Voltage-dependent ionic currents in solitary horizontal cells isolated from cat retina.

1. Horizontal cells of the cat retina were isolated by enzymatic dissociation. Two types of horizontal cells were identified: the axonless (A-type) horizontal cell having four to six thick, long (approximately 100 microns) dendrites, and the short-axon (B-type) horizontal cell having many (> 5) fine, short (approximately 30 microns) dendrites. 2. Membrane properties of isolated horizontal cells were analyzed under current-clamp and voltage-clamp conditions. In the A-type cell, the average resting potential was -55 mV and the mean membrane capacitance was 110 pF, whereas values in the B-type cell were -58 mV and 40 pF, respectively. The A-type cell showed long-lasting Ca spikes, but B-type cells had no Ca spikes. 3. Five types of voltage-dependent ionic currents were recorded: a sodium current (INa), a calcium current (ICa), and three types of potassium currents. Potassium currents consisted of potassium current through the inward rectifier (Ianomal), transient outward potassium current (IA), and potassium current through the delayed rectifier (IK(v)). INa was recorded only from A-type cells. Other currents were recorded from both types of cells. 4. INa activated when cells were depolarized from a holding potential (Vh) of -95 mV, and it was maximal at -25 mV. This current was blocked by tetrodotoxin. Approximately half of the A-type cells had INa, but no B-type cell had this current. 5. L-type ICa, an inward-going sustained current, was activated with depolarization more positive than -25 mV. Current amplitude reached a maximal value near 15 mV and became smaller with further depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary choriocarcinoma and human chorionic gonadotrophin‐producing giant cell carcinoma of the lung: are they independent entities?

AIMS: Human chorionic gonadotrophin (hCG) is a useful marker for chorionic proliferative disorders, such as choriocarcinoma. Although hCG synthesis in lung cancers is frequent, primary pulmonary choriocarcinoma (PCC) is rare. To clarify the differences between primary choriocarcinoma and hCG-producing giant cell carcinoma (GCC) of the lung, we compared the clinicopathological and immunohistochemical findings of these tumours. METHODS AND RESULTS: Three patients, one with PCC and two with hCG-producing GCC, were included in this study. They were all middle-aged men and habitual smokers. The growth of these tumours and the progression of the clinical courses were extremely rapid, and the patients all died within 8 months after the pulmonary tumours were found. Haemorrhagic appearance was a common macroscopic feature of the specimens obtained. Microscopically, both types of tumours mainly consisted of atypical polygonal cells. While PCC contained many syncytial trophoblast-like multinucleated cells that had strong immunoreactivity for anti-hCG, such cells were relatively few in hCG-producing GCC. These histological and immunohistochemical findings reflected the serum test result for hCG, which was higher in the case of PCC. CONCLUSIONS: There are a few differences between PCC and hCG-producing GCC, as described above. Reliable distinction between them seems to be difficult for pathologists and worthless for clinicians.     

Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis

Expression of the Arabidopsis CGS1 gene that codes for cystathionine gamma-synthase is feedback regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, called the MTO1 region, encoded by the first exon of CGS1 itself is involved in this regulation. Here, we demonstrate, using a cell-free system, that AdoMet induces temporal translation elongation arrest at the Ser-94 codon located immediately downstream of the MTO1 region, by analyzing a translation intermediate and performing primer extension inhibition (toeprint) analysis. This translation arrest precedes the formation of a degradation intermediate of CGS1 mRNA, which has its 5' end points near the 5' edge of the stalled ribosome. The position of ribosome stalling also suggests that the MTO1 region in nascent peptide resides in the ribosomal exit tunnel when translation elongation is temporarily arrested. In addition to the MTO1 region amino acid sequence, downstream Trp-93 is also important for the AdoMet-induced translation arrest. This is the first example of nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in eukaryotes. Furthermore, our data suggest that the ribosome stalls at the step of translocation rather than at the step of peptidyl transfer.     

Tumor necrosis factor-alpha in the placenta is not elevated in pre-eclamptic patients despite its elevation in peripheral blood

PROBLEM: Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells at the early and late stages of gestation and promotes the regulation of trophoblast growth and invasion. We evaluated whether TNF-alpha levels in the placenta and blood of pre-eclamptic women differed from those with normal pregnancies. METHOD OF STUDY: The subjects were 39 pregnant women carrying single fetuses (21 normal-pregnant and 18 pre-eclamptic patients). Their average gestational age at entry was 38-39 weeks. Peripheral blood was collected before the onset of labor and separated serum was stored at -20 degrees C. A tissue segment of the placenta was cut and frozen in liquid nitrogen immediately after delivery at -80 degrees C. The frozen placental tissue was added to phosphate-buffered saline. The tissue was fully homogenized and centrifuged. Separated supernatant was stored at -80 degrees C. TNF-alpha levels in separated serum and TNF-alpha and total protein (TP) levels in separated supernatant were measured. The presence of TNF-alpha in the placenta was evaluated by immunohistochemistry in five pre-eclamptic and five normal-pregnant patients. RESULTS: Serum TNF-alpha levels were higher in pre-eclampsia than in normal pregnancies. However, TNF-alpha/TP levels in the placenta did not differ significantly between the two groups. As for TNF-alpha immunostaining of trophoblastic cells in the placenta, it was weak in three and moderate in two of the normal pregnancies, while it was absent in two, weak in one, and moderate in two in the pre-eclampsia group. CONCLUSIONS: We demonstrated no significant increase in TNF-alpha/TP levels in the placenta in pre-eclampsia despite a significant increase in serum TNF-alpha levels. There was no strong immunostaining for TNF-alpha detected by immunohistochemistry in the pre-eclampsia group. These findings suggest that TNF-alpha in the placenta is not a key cytokine to interfere with normal trophoblast invasion into the myometrium in pre-eclampsia, and that sources other than the placenta may contribute to the elevated levels of TNF-alpha found in the circulation of pre-eclamptic patients.     

