Electrogeneration of hydrogen peroxide applied to the peroxide-mediated oxidation of (R)-limonene in organic media

Horse radish peroxidase (HRP) from Armoracia rusticana catalyses the oxidation of (R)-limonene into the oxidized derivatives carveol and carvone. This study compares the direct addition (DA) of hydrogen peroxide with its continuous electrogeneration (EG) during the enzymatic oxidation of (R)-limonene. Reaction mixtures containing HRP, (R)-limonene as substrate, and hydrogen peroxide, added directly or electrogenerated, in 100 mM sodium-potassium phosphate buffer pH 7.0, at 25ºC were studied. Two electrochemical systems for the hydrogen peroxide electrogeneration were evaluated, both containing as auxiliary electrode (AE) a platinum wire and saturated calomel electrode (SCE) as reference. Reticulated vitreous carbon foam (RVCF) and an electrolytic copper web (CW) were evaluated as working electrodes (WE). Results were compared in terms of hydrogen peroxide electrogeneration, (R)-limonene residual concentration or conversion and product selectivity. Best results in terms of maximum H2O2 concentration (1.2 mM) were obtained using the CW electrode at -620 mV SCE, and continuous aeration. Use of the EG system under optimized conditions, which included the use of acetone (30% v/v) as a cosolvent in a 3 hrs enzymatic reaction, lead to a 45% conversion of (R)-limonene into carveol and carvone (2:1). In comparison to the results obtained with DA, the use of EG also improved the half-life of the enzyme.

Effect of Acriflavine-Guanosine Compound (AG60) Treatment on the Gastrointestinal Endocrine Cells of Mouse Inoculated with Ehrlich Carcinoma Cells / 대한해부학회지

To study the tumor-suppression effect of a newly developed anti-tumor agent AG60 [ acriflavine (1) : guanosine (1) composition, Taerim Pharm. Co., Seoul, Korea], each Ehrlich carcinoma (107 cells)-inoculated mouse received the subcutaneous injection of 0.2 ml of saline, 5mg/kg of AG60, and 30 mg/kg of AG60, every other day for two weeks. Animals were sacrificed, and stomach, duodenum, appendix vermiformis and rectal tissues were resected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 microgram-thick sections. For immunocytochemistry, the streptavidine-biotin-peroxidse method was used with a InnoGenex (San Ramon, Calif., USA) staining kit. The tissues were incubated with rabbit antisera against somatostatin (Biogenesis, Poole, England, UK) diluted 1 : 300, secretin (Biogenesis, Poole, England, UK) diluted 1 : 2,400, neurotensin (Biogenesis, Poole, England, UK) diluted 1 : 2,600, or motilin (Biogenesis, Poole, England, UK) diluted 1 : 1,000 for 24 hour at 4dreeges C, followed by incubation in biotinylated antirabbit IgG and horseradish peroxidase-streptavidin conjugate for 1 hour at room temperature. The antigen-antibody reaction sites were visualized by incubating the sections with diaminobezidine tetrahydrochloride (DAB) for 5~15 minutes at room temperature. After mounting in canada balsam, they were examined in a Leica DM RB microscope. The number of the immunoreactive cells in the area of gastrointestinal mucosae (mean number of immunoreactive cells per 0.25mm2) were observed and calculated. The results are as follows : 1. In the fundic gland of normal mouse, somatostatin immunoreactive cells were detected (18.5+/-0.71), but neurotensin, secretin, or motilin immunoreactive cells were not found. In the duodenal mucosa of normal mouse, somatostatin immunoreactive cells were detected (7.0+/-0.10), but neurotensin, secretin or motilin immunoreactive cells were rarely found. 2. Immunoreactivity of somatostatin, secretin, neurotensin or motilin cells was not found in appendix vermiformis and rectum of normal mouse. 3. On immunocytochemical study, somatostatin immunoreactive cells in the fundic glands of normal, experimental control, AG60 (5mg/kg)-treated, AG60 (30 mg/kg)-treated and 5-fluorouracil (60 mg/kg)-treated groups were 18.5+/-0.71, 10.0+/-4.20, 11.5+/-0.71, 13.5+/-2.10, 11.5+/-2.71, respectively. 4. On immunocytochemical study, somatostatin immunoreactive cells in the duodenal mucosae of normal, experimental control, AG60 (5 mg/kg)-treated, AG60 (30 mg/kg)-treated and 5-fluorouracil (60 mg/kg)-treated groups were 7.0+/-2.10, 0.5+/-2.71, 3.0+/-1.41, 0.5+/-0.71, 2.50+/-0.71, respectively. 5. On immunocytochemical study, secretin immunoreactive cells in the duodenal mucosae of normal, experimental control, AG60 (5 mg/kg)-treated, AG60 (30 mg/kg)-treated and 5-fluorouracil (60 mg/kg)-treated groups were rarely found. 6. On immunocytochemical study, neurotensin and motilin immunoreactive cells in the duodenal mucosae of normal groups were detected, but immunoreactivies were not detected in experimental control, AG60 (5 mg/kg)-treated, AG60 (30 mg/kg)-treated or 5-fluorouracil (60 mg/kg)-treated groups.

