Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells

Nanofibrous gelatin substrates are suited for long-term expansion of human pluripotent stem cells (hPSCs) under feeder- and serum-free culture conditions. A combinatorial library with different sets of processing parameters was established to assess the culture performance of hPSCs on nanofibrous substrates in terms of cell adhesion and growth rate, using Matrigel as control. Then, the optimal conditions were applied to long-term expansion of hPSCs with several cell lines, showing a maintained pluripotency over more than 20 passages without introducing any abnormal chromosome. In addition, this approach allowed us to avoid enzymatic disassociation and mechanic cutting during passages, thereby promoting a better hPSC culture and long-term expansion.     

Characterization and Minimization of Block Copolypeptide Vesicle Cytotoxicity Using Different Hydrophobic Chain Lengths

The previously reported amphiphilic block copolypeptide, poly(l‐lysine)₆₀‐block‐poly(l‐leucine)₂₀ (K₆₀L₂₀), is able to form vesicles that can be manipulated to different sizes and be prepared in large quantities. This study expands upon that work by varying the length of the hydrophobic segment to optimize the vesicles so that they are monodisperse and low in toxicity. Copolypeptides with longer oligoleucine segments are found to have fewer toxic micelles, small aggregates, and unstable vesicles, and exhibit lower toxicity than vesicles formed from copolypeptides with shorter hydrophobic domains. Oligoleucine segments that are too long, however, result in rigid hydrophobic membranes that prevent the vesicular assemblies from being extruded into a monodisperse population of nanoscale vesicles.

     

Intrapancreatic gastric duplication cyst mimicking pancreatic cystic tumor

Cystic lesions of the pancreas are sometimes difficult to diagnose. We report a case of a gastric duplication cyst (GDC) of the pancreas in an adult. A 45-year-old woman was admitted to our department for the investigation of anemia. Abdominal ultrasonography revealed a large cystic lesion, measuring about 40 × 70 mm with calcification in the tail of the pancreas. Contrast-enhanced computed tomography of the abdomen revealed a non-enhanced cystic lesion with non-enhanced wall. Endoscopic retrograde pancreatography revealed a mild extended main pancreatic duct and an aberrant pancreatic duct, but there was no communication with the cyst. We could not deny the malignancy, so distal pancreatectomy was performed. The pathological examination revealed that the inner wall of the cyst consisted of columnar epithelium and smooth muscle layer. Immunohistochemical analysis revealed the columnar epithelium to be immunopositive for cytokeratin 7 (CK7) and immunonegative for cytokeratin 20 (CK20) and the glands to be immunonegative for CK 7 and immunopositive for CK 20. Therefore, a diagnosis of GDC of the pancreas was made. On imaging, a GDC usually mimics a pancreatic pseudocyst or a cystic neoplasm of the pancreas. Therefore, even though it is rare, a GDC should be considered in the differential diagnosis of cystic tumors of the pancreas.     

Diabetes attenuates the antidepressant-like effect mediated by the activation of 5-HT1A receptor in the mouse tail suspension test.

Several lines of evidence have indicated that the prevalence of depression in diabetic subjects is higher than that in the general population, however, little information is available on the effects of antidepressants in diabetes. In the present study, the antidepressant-like effect mediated by the activation of 5-HT(1A) receptors was examined using the tail suspension test in streptozotocin-induced diabetic mice. Long-lasting increases in 5-HT turnover rates were observed in the diabetic mouse midbrain and frontal cortex, but not in the hippocampus. Duration of immobility was significantly longer in diabetic than in nondiabetic mice in the tail suspension test. The 5-HT(1A) receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (3-30 microg/kg, i.p.) reduced the duration of immobility in nondiabetic mice, and this effect was completely antagonized by pretreatment with N-[2-[4-(2-methoxyphenil)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY-100635) (30 microg/kg, s.c.), a selective 5-HT(1A) receptor antagonist. In contrast, 8-OH-DPAT (3 microg/kg-3 mg/kg, i.p.) was ineffective in diabetic mice. The selective 5-HT reuptake inhibitor fluoxetine (3-56 mg/kg, i.p.) reduced the duration of immobility in both nondiabetic and diabetic mice. However, fluoxetine was less effective in diabetic mice than in nondiabetic mice. WAY-100635 (30 microg/kg, s.c.) reversed the suppression of the duration of immobility by fluoxetine (30 mg/kg, i.p.) in nondiabetic mice. On the other hand, the anti-immobility effect of fluoxetine (56 mg/kg, i.p.) was not antagonized by WAY-100635 (30 microg/kg, s.c.) in diabetic mice. The selective 5-HT(2) receptor antagonist 6-methyl-1-(1-methylethyl)-ergoline-8beta-carboxylic acid 2-hydroxy-1-methylpropyl ester (LY53,857) (30 microg/kg, s.c.) reversed the anti-immobility effect of fluoxetine in both nondiabetic and diabetic mice. Spontaneous locomotor activity in diabetic mice was not different from that in nondiabetic mice. 8-OH-DPAT (30 microg/kg, i.p.), but not fluoxetine, increased the spontaneous locomotor activity in both nondiabetic and diabetic mice. The number of 5-HT(1A) receptors in the mouse frontal cortex was unaffected by diabetes. Plasma corticosterone levels in diabetic mice were significantly higher than that in nondiabetic mice. These results suggest that the antidepressant-like effect mediated by 5-HT(1A) receptors may be attenuated by diabetes.     

