Thr123 of rat G-substrate contributes to its action as a protein phosphatase inhibitor

Rat G-substrate cDNA was isolated from a cerebellar library and characterized. The deduced amino acid sequence of rat G-substrate contained two putative phosphorylation sites for PKG at Thr72 and Thr123; the amino acid sequences (KPRRKDT(p)PA) around these sites are conserved in human, mouse and rabbit. G-substrate phosphorylated by PKG inhibited the catalytic subunits of both protein phosphatase-1 (IC(50) 14.1 nM) and -2A (IC(50) 5.9 nM). Mutation of Thr123 (site 2) to Ala significantly reduced the inhibition of both PP-1 and PP-2A, while mutation of Thr72 (site 1) to Ala had little effect on inhibitory activity. In situ hybridization analysis revealed that G-substrate mRNA was localized exclusively in cerebellar Purkinje cells. Immunoperoxidase staining showed that in Purkinje cells, G-substrate was present in somata, dendrites and axons. In rat cerebellar slices, activation of PKG with a nitric oxide (NO) donor, NOR3, or 8-Br-cGMP, increased phosphorylation of G-substrate, as demonstrated with a phosphorylation-specific antibody. These results characterize further the inhibition of PP-1 and PP-2A by phospho-G-substrate, and demonstrate its physiological phosphorylation in rat Purkinje cells.     

Ovarian juvenile granulosa cell tumor associated with Maffucci's syndrome.

A 15-year-old girl developed a juvenile granulosa cell tumor associated with Maffucci's syndrome (enchondromatosis + hemangiomas). Clinical manifestations of the disease included an abdominal mass and progressive anemia. She underwent the removal of a Stage Ic juvenile granulosa cell tumor and subsequent adjuvant chemotherapy. On follow-up examination 4 years later, no recurrence of the ovarian tumor was noted. A review of the literature showed 10 previous cases of juvenile granulosa cell tumor associated with enchondromatosis, two associated with Maffucci's syndrome, and the rest with Ollier's disease (enchondromatosis). Ovarian juvenile granulosa cell tumor may occur not infrequently in female patients with enchondromatosis in the first or second decades, in contrast to the widely recognized sarcomatous changes of enchondromas that usually occur after the second decade. Data provided from these cases also emphasize the concept of a generalized mesodermal dysplasia.     

A comparative study of gut‐associated lymphoid tissue in calf and chicken

The calf contains two types of Peyer's patches (PPs): jejunal and ileal. The ileal PP has been thought to be equivalent to the bursa of Fabricius (BF) as a central lymphoid organ. The morphologies of ileal and jejunal PPs in the calf were compared with those of the BF and the caecal tonsil (CT) in the chicken. Immunoglobulin G–positive (IgG⁺) cells appear in the follicles of them all and exhibited a dendritic appearance after birth. We investigated whether the IgG in these follicles was produced in situ. IgG‐producing cells were detected in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. CD4⁺ cells were distributed in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. The data suggest that Ig class switching occurs in both jejunal PP follicles and CT follicles, but does not occur in either the ileal PP follicles or the bursal follicles. Because CD4⁺ T cells would be prerequisite for Ig class switching in these follicles, IgG⁺ cells of the follicular medullas in the ileal PP and the BF would trap immune complexes from the gut lumen. The primary B‐cell repertoire might be selected by gut‐derived antigens in the ileal PP and the BF before seeding the periphery. Anat Rec 266:207–217, 2002. © 2002 Wiley‐Liss, Inc.     

Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test

In order to evaluate the utility of the mouse lymphoma assay(MLA) for detecting in vitro clastogens and spindle poisonsand to compare it with the in vitro chromosomal aberration test(CA), we conducted an international collaborative study of theMLA that included 45 Japanese laboratories and seven overseaslaboratories under the cooperation of the Ministry of Healthand Welfare of Japan and the Japanese Pharmaceutical Manufacturer'sAssociation. We examined 40 chemicals; 33 were reportedly positivein the CA but negative in the bacterial reverse mutation assay,six were negative in both assays and one was positive in both.We assayed mutations of the thymidine kinase (TK) locus (tk)of L5178Y tk^+/– mouse lymphoma cells using the microwellmethod. According to our standard protocol, cells were exposedto the chemical for 3 h, cultured for 2 days and TK-deficientmutants were expressed in 96-well plates under trifluorothymidine.Each chemical was coded and tested by two or three laboratories.Among the 34 CA-positive chemicals, positive MLA results wereobtained for 20 and negative results were obtained for nine.The remaining five chemicals were inconclusive or equivocalbecause of discrepant inter-laboratory results or reproduceddiscrepant results, respectively. Among the six CA-negativechemicals, one was negative in the MLA, two were positive andthree were inconclusive. Thus, the MLA could detect only 59%(20/34) of CA-positive chemicals. We concluded that the MLAwas not as sensitive as the CA. Some MLA-negative chemicalsevoked positive responses in the CA only after long continuoustreatment. These might also be genotoxic in the MLA with longcontinuous treatment. Improvement of the MLA protocol, includingalteration of the duration of the treatment, might render theMLA as sensitive as the CA. ⁸ To whom correspondence should be addressed. Tel: +81 3 37009847; Fax: +81 3 3700 2348; Email: sofuni{at}nihs.go.jp     

Synaptic responses of guinea pig and rat central amygdala neurons in vitro.

1. To investigate postsynaptic potentials (PSPs), we made intracellular recordings from neurons of the amygdaloid central nucleus in slices from the guinea pig and rat brains maintained in vitro. The results from guinea pigs and rats were very similar. 2. In the presence of bicuculline (20 microM), focal electrical stimulation of the amygdaloid basal nucleus with low intensities elicited short-latency excitatory PSPs (EPSPs) followed by long-latency EPSPs. The short-latency EPSP was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 10-20 microM). The long-latency EPSP was preferentially abolished by D,L-2-amino-5-phosphonovaleric acid (D,L-APV; 40 microM) and was augmented by removal of extracellular Mg2+. The compound EPSP reversed at -4 mV, which was close to -1 mV, the reversal potential for pressure-ejected glutamate (Glu). 3. When the intensity of the focal stimulation was increased in the presence of bicuculline (20 microM), CNQX (20 microM), and D,L-APV (50 microM), a second EPSP with a short latency and a prolonged duration could be evoked in approximately 65% of the neurons. The EPSPs were reversibly blocked by d-tubocurarine (50 microM) or hexamethonium (200 microM) but were unaffected by atropine (1 microM) or a 5-hydroxytryptamine type 3 receptor antagonist, ICS-205930 (5-10 microM). In these neurons, acetylcholine (ACh; 1-3 mM) caused a depolarization, associated with a decreased input resistance. 4. In the presence of CNQX (20 microM) and D,L-APV (50 microM), single focal stimulation of the dorsolateral subdivision in the central nucleus with low intensities elicited a depolarizing inhibitory PSP (IPSP). The IPSP was reversibly abolished by bicuculline (20-40 microM). The reversal potential (-63 mV) for the IPSP was similar to the reversal potential (-61 mV) for the response to gamma-aminobutyric acid (GABA) applied by pressure ejection. 5. In the presence of bicuculline (20-40 microM) and CNQX (20 microM), a repetitive focal stimulus with high intensities delivered to the dorsolateral subdivision produced a hyperpolarizing PSP followed by a slow depolarization in most neurons. Of putative inhibitory amino acid transmitters, glycine (Gly; 3 mM) produced only a hyperpolarization, associated with a decrease in input resistance. Strychnine (1-2 microM) reversibly blocked both the Gly hyperpolarization and the synaptically evoked hyperpolarization. The reversal potential of -81 mV for the hyperpolarizing PSP was close to -82 mV for the Gly hyperpolarization. The reversal potential for the Gly response was shifted to less negative values by increasing the external K+ concentration or decreasing the extracellular Cl- concentration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Afterdepolarization mechanism in the in vitro, cesium-loaded, sympathetic preganglionic neuron of the cat