Ultrastructural Analysis of GABA- and Glycine-Immunoreactive Nerve Terminals on Jaw-Closing and Jaw-Opening Motoneurons in the Rat / 대한해부학회지

Previous studies have shown that inhibitory synaptic inputs are different between in spinal and trigeminal motor systems and activities of jaw closing and opening alpha motoneurons are different during a chewing cycle. This study examined the distribution of inhibitory synapses made on masseter and digastric motoneurons by using retrograde tracing of wheat germ agglutinin conjugated to horseradish peroxides (WGA-HRP) combined with postembedding immunogold labeling on serial ultrathin sections.Many boutons IR (immunoreactive) to GABA and/or glycine were found to appose on two kinds of motoneurons, which were containing pleomorphic vesicles (a mixture of round, oval and flattened vesicles) and exhibited symmetrical synaptic contacts on the somata. Packing density and synaptic covering % were higher in digastric than in masseter motoneurons. Of 703 boutons apposing on 12 masseter motoneurons, 6.08+/-3.51, 29.67+/-8.89 and 17.78+/-5.22% were IR to GABA only, glycine only, and both GABA and glycine, respectively. Of 637 boutons apposing on 11 digastric motoneurons, 6.37+/-4.64, 19.74+/-8.25 and 12.01+/-5.38% were IR to GABA only, glycine only, and both GABA and glycine, respectively. Proportions of glycine IR boutons were higher than that of GABA IR boutons in both masseter and digastric motoneurons. Packing density and proportion of boutons IR to GABA and/or glycine were higher in jaw closing than in jaw opening motoneurons (packing density, 12.03+/-1.58 vs 10.28+/-2.99; proportion of IR boutons, 53.54+/-8.94% vs 38.12+/-9.38% in jaw closing and opening motoneurons, respectively). These results provide ultrastructural evidence that GABA and glycine act as important neurotransmitters for modulation of jaw movement and that proportion of inhibitory synapses is higher in jaw closing than in jaw opening motoneurons.

Morphology and Distribution of Motor Neurons in Cat Innervating the Orbicularis Oculi Muscle by Retrograde Horseradish Peroxidase

The facial nerve is mainly composed of motor fibers and is distributed to the muscles of facial expressions. In ophthalmology clinics, orbicularis oculi muscle innervated by the facial nerve is involved in spontaneous and voluntary blinking, winking, and more forceful eyelid closure. To understand pathophysiogy of facial nerve palsy due to brain stem lesion involving nucleus, 50% Horseradish Peroxidase (HRP) was injected into nerve stump innervating orbicularis oculi muscle of cat and serial sections of midbrain were studied with light and dark field of light microscope to examine morphology and distribution of the facial nuclei. The HRP-labelled motor neurons were located exclusively within the intermediate division of the ipsilateral facial nuclei and no labelled neurons were found in the contralateral facial nuclei, in the nuclei of the trigeminal nerve, or any other brain stem nuclei. The mean diameter of HRP-labelled motor neurons was 45 micrometer. Most of them were multipolar in shape containing many dendrites. These result suggest that the intermediate division of ipsilateral facial nuclei play an important role in innervating orbicularis oculi muscle.

Morphology and Distribution of Motoneurons Innervating the Lateral Rectus and the Retractor Bulbi Muscles in Cat : A Retrograde Study Utilizing Horseradish Peroxidase

No Abstract Available.

