Different neural substrates for executive functions in youths with ADHD: a diffusion spectrum imaging tractography study.

BACKGROUND: The relationship between white-matter tracts and executive functions (EF) in attention deficit hyperactivity disorder (ADHD) has not been well studied and previous studies mainly focused on frontostriatal (FS) tracts. The authors explored the microstructural property of several fibre tracts hypothesized to be involved in EF, to correlate their microstructural property with EF, and to explore whether such associations differ between ADHD and typically developing (TD) youths. METHOD: We assessed 45 youths with ADHD and 45 individually matched TD youths with a computerized test battery for multiple dimensions of EF. From magnetic resonance imaging, FS tract, superior longitudinal fasciculus (SLF), arcuate fasciculus (AF) and cingulum bundle (CB) were reconstructed by diffusion spectrum imaging tractography. The generalized fractional anisotropy (GFA) values of white-matter tracts were computed to present microstructural property of each tract. RESULTS: We found lower GFA in the left FS tract, left SLF, left AF and right CB, and poorer performance in set-shifting, sustained attention, cognitive inhibition and visuospatial planning in ADHD than TD. The ADHD and TD groups demonstrated different association patterns between EF and fibre tract microstructural property. Most of the EF were associated with microstructural integrity of the FS tract and CB in TD youths, while with that of the FS tract, SLF and AF in youths with ADHD. CONCLUSIONS: Our findings support that the SLF, AF and CB also involve in a wide range of EF and that the main fibre tracts involved in EF are different in youths with ADHD.

Hypoxia inducible factor 2α/insulin-like growth factor receptor signal loop supports the proliferation and Oct-4 maintenance of mouse germline stem cells.

Hypoxia inducible factor 2α (HIF-2α) is critical for primordial germ cell (PGC) survival as knockout of HIF-2α (HIF-2α(-/-)) decreases both expression of Oct-4 and PGC number in genital ridge. Hypoxia is known to stabilize HIF-2α protein from proteasomal degradation. However, little is known about the hypoxia-associated endocrinal signaling in HIF-2α expression. The current work demonstrates a role for an endocrine insulin-like growth factor-I receptor (IGF-IR)-PI3K/Akt-mTOR-HIF-2α regulatory loop in the proliferation and Oct-4 maintenance of PGC-like alkaline phosphatase positive mouse germline stem cells (AP(+)GSCs). We found that hypoxia greatly increased the cell proliferation and the levels of nuclear Oct-4/HIF-2α protein of AP(+)GSCs. The hypoxic-AP(+)GSCs presented stronger stemness ability for germ cell differentiation than normoxic, with expressions of c-KIT (differentiation germ cell marker), VASA (differentiation germ cell marker) and SCP3 (meiotic marker) using a renal capsule transplantation assay. Meanwhile, hypoxia significantly increased the expression levels of secreted-IGF-I and IGF-IR. The IGF-I dose dependently increased the HIF-2α expression levels in AP(+)GSCs; and, the inhibition of IGF-IR by RNA interference (shIGF-IR) or LY294002 (PI3K inhibitor)/Rapamycin (mTOR inhibitor) effectively suppressed the IGF-I- and/or hypoxia-induced HIF-2α and Oct-4 expression, suggesting that the IGF-IR and its downstream Akt/mTOR signaling are involved in the IGF-I/hypoxia effects. Additionally, knockdown of HIF-2α dramatically suppressed Oct-4 and IGF-IR protein levels in AP(+)GSC cells. In conclusion, the present study demonstrates a regulatory loop of IGF-IR-PI3K/Akt-mTOR-HIF-2α in proliferation and Oct-4 maintenance of PGC-like AP(+)GSCs under hypoxia. This finding provides insights into the niche endocrinology underlying early germ cell development.

Are there ethnic differences in impulsive/compulsive behaviors in Parkinson's disease?