Intracellular recordings were performed in Cs-loaded sympathetic preganglionic neurons (SPNs) of the intermediolateral nucleus, identified by antidromic stimulation, in the slice of the T2 or T3 segment of the cat spinal cord. Loading the neurons with Cs resulted in broadening of the action potential, depression of the fast component of the afterhyperpolarization (AHP), and appearance of an afterdepolarization (ADP). A typical ADP in a Cs-loaded neuron had time to peak of 45-110 ms, half-decay time of 70-250 ms, and amplitude of 2-10 mV at membrane potentials between -60 and -70 mV and at a Ca and K concentration of 2.5 and 3.6 mM, respectively, in the superfusion medium. The ADP was associated with a decrease in neuron input resistance and increased in magnitude with hyperpolarization of the cell membrane. The relation between peak ADP amplitude and membrane potential was linear within the range of membrane potentials from -60 to -100 mV. The ADP was reversibly suppressed by the Ca-channel blocker cobalt (2 mM) or by low Ca Krebs solution (0.25 mM). Superfusion with BaCl2 (1.0 mM) or tetraethylammonium (TEA) (10-20 mM) caused an increase in amplitude of the ADP and an increase in action potential duration. Hyperpolarizing pulses, delivered during the course of the spike shoulder, resulted in a decrease of spike duration and ADP amplitude. The ADP was not affected by tetrodotoxin, at a dose blocking the Na-spike, and was enhanced, in association with an increase in action potential duration, when NaCl in the Krebs solution was replaced with choline chloride. Increasing intracellular Cl concentration or decreasing extracellular Cl concentration had no effect on the ADP. Changes in external K concentration from 3.6 to 10 or 0.36 mM increased and decreased, respectively, the amplitude of the ADP. In the absence of Cs, and ADP, with similar time course to that recorded in Cs-loaded SPNs, was recorded when CaCl2 was replaced by BaCl or NaCl was replaced by TEAC1. It is concluded that the SPN afterpotential includes a Ca-dependent inward current, in addition to the already described fast and slow outward K currents of the AHP.     

Experimental urinary tract infection with Pseudomonas aeruginosa in mice.

Urinary tract infection with Pseudomonas aeruginosa was induced in mice by transurethral inoculation of the organism into the bladder, followed by urethral obstruction for 6 h. The infection was mostly localized in the urinary organs. P. aeruginosa P9 was selected as the challenge organism from 10 laboratory strains of P. aeruginosa. After the inoculation of 10(7) colony-forming units of P. aeruginosa P9, transient bacteremia was observed in some of the mice from 6 h to 1 day after the inoculation. The number of organisms in the bladder tissue gradually decreased, whereas that in the kidneys increased to levels of 10(6) to 10(7) colony-forming units in 3 days, and these levels remained up to 2 weeks after the inoculation. The organisms gradually disappeared thereafter, and spontaneous recovery took place. The organisms could be recovered from the kidneys of 95% of the mice, and the gross lesions in the kidneys were observed in 77% of the mice 1 week after inoculation. The method developed here is simple and may be useful in the study of urinary tract infections due to P. aeruginosa and other species of bacteria.20     

Rhythmic hyperpolarizations and depolarization of sympathetic ganglion cells induced by caffeine.

