Blockade of the KATP channel Kir6.2 by memantine represents a novel mechanism relevant to Alzheimer's disease therapy.

Here, we report a novel target of the drug memantine, ATP-sensitive K+ (KATP) channels, potentially relevant to memory improvement. We confirmed that memantine antagonizes memory impairment in Alzheimer's model APP23 mice. Memantine increased CaMKII activity in the APP23 mouse hippocampus, and memantine-induced enhancement of hippocampal long-term potentiation (LTP) and CaMKII activity was totally abolished by treatment with pinacidil, a specific opener of KATP channels. Memantine also inhibited Kir6.1 and Kir6.2 KATP channels and elevated intracellular Ca2+ concentrations in neuro2A cells overexpressing Kir6.1 or Kir6.2. Kir6.2 was preferentially expressed at postsynaptic regions of hippocampal neurons, whereas Kir6.1 was predominant in dendrites and cell bodies of pyramidal neurons. Finally, we confirmed that Kir6.2 mutant mice exhibit severe memory deficits and impaired hippocampal LTP, impairments that cannot be rescued by memantine administration. Altogether, our studies show that memantine modulates Kir6.2 activity, and that the Kir6.2 channel is a novel target for therapeutics to improve memory impairment in Alzheimer disease patients.

Endocytosis following dopamine D2 receptor activation is critical for neuronal activity and dendritic spine formation via Rabex-5/PDGFRß signaling in striatopallidal medium spiny neurons.

Aberrant dopamine D2 receptor (D2R) activity is associated with neuropsychiatric disorders, making those receptors targets for antipsychotic drugs. Here, we report that novel signaling through the intracellularly localized D2R long isoform (D2LR) elicits extracellular signal-regulated kinase (ERK) activation and dendritic spine formation through Rabex-5/platelet-derived growth factor receptor-ß (PDGFRß)-mediated endocytosis in mouse striatum. We found that D2LR directly binds to and activates Rabex-5, promoting early-endosome formation. Endosomes containing D2LR and PDGFRß are then transported to the Golgi apparatus, where those complexes trigger Gαi3-mediated ERK signaling. Loss of intracellular D2LR-mediated ERK activation decreased neuronal activity and dendritic spine density in striatopallidal medium spiny neurons (MSNs). In addition, dendritic spine density in striatopallidal MSNs significantly increased following treatment of striatal slices from wild-type mice with quinpirole, a D2R agonist, but those changes were lacking in D2LR knockout mice. Moreover, intracellular D2LR signaling mediated effects of a typical antipsychotic drug, haloperidol, in inducing catalepsy behavior. Taken together, intracellular D2LR signaling through Rabex-5/PDGFRß is critical for ERK activation, dendritic spine formation and neuronal activity in striatopallidal MSNs of mice.

Visualization of lower extremity lymphedema in the same cohort using 99mTc-human serum albumin and 99mTc-phytate lymphoscintigraphy with SPECT-CT.

Lymphoscintigraphy with single-photon emission computed tomography (SPECT-CT) is useful in diagnosing lymphedema. However, there are multiple timings, techniques, and tracers utilized worldwide without any comparison. We examined and compared the image clarity with two different radiotracers, 99mTc human serum albumin (HSA) and 99mTc phytate (phytate), in the same patients. The study retrospectivity examined 46 limbs of 36 patients who underwent lymphoscintigraphy using HSA and phytate from January 2013 to September 2018. Tracer accumulation in the lymph nodes, linear pattern (LP), and dermal backflow (DBF) were qualitatively analyzed; contrast-to-noise ratios (CNR) of DBF and standardized uptake value ratio (SUVR) of LP were also quantitatively analyzed. Neither lymph node accumulation nor DBF identification showed significant difference. However, a significant difference was observed between the LP identification of the unaffected (p<0.001) and affected sides (p<0.001). On quantitative evaluation, CNR and SUVR of LP was significantly higher with HSA than with phytate (p<0.001). SUVR of LP was also significantly higher with HSA than with phytate in both unaffected (p=0.002) and affected (p=0.005) sides. Overall, images acquired with HSA were clearer than that with phytate, and the identification of LP was particularly better with HSA than with phytate. Thus, lymphoscintigraphy using HSA is preferred over phytate for both diagnosis and evaluation of disease severity and surgical site selection.

Classification of lymphoscintigraphy and relevance to surgical indication for lymphaticovenous anastomosis in upper limb lymphedema.

