA titers < or = 1:80, nevertheless 36 patients (47.9%) have high ANA titers > or = 1:160. ANA fluorescent patterns were homogeneous and speckled in 75.3%, speckled in 17.6% and others in 6.8%. ANA positive patients tended to have general fatigue and low grade fever, while gastrointestinal problems such as abdominal pain and diarrhea and orthostatic dysregulation symptoms were commonly seen in ANA negative patients. Children who were unable to go to school more than 1 day a week were seen significantly more in ANA positive patients than in negative patients. Autoantibody analysis using Western immunoblot revealed that 26 out of 63 ANA positive sera (41.3%) had antibodies to the 62 kD protein which had not been previously noticed. These data suggest that autoimmune mechanism may play a role in childhood chronic nonspecific symptoms. We therefore propose a new disease entity of the autoimmune fatigue syndrome in children. When chronically complaining children visit a pediatric out-patient clinic, immunological approaches should be considered before they are discriminated as school refusal or having psychogenic disorders.

Fas-independent apoptosis of photoreceptor cells in C3H mice.

The morphogenesis of the photoreceptor cells in the retinas of C3H mice carrying the rd gene and C57BL mice carrying the normal gene was compared, and retinas of the C3H mutants (C3H-lpr/lpr, -lprcg/lprcg, and -lpr/lpr-gld/gld) defective in apoptosis through the Fas system were examined. In the C57BL retina, the inner and outer nuclear layers were separated at 8 days of age, and the photoreceptor inner and outer segments began to grow between 8-11 days after birth with their most rapid growth occurring between 14-17 days of age. In the C3H retina, the development was comparable to that of the C57BL retina at 8 days of age but the reduction in thickness of the outer nuclear and photoreceptor layers was noted at 11 days of age, and the outer nuclear layer became reduced to only a few nuclei in thickness at 14 days, being completely missing or reduced to a single row of cells at 20 days. The degeneration was by an apoptotic mechanism as confirmed morphologically and by the TUNEL method. In all the C3H mutant retinas examined over 24 days of age, the complete depletion of the outer nuclear layer or reduction to a single row comparable to 20-day-old C3H mice was seen. The rd gene action is therefore independent of Fas/Fas ligand-medicated apoptosis.

Treatment with bifemelane for optic nerve damage following high intraocular pressure in rat eyes.

This study found that pretreatment with 4-(o-benzylphenoxy)-N- methylbuty-lamine hydrochloride (bifemelane hydrochloride, Celeport) reduced ischemia-reperfusion injury in rat eyes. Bifemelane (25 mg/kg) was injected intraperitoneally 30 minutes before an ischemic insult, then acute ischemia of the retina and optic disc was induced by increasing intraocular pressure to 110 mmHg for 45 minutes. After one week, the axonal count of the optic nerve was investigated using electron microscopy. The control group consisted of vehicle-treated eyes which received normal saline. The axon count was 93.4 +/- 7.9 for the bifemelane treated group, and 79.2 +/- 6.4 for the controls. The axon count in the treated group was significantly higher. These results suggest that bifemelane, which prevents cerebral nerve cell damage from ischemia, can reduce ischemic retinal nerve cell injury.

N-methyl-N-nitrosourea-induced photoreceptor apoptosis in the mouse retina.

Retinal degeneration induced by a single intraperitoneal injection of N-methyl-N-nitrosourea (MNU) in male and female albino (GRS/A and DDD/1) and colored (C57BL) mice at 7 weeks of age was examined morphologically 1, 3, 7, 14 and 21 days after the treatment. A dose of 60 mg/kg body weight evoked progressive retinal degeneration in all mice. All albino and colored mice had a comparable progression of photoreceptor cell degeneration by an apoptotic mechanism, as confirmed by morphological and TUNEL methods. Apoptosis had already taken place 1 day after the treatment and was completed by Day 7. This process resulted in a thin remnant of retina with complete loss of photoreceptor cells-21 days after the treatment. During the course of apoptosis, the pigment epithelial cells were maintained in a continuous layer in all strains of mice. In colored mice, several layers of the swollen pigment-enriched cells were seen between the inner nuclear layer and the pigment epithelial layer 14 and 21 days after the treatment. In summary, the destruction of photoreceptor cells by the apoptotic process was the mechanism by which retinal degeneration was induced by MNU.

Evaluation of myocardial ischemia and infarction by signal-averaged electrocardiographic late potentials in children with Kawasaki disease.

