Dry blending process scale-up for a very low dose drug candidate.
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Capsules↗
Biomedical subjects
J G Chen
Publications and source records attributed to J G Chen.
Permeation and gating residues in serotonin transporter.
The third transmembrane domain (TM3) of serotonin transporter (SERT) contains two isoleucine residues previously proposed to be involved in binding and transport of serotonin. When Ile-172 was replaced with cysteine, SERT became sensitive to inactivation by externally added [2-(trimethylammonium)ethyl]methanethio-sulfonate (MTSET). The disulfide product of this inactivation was not sensitive to reduction by externally added sulfhydryl compounds, but apparently reacted with intracellular reducing agents to spontaneously regenerate active SERT. The apparent accessibility of this residue to both external and cytoplasmic reagents is consistent with its localization near a serotonin binding site that is alternately exposed to both internal and external media. In another SERT mutant, I179C, transport also was inactivated by MTSET but substrate binding was resistant. External substrate bound to the inactivated I179C and enhanced its reactivation by free thiols. In norepinephrine transporter (NET), cysteine replacement of Ile-155 (corresponding to SERT Ile-179) also rendered the transporter sensitive to MTSET inactivation. In NET I155C, cocaine enhanced this inactivation, and the substrate, dopamine, apparently protected against inactivation. The characteristics of this protection suggest that dopamine was transported, converting NET to a form in which Ile-155 was occluded. The results support the proposal that TM3 of SERT and NET constitute part of the substrate permeation pathway, and that Ile-172 in SERT resides close to the substrate binding site. They also suggest that Ile-179 in SERT (and Ile-155 in NET) is in a conformationally sensitive part of TM3, which may act as part of an external gate.
Allosteric Regulation↗
Magnetic resonance spectroscopy in Alzheimer's disease: focus on N-acetylaspartate.
This paper reviews published post-mortem brain and in-vivo proton magnetic resonance spectroscopy (1H-MRS) studies in Alzheimer's disease (AD) and focuses on the emerging role of N-acetylaspartate (NAA) as a prognostic marker of neuronal function. Post-mortem brain studies have reported significantly lower NAA levels in AD brains than in control brains, and some have correlated the low levels with neuropathological findings (i.e. amyloid plaques and neurofibrillary tangles). Similarly, almost all published in-vivo studies have reported lower NAA levels in AD patients compared to elderly controls. While some studies have found changes in metabolite levels that were considered useful for the diagnosis of AD, most have found that 1H-MRS provided little or no advantages over other, more common diagnostic tools. Instead, recent studies in AD and other neuropsychiatric disorders suggest that NAA may be more useful as a prognostic marker for monitoring neurodegeneration, stabilization, or improvement, and for evaluating therapeutic response to novel drugs.
Aged↗
Degradation of a fluoropyridinyl drug in capsule formulation: degradant identification, proposed degradation mechanism, and formulation optimization.
The purpose of this paper was to investigate the degradation chemistry of a fluoropyridinyl drug candidate in capsule formulation and to optimize the formulation based on a proposed degradation mechanism. Small developmental batches of capsules were made by tituration of drug substance and excipients using a mortar and pestle, followed by manual encapsulation. Degradants were identified by LC-MS/MS and LC-photodiode array detector (PDA) and were monitored by LC-ultraviolet detector (UVD) during stability studies. It was found that the drug could undergo a nucleophilic substitution reaction in which hydroxyl groups replace the fluorine substituents on the pyridine rings. The initial degradation rate is independent of the drug concentration but dependent on the temperature, the pH of the microenvironment, and the excipient type. On the basis of these experimental results, a nucleophilic substitution reaction mechanism for the degradation was proposed and a successful capsule formulation was developed.
Anthracenes↗
Crystallization and preliminary X-ray analysis of the auxin receptor ABP1.
Auxin-binding protein (ABP1) is an important receptor for the plant hormone auxin that is involved with many growth and developmental responses in plants. The maize ABP1 gene has been expressed in insect cells, purified and crystallized. Type II crystals are monoclinic, with two glycosylated homodimers in the asymmetric unit, and diffract to 1.9 A using synchrotron radiation.
Crystallization↗
[Condyle process hypertrophy combined with beak malformation: report of one case].
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Journal Article↗
An integrated logistics support system for training crew medical officers in advanced cardiac life support management.