BACKGROUND AND PURPOSE: Recent studies have suggested increased prevalence of impulsive/compulsive behaviors (ICB) in patients with Parkinson's disease (PD) as compared to general population in different ethnic groups. The spectrum of these behaviors includes dopamine dysregulation syndrome (DDS), punding, pathological gambling (PG), hypersexuality (HS), binge eating (BE), and compulsive shopping (CS). METHODS: Two hundred and seventy-eight consecutive patients with idiopathic PD regularly followed-up at an outpatient clinic were interviewed and screened for the ICB between September 2008 and December 2008 using designated diagnostic criteria. All patients who screened positive for ICB or obsessive-compulsive disorder (OCD) were further confirmed by an experienced psychiatrist. RESULTS: Of all the studied patients, 15 patients confirmed to have ICB (lifetime prevalence: 5.60%), 3 (1.12%) were diagnosed to have DDS, 1 (0.37%) punding, 4 (1.49%) PG, 8 (2.99%) HS, 1 (0.37%) BE, 0 (0%) CS. OCD was found in one patient (0.37%). CONCLUSIONS: The prevalence of ICB is lower in Taiwan as compared with the Caucasians, with similar risk factors. The possible reasons include differences in ethnicity, environmental, cultural, and social factors as well as the dosage and selection of dopaminergic medications.

Rapid dose-dependent effect of clozapine on a schizophrenic patient's lipid profile.

Clozapine is known to be associated with higher risk of metabolic syndrome, including dyslipidaemia, but the mechanisms of such a relationship are still under debate. The case we reported shows a seemingly dose-dependent effect of clozapine on a patient's lipid profiles with no influence on his blood sugar in a relatively short period of time. A direct effect of clozapine on lipid metabolism, independent of insulin or obesity-related metabolic changes, is suggested. The rapid effect of clozapine on lipid profile allows fine-tuning in dose adjustment while treating refractory schizophrenia complicated with drug-related dyslipidaemia but worrying about psychotic rebound during clozapine discontinuation.

Epidural abscess in an obstetric patient with patient-controlled epidural analgesia--a case report.

We present the case of a 37-year-old pregnant woman who underwent a cesarean section due to previous cesarean delivery. Spinal anesthesia was performed at the L2-3 intervertebral space with an epidural catheter inserted at L1-2 for postoperative patient-controlled epidural analgesia. When the epidural catheter was removed on day three, an area of redness round the entry point was noted and the patient complained of low back pain, but was discharged from hospital. Later the same day, she felt backache so severe that she was unable to stand up or bend her body. She called for help and was sent to our emergency room. Physicians noted a small amount of discharge from the insertion site, and the body temperature was elevated to 38 degrees C. An anesthesiologist and an infectious disease specialist were consulted, and an epidural abscess was suspected. Urgent magnetic resonance imaging revealed an epidural abscess at L1-2. After five days of unsuccessful treatment with oxacillin, a 28-day course of vancomycin, followed by two months of oral fusidic acid, resulted in complete remission of the epidural abscess. The patient has remained free of neurologic deficit.

Randomized, double-blind study comparing postoperative effects of treatment timing with histamine H-receptor antagonist cimetidine.

BACKGROUND: The purpose of this study was to evaluate the effect of timing of preoperative, postoperative, and placebo administration of the H(2)-antagonist cimetidine on postoperative pain management and the incidence of side-effects. METHODS: One hundred and twenty ASA I to II patients, undergoing major gynaecological abdominal surgery, were randomly divided into three groups, and received a standardized general anaesthesia. The patients in the preoperative ('Pre') group received an intravenous infusion of cimetidine 4 mg kg(-1) prior to anaesthesia induction; the postoperative ('Post') group received the same volume of normal saline. Postoperatively, the patients in the Post group received an intravenous infusion of cimetidine 4 mg kg(-1); the patients in the Pre group received the same volume of normal saline. The Control group received the same volume of normal saline prior to anaesthesia induction and after the end of operation. Postoperatively, all patients were treated with a patient-controlled intravenous analgesia system, which was programmed to deliver 1 mg of morphine on demand for three consecutive days. RESULTS: Pain intensity, morphine consumption, sedation score, and side-effects were recorded and evaluated. We found no difference among the three groups with respect to pain intensities, morphine usage, sedation scores, and the incidence of nausea, vomiting, pruritus, or dizziness. CONCLUSIONS: Our results suggested that neither preoperative nor postoperative administration of cimetidine 4 mg kg(-1) provided a pre-emptive or preventive analgesic advantage for postoperative pain or morphine consumption, and that the use of cimetidine failed to reduce the incidence of nausea or vomiting.