We investigated myocardial ischemia and old myocardial infarction noninvasively using signal-averaged electrocardiographic late potentials (LPs) in patients with Kawasaki disease. Patients were divided into 4 groups: a noncoronary artery lesion group (n=136), a coronary artery lesion group (without myocardial ischemia and an old myocardial infarction; n=33), an ischemia group (n=16), and an old myocardial infarction group (n=13). Grouping was based on exercise thallium-201 myocardial scintigraphy, thallium-201 myocardial scintigraphy, exercise electrocardiography, coronary angiography, left ventriculography, and echocardiography. Signal-averaged electrocardiograms were recorded using a high-resolution system. Values of filtered QRS duration (f-QRSd), root-mean-square voltage, and duration of low-amplitude signal were judged using our own body surface area-related criteria (n=205) to determine positive rates of LPs and sensitivities and specificities to ischemia and infarction. These data were also interpreted using published criteria for adults and compared with those interpreted by our criteria. Positive rates by our criteria were 0% in the noncoronary artery lesion group, 9.1% in the coronary lesion group, 56.3% in the ischemia group, and 69.2% in the old myocardial infarction group. However, using the criteria for adults, these values were 0%, 3.0%, 25%, and 46.2%, respectively. Sensitivities to ischemia and infarction using our criteria were significantly higher (56.3% and 69.2%) than those using the criteria for adults (p < 0.05). Moreover, specificities to ischemia and infarction were very high (93.4% and 93.5%, respectively) using our criteria, and there were no significant differences from specificities using the criteria for adults. Also, we examined the reproducibility of values of LPs and LP parameters. The values of filtered QRS duration showed a high reproducibility in both LP-positive and -negative groups, followed by low-amplitude signal and then root-mean-square voltage. The results of LP presence or absence showed 100% reproducibility for both the LP-positive and -negative groups, supporting the utility of LPs for clinical applications. Thus, LPs provide useful information in a noninvasive manner for clarifying ischemia and infarction in patients with Kawasaki disease.

Photoreceptor apoptosis induced by a single systemic administration of N-methyl-N-nitrosourea in the rat retina.

Retinal degeneration was induced by a single intraperitoneal injection of N-methyl-N-nitrosourea in female Sprague-Dawley albino rats at 50 days of age by two dose regimens, which were observed sequentially at 24, 48, and 72 hours and 7, 21, and 35 days after the treatment. After a dose of 75 mg/kg, methylnitrosourea evoked progressive retinal degeneration in all treated rats whereas a dose of 50 mg/kg was less effective. The 75-mg/kg-treated rats showed selective destruction of the photoreceptor cells by an apoptotic mechanism, as confirmed morphologically and by the terminal dUTP nick end labeling method. Apoptosis had already started at 24 hours after the treatment and was completed by day 7. During the photoreceptor degeneration, proliferation of glial fibrillary acidic protein and vimentin-positive Müller cells as detected by proliferating cell nuclear antigen labeling appeared at 48 hours and was prominent 72 hours after the treatment, and macrophage infiltration within the retina as recognized by ED1 positivity was maximal 7 and 21 days after the treatment. Retinal degeneration was also induced in female Brown-Norway colored rats in a similar dose-dependent manner. Pigment epithelium was discontinuous above Bruch's membrane, and migration of the swollen pigment epithelium toward the inner nuclear layer was seen 7 days after the treatment. Therefore, as also confirmed electron microscopically, the most striking change was the destruction of photoreceptor cells by the apoptotic process, followed by Müller cell proliferation, pigment epithelium migration, and macrophage infiltration for cell debris phagocytosis, resulting in a thin remnant of retina with attenuated inner nuclear cells in direct contact with Bruch's membrane or with the pigment epithelium and/or with the Müller cells 35 days after the treatment.

Neonatal cholestasis in two siblings: a variant of Dubin-Johnson syndrome?

OBJECTIVE: Two Japanese brothers with neonatal cholestasis associated with pigment granules in the hepatocytes and hepatosteatosis were evaluated for the possible role of hepatosteatosis in the Dubin-Johnson syndrome. METHODOLOGY AND RESULTS: The morphology of pigment accumulation and the laboratory data in these cases were examined. The elevation of urinary coproporphrin isomer I to more than 90% and the presence cholestasis resembled that in the Dubin-Johnson syndrome, but the hypertriglyceridaemia ( > 1.13 mmol/L as triolein) and the hepatosteatosis differed. Both infants were thought to have familial hypertriglyceridaemia. However, this diagnosis was difficult to confirm in the absence of data on the normal values of apolipoprotein and lipoprotein isomer for infants. CONCLUSIONS: A neonatal variant of the Dubin-Johnson syndrome may account for the unusual findings in these infants.

Immunohistochemical features of the human retina and retinoblastoma.