Upper limb lymphedema that develops after breast cancer surgery causes physical discomfort and psychological distress, and it can require both conservative and surgical treatment. Lymphaticovenous anastomosis has been reported to be an effective treatment; however the disease severity criteria that define indications for this treatment remain unclear. Here, we examined lymphoscintigraphic findings in 78 patients with secondary upper limb lymphedema and classified them into 5 major types (Type I-V) and 3 subtypes (Subtype E, L, and 0). Results revealed that this classification is related to the clinical stage scale of the International Society of Lymphology. Based on intraoperative examination findings in 20 of the 78 patients, lymphatic pressure is likely to be further elevated in Type II-V cases which are characterized by the presence of dermal back flow. Therefore, lymphaticovenous anastomosis should be considered as a treatment option for lymphedema in Type II-V cases. Furthermore, there are only limited lymph vessel sites usable for lymphaticovenous anastomosis in more severe lymphedema types [Types IV and Type V (which is characterized by dermal backflow only in the hand)]. The findings in Type IV-V cases suggest that therapeutic strategies for severe upper limb lymphedema need further consideration.

Purification of a kininogenase (kininogenase-2) from the venom of Agkistrodon caliginosus (Kankoku-Mamushi).

From the partially purified capillary permeability-increasing enzyme obtained from A. caliginosus venom, another kininogenase (kininogenase-2) was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50, S-Sepharose Fast Flow and Q-Sepharose Fast Flow. The purified enzyme was homogeneous by polyacrylamide gel electrophoresis at pH 8.3 and SDS-gel electrophoresis. The kininogenase-2 had arginine ester hydrolytic and capillary permeability-increasing activities, and did not show any caseinolytic or clotting activity in a similar manner to a previously purified kininogenase (kininogenase-1). The purified kininogenase-2 liberated bradykinin on incubation with purified bovine high mol. wt kininogen. The rate of bradykinin release from the kininogen by kininogenase-2 was slower than that by the kininogenase-1, although both enzymes rapidly cleaved the peptide bonds in the kininogen molecule.

Purification of a kininogenase from the venom of Agkistrodon caliginosus (Kankoku-Mamushi).

During the isolation of a capillary permeability-increasing enzyme from the venom of A. caliginosus, a kininogenase was also purified from the venom by gel filtration on Sephadex G-100, ion-exchange chromatographies on CM-Sephadex C-50 and DEAE-Sephadex A-50, and gel filtration on Sephadex G-75. By this procedure, 11 mg of the purified enzyme were obtained from 4 g of the venom. The purified enzyme was homogeneous by polyacrylamide disc gel electrophoresis at pH 8.3 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and did not show any caseinolytic or clotting activity. The purified enzyme released bradykinin from purified bovine high mol.wt kininogen. The capillary permeability was increased by injection of the purified enzyme into the depilated skin of the back of a rabbit. It is supposed that the capillary permeability-increasing activity exerted by the enzyme is due to the release of bradykinin.

Some properties of a kininogenase from the venom of Agkistrodon caliginosus (Kankoku-Mamushi).

A kininogenase (bradykinin-releasing enzyme) from the venom of A. caliginosus, is a single polypeptide-chain glycoprotein with a mol.wt of about 33,500, which contains 10.1% carbohydrate. The isoelectric point of the enzyme is 3.5 and the enzyme has 274 amino acid residues based on the mol.wt of 33,500. The enzyme hydrolyzed arginine esters more readily than lysine esters, but did not hydrolyze tyrosine ester. The activity of the enzyme on hydrolysis of arginine ester or on liberation of kinin from purified bovine high mol.wt kininogen was inhibited by diisopropylfluorophosphate, indicating that the serine hydroxyl group is involved in enzymatic activity. Moreover, the enzyme split N-alpha-carbobenzoxy-Gly-Pro-Arg-p-nitroanilide (PNA), H.D.Val-Leu-Arg-PNA, H.D.Pro-Phe-Arg-PNA, H.D.Phe-pipecolyl-Arg-PNA and Pro-Phe-Arg-4-methylcoumaryl-7-amide more readily than the other chromogenic or fluorogenic substrates. This result indicates that the substrate specificity of the enzyme is broader than that of mammalian serine proteinases.

Histopathological evaluation of disseminated intravascular coagulation in Haemophilus somnus infection in cattle.

