Ichiro Kimura

A myotrophic protein from chick embryo extract: Its purification, identity to transferrin, and indispensability for avian myogenesis☆

Abstract Chick embyro extract (EE) has been widely employed as a growth-promoting supplement in avian myogenic cell cultures. We have purified a myotrophic substance from EE with ammonium sulfate precipitation, CM-Sephadex and DEAE-cellulose chromatography. Salt gradient elution from DEAE-cellulose columns yielded three active peaks with a protein of 80K daltons. The proteins have different isoelectric points of 6.1, 5.9, and 5.7, respectively. They promoted chick myoblasts to proliferate and myotubes to grow when added in the place of EE to a basal culture medium (BCM) composed of Eagles minimal essential medium and horse serum. Their myotrophic activities were the same and reversibly lost by removal of protein-bound Fe. They were identified as transferrin (Tf) species of differing numbers of sialic acid residues, on the basis of physicochemical and immunological analyses. Tf in EE consisted of species of fewer sialic acid residues than adult serum Tf. Indispensability of Fe-bound Tf for EE to exert myotrophic activity was demonstrated by experiments to remove Tf by immunoprecipitation and to remove Fe from Tf in EE. Either treatment led to a complete loss of the myotrophic activity, which was restored by supplementation of Fe-bound Tf or Fe3+. Comparison of myotrophic activity of EE with that of Tf indicated the presence of other factors in EE which promote myogenic cell growth synergistically with Tf. From the results and on the basis of the class-specific function of Tf on the cells, we discuss the relation of Tf to nerve-derived myotrophic proteins and other factors in EE.

Comparison of mice deficient in the high- or low-affinity neurotensin receptors, Ntsr1 or Ntsr2, reveals a novel function for Ntsr2 in thermal nociception.

Neurotensin (NT) is a neuropeptide that induces a wide range of biological activities including hypothermia and analgesia. Such effects are mediated by the NT receptors Ntsr1, Ntsr2 and Ntsr3, although the involvement of each receptor in specific NT functions remains unknown. To address nociceptive function in vivo, we generated both Ntsr1-deficient and Ntsr2-deficient mice. In addition, histochemical analyses of both Ntsr1 and Ntsr2 mRNAs were performed in the mouse brain regions involved in NT-related nociception. The expression of Ntsr2 mRNA was greater than that of Ntsr1 in the periaqueductal gray (PAG) and the rostral ventral medulla (RVM). The mutant and control mice were subjected to the examination of thermal nociception, and in the hot plate test, a significant alteration in jump latency was observed in Ntsr2-deficient mice compared to Ntsr1-deficient or wild-type control mice. Latencies of tail flick and hind paw licking of the mutant mice were not affected compared to control mice. These results suggest that Ntsr2 has an important role in thermal nociception compared to Ntsr1, and that these mutant mice may represent a useful tool for the development of analgesic drugs.

A balanced diet is necessary for proper entrainment signals of the mouse liver clock.

Background The peripheral circadian clock in mice is entrained not only by light-dark cycles but also by daily restricted feeding schedules. Behavioral and cell culture experiments suggest an increase in glucose level as a factor in such feeding-induced entrainment. For application of feeding-induced entrainment in humans, nutrient content and dietary variations should be considered. Principal Finding To elucidate the food composition necessary for dietary entrainment, we examined whether complete or partial substitution of dietary nutrients affected phase shifts in liver clocks of mice. Compared with fasting mice or ad libitum fed mice, the liver bioluminescence rhythm advanced by 3–4 h on the middle day in Per2::luciferase knock-in mice that were administered a standard mouse diet, i.e. AIN-93M formula [0.6–0.85 g/10 g mouse BW] (composition: 14% casein, 47% cornstarch, 15% gelatinized cornstarch, 10% sugar, 4% soybean oil, and 10% other [fiber, vitamins, minerals, etc.]), for 2 days. When each nutrient was tested alone (100% nutrient), an insignificant weak phase advance was found to be induced by cornstarch and soybean oil, but almost no phase advance was induced by gelatinized cornstarch, high-amylose cornstarch, glucose, sucrose, or casein. A combination of glucose and casein without oil, vitamin, or fiber caused a significant phase advance. When cornstarch in AIN-93M was substituted with glucose, sucrose, fructose, polydextrose, high-amylose cornstarch, or gelatinized cornstarch, the amplitude of phase advance paralleled the increase in blood glucose concentration. Conclusions Our results strongly suggest the following: (1) balanced diets containing carbohydrates/sugars and proteins are good for restricted feeding-induced entrainment of the peripheral circadian clock and (2) a balanced diet that increases blood glucose, but not by sugar alone, is suitable for entrainment. These findings may assist in the development of dietary recommendations for on-board meals served to air travelers and shift workers to reduce jet lag-like symptoms.