Involvement of diazepam-insensitive benzodiazepine receptors in the suppression of DOI-induced head-twitch responses in diabetic mice

Rationale We previously reported that the head-twitch responses induced by the 5-HT₂ receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) (DOI-HTRs) were decreased in streptozotocin-induced diabetic mice.Objectives We examined the involvement of γ-aminobutyric acid (GABA)/benzodiazepine system on the suppression of DOI-HTRs in diabetic mice.Results The benzodiazepine receptor antagonist flumazenil (0.1–1 mg/kg, i.v.) dose-dependently and significantly increased DOI-HTRs in diabetic mice to the same levels as in nondiabetic mice. However, flumazenil (0.1–1 mg/kg, i.v.) did not affect DOI-HTRs in nondiabetic mice. The benzodiazepine receptor agonist diazepam (0.1–1 mg/kg, i.p.) had no effect on DOI-HTRs in either nondiabetic or diabetic mice. The GABA_A receptor antagonist bicuculline (0.1–1 mg/kg, i.p.) and the benzodiazepine receptor partial inverse agonist Ro 15-4513 (0.1–1 mg/kg, i.v.) dose-dependently and significantly suppressed DOI-HTRs in nondiabetic mice to the same levels as in diabetic mice. Ro 15-4513-induced reduction of DOI-HTRs in nondiabetic mice was completely antagonized by flumazenil (1 mg/kg, i.v.), but not diazepam (0.3 mg/kg, i.p.).Conclusions We suggest that the abnormal diazepam-insensitive benzodiazepine receptor function partly underlies the suppression of DOI-HTRs in diabetic mice.     

Phencyclidine impairs latent learning in mice: interaction between glutamatergic systems and sigma(1) receptors.

The effect of phencyclidine (PCP) on latent learning was investigated using a one-trial water-finding task in mice. Mice without water deprivation were given PCP or saline before a training trial, which consisted of exposure to a novel open-field environment with an alcove containing a water tube. Twenty to twenty-four hours after water deprivation, animals were placed in the same apparatus and the time required to find the water tube measured (test trial). Saline-treated trained mice showed a significantly shorter time to find the water tube during the test trial (finding latency) than naive mice that had not been trained. When PCP (1mg/kg i.p.) was administered before the training trial, the finding latency was significantly prolonged in comparison with that in the saline-treated mice, indicating that PCP induced impairment of latent learning. 1-(3,4-Dimethoxy-phenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (SA4503: 0.3 mg/kg s.c.) and (+)-pentazocine (1 mg/kg s.c.), selective sigma(1) receptor agonists, or D-cycloserine (10 and 30mg/kg, s.c.), a glycine binding site agonist, significantly counteracted the PCP-induced impairment of latent learning, whereas (+)-SKF-10,047 (0.1-3 mg/kg s.c.), a putative sigma(1) receptor agonist, did not. The ameliorating effects of SA4503 and (+)-pentazocine were antagonized by N,N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy) phenyl) ethylamine (NE-100: 1 mg/kg i.p.), a selective sigma(1) receptor antagonist. SA4503 also ameliorated the impairment of latent learning induced by dizocilpine, a non-competitive N-methyl-D-aspartate receptor antagonist, the effect being antagonized by NE-100. These results suggest that PCP induces an impairment of latent learning, this effect being mediated via glutamatergic systems, and that activation of sigma(1) receptors ameliorates impairment of latent learning induced by PCP.     

Effects of Antiepileptics on Lateral Geniculate Nucleus–Kindled Seizures in Rats

The present study was undertaken to clarify the characteristics of lateral geniculate nucleus (LGN) kindling in rats, especially the efficacies of antiepileptics, in comparison with those of amygdala (AMG) kindling. Daily electrical stimulation of the LGN led to the development of a generalized convulsion (kangaroo posture and falling back) in all subjects, similar to AMG kindling. The kindling response of the LGN differed from that of the AMG in a number of respects, that is, a high after-discharge (AD) threshold, a large number of stimulations for completion of kindling, and a different pattern of electroencephalogram (EEG) development. On the other hand, the oral administration of sodium valproate, carbamazepine, clobazam, or zonisamide caused dose-dependent inhibitions of both seizure stage and AD duration of LGN-kindled seizures, whereas ethosuximide had no significant effects. In addition, seizure stage was more potently inhibited than AD duration by these antiepileptics, particularly with clobazam. In conclusion, LGN kindling possesses characteristics that are different from AMG kindling. In addition, it was demonstrated that LGN kindling is a useful model, similar to other types of limbic system kindling, for the evaluation of antiepileptics.     

Reexamination of Coccidioides spp. reserved in the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, based on a multiple gene analysis.

The Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University is the only organization in Japan to possess a series isolates of Coccidioides spp., which are the most virulent pathogenic fungi and which are treated as biosafety level 3 microorganisms. Recently, the genus Coccidioides has been classified into two species, C. immitis and C. posadasii, based on their endemic areas and genotyping; the former species is endemic to the state of California, and the latter is endemic to other parts of North and South America. We reevaluated 19 isolates of Coccidioides immitis stored in our center using a multiple gene analysis. Five isolates were identified as C. immitis and 14 as C. posadasii. Their sequence information in GenBank will help to identify the two genospecies of Coccidioides spp.