Comparison of motor conduction techniques in the diagnosis of carpal tunnel syndrome.

OBJECTIVE: To compare the sensitivities of motor wrist-to-palm (W-P) conduction velocity and two median-ulnar motor latency differences with that of sensory W-P conduction velocity in the diagnosis of carpal tunnel syndrome (CTS). METHODS: This study included 116 consecutive patients with CTS (160 hands) referred for evaluation and 100 volunteers who served as controls. Median motor and sensory nerve responses with wrist and palm stimulation allowed for the determination of motor and sensory W-P CV (W-P MCV and SCV). Two motor distal latency (MDL) differences between the median-thenar and ulnar-hypothenar (M-U) muscles and between the median-second lumbrical and ulnar-interossei muscles (2L-INT) were measured and calculated. The mean values of controls plus or minus 2.5 SD served as the normal limits. RESULTS: Among the 160 hands with suspected CTS, 11 (6.88%) had normal electrodiagnostic studies and 149 (93.1%) had at least one abnormal electrodiagnostic study. Among the 149 hands with an abnormality, 139 (86.88%) had abnormal W-P MCV and 129 (80.63%) had abnormal W-P SCV. The sensitivity for 2L-INT was 77.5%, and it was 70% for M-U, 68.75% for median MDL, and 73.75% for sensory distal latency. Combining W-P MCV and W-P SCV allowed for the detection of abnormalities in 147 hands (91.88%) and yielded a markedly improved diagnostic rate compared with W-P SCV alone. CONCLUSION: Motor W-P conduction study is more valuable and no more difficult than sensory W-P conduction study for the diagnosis of CTS. In patients with suspected CTS in whom the results of conventional nerve conduction studies are normal, studying both motor and sensory W-P conduction increases the diagnostic yield.

The adsorption of benzene and methylethylketone onto activated carbon: thermodynamic aspects.

The adsorption of volatile organic compounds (VOCs), exemplified by benzene and methylethylketone (MEK), onto seven different types of activated carbon was investigated. Results show that for benzene adsorption the adsorption characteristic energy, enthalpy, free energy and entropy are in the range 17.12-36.86, -20.8 to -44.7, -11.89 to -16.22 kJ/mole and -29.4 to -85.3 J/mole/K, respectively. For the adsorption of MEK, the adsorption characteristic energy, enthalpy, free energy and entropy are in the range 14.47-32.34, -18.3 to -40.8, -10.78 to -15.56 kJ/mole and -24.8 to approximately -60.3 J/mole/K, respectively. The adsorption enthalpy can be calculated indirectly from statistical thermodynamic method and directly from the immersion enthalpy method. The adsorption characteristic energy is calculated by the Dubinin-Astokhov equation. The free energy is calculated by the measured equilibrium adsorption constant.

Cyclophilin A mediates Vid22p function in the import of fructose-1,6-bisphosphatase into Vid vesicles.