The frequency and distribution of fibrin thrombi (microthrombi) in the main organs of spontaneously infected cattle were investigated to evaluate disseminated intravascular coagulation (DIC) in Haemophilus somnus infection. This infection is well known as infectious thrombo-embolic meningo-encephalitis (ITEME) and is characterized histopathologically by formation of thrombi, necrosis of blood vessels and neutrophil infiltration. The precise pathogenic mechanism of this disease has not yet been fully elucidated. The liver, spleen, kidney, lung, heart and brain of 11 cattle showing thromboembolic meningo-encephalitis were examined histopathologically and special attention was paid to fibrin thrombi. PTAH staining showed a high frequency of fibrin thrombi in the small vessels and capillaries in more than 3 organs and all the cases were regarded as falling within the histopathological criterion of DIC. The results of the present study indicate that the pathogenesis of the infection is closely related to the DIC.

Myofibroblastoma of the neck in a heifer.

A 1.5-year-old Holstein heifer had a subcutaneous tumor mass (20 cm diameter) on the ventral portion of the neck, and the tumor was diagnosed as a locally invasive myofibroblastoma. It consisted of moderately cellular fibrous tissue, and the interlobular septum of the thymus was invaded by tumor cells. The neoplastic cells were positive for alpha smooth muscle actin and vimentin, but not for desmin. Electron microscopy disclosed the presence of moderately developed rough endoplasmic reticulum and microfilaments with focal densities.

[Clinical evaluation of sulbactam/cefoperazone in the field of obstetrics and gynecology].

One or 2 grams of sulbactam/cefoperazone (SBT/CPZ) in 250 ml of 5% glucose solution was administered twice daily by drip infusion to 10 patients with female genital organ infections. The clinical effectiveness was seen in 8 patients but not in 2 patients associated with emergence of replaced organism or non-eradicated causative organism. Antibacterial activity of SBT/CPZ vs. CPZ in the 20 clinical isolates revealed that SBT/CPZ was 1 or 3 tubes superior to CPZ alone in the beta-lactamase producing organisms, but, 1 or 2 tubes inferior to CPZ in the beta-lactamase non-producing organisms. In 1 case increases in GOT and GPT, and in another increases in GOT and GPT, and a decrease in platelets were observed even though their degrees were mild.

[Therapeutic effect of T-1982 (cefbuperazone) on the gynecologic infectious diseases].

T-1982 (cefbuperazone), a new cephamycin antibiotic with broad spectrum against Gram-positive, negative aerobic and anaerobic organisms, was clinically and bacteriologically evaluated on the gynecologic infectious diseases. Fourteen cases hospitalized at Kanazawa Medical University Hospital and the affiliated hospitals from October 1981 to July 1982 were treated with T-1982. By clinical symptoms, signs and bacteriological examinations the patients were diagnosed as pelveoperitonitis (1), intrauterine infection (3), adnexitis (3), infectious diseases of external genitalia (4), infectious abortion (1), vulvar hematoma (1), and rectovaginal fistula (1). T-1982 was administered intravenously or by drip infusion at a dose of 0.5-2.0 g twice a day after dissolved in a saline solution or a 5% glucose solution. Based on the improvement of clinical findings and antibacterial effect of T-1982, results were evaluated as excellent, good, poor and unknown. Clinical effects more than good were shown in 9 of 11 cases which could be followed up exactly on the therapeutic of T-1982. Two cases showing poor response were pelveoperitonitis and pyometra under carcinoma colli uteri stage IIIb, respectively. In 8 of 11 cases, antibacterial effect of T-1982 could be evaluated. Thirteen strains of aerobic and anaerobic bacteria were disappeared by T-1982 therapy, while no effect was seen on 5 strains. On the side effect of T-1982, exanthema was observed in 1 case but disappeared soon after the cessation of administration.

[Decreasing seropositivity of cytomegalovirus of pregnant women in Japan].

We examined seroprevalence of cytomegalovirus (CMV) of pregnant women for 18 years since 1980 with complement-fixing antibody (CF) and specific IgG antibody. CF seropositive rate decreased gradually from 93.2% to 66.7%. CMV-IgG Seropositive rates were 87.4% in 1985, and 75.2% in 1996 to 1997. Age and parity of pregnant women associated with the immune status; 35.6% of recent young primipara were susceptible.

Nobiletin treatment improves motor and cognitive deficits seen in MPTP-induced Parkinson model mice.