Quantitative Analysis of GABA and/or Glycine Like Immunoreactive Synaptic Terminals on Masseter gamma-motoneurons for Jaw Closing Muscle of the Rat / 대한해부학회지

The distribution of GABA and/or glycine like immunoreactive nerve terminals on the soma of the masseteric gamma motoneurons were investigated using retrograde tracing of WGA-HRP (wheat germ agglutinin conjugated horseradish peroxidase) and postembedding immunogold labeling methods in serial ultrathin sections. Quantitative analysis of 140 nerve terminals apposing on somata of gamma motoneuron size less than 21 mm in average diameter was performed. The results obtained were as follows. 1. Synaptic covering % of apposing nerve terminals was 21.45+/-11.48% and packing density was 12.85+/-6.17. 2. Nerve terminals immunoreactive (IR) to GABA or glycine were F type containing pleomorphic vesicles with round shape predominant. Majority of nerve terminals immunonegative to GABA or glycine were S type containing spherical vesicles and few of them were F type. 3. 11.42+/-10.00% of examined nerve terminals were IR to GABA only, and 12.71+/-9.85% were IR to GABA and glycine, and 15.21+/-9.58% were IR to glycine only. 4. Synaptic covering % of nerve terminals IR to glycine only was highest (4.58+/-4.50%), followed in order by GABA and glycine (3.18+/-2.77%), and GABA only (2.38+/-2.06%). 5. Among all terminals, immunonegative nerve terminals (60.66+/-14.65%) were much more than nerve terminals immunoreactive to GABA and/or glycine (39.34+/-14.65%) These results show that inhibitory synaptic input and synaptic organization of the masseteric gamma motoneurons reveal characteristic features in contrast to that of alpha motoneurons and which may correlated to the electrophysi-ological characteristics of masseteric gamma motoneurons.

Origings of Dorsal Root Ganaglion Cells Innervating Anterior and Posterior Cruciate Ligaments of the Rat Knee Joint

PURPOSE: The present study was designed to examine the distribution of dorsal root ganglion(DRG) cells innervating the anterior and posterior cruciate ligaments of the Sprague-Dawley rat knee joint. MATERIALS AND METHODS: Fluoro-gold(FG) was used to identify the distribution of DRG cells innervating the ligaments, and horseradish peroxidase(HRP) was used to measure the DRG cell size innervating the ligaments. RESULTS: Neural tracers-labelled DRG cells were found ipsilaterally only in the lumbosacra1 DRGs. FG-labelled DRG cells innervating the anterior and posterior cruciate ligaments were found from the 1st lumbar DRG to the 1st sacral DOR(L1-Sl). The majority of FG-labelled DRG cells innervating the poste-rior cruciate ligaments were located in the L4, and the majority innervating the anterior cruciate ligaments were found in the L3, The size of HRP-labelled DRG cells innervating the cruciate ligaments was below 800 micromiter (c), showing that these cells were small. CONCLUSION: This study indicates that the DRG origin of sensory nerves is different in each cruciate ligament of the knee joint. But the size and the type innervating each ligament is similar.

Bilateral Innervations to Superior Oblique by Trochlear Nucleus in cats: Retrograde Tracer Study by Horseradish Peroxidase

Trochlear motorneurons were identified by applying horseradish peroxidase(HRP) to superior oblique muscle in cats. Ninety five to ninety seven percent of the contralateral trochlear nucleus were stained 3-5% of ipsilateral side also labelled by HRP. These findings showed that superior oblique muscle was innervated by trochlear nuclei on both side.

Changes of Blood-Retinal Barrier Induced by Destruction of the Retinal Pigment Epithelium

The author studied the functional derangement of blood-retinal barrier induced by destruction of the pigment epithelial cells of the retina. Sodium iodate, which was well known to exert a selectively destructive action to the retinal pigment epithelial cells, was injected to the rabbits intravenously(60mg/kg of body weight). Eyes were enucleated 2 days and 4 days after sodium iodate injection and examined by electron microscope. Some of the tissue were fixed in colloid lanthanum, to investigate the changes of the permeability of plasma membrane in accordance with cellular damages induced by sodium iodate. The permeability of the blood-retinal barrier was also studied after intravenous(200mg/kg) or intraocular(4 microgram/20ml of saline) injection of horseradish peroxidase(HRP). The results obtained were summarized as the following: Sodium iodate induced patchy areas of loss of pigment epithelial cells after 2 days, which were more widespread and severe after 4 days with regenerative activities. Loss of outer segment and mitochondrial swelling of the inner segment of visual cells were also noted after 4 days. Colloidal lanthanum penetrated into the mitochondria of pigment epithelial cells at 2 days after sodium iodate injection, which was extended to the mitochondria of inner segment of visual cells after 4 days. Intraocularly injected HRP appeared from the internal limiting membrane to Bruch's membrane after 2 days. Intravenously injected HRP appeared from the Bruch's membrane to ganglion cell layer after 2 days, which were extended to the vitreal cavity. The results suggested that the damage of the pigment epithelial cells induced by sodium iodate destroy blood-retinal barrier. HRP exudation is more extensive in direction of retina to choroid than choroid to retina.