Predominant melanogenesis and lentoidogenesis in vitro from multipotent pineal cells by dimethyl sulfoxide and hexamethylene bisacetamide.

Pineal cells of the 8-day embryonic quail are multipotent cells which differentiate in vitro into skeletal muscle fibers, pigmented epithelial cells (PECs), lens cells and neurons. However, it was not yet clear whether precursor cells which gave such a wide repertoire of differentiation were single type or not. The present culture studies revealed that pineal cells were exclusively directed to ocular differentiation pathways by dimethyl sulfoxide (DMSO) and hexamethylene bisacetamide (HMBA), suggesting a single type of precursor cell in the pineal body. DMSO directed pineal cells to differentiate into PECs. Co-administration of basic fibroblast growth factor (bFGF) with DMSO partially inhibited PEC differentiation and promoted lens cell differentiation. Northern blot analysis using cDNAs specific to PEC and lens cell confirmed this morphological observation. HMBA completely inhibited pigmentation of cultured pineal cells and markedly promoted lens cell differentiation. Ocular differentiation of pineal cells was accompanied with the loss of myogenicity. We discuss three possible pathways of lens cell differentiation from pineal cells. The agents which affect pineal cell differentiation seemed to modulate the cell-substrate interaction. And the interaction was suggested to be one of the environmental cues in the differentiation.

Reno-submandibular axis controls release of extrarenal inactive renin.

The mechanisms causing the release of plasma inactive renin (PIR) area still unclear. We have investigated the role of the kidney in the release of trypsin-activable PIR from extrarenal sources in the rat, with special reference to the submandibular gland. The activation of PIR was performed by incubation with 20 mg/ml trypsin at 4 degrees C for up to 10 min; the reaction was then terminated by addition of 20 mg/ml of soybean trypsin inhibitor. Bilateral nephrectomy resulted in a gradual, marked, sex-independent increase in PIR concentration, reaching levels 4.5 times higher than basal in 24 h (time 0: 14.8 +/- 1.0 ng/ml per h; 24 h: 66.8 +/- 3.4 ng/ml per h, mean +/- s.d., P less than 0.001). This increase was not altered by the concomitant intravenous infusion of pressor doses of either angiotensin (Ang) II (30 ng/min) or pure mouse submandibular renin (a 20-ng intravenous bolus followed by intravenous infusion at the rate of 50 ng/h) for 4 h, but was completely prevented by prior removal of the submandibular glands, in which no activity of active renin and no inactive renin was detected. These results suggest that the post-nephrectomy increase of PIR is not dependent on feedback mechanisms of the suppressed renin-angiotensin system, but is controlled by the presence of submandibular glands in the rat.

Survival of axotomized retinal ganglion cells in peripheral nerve-grafted ferrets.

PURPOSE: Peripheral nerve (PN) grafting to the optic nerve stump stimulates not only axonal regeneration of the axotomized retinal ganglion cells (RGCs) into the grafted PN but also their survival. The purpose of the present study was to determine the number, distribution, and soma diameter of only surviving RGCs without regenerated axons and surviving RGCs with regenerated axons in PN-grafted mammals. METHODS: A segment of PN was grafted to the optic nerve stump of adult ferrets. Two months after the PN grafting, surviving RGCs with regenerated axons were retrogradely labeled with granular blue (GB) and stained with RGC-specific antibody C38. Surviving RGCs without regenerated axons were identified as C38-positive cells without GB labeling. RESULTS: Twenty-one percent of RGCs survived axotomy after PN grafting in the area centralis (AC), whereas 47% survived in the peripheral retina. Twenty-six percent of surviving RGCs in the AC exhibited axonal regeneration, which was higher than that in the peripheral retina. Soma diameter histograms revealed that RGCs with regenerated axons showing both GB and C38 positivity were in the large soma diameter ranges. In contrast, the soma diameter distribution of surviving RGCs that did not have regenerated axons showed a peak in the smaller soma diameter ranges. CONCLUSIONS: The present data suggest that PN grafting promotes survival of axotomized RGCs more effectively in the peripheral retina than in the AC. Among surviving RGCs, the larger cells exhibited axonal regeneration into the grafted PN, whereas the axons of smaller cells did not to regenerate in either the AC or the peripheral retina.

Nitric oxide-induced cytotoxicity attenuation by thiopentone sodium but not pentobarbitone sodium in primary brain cultures.

