New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro.

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (ß-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase.

Ferroportin-Dependent Iron Homeostasis Protects against Oxidative Stress-Induced Nucleus Pulposus Cell Ferroptosis and Ameliorates Intervertebral Disc Degeneration In Vivo.

Ferroptosis is a specialized form of regulated cell death that is charactered by iron-dependent lethal lipid peroxidation, a process associated with multiple diseases. However, its role in the pathogenesis of intervertebral disc degeneration (IVDD) is rarely investigated. This study is aimed at investigating the role of ferroptosis in oxidative stress- (OS-) induced nucleus pulposus cell (NPC) decline and the pathogenesis of IVDD and determine the underlying regulatory mechanisms. We used tert-butyl hydroperoxide (TBHP) to simulate OS conditions around human NPCs. Flow cytometry and transmission electron microscopy were used to identify ferroptosis, while iron assay kit, Perl's staining, and western blotting were performed to assay the intracellular iron levels. A ferroportin- (FPN-) lentivirus and FPN-siRNA were constructed and used to explore the relationship between FPN, intracellular iron homeostasis, and ferroptosis. Furthermore, hinokitiol, a bioactive compound known to specifically resist OS and restore FPN function, was evaluated for its therapeutic role in IVDD both in vitro and in vivo. The results indicated that intercellular iron overload plays an essential role in TBHP-induced ferroptosis of human NPCs. Mechanistically, FPN dysregulation is responsible for intercellular iron overload under OS. The increase in nuclear translocation of metal-regulatory transcription factor 1 (MTF1) restored the function of FPN, abolished the intercellular iron overload, and protected cells against ferroptosis. Additionally, hinokitiol enhanced the nuclear translocation of MTF1 by suppressing the JNK pathway and ameliorated the progression of IVDD in vivo. Taken together, our results demonstrate that ferroptosis and FPN dysfunction are involved in the NPC depletion and the pathogenesis of IVDD under OS. To the best of our knowledge, this is the first study to demonstrate the protective role of FPN in ferroptosis of NPCs, suggesting its potential used as a novel therapeutic target against IVDD.

Effect of U-92032, T-Type Ca2+ Channel Blocker, on Rats with Genetic Absence Epilepsy.

INTRODUCTION: Absence epilepsy is associated with diffuse spike-and-wave discharges (SWD) on the electroencephalogram (EEG). Recent studies have demonstrated that the primary somatosensory cortex is also implicated in the generation of the SWDs. OBJECTIVE: This study investigated the effects of systemic and local administrations of U-92032 into the brain of Genetic Absence Epilepsy Rats from Strasbourg (GAERS). METHODS: GAERS animals underwent stereotaxic surgery for the placement of EEG recording electrodes and guide cannulas for U-92032 administration into the lateral ventricle (intracerebroventricular [i.c.v.]), upper lips area (S1Ulp) or barrel field area (S1B) of primary somatosensory cortex. Following 7 days of recovery, electrical activity was recorded continuously for 1 h before and 6 h after intraperitoneal (0.25; 1; 5 mg/kg i.p.) or local U-92032 or dimethyl sulfoxide (DMSO) injections. RESULTS: No changes were detected in the cumulative duration, mean duration, and number of SWDs following i.p. U-92032 injections. Local i.c.v. injections of U-92032 caused a significant decrease in the cumulative duration (i.c.v., 50 and 100 nmol/L), mean duration (i.c.v., 50, 100, and 250 nmol/L), and the number (i.c.v., 250 nmol/L) of SWDs compared to DMSO groups. Intra-cortical (S1Ulp and S1B) U-92032 injections caused a significant decrease in all 3 parameters compared to DMSO groups, as well. CONCLUSION: Intra-cortical injection of U-92032 caused almost complete removal of SWDs in GAERS and i.c.v. administration resulted in a significant reduction. However, systemic i.p. administration did not cause a significant change with the applied -doses.

ß-Thujaplicin inhibits basal-like mammary tumor growth by regulating glycogen synthase kinase-3ß/ß-catenin signaling.