Immunohistochemical localization of gastrin-releasing peptide receptor in the mouse brain

Gastrin-releasing peptide (GRP) is a mammalian bombesin (BN)-like peptide that binds with high affinity to the GRP receptor (GRP-R). Previous behavioral studies using mice and rats showed that the GRP/GRP-R system mediates learning and memory by modulating neurotransmitter release in the local GABAergic network of the amygdala and the nucleus tractus solitarius (NTS). To date, the precise distribution of GRP-R in the brain has not been elucidated. We used a synthetic peptide derived from mouse GRP-R to generate affinity-purified antibodies to GRP-R and used immunohistochemistry to determine the distribution of GRP-R in the mouse brain. The specificity of anti-GRP-R antibody was confirmed in vitro using COS-7 cells transiently expressing GRP-R and in vivo using GRP-R-deficient and wild-type mouse brain sections. GRP-R immunoreactivity was widely distributed in the isocortex, hippocampal formation, piriform cortex, amygdala, hypothalamus, and brain stem. In particular, GRP-R immunoreactivity was observed in the lateral (LA), central, and basolateral amygdaloid (BLA) nuclei and NTS, which are important regions for memory performance. Double-labeling immunohistochemistry demonstrated that subpopulations of GRP-R are present in GABAergic neurons in the amygdala. Consequently, GRP-R immunoreactivity was observed in the GABAergic neurons of the limbic region. These anatomical results provide support for the idea that the GRP/GRP-R system mediates memory performance by modulating neurotransmitter release in the local GABAergic network.

Indispensability of Iron‐bound Chick Transferrin for Chick Myogenesis in Vitro

Myotrophic activity of highly purified chick transferrins (Tfs) to chick primary myogenic cells has been studied in a culture medium containing horse serum. Iron‐binding to Tfs is indispensable for the activity. The removal of iron from Tfs gives rise to a complete loss of the activity and it is restored by iron‐rebinding depending on the amount of bound iron. This result, combined with other physicochemical and immunological data, strongly, confirms that the myotrophic activity is exerted by the Tfs themselves, not by a contaminating material(s). It has been found that culture medium containing horse Tf which seems inadequate for the study of the biological effects of Tfs is, however, suitable for studies on chick Tfs, since horse Tf is inactive in promoting chick myogenesis. Terminal sialic acid residues are unrelated to myotrophic activity since Tfs with different numbers of residues (0, 1, and 2 moles/Tf molecule) are comprable in their activities. The mechanism of Tf action on cells and contradictions among previous papers as to the requirement of Tf for cell growth have been discussed from the viewpoint of an iron‐donor with class‐specificity.

Chick Embryo Extract, Muscle Trophic Factor and Chick and Horse Sera as Environments for Chick Myogenic Cell Growth

Chick myogenic cells grew in a medium composed of Eagles minimum essential medium (MEM), horse serum (HS), and one of the essential factors needed for myogenic cell growth (EFMG), that is, chick embryo extract (EE), chick serum (CS), or the muscle trophic factor (MTF). But they did not grow in the absence of the EFMG. In the absence of HS, they scarcely grew in a medium composed of MEM, and EE or MTF. They grew in a medium composed of MEM and CS; they grew much better in a medium composed of MEM, CS, and HS.

The AMPA receptor allosteric potentiator PEPA ameliorates post-ischemic memory impairment

PEPA (4-[2-(Phenylsulphonylamino)ethylthio]-2,6-difluorophenoxyacetamide) is a recently developed allosteric potentiator of AMPA receptors that preferentially affects flop splice variants. We tested the effects of PEPA on ischemia-induced memory deficit in rats. Permanent unilateral occlusion of the middle cerebral artery induced severe impairment of performance of rats in the Morris water maze test. Repeated intravenous administration of PEPA (1, 3, 10 mg/kg/day for 10 days) improved test performance. In contrast, a corresponding dose of aniracetam, a representative potentiator of AMPA receptor, did not significantly improve test performance. Thus, PEPA is more effective than aniracetam in reversing impaired memory function as assessed by the Morris water maze test; and PEPA may be an effective compound for the treatment of impaired memory.

Further purification of a fibroblast growth factor-like factor from chick embryo extract by heparin-affinity chromatography

SummaryA mitogenic factor which promotes quail myoblast proliferation has been purified some 105-fold from chick embryo extract by a combination of cation-exchange chromatography and heparin-affinity chromatography. The factor is eluted from heparin-Sepharose with 2M NaCl and is a single-chain polypeptide with an apparent molecular weight of 15000 to 17000. It is active at subnanogram level in triggering the proliferation and thereby delaying temporarily fusion of myoblasts. It also stimulates the proliferation of quail fibroblasts in a similar effective concentration range. For both myoblasts and fibroblasts the dose-response to the factor is quantitatively and qualitatively comparable with that of bovine pituitary fibroblast growth factor. These observations strongly suggest that the factor very probably corresponds to chicken fibroblast growth factor or to a closely related molecule(s) and that it is possibly involved in the regulation of myogenesis.

Developmental Change in Microheterogeneity of Serum Transferrin of Chickens

Developmental change in microheterogeneity of chicken serum transferrin (Tf) was investigated by polyacrylsmide‐gel isoelectric focusing, direct immunofixation, and densitometry. Three main Tf species (Tf‐0, Tf‐1, and Tf‐2, which have 0, 1, and 2 sialic acid residues per molecule, respectively) were resolved and their relative ratios were determined. As development proceeded, a relative increase occurred in the most acidic species (Tf‐2) with decreases in the less acidic ones (Tf‐0 and Tf‐1).

Further Studies on the Developmental Change in Myotrophic Activity of Chicken Serum: Relation between Activity and Transferrin

As an extension of previous studies, we reexamined the developmental change in trophic activity of chicken serum on chicken myogenic cells in vitro and attempted to elucidate it on the basis of possible changes in serum transferrin (Tf), the myotrophic activity of which depends both on its concentration and on the level of its iron‐saturation. The myotrophic activity was found to be low until the second week in ovo, then to increase rather abruptly to a plateau at about the time of hatching, and then to decrease to the adult level. Determination of the concentration and level of iron‐saturation of serum Tf suggested that the change in myotrophic activity was mainly caused by these two parameters, though another factor(s) may also be involved.