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Microstimulation of primate motor thalamus: somatotopic organization and differential distribution of evoked motor responses among subnuclei.

1. The functional organization of motor responses to microstimulation throughout the primate "motor" thalamus including nucleus ventralis lateralis, pars oralis (VLo); nucleus ventralis posterior lateralis, pars oralis (VPLo); nucleus ventralis lateralis, pars caudalis (VLc); and portions of ventralis anterior (VA) and area X, was systematically studied in awake monkeys. A total of 2,021 sites were examined for their response to microstimulation. Of these, 1,123 were histologically verified as to their location within the motor thalamus. At or near each site, isolated neurons were examined for their responses to somatosensory examination and active movement (n = 1,272). This study was carried out as part of a larger study examining the responses of neurons in the motor thalamus to somatosensory examination, torque-induced limb perturbations, and active movement in a visuomotor step-tracking task. 2. Microstimulation at < or = 40 microA evoked movements in the contralateral limbs, trunk, or face. Evoked movements of the limb were generally maximal about a single joint. 3. There was a differential response to microstimulation between subnuclei of the motor thalamus. In order of decreasing frequency, the percentages of sites within each subnucleus from which movements were evoked were as follows: VPLo, 93% (449 of 483); VLo, 21% (57 of 272); VLc, 11% (15 of 140); VA, 1% (1 of 85); and reticular nucleus, 0% (0 of 65). In VPLc, 44% (34 of 78) of sites examined were microexcitable. However, these were almost all within 500 microns of the border of VPLo, suggesting they may have occurred as a result of current spread to adjacent VPLo. Although area X was not sampled in its entirety, it did not appear to be microexcitable. 4. Microexcitable responses had a somatotopic organization, similar to that for neuronal responses to sensorimotor examination, with leg responses found most laterally and arm and face responses found progressively more medially. 5. Zones in VPLo generally ranging from 500 to 1,500 microns were found in which microstimulation resulted in the same motor response. These microexcitable zones resemble those described for the striatum and were termed thalamic microexcitable zones (TMZ). TMZs also resemble cortical efferent zones in that both are somatotopically organized, may affect a single muscle or group of muscles, have low thresholds for microstimulation with sharp boundaries that lie adjacent to other microexcitable zones with the opposite effects, and are of approximately the same dimension. 6. This study suggest that a fundamental unit of motor organization, i.e., single muscle or joint, is preserved at the thalamic level in the form of TMZs, and that these fundamental units of organization may contribute to the modular organization of the cortex.

Animals↗

Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells.

Successful gene therapy of beta-thalassemia will require replacement of the abnormal erythroid compartment with erythropoiesis derived from genetically corrected, autologous hematopoietic stem cells (HSCs). However, currently attainable gene transfer efficiencies into human HSCs are unlikely to yield sufficient numbers of corrected cells for a clinical benefit. Here, using a murine model of beta-thalassemia, we demonstrate for the first time that selective enrichment in vivo of transplanted, drug-resistant HSCs can be used therapeutically and may therefore be a useful approach to overcome limiting gene transfer. We used an oncoretroviral vector to transfer a methylguanine methyltransferase (MGMT) drug-resistance gene into normal bone marrow cells. These cells were transplanted into beta-thalassemic mice given nonmyeloablative pretransplantation conditioning with temozolomide (TMZ) and O6-benzylguanine (BG). A majority of mice receiving 2 additional courses of TMZ/BG demonstrated in vivo selection of the drug-resistant cells and amelioration of anemia, compared with untreated control animals. These results were extended using a novel gamma-globin/MGMT dual gene lentiviral vector. Following drug treatment, normal mice that received transduced cells had an average 67-fold increase in gamma-globin expressing red cells. These studies demonstrate that MGMT-based in vivo selection may be useful to increase genetically corrected cells to therapeutic levels in patients with beta-thalassemia.

Animals↗

Inhibition of the human apurinic/apyrimidinic endonuclease (APE1) repair activity and sensitization of breast cancer cells to DNA alkylating agents with lucanthone.