ß-Thujaplicin, a natural monoterpenoid, has been demonstrated to exert health beneficial activities in chronic diseases. However, it has not been studied in regulating estrogen receptor (ER) negative breast cancer. Here, we investigated the effect of ß-thujaplicin on inhibiting ER-negative basal-like breast cancer and the underlying mechanism of action using an in vitro and in vivo xenograft animal model. ß-Thujaplicin induced G0/G1 phase cell cycle arrest and regulated cell cycle mediators, cyclin D1, cyclin E, and cyclin-dependent kinase 4 (CDK 4), leading to the inhibition of the proliferation of ER-negative basal-like MCF10DCIS.com human breast cancer cells. It also modulated the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase (GSK-3ß) and the protein level of ß-catenin. In an MCF10DCIS.com xenograft animal model, ß-thujaplicin significantly inhibited tumor growth, reduced tumor weight, and regulated the expression of cell cycle proteins, phosphorylation of AKT and GSK-3ß, and protein level of ß-catenin in the tumor tissues. These results demonstrate that ß-thujaplicin can suppress basal-like mammary tumor growth by regulating GSK-3ß/ß-catenin signaling, suggesting that ß-thujaplicin may be a potent chemopreventive agent against the basal-like subtype of breast cancer.

Hinokitiol Offers Neuroprotection Against 6-OHDA-Induced Toxicity in SH-SY5Y Neuroblastoma Cells by Downregulating mRNA Expression of MAO/α-Synuclein/LRRK2/PARK7/PINK1/PTEN Genes.

Parkinson's disease (PD) remarks its pathology by affecting the patient's movements and postural instability by dopaminergic loss in the substantia nigra of midbrain. The disease is characterized by the accumulation of alpha-synuclein protein followed by dementia symptoms. Moreover, the pathology enhances the production of monoamine oxidases A and B (MAO A and B), leucine-rich repeat kinase 2 (LRRK2), phosphate and tensin homolog (PTEN), PTEN-induced putative kinase 1 (PINK1), and PARK7 (deglycase 1 (DJ-1)). Hinokitiol (HIN), a tropolone-related compound, has widely been reported as an antioxidant, antineuralgic as well as a neuroprotective agent. Hence, in this study, we have examined the effect of hinokitol to act as a neuroprotective agent against 6-OHDA-induced toxicity in SH-SY5Y neuroblastoma cells through downregulation of the mRNA expression of PD pathological proteins like alpha-synuclein, MAO A and B, LRRK2, PTEN, PINK1, and PARK7 (deglycase 1 (DJ-1)). The study revealed that the 6-OHDA-induced elevation in the mRNA expression of the pathology marker proteins was subsequently downregulated by the treatment with HIN and was referenced with the positive control, amantadine (AMA), widely used nowadays as a treatment drug for PD symptoms. Thus, the study suggests that hinokitiol could be a drug of choice against 6-OHDA-induced neurotoxicity in SH-SY5Y neuroblastoma cells.

Treatment and Prevention of Oral Candidiasis in Elderly Patients.

The incidence of oral candidiasis has increased in the elderly in recent years. Although the increase of the elderly population plays a big role in this rise of oral candidiasis, the broader recognition that elderly people have higher infection rates for oral candidiasis is considered to be also an important factor. Oral candidiasis can be categorized into three types. Pseudomembranous oral candidiasis is characterized by the appearance of white moss, erythematous oral candidiasis by the eruption of erythema, and hyperplastic oral candidiasis by mucosal hyperplasia. Miconazole has been commonly used when treating oral candidiasis. Elderly patients, however, have a tendency to develop oral candidiasis repeatedly. It is therefore critical to take measures to prevent recurrence. We recommend the use an oral moisturizer containing hinokitiol, an antifungal substance, on a regular basis, to help prevent recurrence of oral candidiasis.

Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin.

The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3-0.5 µg/mL (1.4-2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca(2+)-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death.

Hinokitiol reduces matrix metalloproteinase expression by inhibiting Wnt/ß-Catenin signaling in vitro and in vivo.

OBJECTIVE: In this study, we investigated the effects of hinokitiol on matrix metalloproteinase (MMP)-1, -3, -13, collagen type II (Col2a1) and ß-catenin expressions in rat chondrocytes induced by interleukin-1ß and in an experimental rat model induced by intra-articular injection of mono-iodoacetate (MIA) into the knee. METHODS: Chondrocytes were cultured from the articular cartilage of 2-week-old rats. Passaged chondrocytes were pretreated with hinokitiol for 2h followed by co-incubation with IL-1ß for 24h. Quantitative real-time polymerase chain reaction and Western blotting were used to assess the expression of MMP-1, -3, -13, Col2a1 and ß-catenin. Chondrocytes were also treated with Licl, Dickkopf-1, and/or hinokitiol for 24h, the MMP-1, -3, -13 and ß-catenin protein levels determined by Western blotting. The in vivo effects of hinokitiol were assessed by morphological and histological analyses following MIA injection. RESULTS: Hinokitiol inhibited IL-1ß-stimulated MMP-1,-3 and -13 expressions and IL-1ß-induced activation of intracellular ß-catenin proteins in cultured chondrocytes. In vivo, morphological and histological examinations demonstrated that hinokitiol significantly ameliorated cartilage degeneration. CONCLUSIONS: Hinokitiol is an effective anti-inflammatory reagent that acts by inhibiting the Wnt/ß-catenin signaling pathway and could be a promising therapeutic agent for the prevention and treatment of osteoarthritis.