Nobiletin, a polymethoxylated flavonoid found in citrus fruit peel, reportedly improves memory impairment in rodent models. Here we report its effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced motor and cognitive deficits. Nobiletin administration (50mg/kg i.p.) for 2 consecutive weeks improved motor deficits seen in MPTP-induced Parkinson model mice by 2weeks, an effect that continued until 2weeks after drug withdrawal. Drug treatment promoted similar rescue of MPTP-induced cognitive impairment at equivalent time points. Nonetheless, nobiletin treatment did not block loss of dopaminergic neurons seen in the MPTP-treated mouse midbrain, nor did it rescue decreased tyrosine hydroxylase (TH) protein levels seen in the striatum or hippocampal CA1 region of these mice. Interestingly, nobiletin administration (50mg/kg i.p.) rescued reduced levels of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and phosphorylation at Thr-34 of dopamine- and cAMP-regulated phosphoprotein-32 (DARPP-32) in striatum and hippocampal CA1 to levels seen in sham-operated mice. Likewise, CaMKII- and cAMP kinase-dependent TH phosphorylation was significantly restored by nobiletin treatment. MPTP-induced reduction of dopamine contents in the striatum and hippocampal CA1 region was improved by nobiletin administration (50mg/kg i.p.). Acute intraperitoneal administration of nobiletin also enhanced dopamine release in striatum and hippocampal CA1, an effect partially inhibited by treatment with nifedipine (a L-type Ca(2+) channel inhibitor) or NNC 55-0396 (a T-type Ca(2+) channel inhibitor) and completely abolished by combined treatment with both. Overall, our study describes a novel nobiletin activity in brain and suggests that nobiletin rescues motor and cognitive dysfunction in MPTP-induced Parkinson model mice, in part by enhancing dopamine release.

Oral administration of glutathione improves memory deficits following transient brain ischemia by reducing brain oxidative stress.

Oxidative stress aggravates brain injury following ischemia. The glutathione (GSH) system plays a pivotal role in combating oxidative stress in various cell types. To determine whether oral GSH administration elicits anti-oxidative effects, we assessed its potential neuroprotective effects in transient bilateral common carotid artery occlusion (BCCAO) mice. In naïve mice, acute oral administration of GSH significantly increased GSH levels by 1h in the cortex and hippocampus. Eleven days after BCCAO, untreated mice showed significantly decreased GSH levels and an inverse elevation of glutathione-disulfide (GSSG) levels in both the cortex and hippocampus. Oral administration of GSH (100 and 500 mg/kg p.o.) for 10 consecutive days after ischemia restored reduced GSH levels and inhibited GSSG elevation. Notably, post-administration of GSH (100 and 500 mg/kg p.o.) significantly prevented neuronal cell death in the hippocampal CA1 region in BCCAO mice, an effect closely correlated with decreased levels of oxidative markers such as 4-hydroxy-2-nonenal (4-HNE), 8-hydroxy-2-deoxyguanosine (8-OHdG) and nitrotyrosine in that region. Finally, GSH administration for 10 days improved memory deficits observed in BCCAO mice. Taken together, our findings indicate that the anti-oxidative effect of oral GSH administration ameliorates post-ischemia neuronal cell death and, in turn, may improve memory.

Decreased CaMKII and PKC activities in specific brain regions are associated with cognitive impairment in neonatal ventral hippocampus-lesioned rats.

Neonatal ventral hippocampus (NVH)-lesioned rats represent a neurodevelopmental impairment model of schizophrenia. Previous observations indicate that postpubertal NVH-lesioned rats exhibit impairments in prepulse inhibition (PPI), spontaneous locomotion and social interaction behavior. Here, we document the neurochemical basis of those defects. PPI impairment but not cognitive impairment was improved by acute risperidone treatment (0.30mg/kgi.p.). Immunohistochemical analyses using anti-autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) antibody indicated significantly reduced CaMKII autophosphorylation, especially in the medial prefrontal cortex (mPFC), striatum and hippocampal CA1 region, of NVH-lesioned rats relative to control animals. We also confirmed that reduced CaMKII autophoshorylation in the mPFC, striatum and hippocampal CA1 region causes decreased phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid-type glutamate receptor subunit 1 (GluR1) (Ser 831), a CaMKII substrate. Like CaMKII, PKCα (Ser 657) autophosphorylation and NR1 (Ser 896) phosphorylation were decreased both in the mPFC and CA1 region. Interestingly, phosphorylation of DARPP-32 (Thr 34) was decreased in the mPFC but increased in the striatum and CA1 region of NVH-lesioned rats compared to controls. Risperidone treatment restored increased DARPP-32 phosphorylation in the striatum and CA1 regions of NVH-lesioned rats but did not rescue CaMKII and PKCα autophosphorylation. Taken together, we find that impaired cognition observed in NVH-lesioned rats is associated with decreased CaMKII and PKCα activities in memory-related brain regions, changes not rescued by risperidone treatment.