1. We describe the effects of barbiturates on the neurotoxicity induced by nitric oxide (NO) on foetal rat cultured cortical and hippocampal neurones. Cessation of cerebral blood flow leads to an initiation of a neurotoxic cascade including NO and peroxynitrite. Barbiturates are often used to protect neurones against cerebrovascular disorders clinically. However, its neuroprotective mechanism remains unclear. 2. In the present experiment, we established a new in vitro model of brain injury mediated by NO with an NO-donor, 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5) on grid tissue culture wells. We also investigated the mechanisms of protection of CNS neurones from NO-induced neurotoxicity by thiopentone sodium, which contains a sulphydryl group (SH-) in the medium, and pentobarbitone sodium, which does not contain SH-. 3. Primary cultures of cortical and hippocampal neurones (prepared from 16-day gestational rat foetuses) were used after 13-14 days in culture. The cells were exposed to NOC-5 at the various concentrations for 24 h in the culture to evaluate a dose-dependent effect of NOC-5. 4. To evaluate the role of the barbiturates, neurones were exposed to 4, 40 and 400 microM of thiopentone sodium or pentobarbitone sodium with or without 30 microM NOC-5. In addition, superoxide dismutase (SOD) at 1000 u ml(-1) and 30 microM NOC-5 were co-administered for 24 h to evaluate the role of SOD. 5. Exposure to NOC-5 induced neural cell death in a dose-dependent manner in both cortical and hippocampal cultured neurones. Approximately 90% of the cultured neurones were killed by 100 microM NOC-5. 6. This NOC-5-induced neurotoxicity was significantly attenuated by high concentrations of thiopentone sodium (40 and 400 microM) as well as SOD, but not by pentobarbitone sodium. The survival rates of the cortical neurones and hippocampal neurones that were exposed to 30 microM NOC-5 were 11.2+/-4.2% and 37.2+/-3.0%, respectively, and in the presence of 400 microM thiopentone sodium, the survival rate increased to 65.3+/-3.5% in the cortical neurones and 74.6+/-2.2% in the hippocampal neurones. 7. These findings demonstrate that thiopentone sodium, which acts as a free radical scavenger, protects the CNS neurones against NO-mediated cytotoxicity in vitro. In conclusion, thiopentone sodium is one of the best of the currently available pharmacological agents for protection of neurones against intraoperative cerebral ischaemia.

Expression and localization of gamma-aminobutyric acid A (GABAA) receptor alpha 1 subunit and L-glutamate decarboxylase (GAD) mRNAs in rat retina: an analysis by in situ hybridization.

The localization of the mRNAs encoding gamma-aminobutyric acidA receptor alpha 1 subunit (GABAA alpha 1) and L-glutamate decarboxylase (GAD) was elucidated in the rat retina by in situ hybridization. Soma diameter analysis of signal positive cells in the ganglion cell layer demonstrated that a subpopulation including alpha-cells of retinal ganglion cells expressed GABAA alpha 1 mRNA and a subpopulation of ganglion cells smaller than alpha-cells expressed GAD mRNA.

Monoclonal antibody C38 labels surviving retinal ganglion cells after peripheral nerve graft in axotomized rat retina.

C38 is a monoclonal antibody that labels retinal ganglion cells in both intact and axotomized rat retinas. We report here that C38 labeled retinal ganglion cells that survived after optic nerve section and peripheral nerve graft in rats. Furthermore, with combination of the retrograde labeling, we succeeded to distinguish surviving retinal ganglion cells without axonal regeneration from those with regenerating axon.

Adenovirus vector-mediated gene transfer into rat retinal neurons and Müller cells in vitro and in vivo.

The ability and efficiency of human type V adenovirus-originated vector with CAG promoter to transfer foreign genes was examined in the rat retina. We introduced the adenovirus vector, AxCALacZ with the reporter gene LacZ (beta-galactosidase gene) into cultured retinal cells, and then injected the vector suspension into the vitreous cavity of the eye. Beta-galactosidase staining was observed in both glial and neuronal cells in vitro and in Müller cells near the injection site in vivo. Adenovirus vectors with CAG promoter are useful and efficient for the expression of foreign genes in retinal cells.

Demonstration of retinal cells expressing messenger RNAs of the c-kit receptor and its ligand.

We found cells expressing c-kit receptor and its ligand (Sl factor, SLF) in the retina of rats and mice. c-kit messenger RNA (mRNA) was detected in a limited number of cells in the inner nuclear layer of rats and mice by in situ hybridization. The c-kit-expressing cells were assumed to be a subpopulation of the amacrine cells on the basis of their size and distribution pattern. c-kit protein-containing cells were demonstrated in the mouse retina by using ACK2 monoclonal antibody against the extracellular domain of the mouse c-kit receptor. The c-kit protein was detected in cell bodies and processes of the presumed amacrine cells. Hybridization signals for SLF mRNA were observed in the retinal ganglion cells. The results suggest that the c-kit receptor and its ligand may play some roles in the formation of junctions between the amacrine and retinal ganglion cells.