The purpose of this paper is to present an approach for the development of an integrated logistics support system for training space flight crew medical officers in advanced cardiac life support (ACLS). The most practical approach to deal with this problem has been determined to be the training of one or more of the crew members on the mission as crew medical officers (CMO's). If this approach is taken, there are a number of problems that must be addressed. The basic approach presented in this paper is to develop a performance quality index for ACLS tasks and use this index to monitor and control the performance of crew members throughout their tenure as CMO's. The control tool of the system is based on an integrated learning and forgetting model used to forecast CMO performance level at a given point during flight training. The model represents an aid for trainers in determining a training regime and maintaining the performance standards. A performance evaluator and trainer is also developed to help in the establishment of trainee performance level during training or retraining. All of these tools were evaluated using either expert opinion questionnaires or experimental results. In conclusion, the results presented provide the tools required for an integrated logistics support system for training ACLS personnel.
Aerospace Medicine↗
The gatekeeper model is inefficient for the delivery of dermatologic services.
BACKGROUND: Gatekeeper-paradigm managed care systems may discourage the use of dermatologists in the management of skin conditions by limiting direct access. This may limit the quality of care patients with skin disorders receive and may be an inefficient use of medical resources. OBJECTIVE: The purpose of this study was to determine the likelihood that patients with dermatologic conditions who see a primary care provider will be referred to a dermatologist. METHODS: Data on the disposition of outpatient visits to primary care physicians for one and only one dermatologic diagnosis were obtained from the 1990-1994 National Ambulatory Medical Care Survey. These data were used in an econometric model to estimate the likelihood of referral to a dermatologist for an episode of care. RESULTS: Of all visits for a single dermatologic diagnosis, 39% were to primary care physicians. The disposition of referral was more common for these dermatology-related visits than for all office visits to primary care physicians (5.8% vs 4.5%, P < .001). The most frequent diagnoses associated with referral were common dermatologic problems, not rare disorders. The number of visits per episode of care was highly dependent on the assumptions of the analysis, resulting in estimate ranges for referral rates per episode between 6.8% and 18.5% for pediatricians, 8.2% and 23% for family and general practitioners, and 16.6% and 46.5% for internists. CONCLUSION: The relative difficulty for primary care providers of managing skin problems is reflected by their frequent need to refer patients with common skin problems and by the greater likelihood of referral for skin disorders than for other medical conditions. The high rates of referral per episode of care supports the cost-effectiveness of direct access to dermatologists.
Costs and Cost Analysis↗
[Effect of endothelin-1 injected into rostral ventrolateral medulla on cardiovascular responses in cats].
AIM: To study the effect of endothelin-1 (ET-1) in the rostral ventrolateral medulla (rVLM) on cardiovascular responses in cats. METHODS: The stereotatic technique and microinjection method were used. RESULTS: ET-1 (4 mumol.L-1 0.5 microL) microinjected into rVLM induced mean arterial pressure (MAP) increasing (3.7 +/- 1.3) kPa, heart rate (HR) accelerating (29 +/- 7) beats.min-1, and renal nerve activity (RNA) intensifying 45% +/- 10%. The effects were dose-dependent. Before and after bilateral vagotomy, there was no significant difference in the reaction of MAP, HR, and RNA. After intravenous injection with phentolamine (5 mg.kg-1, alpha-blocker), ET-1 did not induce significant change of MAP. ET-1 raised the content of peripheral plasma argipressin (Arg) from (12.4 +/- 6.5) to (70.3 +/- 24.2) ng.L-1 with radioimmunoassay, and showed a correlation with MAP changes. ET-1 induced heart rhythm disorder (HRhD) in acute myocardiac ischemia, the occur time of HRhD was (4.8 +/- 2.9) min, and the score was 4.4 +/- 1.6, and it was significantly different from control. CONCLUSION: ET-1 microinjected into rVLM could involve with control regulation of cardiovascular and sympathetic nerve activity.
Animals↗
[Immunohistochemical study on myocardial acute ischemia reperfusion injury in rats with anti-FOS protein antibody].
To investigate law of FOS protein induced by myocardial ischemia reperfusion (MI/R) in acute period, a model in 20 anaethetized SD rats was established. Rats with normal and ischemia were used as control groups. Specimens were studied immunohistochemically with c-fos antibody. After ischemia 20 minutes, followed by 30 minutes reperfusion, the area of MI/R showed nuclei of myocytes positive staining in cryosection slides. In C2 group, the area showed nuclei of myocytes (37.76% +/- 9.66%) positive staining weakly. In C3 group, nuclei of cardiac myocytes(40.34% +/- 3.32%) was significantly positive. In C4 group it began to attenuate(35.36% +/- 4.81%). The myocardium in normal and ischemic control groups showed negative staining. No changes were seen with HE staining. Our data indicated that immunohistochemical method may reveal acute MI/R injury of ischemia 20 min and reperfusion 30 min with anti-FOS protein staining and there is problely a peak between 60-120 min after reperfusion. It is possible that the method be used to diagnose sudden cardiac death in forensic medicine.