Human elastic cartilage engineering from cartilage progenitor cells using rotating wall vessel bioreactor.

Transplantation of bioengineered elastic cartilage is considered to be a promising approach for patients with craniofacial defects. We have previously shown that human ear perichondrium harbors a population of cartilage progenitor cells (CPCs). The aim of this study was to examine the use of a rotating wall vessel (RWV) bioreactor for CPCs to engineer 3-D elastic cartilage in vitro. Human CPCs isolated from ear perichondrium were expanded and differentiated into chondrocytes under 2-D culture conditions. Fully differentiated CPCs were seeded into recently developed pC-HAp/ChS (porous material consisted of collagen, hydroxyapatite, and chondroitinsulfate) scaffolds and 3-D cultivated utilizing a RWV bioreactor. 3-D engineered constructs appeared shiny with a yellowish, cartilage-like morphology. The shape of the molded scaffold was maintained after RWV cultivation. Hematoxylin and eosin staining showed engraftment of CPCs inside pC-HAp/ChS. Alcian blue and Elastica Van Gieson staining showed of proteoglycan and elastic fibers, which are unique extracellular matrices of elastic cartilage. Thus, human CPCs formed elastic cartilage-like tissue after 3-D cultivation in a RWV bioreactor. These techniques may assist future efforts to reconstruct complicate structures composed of elastic cartilage in vitro.

[Establishment of persistent cytomegalovirus infection in primary cultured trophoblastic cells (author's transl)].

The primary cultured cells (Ch) derived from chorionic villi were infected with human cytomegalovirus (CMV). The main results obtained are as follows. 1) The primary cultured trophoblastic cell (Ch) infected with CMV have been maintained in the state of CMV persistent infection for over 6 months. These cells (Ch/CMV) were the CMV-carrier cells in a balance between the cytolysis by CMV-specific cytopathic effect (CPE) and the growth of uninfected cells, showing the CMV persistent infection at the cell population levels. 2) The Ch/CMV cells have persistently released infectious CMV with titers ranging from 1.6 X 10(2) to 1.2 X 10(4) PFU/ml into the culture fluid. CMV-specific antigens were detectable by immunofluorescent antibody staining of the virus releasing cells. 3) These Ch/CMV cells still maintained an ability to secret estrone and estradiol. When these abilities lowered in the successive cultures, the cells encountered the cytolysis by CMV release. 4) Infectious titer of CMV released from Ch/CMV cells reduced by the addition of estrogen in the culture media. In this case, however CMV-specific CPE occurred with a similar pattern as the control one lacked an addition of estrogen. 5) It was suggested that estrogen synthesis in the cells may play an important role in an establishment or a maintenance of CMV persistent infection.

Evidence for early membrane antigens in cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) induces membrane antigens (MA) which are detectable by immunofluorescent techniques as early as 24 h after infection. These antigens appear on the surface of infected cells in non-permissive human epithelioid and animal cells in addition to permissive human cells. The synthesis of the MA occurs in the presence of a DNA synthesis inhibitor, whereas RNA and protein synthesis inhibitors prevent its formation completely. These results demonstrate that the MA is a newly synthesized protein coded by an early function of the viral genome.

[Avidity of rubella IgG antibody in rubella virus infection].

The avidity assay of rubella IgG antibody with urea was evaluated to distinguish the early convalescent phase of primary rubella from the late one and reinfection. We examined 116 sera from 23 patients with primary infection collected 7 to 1,477 days after the rash appeared and 25 sera from 8 patients with rubella reinfection and 16 sera from 15 pregnant women without fetal infection having high hemagglutination inhibition (HI) antibody or weak positive rubella IgM antibody. The avidity index (AI) was calculated as the ratio of the optical density from the urea-washed well to that of the non-treated well, expressed as a percentage. In primary infection AI were lower than 30% within 30 days after the rash appeared, then gradually increased and reached a plateau of 60 to 80% three months later, whereas the AIs of patients after reinfection, with high HI antibody and weak positive IgM antibody were as high as 79.2 +/- 7.5%, 93.5 +/- 4.1% and 90.6 +/- 5.0%, respectively. These results indicate that the measurement of avidity in rubella IgG antibody is valuable in diagnosing recent primary rubella in pregnant women having a high HI antibody or IgM antibody.