Design and preparation of single-chain nanocarriers mimicking disordered proteins for combined delivery of dermal bioactive cargos.

Inspired by the multifunctionality of vitamin D-binding protein and the multiple transient-binding behavior of some intrinsically disordered proteins (IDPs), a polymeric platform is designed, prepared, and characterized for combined delivery of dermal protective and anticancer bioactive cargos on the basis of artificial single-chain nano-objects mimicking IDPs. For the first time ever, simultaneous delivery of folic acid or vitamin B9 , and hinokitiol, a relevant natural bioactive compound that exhibits anticancer activity against human malignant melanoma cells, from these multidirectionally self-assembled unimolecular nanocarriers is illustrated.

Stress-related activities induced by predator odor may become indistinguishable by hinokitiol odor.

Predator odors, such as 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), induce a stress-like behavior in some rodents, and there is activation of a complex mix of brain regions including the anterior piriform cortex (APC) and the bed nucleus of stria terminalis (BST). In contrast, rose odor can counteract TMT-induced activation of the ventrorostral part of APC and the medial part of BST. In the present study, two novel odors, woody (hinokitiol) and caraway [S(+)-carvone] odors, were evaluated to determine whether they have an antistress effect. Plasma adrenocorticotropic hormone levels, a marker of stress, and the number of c-Fos-immunopositive cells were determined in APC and BST. Plasma adrenocorticotropic hormone levels were increased by TMT alone and in combination with S(+)-carvone; however, hinokitiol with or without TMT did not have an effect. The number of activated cells in the medial part of BST was increased by TMT alone and in combination with S(+)-carvone or hinokitiol. Although TMT alone activated the medial part of BST, a mixture of TMT and hinokitiol activated both the medial and the lateral part of BST. These data suggest that the selective responses to TMT in the medial part of BST were obscured by activation of more odor-related regions by hinokitiol with TMT. In addition, the ratio of medial to lateral BST activation may be critical in stress-related behavior. In conclusion, hinokitiol can alleviate TMT-induced stress; however, the underlying mechanism appears to be different from that of the rose odor, as found in our previous study.

In vitro and in vivo anti-plasmodial activity of essential oils, including hinokitiol.

Abstract. The anti-plasmodial activity of 47 essential oils and 10 of their constituents were screened for in vitro activity against Plasmodium falciparum. Five of these essential oils (sandalwood, caraway, monarda, nutmeg, and Thujopsis dolabrata var. hondai) and 2 constituents (thymoquinone and hinokitiol) were found to be active against P. falciparum in vitro, with 50% inhibitory concentration (IC50) values equal to or less than 1.0 microg/ml. Furthermore, in vivo analysis using a rodent model confirmed the anti-plasmodial potential of subcutaneously administered sandalwood oil, and percutaneously administered hinokitiol and caraway oil against rodent P. berghei. Notably, these oils showed no efficacy when administered orally, intraperitoneally or intravenously. Caraway oil and hinokitiol dissolved in carrier oil, applied to the skin of hairless mice caused high levels in the blood, with concentrations exceeding their IC50 values.

A 13-week subchronic toxicity study of hinokitiol administered in the diet to F344 rats.

Myocarditis has been reported in male F344 rats given a diet containing hinokitiol (HT). A subchronic toxicity study was here performed to re-evaluate toxic effects of HT in both sexes of F344 rats with dietary administration at concentrations of 0%, 0.02%, 0.07% and 0.2% for 13 weeks. Significant reduction of body weight gain was noted in 0.2% males and 0.07% and above females. Significant decrease in RBC counts, hemoglobin and hematocrit was detected in 0.07% and 0.2% females. Significant increase in MCV was observed in 0.07% and above males and 0.2% females. In the rats given 0.07% and 0.2%, significant increase in total protein and albumin were detected in males, and in total cholesterol in females. Significant increases in total cholesterol, urea nitrogen and creatinine were also detected in the 0.2% males. Significant increase in relative liver weights was detected in the 0.07% and above males and females. Absolute and relative heart weights were significantly decreased in the 0.07% and above males. Based on the above findings the no-observed-adverse-effect level (NOAEL) of HT for both male and female rats was estimated to be 0.02%, translating into 12.7 and 14.8 mg/kg b.w./day, respectively. Myocarditis was not evident in the present study.