Animals↗
Determination of external loop topology in the serotonin transporter by site-directed chemical labeling.
The transmembrane topology of the serotonin transporter (SERT) has been examined by measuring the reactivity of selected lysine and cysteine residues with extracellular reagents. An impermeant biotinylating reagent, sulfosuccinimidyl 2-(biotinamido)ethyl-1, 3-dithiopropionate (NHS-SS-biotin), was shown to label SERT transiently expressed in cultured cells. Replacement of four lysine residues that were predicted to lie in external hydrophilic loops (eK-less) largely prevented the biotinylation reaction. Likewise, the cysteine-specific biotinylation reagent N-biotinylaminoethylmethanethiosulfonate (MTSEA-biotin) labeled wild type SERT but not a mutant in which Cys-109, predicted to lie in the first external loop, was replaced with alanine. These two mutant transporters reacted with the biotinylating reagents in digitonin-permeabilized cells, demonstrating that the abundant lysine and cysteine residues predicted to lie in intracellular hydrophilic domains were reactive but not accessible in intact cells. Mutants containing a single external lysine at positions 111, 194, 243, 319, 399, 490, and 571 reacted more readily with NHS-SS-biotin than did the eK-less mutant. Similarly, mutants with a single cysteine at positions 109, 310, 406, 489, and 564 reacted more readily with MTSEA-biotin than did the C109A mutant. All of these mutants were active and therefore likely to be folded correctly. These results support the original transmembrane topology and argue against an alternative topology proposed recently for the related glycine and gamma-aminobutyric acid transporters.
Biotin↗
Population-based cancer survival in Qidong, People's Republic of China.
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Adolescent↗
[The assembly of low molecular weight neurofilament protein (NF-L) in vitro].
Neurofilaments were isolated from bovine spinal cords by ultra-speed centrifugation and examined by negative staining. The neurofilament triplet proteins: NF-L, NF-M and NF-H were purified by DE-52 anion exchange chromatography in the presence of 6 mol/L urea. The reassembly of NF-L under controlled conditions was studied. NF-L can reassemble into 10 nm width filaments within 60 minutes at physiological condition of around 0.15 mol/L NaCl, 2 mmol/L MgCl2, neutral pH(pH 6.8) and 37 degrees C. In 6 mol/L urea, NF-L was examined as 12 nm-diameter particle by low angle rotary shadowing. When dialyzed against reassembly buffer for 20 minutes, some irregular filaments were formed. Further dialyzed for another 40 minutes, the long smooth filaments appeared. Some filaments were unraveled at the end regions, where existed 2-4 subfilaments. Four subfilaments were more often observed. That is to say, the 10 nm-width filament was composed of 4 subfilaments. While dialyzed against the alkaline buffer containing 0.15 mol/L NaCl, NF-L reconstituted into 45-180 nm-long, 10 nm-width filaments, which were not able to elongate into long filaments.
Animals↗
[Advance in physiological study on taste].
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Humans↗
The third transmembrane domain of the serotonin transporter contains residues associated with substrate and cocaine binding.
Twenty residues in the third transmembrane domain of the serotonin transporter (SERT) were mutated, one at a time, to cysteine. Almost all of these mutants were fully active for serotonin (5-HT) transport and insensitive to inactivation by the positively charged cysteine reagent [2-(trimethylammonium)ethyl]methanethiosul-fonate (MTSET). Two active mutants, I172C and I179C, were sensitive to rapid inactivation by MTSET but were relatively insensitive to the negatively charged reagent (2-sulfonatoethyl)methanethiosulfonate (MTSES). Inactivation of I172C was blocked by 5-HT and cocaine, but I179C was not similarly protected. Replacement of Tyr-175 with cysteine resulted in a mutant with low transport activity, and, at the neighboring Tyr-176, cysteine replacement completely blocked transport. The Y175C and Y176C mutants were expressed on the cell surface at levels 84% and 69%, respectively, that of wild type (C109A) SERT. Mutants Y175C and Y176C had lower cocaine affinity than C109A, as measured by displacement of the high affinity cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT). For Y176C, 5-HT affinity also was decreased. MTSET inactivated beta-CIT binding to I172C and Y176C, but only slightly inhibited binding to I179C and C109A. The MTSET sensitivity of cysteine replacements at positions 172, 176, and 179 was not observed when these positions were replaced with alanine, serine, or methionine. The results suggest that Ile-172, Tyr-176 and Ile-179 are on one face of an alpha-helical transmembrane element, and that Ile-172 and Tyr-176 are in proximity to the binding site for 5-HT and cocaine.