B-cell repertoire specific for an unfolded self-determinant of mouse lysozyme escape tolerance and dominantly participate in the autoantibody response

We previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant T-cell epitope of hen egg lysozyme (HEL), HEL 107-116. As T cells specific for HEL 107-116 were primed in these mice, the anti-ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107-116. Serum IgG responses against ML were dominantly focused on the ML 14-69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14-69 and examined their antigen specificities in detail, to characterize the nature of the remaining B-cell repertoire specific for ML. mAbs specific for ML 14-69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six- to 80-fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B-cell repertoire in normal mice.     

BRONCHIAL PLASMACYTOMA IDENTIFIED BY IMMUNOPEROXIDASE TECHNIQUE ON PARAFFIN EMBEDDED SECTION

A case of bronchial plasmacytoma occurring in a 57- year-old housewife is reported. She had had the productive cough and the "abnormal shadow" in the right lower lobe for three years before admission. On bronchocopy, a tumor was found in the right main bronchus, large enough to obstruct the air way. The tumor was resected through rigid bronchoscope. Histological impression was "plasmacytoma with local amyloid deposit." M-protein was never detected in the serum or urine. Applying the immunoperoxidase technique for the paraffin section, the plasma cells were found to contain only a single type of immunoglobulin, Ig G-L. The differential diagnosis between plasmacytoma and plasma cell granuloma was made, and plasmacytoma was considered to be one type of extranodal malignant lymphoma.     

Relative frequency of VP4 gene alleles among human rotaviruses recovered over a 10-year period (1982-1991) from Japanese children with diarrhea.

The relative frequencies of the Wa (corresponding to serotype P1A), DS-1(P1B), M37(P2), and AU-1(P3) alleles of the VP4 gene from rotaviruses collected from the stools of individuals in Japan between 1982 and 1991 were determined to be 83.1, 15.6, 0, and 1.3%, respectively, by a polymerase chain reaction-based typing assay.     

Characterization of rabies virus glycoprotein expressed by recombinant baculovirus.

A cDNA of the glycoprotein (G protein) gene of rabies virus Nishigahara strain was cloned and inserted into a baculovirus genome under the control of the polyhedrin promoter. Infection of Spodoptera frugiperda cells with this recombinant virus produced a large quantity of new protein instead of the parental polyhedrin protein. By immunofluorescent and immunoblotting analyses, the recombinant protein was antigenically similar to the authentic G protein. Its molecular mass estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, however, was slightly smaller than that of the authentic one, and this observation was suggested to be due to the difference in glycosylation level between the two G proteins. The recombinant G protein expressed on the cell surface of the insect cells showed a fusion activity at low pH. The fusion activity was inhibited by antiserum against either whole virions or G protein of rabies virus.     

Enhancement of N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials in the neostriatum after methamphetamine sensitization. An in vitro slice study

It has been suggested that behavioral methamphetamine sensitization involves changes in cortical excitatory synaptic inputs to neostriatal (Str) projection neurons. To test this, we performed blind whole-cell recording of medium spiny neurons in Str slice preparations. In Str neurons of naive rats, the amplitude of the subcortical white matter stimulation-induced N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials (NMDA-EPSPs) was decreased upon hyperpolarization, owing to the voltage-dependent Mg(2+) blockade of NMDA receptor channels. In contrast, the amplitude of the NMDA-EPSPs in Str neurons of rats undergoing methamphetamine withdrawal (MW) did not show the Mg(2+) blockade and was nearly voltage independent over the membrane potential range of -70 to -110 mV. Application of the specific protein kinase C (PKC) activator, phorbol 12, 13- DL-acetate, blocked the voltage-dependent Mg(2+) blockade of NMDA receptor channels in Str neurons of naive rats. Application of the specific activator of cAMP-dependent protein kinase A (PKA), Sp-cAMPS-triethylamine salt, increased the amplitude of the NMDA receptor-mediated EPSPs at the rest but not at hyperpolarized potentials. Coapplication of the PKC and PKA activators yielded NMDA-EPSPs similar to those seen in Str neurons of MW rats. In Str slices of naive rats, tetanic subcortical white matter stimulation induced long-term depression of field potentials. In Str slices treated with the PKC and/or PKA, the same stimulation induced long-term potentiation of field potentials similar to those observed in slices obtained from MW rats. These results suggest that the enhancement of the NMDA receptor-mediated corticostriatal synaptic transmission plays an important role in behavioral methamphetamine sensitization. This enhancement is probably associated with phosphorylation of NMDA receptors mediated by the simultaneous activation of PKC and PKA.