Superfusion of the isolated sympathetic ganglion of the bullfrog with a caffeine-containing (1-6 mM) solution caused in many cells an initial slow hyperpolarization which was followed by a subliminal depolarization interruped by rhythmic hyperpolarizations. A hyperpolarization, similar to one of the rhythmic hyperpolarizations, could be triggered by an action potential in the presence of caffeine. The action potential itself was not markedly affected by caffeine except for its afterhyperpolarization which was prolonged. All these caffeine-induced hyperpolarizations were associated with a marked reduction of the membrane resistance, their amplitude was increased in a K+-free solution and decreased in a high-K+ solution, and their polarity was reversed at the same level at which the afterhyperpolarization was also inverted. This reversal level was not altered by omission of Na+ or C1- from the external medium. These hyperpolarizations were reversibly abolished by depletion of external Ca2+ or replacement of external Ca2+ by Mg2+. Excess of external Ca2+ caused a shortening of the interval between rhythmic hyperpolarizations. Furthermore, iontophoretic injection of EDTA into the cytoplasm markedly depressed the initial caffeine hyperpolarizatin and abolished both the rhythmic and evoked caffeine hyperpolarizations. The caffeine-induced depolarization was not affected by omission of external Cl-. It was decreased in a Na+-free medium, but completely eliminated by omission of both Na+ and Ca2+ from the external medium. Tetrodotoxin did not impair the production of the initial and the rhythmic hyperpolarizations. A strong depolarizing pulse could evoke a typical hyperpolarizing response in the presence of this compound. Dibutyryl cyclic AMP, d-tubocurarine, atropine, and phenoxybenzamine were without effect on the caffeine-induced hyperpolarizations and depolarization. It was concluded that each caffeine-induced hyperpolarization is the result of an increased K+ permeability, which is probably caused by a rise in the internal Ca2+ concentration. It was also concluded that the caffeine-induced depolarization is due to an increased membrane permeability to Ca2+ and Na+.     

Lectin affinity electrophoresis in a yolk sac tumour in the vagina with yolk sac tumour-type glycoform of alpha fetoprotein.

AIMS: To investigate a potential diagnostic use of alpha fetoprotein (alpha FP) isoform analysis by lectin affinity electrophoresis to distinguish between endodermal sinus tumours arising in the vagina in infants from those at other sites. METHODS: alpha FP in the serum of a patient with a vaginal endodermal sinus tumour was analysed for its isoforms by lectin affinity electrophoresis. The isoforms were compared with that of cord serum, sera of hepatoid adenocarcinoma of the uterus, and endodermal sinus tumour of the ovary. RESULTS: The isoforms of alpha FP obtained by lectin affinity electrophoresis in the serum of the patient with vaginal endodermal sinus tumour differed from the isoforms of alpha FP in the cord serum of normal neonates, and sera of patients with hepatoid adenocarcinoma of the uterus or endodermal sinus tumour of the ovary. CONCLUSIONS: Endodermal sinus tumour arising in the vagina could be distinguished from that in the ovary by the lectin affinity electrophoresis, and a potential diagnostic use of alpha FP isoform analysis by the lectin affinity electrophoresis for the detection of the endodermal sinus tumour in infants was demonstrated.     

Electrophysiological properties of sympathetic preganglionic neurons in the cat spinal cord in vitro

Intracellular recordings were obtained from sympathetic preganglionic neurons of the intermedio-lateral nucleus of the adult cat in slices of upper thoracic spinal cord maintained in vitro. The neurons were identified by their antidromic responses to stimulation of various ipsilateral sites. Sites from which antidromic responses could be evoked were the white ramus, the ventral root, the ventral root exit zone, the white matter between the latter and the outer edge of the tip of the ventral horn, the lateral edge of the ventral horn. Resting membrane potential was –61.3±1.6 mV (mean±SEM), input resistance 67.5±3.7 M, time constant 11.5±1.2 ms. The amplitude of the action potential generated by antidromic or direct stimulation was 77.4±2.3 mV. Threshold for direct spikes was 18.2±1.8 mV. The action potential had an average duration of 3.03±0.16 ms. It showed a prominent hump on the falling phase. The action potential had a tetrodotoxin (TTX)-sensitive and a TTX-resistant component. The latter was abolished by cobalt.Tetraethylammonium, cesium and barium prolonged the action potential duration which acquired a plateau-shape. A prolonged after-hyperpolarization (AHP) followed the sympathetic preganglionic neuron spike. Following a single spike, AHP duration and peak amplitude were 2.8±0.3 s and 16.6±0.7 mV, respectively. The AHP was abolished by cesium or barium, but enhanced by tetraethylammonium. An AHP followed the TTX-resistant spike. EPSPs and IPSPs could be generated by focal stimulation. The EPSP triggered spikes when threshold (15.0±2.0 mV) was reached. The slice of the thoracic spinal cord provides a useful experimental preparation for analysis of cellular properties and synaptic mechanisms of the sympathetic preganglionic neuron.