The influence of the timing of administration of thiopentone sodium on nitric oxide-mediated neurotoxicity in vitro.

Thiopentone sodium is a highly useful pharmacological agent that provides a neuroprotection against cerebral ischaemia. Since not all patients can receive thiopentone sodium before cerebral ischaemia occurs, we investigated the influence of timing of thiopentone sodium administration on the neurotoxicity induced by nitric oxide (NO) using Shibuta's established model of primary brain cultures. Cortical neurones prepared from 16-day gestational rat foetuses were used after 13-14 days in culture. The cells were exposed to an NO-donor, NOC-5 at 30 microM. Thiopentone sodium administered at 30 and 10 min before or 5, 10 and 15 min after exposure to NOC-5, but not thereafter, significantly attenuated NO-induced neurotoxicity compared with controls. The survival rate of the neurones in which thiopentone sodium was administered at 15 min after exposure to NOC-5 was 55.7+/-2.4%, compared to a 10.0+/-1.6% survival rate in neurones when thiopentone sodium was administered at 30 min after exposure to NOC-5. These findings demonstrate that thiopentone sodium, which protects cerebral cortical neurones against NO-mediated cytotoxicity, should be given as soon as possible in case ischaemic or hypoxic neuronal damage is predicted.

Monoclonal antibody C38 recognizes retinal ganglion cells in cats and rats.

We developed monoclonal antibody C38 which specifically recognizes retinal ganglion cells (RGCs) in flatmount preparations of cat and rat retinas. We first induced immunological tolerance in Balb/c mice against axotomized rat retinas which lack most of the RGCs. Then the mice were immunized with intact rat retinas to produce antibodies against RGCs. Monoclonal antibody C38 appeared to be specific for cat RGCs based on immunoreactivities seen in flatmounts and vertical sections of the retina. In rats, we verified that over 90% of retrogradely labeled RGCs were immunoreactive for C38 antibody. In axotomized rat retinas, surviving RGCs were labeled with C38 without erroneous labeling of glial cells. The antigen that C38 recognized was 24 kDa in molecular weight and found in cerebrum, cerebellum, and spinal cord as well as retina. It is suggested that monoclonal antibody C38 is a useful label for RGCs.

Transdifferentiation of chicken retinal pigmented epithelial cells in serum-free culture.

A serum-free culture of chicken retinal pigmented epithelial cells has been established in order to analyse how cell-substrate interactions or environmental factors affect the process of transdifferentiation into lens cells from pigmented epithelial cells. The serum-free culture medium for chicken pigmented epithelial cells was Eagle's minimum essential medium, supplemented with chicken transferrin, soybean trypsin inhibitor and bovine insulin. Pigmented epithelial cells were able to survive and grow in the medium for longer than 2 weeks. Collagen did not promote initial cell attachment, but this material effectively supports pigmented epithelial cells to organize monolayer structure characteristics to pigmented epithelium in situ in comparison with the plastic substrate of culture dishes. The process of lens transdifferentiation of chicken pigmented epithelial cells in serum-free conditions was also enhanced with the aid of phenylthiourea and testicular hyaluronidase, which had already been known to promote the transdifferentiation of pigmented epithelial cells in the serum-supplemented condition. Typical lentoid bodies were developed after about 2 weeks of serum-free culture. Thus, we can clearly demonstrate that the chicken embryonic pigmented epithelial cells do not always require a full set of serum factors for their transdifferentiation to lens cells in vitro.

Silent thyroiditis in an eleven-year-old girl, associated with transient increase in serum IgM and thyroid hormone.

An 11-year-old-girl with silent thyroiditis associated with a transient increase in serum IgM and thyroid hormone is described. The levels of serum IgM decreased from 4.38 g/L to 3.35 g/L after 1.5 months at the same time as thyroid hormones returned to normal. An unidentified antecedent infection or exposure to antigen causing the increase in serum IgM might have triggered the occurrence of silent thyroiditis in this patient, although a search for viral antibodies revealed no significant titer changes during the course of the disease.

Differential localization and expression of alpha and beta isoenzymes of protein kinase C in the rat retina.