Asparagine↗
Low-frequency stimulation of afferent Adelta-fibers induces long-term depression at primary afferent synapses with substantia gelatinosa neurons in the rat.
Impulses in primary afferent nerve fibers may produce short- or long-lasting modifications in spinal nociception. Here we have identified a robust long-term depression (LTD) of synaptic transmission in substantia gelatinosa neurons that can be induced by low-frequency stimulation of primary afferent Adelta-fibers. Synaptic transmission between dorsal root afferents and neurons in the substantia gelatinosa of the spinal cord dorsal horn was examined by intracellular recording in a transverse slice dorsal root preparation of rat spinal cord. Conditioning stimulation of dorsal roots with 900 pulses given at 1 Hz (10 V, 0.1 msec) produced LTD of EPSP amplitudes in substantia gelatinosa neurons to 41 +/- 10% of control that lasted for at least 2 hr. When A- and C-fibers were recruited, conditioning stimulation was as effective as A-fiber stimulation alone. After LTD, synaptic strength could be increased to its original level by applying a second, high-frequency tetanic stimulus to the dorsal root, indicating that LTD is reversible and not attributable to damage of individual synapses. Bath application of the GABAA receptor antagonist bicuculline and glycine receptor antagonist strychnine did not affect LTD. When NMDA receptors were blocked by bath application of D-2-amino-5-phosphonovaleric acid, LTD was abolished or strongly reduced. Loading substantia gelatinosa neurons with Ca2+ chelator BAPTA also blocked or reduced LTD. After incubation of slices with calyculin A, a selective and membrane permeable inhibitor of protein phosphatases 1 and 2A, LTD was not attenuated. We propose that this form of LTD may be relevant for long-lasting segmental antinociception after afferent stimulation.
2-Amino-5-phosphonovalerate↗
External cysteine residues in the serotonin transporter.
Hydropathy analysis predicts three cysteines (C109, C200, and C209) in extracellular loops of the rat serotonin transporter (SERT). We mutated these residues, singly and in combination, to either alanine or serine and expressed the mutant transporters in HeLa cells using the vaccinia-T7 transient expression system. Mutation of C109 to alanine had no effect on transport activity or surface expression of the transporter. In Na+-containing solutions, methanethiosulfonate (MTS) reagents had little effect on transport activity in the wild type or in the C109A mutant. When Na+ was replaced with Li+, inactivation of wild type by MTS reagents increased dramatically, but C109A was still resistant. The results suggest that C109 is exposed to the external medium in a manner dependent on cation binding. Replacing either C200 or C209 with serine resulted in either a partial (C200S) or almost total (C209S) loss of transport activity. MTS reagents rapidly inactivated transport activity in mutant C200S, suggesting increased accessibility of a previously unreactive cysteine residue. The double mutants C200S-C109A and C200S-C209S each retained partial activity. C200S-C109A was very sensitive to MTS reagents, but the C200S-C209S mutant was much less sensitive, similar to the wild type transporter. Replacement of C200 or C209 with serine dramatically decreased surface expression of the fully glycosylated transporter. Expression was normal, however, in the C200S-C209S double mutant. The Na+ dependence of transport and ligand binding was abnormal in both C200S and C200S-C209S mutants. Replacing C200 or C209 had similar effects on Na+ dependence and surface expression. Together with the increased MTS reactivity of C200S, these results support the possibility that C200 and C209 may be linked by a disulfide bond in the second external loop of SERT.
Animals↗
Dual action of colchicine on hypertonic activation of system A amino acid transport in vascular smooth muscle cells.
Amino acid transport system A is present in many cells and tissues and is regulated by hormones and other factors, including hypertonic stress. System A in vascular smooth muscle cells is also activated when microtubules are disrupted by drugs such as colchicine. The present study examined the action of colchicine on hypertonic activation of system A in smooth muscle cells. In serum-free medium, activation of system A by modest (340 mOsm) hypertonicity was not affected by colchicine addition. However, at high osmotic stress (460 mOsm) the addition of colchicine partially blocked the activation of system A. Addition of colchicine alone, at normal osmolarity, produced activation of system A. In the presence of serum, colchicine action was markedly different. Colchicine consistently inhibited hypertonic activation of system A at any degree of hypertonic stress but had no effect on system A at normal osmolarity. The action of colchicine as both an activator and inhibitor of system A implies microtubule involvement at more than one step in the intracellular regulation of system A.
Amino Acids↗