Fructose-1,6-bisphosphatase (FBPase) is synthesized in yeast during glucose starvation but is rapidly degraded in the vacuole following the addition of glucose. FBPase trafficking to the vacuole involves two distinct steps, import into intermediate transport vesicles (Vid vesicles) and Vid vesicle trafficking to the vacuole. FBPase import into Vid vesicles requires the VID22 gene. However, VID22 affects FBPase import indirectly through a cytosolic factor. To identify the required cytosolic component, wild type cytosol was fractionated and screened for proteins that complement Deltavid22 mutant cytosol using an in vitro assay that reproduces FBPase import into Vid vesicles. Cyclophilin A (Cpr1p) was identified as a cytosolic protein that mediates Vid22p function in FBPase import. Mutants lacking Cpr1p were defective in FBPase import. Furthermore, the addition of purified Cpr1p restored FBPase import in both the Deltacpr1 and the Deltavid22 mutants. The cyclosporin A binding pocket is important for Cpr1p function, since cyclosporin A binding-deficient mutants failed to complement FBPase import in Deltacpr1 and Deltavid22 mutants. The levels of Cpr1p were reduced in the Deltavid22 mutants, implying that the expression of Cpr1p is regulated by Vid22p. Our results suggest that Cpr1p mediates Vid22p function and is directly involved in the import of FBPase into Vid vesicles.

Cytochrome b gene for species identification of the conservation animals.

A partial DNA sequence of cytochrome b gene was used to identify the remains of endangered animals and species endemic to Taiwan. The conservation of animals species included in this study were: the formosan gem-faced civets, leopard cats, tigers, clouded leopards, lion, formosan muntjacs, formosan sika deers, formosan sambars, formosan serows, water buffalo, formosan pangolins and formosan macaques. The control species used included domestic cats, domestic dogs, domestic sheeps, domestic cattles, domestic pigs and humans. Heteroplasmy was detected in the formosan macaque, domestic pig and domestic cats. The frequencies of heteroplasmy in these animals were about 0.25% (1 in 402bp). Sequences were aligned by Pileup program of GCG computer package, and the phylogenetic tree was constructed by the neighbor-joining method. The results of sequence comparison showed that the percentage range of sequence diversity in the same species was from 0.25 to 2.74%, and that between the different species was from 5.97 to 34.83%. The results of phylogenetic analysis showed that the genetic distance between the different species was from 6.33 to 40.59. Animals of the same species, both the endangered animal species and domestic animals, were clustered together in the neighbor-joining tree. Three unknown samples of animal remains were identified by this system. The partial sequence of cytochrome b gene adopted in this study proved to be usable for animal identification.

Biochemical analysis of fructose-1,6-bisphosphatase import into vacuole import and degradation vesicles reveals a role for UBC1 in vesicle biogenesis.

When Saccharomyces cerevisiae are shifted from medium containing poor carbon sources to medium containing fresh glucose, the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is imported into Vid (vacuole import and degradation) vesicles and then to the vacuole for degradation. Here, we show that FBPase import is independent of vacuole functions and proteasome degradation. However, FBPase import required the ubiquitin-conjugating enzyme Ubc1p. A strain containing a deletion of the UBC1 gene exhibited defective FBPase import. Furthermore, FBPase import was inhibited when cells overexpressed the K48R/K63R ubiquitin mutant that fails to form multiubiquitin chains. The defects in FBPase import seen for the Deltaubc1 and the K48R/K63R mutants were attributed to the Vid vesicle fraction. In the Deltaubc1 mutant, the level of the Vid vesicle-specific marker Vid24p was reduced in the vesicle fraction, suggesting that UBC1 is required for either Vid vesicle production or Vid24p binding to Vid vesicles. However, the K48R/K63R mutant did not prevent Vid24p binding to Vid vesicles, indicating that ubiquitin chain formation is dispensable for Vid24p binding to these structures. Our results support the findings that ubiquitin conjugation and ubiquitin chain formation play important roles in a number of cellular processes including organelle biogenesis.

Residue characteristics and pore development of petrochemical industry sludge pyrolysis.

Petrochemical industry bio-sludge was pyrolyzed to investigate the composition and pore size distribution of pyrolytic residue. Results indicated that the carbon, nitrogen, and hydrogen concentrations could be reduced after an increase in pyrolytic temperature. The trace element analysis indicated that Al, Ca, Fe, Mg. K, Cu, Sr, and Sb concentrated during the pyrolytic process. When forty grams of pre-dried sludge were pyrolyzed at various pyrolytic temperatures, the transfers from the gas phase to liquid phase to residue were from 21.2 to 36.0%, from 49.0 to 70.0%, and from 8.3 to 16.5%. Results of the pore size distribution examination indicated that the mesopore had the greatest effect on the bio-sludge pyrolysis. The optimal pyrolytic temperatures and times were approximately 800 degrees C for 30 min and 900 degrees C for 10 min. The conceptual model can reasonably explain the pore structure development during the pyrolysis process.