Expression and cellular localization of three isoenzymes of Ca2+-dependent protein kinase C (PKCalpha, PKCbeta, and PKCgamma) in the adult rat retina were revealed by immunohistochemistry and in situ hybridization histochemistry with isoenzyme-specific antibodies and cRNA probes. Immunoreactivities and mRNA signals for PKCalpha were conspicuous in rod bipolar cells. A subgroup of amacrine cells expressed PKCalpha. The cells in the ganglion cell layer also displayed PKCalpha gene products. Positive immunoreactivities for PKCbeta were localized as stripe patterns in the inner plexiform layer, corresponding to the stratification levels of axon terminals of cone bipolar cells. The somata of cone bipolar cells expressed PKCbeta. Amacrine cells and retinal ganglion cells also displayed PKCbeta gene products. The results obtained by immunohistochemistry were confirmed with colocalization of mRNA signals for PKCalpha and PKCbeta on the somata. The cell membranes showed stronger immunoreactivities than did the cytoplasms for both PKCalpha and PKCbeta. Neither immunoreactivities nor mRNA signals for PKCgamma were detected in all retinal regions. The differential roles of Ca2+-dependent PKC isoenzymes could be revealed in physiological defined retinal neurons.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) activity in patients with Graves' disease, subacute thyroiditis, and silent thyroiditis: a longitudinal study.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) activity was measured longitudinally in 12 patients with Graves' disease, 5 patients with subacute thyroiditis, and 1 patient with silent thyroiditis, and compared with that of 36 normal controls. The patients with Graves' disease and subacute thyroiditis were treated with anti-thyroid drug (methimazole or propylthiouracil) and prednisolone, respectively. On the other hand, no treatment was given to the patient with silent thyroiditis. Since two patients with Graves' disease clearly showed transient deterioration of the thyroid function during the treatment period, data from these two patients were separately investigated. Urinary levels of NAG in the remaining ten patients with Graves' disease before, 1, 3, 6 and 12 months after the treatment were 15.59 +/- 7.93 (SD), 8.96 +/- 6.82, 4.39 +/- 2.33, 3.46 +/- 2.24, and 3.63 +/- 2.38 U/g.creatinine (g.Cr.), respectively. Those obtained before, 1 and 3 months after the treatment were significantly higher than those of the controls (2.85 +/- 1.12 U/g.Cr.). Free thyroid hormone levels became normal or low 3 months after the treatment. The two Graves' patients mentioned above showed a transient increase in urinary NAG with concomitant changes in free thyroid hormone levels. Urinary NAG levels in the patients with subacute thyroiditis before, 2, 4, and 6 weeks after the treatment were 16.56 +/- 10.97, 6.76 +/- 2.79, 3.14 +/- 0.48 and 3.70 +/- 1.44 U/g.Cr., respectively. Those obtained before and 2 weeks after the treatment were significantly higher than those of the controls. Free thyroid hormones were normal 2 weeks after therapy. Urinary NAG in the patient with silent thyroiditis was 9.60 U/g.Cr. on the first visit and gradually decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

The significance of autonomic neuropathy in the elevation of inactive renin in diabetes mellitus.

Plasma renin activity (PRA) and inactive renin(IR, activated by trypsin) were measured in the plasma of 15 type II diabetics with autonomic neuropathy (group 3), 15 type II diabetics without (group 2), and 14 nondiabetic control subjects (group 1) in the recumbent position. There were no significant differences between the 3 groups with respect to age, ideal body weight, supine resting mean blood pressure, serum creatinine, daily urinary excretion of sodium, or renin substrate at the time of study. Autonomic neuropathy (AN) was assessed by measurement of the ratio of the longest to the shortest R-R interval during deep breathing (E/I-ratio) and by postural hypotension. PRA was significantly lower in group 3 than in group 1 (p less than 0.05). The IR level was significantly higher in group 3 than in groups 2 and 1 (p less than 0.005 for both comparisons). The ratio of active renin to total renin (TR) (PRA/(IR + PRA)) was significantly lower in group 3 than in groups 2 and 1 (p less than 0.001 for both comparisons). The IR level and PRA/(IR + PRA) were significantly correlated with E/I-ratio (r = -0.498, p less than 0.01 and r = 0.588, p less than 0.001, respectively) and with the severity of postural hypotension (r = 0.383, p less than 0.05 and r = 0.401, p less than 0.05, respectively), but not with the daily urinary excretion of protein or 24 h-creatinine clearance (24 h-Ccr) in the whole diabetics. From these results, we conclude that autonomic neuropathy might be a more important factor than nephropathy in the lower PRA and higher IR level in type II diabetics with AN.

Structural diversity of murine serum amyloid A genes. Evolutionary implications.

Mouse serum amyloid A (SAA) gene family comprises four members that are closely linked in the chromosome 7. Two of these genes encoding major mouse SAA isotypes (SAA1 and SAA2) are highly homologous not only in exons but also in introns and flanking regions; this sequence homology extends 280 base pairs upstream of major cap sites and 430 base pairs downstream of polyadenylation sites, and the 5' boundary of this homology unit is marked by the CA/GT repeat. Sequence comparison also shows that one (SAA4) of the other two genes is related to the SAA1/2 gene, whereas the other gene (SAA3) evolved independently. Based on these results and the SAA gene arrangement, we discussed mouse SAA gene evolution.