In vitro skin permeation of hinokitiol loaded in vesicles composed of behenyltrimethylammonium chloride and stearic acid.

INTRODUCTION: Behenyltrimethylammonium chloride (BTAC) and stearic acid (SA) could be associated with each other through salt bridges, and the associated BTAC/SA could build bilayer vesicles with the aid of hinokitiol (HKL). METHOD: The vesicles were prepared by a precipitation method and used to enhance the skin permeation of HKL. RESULTS: In case the molar ratio of BTAC/SA/HKL was 1/1/0, no vesicle was observed on transmission electron microscope photos. When the molar ratio of BTAC/SA/HKL was 1/1/0.4, vesicle was observed together with some agglomerates. When the content of HKL increased to the ratios of 1/1/0.8 and 1/1/1.2, vesicles were exclusively observed. In vitro fluxes for 18 hours through hairless mouse skin of HKL dissolved in alcoholic solutions were less than 1 mg/cm2/h. Whereas the fluxes of HKL encapsulated in the vesicles were about three times higher than that of HKL in the alcoholic solutions. CONCLUSION: The vesicles could be used for the hair growth promotion.

In vitro permeation study of hinokitiol: effects of vehicles and enhancers.

In vitro permeation of hinokitiol (HKL) through hairless mouse skin was investigated using a diffusion cell. Either propylene glycol (PG) or ethanol (EtOH) was used as a vehicle for HKL. After applying the HKL solutions of 0.5%. 1%, 2%, and 5% onto the skin, the amount of HKL transferred through the skin into the receptor solution, phosphate-buffered saline (PBS, pH7.4), was determined at a predetermined time intervals for 18 hr using a high performance chromatography (HPLC). EtOH was more effective than PG in terms of in vitro permeation of HKL. This is possibly because EtOH acts as a permeation enhancer. Another reason would be related to the higher thermodynamic activity of HKL in ethanol. To investigate the effect of an enhancer on the in vitro permeation, oleyl alcohol, 1-dodecyl-2-pyrrolidone (DP), and lauric acid were used as enhancers. Each was added to the HKL solution (1%) so that the concentration of the enhancer was 1%. Among the enhancers, DP was the most effective and it enhanced the permeation of HKL approximately 5-10 times.

Hinokitiol, a metal chelator derived from natural plants, suppresses cell growth and disrupts androgen receptor signaling in prostate carcinoma cell lines.

Hinokitiol (beta-thujaplicin), a troplone-related compound found in the heartwood of cupressaceous plants, strongly inhibits the proliferation of a broad range of tumor cell lines. This is the first report to demonstrate that hinokitiol, a metal chelator derived from natural plants, suppresses cell growth and disrupts AR signaling in prostate carcinoma cell lines. Our present studies indicate that hinokitiol suppresses androgen/AR-mediated cell growth and androgen-stimulated DNA synthesis by [(3)H]thymidine incorporation in a dose- and time-dependent manner. Hinokitiol simultaneously suppresses the intracellular and secreted PSA levels, a marker for the progression of prostate cancer. Hinokitiol significantly represses the AR mRNA and protein expression in a dose- and time-dependent manner. Additionally, the ligand-binding assay shows that hinokitiol blocks binding of the synthetic androgen [(3)H]R1881 to AR in LNCaP cells. These findings collectively suggest that hinokitiol is potentially effective against prostate cancer in vitro, and thus it might become a novel chemopreventive or chemotherapeutic agent for prostate cancer.

Labelling of human mesenchymal stem cells with indium-111 for SPECT imaging: effect on cell proliferation and differentiation.

PURPOSE: Stem cell therapy seems to be a new treatment option within cardiac diseases to improve myocardial perfusion and function. However, the delivery and traceability of the cells represent a problem. Radioactive labelling with 111In could be a method for tracking mesenchymal stem cells (MSCs). However, 111In could influence the viability and differentiation capacity of MSCs, which would limit its use. Therefore, the aim of this study was to evaluate the influence of 111In labelling in doses relevant for SPECT imaging in humans on the viability and differentiation capacity of human MSCs. METHODS AND RESULTS: Human MSCs isolated from bone marrow were incubated with 111In-tropolone (15-800 Bq/cell). The labelling efficiency was approximately 25% with 30 Bq/cell 111In. The MSC doubling time was 1.04+/-0.1 days and was not influenced by 111In within the range 15-260 Bq/cell. Using 30 Bq 111In/cell it was possible to label MSCs to a level relevant for clinical scintigraphic use. With this dose, 111In had no effect on characteristic surface and intracellular markers of cultured MSCs analysed both by flow cytometry and by real-time polymerase chain reaction. Further, the labelled MSCs differentiated towards endothelial cells and formed vascular structures. CONCLUSION: It is possible to label human MSCs with 111In for scintigraphic tracking of stem cells delivered to the heart in clinical trials without affecting the viability and differentiation capacity of the MSCs. This creates an important tool for the control of stem cell delivery and dose response in clinical cardiovascular trials.