Diffusion of hydrogen sulfide and methyl mercaptan onto microporous alkaline activated carbon.

Activated carbon kinetic studies show that both H2S and CH3SH yielded pore diffusion coefficients from 10(-6) to 10(-8) cm2/s. Results indicated that pore structures could influence effective diffusivity. Under the same adsorbate concentration, CH3SH exhibited a greater effective pore diffusion coefficient than H2S. This may be attributed to the fact that CH3SH has both polar (-SH) and non-polar (-CH3) functional groups and dissolves into water easier, thus providing more attraction for the activated carbon surface. In addition, the saturation vapor pressure of CH3SH is lower than that of H2S. Therefore, CH3SH is easier to adsorb onto activated carbon than H2S.

Adsorption/desorption properties of copper ions on the surface of iron-coated sand using BET and EDAX analyses.

This study was conducted to develop a heating process for coating hydrated iron oxide on the sand surface to utilise the adsorbent properties of the coating and the filtration properties of the sand. BET and scanning electron microscope (SEM) analyses were used to investigate the surface properties of the coated layer. An energy dispersive X-ray (EDAX) technique of analysis was used for characterising metal adsorption sites on the iron-coated sand surface. The results indicated that the iron-coated sand had more micropores and higher specific surface area because of the attachment of iron oxide. Copper ions could penetrate into the micropores and mesopores of iron oxide on sand surface, and the regeneration of the iron-coated sand could be achieved by soaking with pH = 3.0 acid solution. Besides, the results of EDAX analysis showed that copper ions were chemisorbed on the surface of iron-coated sand. Results of the study developed an innovative technology for coating iron oxide on sand surface for the treatment of heavy metal in water.

The heat shock protein Ssa2p is required for import of fructose-1, 6-bisphosphatase into Vid vesicles.

Fructose-1,6-bisphosphatase (FBPase) is targeted to the vacuole for degradation when Saccharomyces cerevisiae are shifted from low to high glucose. Before vacuolar import, however, FBPase is sequestered inside a novel type of vesicle, the vacuole import and degradation (Vid) vesicles. Here, we reconstitute import of FBPase into isolated Vid vesicles. FBPase sequestration into Vid vesicles required ATP and cytosol, but was inhibited if ATP binding proteins were depleted from the cytosol. The heat shock protein Ssa2p was identified as one of the ATP binding proteins involved in FBPase import. A Deltassa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Deltassa1, Deltassa3, or Deltassa4 strains. Likewise, in vitro import was impaired for the Deltassa2 strain, but not for the other Deltassa strains. The cytosol was identified as the site of the Deltassa2 defect; Deltassa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Deltassa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Deltassa2 Vid vesicles, providing Deltassa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.

Pyrolysis kinetics and residue characteristics of petrochemical industrial sludge.

This study investigated the pyrolysis characteristics of sludge from wastewater treatment plants in the petrochemical industry and focused on the pyrolysis kinetics, elemental composition of residue, and volatile organic compounds (VOCs) of exhaust gas. As pyrolysis temperature increased to 773 K, the increasing rate of crude oil production tended to a stable condition. The result indicated that the optimal temperature of crude oil and water mixed production was 773 K. When pyrolysis temperature increased from 673 to 973 K, carbon, oxygen, nitrogen, and hydrogen concentrations of residue decreased and the sulfur concentration of residue increased. The concentrations of benzene, toluene,ethylbenzene, and styrene increased by the increasing pyrolysis temperature. We found that the reaction order of sludge pyrolysis was 2.5 and the activation energy of the reaction was 11.06 kJ/mol. We believe that our pyrolysis system is transitional between devolatilization and combustion.