The immunohistochemical features of 24 retinoblastoma specimens from 22 patients, 15 with unilateral and 7 with bilateral disease, were examined by the labelled streptavidin biotin (LSAB) method and compared with those of specimens from the remaining morphologically normal retina. In the normal retina, S-100 protein, glial fibrillary acidic protein (GFAP) and vimentin were detected in astrocytes and/or Müller cells. Neurofilament protein was seen in axons of the ganglion cells, synaptophysin was present in both plexiform layers, bcl-2 oncoprotein was seen in ganglion cells and bipolar cells, and neuron-specific enolase (NSE) was detected in ganglion cells, bipolar cells and photoreceptor cells and in their cell processes. While retinoblastoma (Rb) protein expression was noted in ganglion cells, bipolar cells, and some photoreceptor cells, p53 protein was not expressed at all. In all retinoblastomas, strong NSE expression and weak bcl-2 expression was observed in almost all tumour cells and synaptophysin was localized in rosette-forming cells, while tumour cells were devoid of S-100, GFAP, vimentin and neurofilament protein. These findings support the view that retinoblastomas are composed of neuron-committed cells. In addition, no Rb protein expression was detected in retinoblastomas, whereas p53 expression was found in 18 cases (75%).

A Ro/SS-A auto-antibody positive mother's infant revealed congenital complete atrioventricular block, followed by insulin dependent diabetes mellitus and multiple organ failure.

We experienced a congenital complete atrioventricular block infant who was born from a Ro/SS-A antibody positive mother. Ro/SS-A antibody was also found in this baby which was presumed to be mediated by the maternal placenta. Temporary cardiac pacing was required at birth and pacemaker implantation was performed at 9 months. At 11 months of age, the baby fell into shock and experienced multiple organ failure because of diabetes mellitus-induced coma. The association between congenital complete heart block and the Ro/SS-A antibody is well known. However, the accompaniment of insulin-dependent diabetes mellitus has not been reported previously. As the Ro/SS-A antigen appears in the cytoplasm of many tissues, the possibility of an association between Ro/SS-A antibody and diabetes mellitus is difficult to deny. We report this rare case to draw attention to the possibility that babies who are born from an Ro/SS-A antibody positive mother may develop diabetes mellitus as well as congenital complete heart block.

Functional oligomerization of purified human papillomavirus types 16 and 6b E7 proteins expressed in Escherichia coli.

Purified non-fused soluble human papillomavirus type 16 and 6b E7 proteins expressed in Escherichia coli were found to form oligomers. For both proteins, several degrees of oligomerization were demonstrated by gel filtration, dynamic laser light scattering and scanning electron microscopy. Oligomerization was dependent on the concentration of E7 protein. Oligomerized E7 proteins were able to bind the retinoblastoma gene product pRB and stimulated DNA synthesis when introduced into cells.

Transformation assays of the cell mitotic and proliferation activities of purified oncogene products.

Using two direct introduction methods, DNA synthesis or cell proliferation activities of three purified proteins from E. coli, namely, human papillomavirus (HPV) E7 proteins of type 16, a mutant type 16 (24 C-G) (transformation defective) and type 6b, were measured in mouse fibroblast, C127 cells. By a microinjection method, the order of the cell mitotic indexes for the three E7 proteins as determined by 5-bromo-2'-deoxy-uridine (BrdU) staining was type 16, 6b and 16 (24 C-G). By the osmotic shock method, the 3H-TdR incorporation and coloration by (3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetolazorium (MTS) for the three proteins correlated with the pRb binding and focus forming activities previously reported (Munger et al. 1991). These results indicate that the simple osmotic shock method for direct protein introduction may be generally useful for transformation assays of oncoproteins.

[Innervation of the external ear in humans and the musk shrew].

The external ear appears only in mammals and possesses a great diversity of forms. In addition, multiple nerves are distributed in the external ear. The nerves which are distributed in the external ear were investigated in humans and in the musk shrew (Suncus murinus) in a macroscopic study. The following results were obtained. Cranial nerves (CN) V, VII, X and cervical nerves supply the auricle and the external meatus in both humans and the musk shrew. Branches of the third division of CN V supply an anterior part of the external meatus and the anterior part of the auricle in both humans and the musk shrew. A branch of CN X, together with a branch of CN VII, supply the posterior parts of the external meatus in humans. In the musk shrew, a branch of CN X, together with a branch of CN VII, supplies the posterior part of the external meatus and the central part and the anterior portion of the auricle. Branches of the cervical nerve supply the posterior part of the auricle. Branches of the cervical nerve supply the posterior part of the external meatus and the central and posterior parts of the auricle in the musk shrew.