Therapeutic effects of beta-thujaplicin eardrops on canine Malassezia-related otitis externa.

An eardrop solution of beta-thujaplicin was examined for therapeutic effects on canine Malassezia-related otitis externa. Half to one ml of beta-thujaplicin solution of 100 microg/ml including DMSO 2% was injected everyday into both external ear canals of 31 cases for test-of-cure agreement. Fifteen score phases were established from the symptoms and cerumen smear biopsy findings, and score changes were recorded at least once a week. The means of the second and the third inspection day scores decreased significantly more than the previous value of each. In addition, the numbers of yeast-like organisms clearly decreased. These results suggest that beta-thujaplicin eardrops are effective for Malassezia-related otitis externa in dogs.

Potential antimalarial lead structures from fungi of marine origin.

Antiplasmodial and cytotoxicity testing of five highly oxygenated natural products (6R,12R,14R-colletoketol, 6R,11R,12R,14R-colletoketodiol, dihydrobotrydial, pycnidione, and 3R,4S-hydroxymellein), all derived from fungi of marine origin, showed one of them, pycnidione, to have activities against three different strains of Plasmodium falciparum in the sub-micromolar (microM) range. Although the mean selectivity index of 1 for the observed antiplasmodial activity of 4 is low, pycnidione's usefulness as a potential lead structure should not be ignored.

Evaluation of developmental toxicity of beta-thujaplicin (hinokitiol) following oral administration during organogenesis in rats.

The objective of this study was to evaluate the developmental toxicity of beta-thujaplicin (TP) in rats. Pregnant rats were given TP by gastric intubation at 15, 45, or 135 mg/kg on days 6-15 of pregnancy. The maternal body weight gain during administration at 45 and 135 mg/kg and after administration at 136 mg/kg and adjusted weight gain at 45 and 135 mg/kg were significantly reduced. A significant decrease in food consumption during and after administration was found at 45 and 135 mg/kg. A significant increase in the incidence of postimplantation loss was found in pregnant rats given TP at 135 mg/kg. A significantly lower weight was found in female fetuses at 45 and 135 mg/kg and in male fetuses at 135 mg/kg. Although a significantly increased incidence of fetuses with skeletal variations and decreased degree of ossification were found at 135 mg/kg, no significant increase in external, skeletal and internal malformations was detected after administration of TP. The data demonstrated that TP had adverse effects on embryonic/fetal survival and growth only at maternal toxic doses. No adverse effects on morphological development were found in rats fetuses. Based on the significant decreases in maternal body weight gain and weight of female fetuses at 45 mg/kg and higher, it is concluded that the no-observed-adverse-effect levels (NOAELs) of TP for both dams and fetuses are considered to be 15 mg/kg in rats.

Homogeneous or heterogeneous distribution of systemically administered adrenaline: organ dependence.

In incubation experiments it was shown that exogenous adrenaline or noradrenaline does not distribute homogeneously into the adrenergic varicosities of the rat vas deferens (wall with thick and compact muscle layer) but does distribute homogeneously in the rat spleen capsule (thin and loose muscle layer, containing more extracellular space than the vas deferens). To circumvent any hypothetical role of the muscular layer in the distribution of the amine, 100 micrograms.kg-1.h-1 adrenaline was administered to rats in vivo either i.v. (during 90 min) or i.p. (under pentobarbital anaesthesia, an Alzet minipump was implanted which delivered that dose during 6 days). The rats also received 100 mg.kg-1 pargyline (to inhibit MAO) and 100 mg.kg-1 tropolone (to inhibit COMT). At the end of adrenaline administration, vasa deferentia and spleen capsule were removed, washed and then exposed to 100 mumol.l-1 tyramine for 20 min. At the end of this exposure, the ratio noradrenaline/adrenaline in the tissue and in the medium was compared. In the vas deferens both after i.v. and i.p. administration of adrenaline, the ratio noradrenaline/adrenaline was about 3 times higher in the medium than in the tissue, while in the spleen capsule the ratio noradrenaline/adrenaline was not significantly different in the medium and in the tissue. We conclude that, even when the amine reaches the storage sites from the blood, it distributes homogeneously in the spleen capsule and heterogeneously in the vas deferens, perhaps because there are more than one kind of storage vesicles in the vas deferens.