Vid24p, a novel protein localized to the fructose-1, 6-bisphosphatase-containing vesicles, regulates targeting of fructose-1,6-bisphosphatase from the vesicles to the vacuole for degradation.

Glucose regulates the degradation of the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), in Saccharomyces cerevisiae. FBPase is targeted from the cytosol to a novel type of vesicle, and then to the vacuole for degradation when yeast cells are transferred from medium containing poor carbon sources to fresh glucose. To identify proteins involved in the FBPase degradation pathway, we cloned our first VID (vacuolar import and degradation) gene. The VID24 gene was identified by complementation of the FBPase degradation defect of the vid24-1 mutant. Vid24p is a novel protein of 41 kD and is synthesized in response to glucose. Vid24p is localized to the FBPase-containing vesicles as a peripheral membrane protein. In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole, suggesting that Vid24p regulates FBPase targeting from the vesicles to the vacuole. FBPase sequestration into the vesicles is not affected in the vid24-1 mutant, indicating that Vid24p acts after FBPase sequestration into the vesicles has occurred. Vid24p is the first protein identified that marks the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.

In vitro reconstitution of glucose-induced targeting of fructose-1, 6-bisphosphatase into the vacuole in semi-intact yeast cells.

Fructose-1,6-bisphosphatase (FBPase), the key enzyme in gluconeogenesis in the yeast Saccharomyces cerevisiae, is induced when cells are grown in medium containing poor carbon sources. FBPase is targeted from the cytosol to the vacuole for degradation when glucose-starved yeast cells are replenished with fresh glucose. In this study, we report the reconstitution of the glucose-induced import of FBPase into the vacuole in semi-intact yeast cells using radiolabeled FBPase, an ATP regenerating system and cytosol. The import of FBPase was defined as the fraction of the FBPase that was sequestered inside a membrane-sealed compartment. FBPase import requires ATP hydrolysis and is stimulated by cytosolic proteins. Furthermore, the import of FBPase is a saturable process. FBPase import is low in the glucose-starved cells and is stimulated in the glucose-replenished cells. FBPase accumulates to a higher level in the pep4 cell, suggesting that FBPase is targeted to the vacuole for degradation. Indirect immunofluorescence microscopy studies demonstrate that the imported FBPase is localized to the vacuole in the permeabilized cells. Thus, the glucose-induced targeting of FBPase into the vacuole can be reproduced in our in vitro system.

Identification of novel vesicles in the cytosol to vacuole protein degradation pathway.

The key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose. FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated. In some vid mutants, FBPase is found in punctate structures in the cytoplasm. When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants. In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells. We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity. Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway. Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles. Under EM, these vesicles are 30-40 nm in diam. Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles. We propose that FBPase is imported into these vesicles before entering the vacuole.

Isolation of degradation-deficient mutants defective in the targeting of fructose-1,6-bisphosphatase into the vacuole for degradation in Saccharomyces cerevisiae.

The key regulatory enzyme in the gluconeogenesis pathway, fructose-1, 6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are grown in medium containing a poor carbon source. FBPase is targeted to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. To identify genes involved in the FBPase degradation pathway, mutants that failed to degrade FBPase in response to glucose were isolated using a colony-blotting procedure. These vacuolar import and degradation-deficient (vid) mutants were placed into 20 complementation groups. They are distinct from the known sec, ups or pep mutants affecting protein secretion, vacuolar sorting and vacuolar proteolysis in that they sort CpY correctly and regulate osmotic pressure normally. Despite the presence of FBPase antigen in these mutants, FBPase is completely inactivated in all vid mutants, indicating that the c-AMP-dependent signal transduction pathway and inactivation must function properly in vid mutants. vid mutants block FBPase degradation by accumulating FBPase in the cytosol and also in small vesicles in the cytoplasm. FBPase may be targeted to small vesicles before uptake by the vacuole.