Cells repair DNA damage via four main mechanisms, however, damage induced by alkylators and oxidative damage is predominantly repaired by the DNA base excision repair (BER) pathway. The AP endonuclease, APE1, is one of the main enzymes in the BER pathway. It is abundant in human cells and accounts for nearly all of the abasic site cleavage activity observed in cellular extracts. APE1 expression is elevated in a variety of cancers and a high APE1 expression has been associated with poor outcome to chemoradiotherapy. The small molecule lucanthone has been shown to enhance the killing ability of ionizing radiation in cells and preliminary evidence suggests that lucanthone may inhibit AP endonuclease. Given the role APE1 plays in repairing oxidative and ionizing radiation DNA damage, the reports of lucanthone as an ionizing radiation enhancer and the potential use of lucanthone as an AP endonuclease inhibitor, we examined whether lucanthone could inhibit APE1 endonuclease activity. We report that lucanthone inhibits the repair activity of APE1, but not its redox function or exonuclease activity on mismatched nucleotides. Lucanthone also appears to inhibit exonuclease III family members (APE1 and ExoIII), but not endonuclease IV AP endonucleases, nor bifunctional glycosylase/lyases such as endonuclease VIII or formamidopyrimidine-DNA glycosylase (Fpg). Furthermore, the addition of lucanthone inhibits APE1 repair activity from cellular extracts and enhances the cell killing effect of the laboratory alkylating agent methyl methanesulfonate (MMS) and the clinically relevant agent temozolomide (TMZ). Given these initial findings, it would be of interest to further develop lucanthone as an APE1 inhibitor through the use of structure-function studies as a means of enhancing the sensitization of tumors to chemotherapeutic agents.

Antineoplastic Agents, Alkylating↗

[Temozolomide--a new antitumor preparation in the treatment of central nervous system malignant tumors].

Treatment results of the patients with central nervous system primary tumors are unsatisfactory. Nytrosomethylureal and vincaalkaloid preparations are rather ineffective in such cases. In recent years clinicians paid great attention to a new-generation chemotherapy drug, temozolomide (temozolomide - TMZ). Temozolomide is used for the treatment of primary malignant tumors of central nervous system, as well as for brain metastatic tumors and melanoma. In the National Cancer Center from 1996 to 2005, 140 patients with central nervous system primary tumors were selected and randomized into 2 groups. In these patients complex therapy using standard schemes of chemotherapy and complex treatment with temozolomide was conducted. 88 patients entered to the control group. 52 patients of the prospective group received temozolomide. Thus, clinical analysis is based on the treatment results of 140 patients. Data obtained have shown that median survival rate of the prospective group patients receiving temozolomide is 2,23 times better in comparison to that of the control group patients. On the basis of the detailed analysis of both retrospective and prospective materials it has been shown that histological type of the tumor is of great importance for the remote treatment results. Of all malignant tumors of central nervous system temozolomide is more effective in the treatment of astrocytomas in comparison with the tumors of other histological types. The use of temozolomide in complex treatment of the patients with malignant tumors of central nervous system resulted in marked effect. Median survival of the patients is 22,35 months.

Antineoplastic Agents, Alkylating↗

[Effect of adenoviral N-methylpurine DNA glycosylase overexpression on chemosensitivity of human osteosarcoma cells].

OBJECTIVE: The overexpression of N-methylpurine DNA glycosylase (MPG) may imbalance the DNA base excision repair (BER) to sensitize tumor cells to current DNA damage chemotherapy. In an effort to improve the efficacy of cancer chemotherapy, we have constructed adenoviral vector of MPG, to study its ability to sensitize human osteosarcoma cell HOS to DNA damage agents. METHODS: The adenoviral infection and MPG expression, as well as enzyme activity were determined by flow cytometry, Western blot, and HEX labeled oligonucleotide-based assay respectively. The cell survival/proliferation was measured using MTS, SRB, and [(3)H] thymidine incorporation assay. Apoptosis cell death was assayed by flow cytometry after treatment using phycoerythin (PE)-conjugated Annexin V and 7-amino-actinomycin (7-AAD). RESULTS: A 10 MOI of recombinant nonreplicating adenovirus was found to infect more than 90% of HOS cells within 24 hours by EGFP fluorescence, in which the MPG overexpression and MPG enzyme activity were also detected. The MPG overexpression HOS cells were significantly more sensitive to the DNA damage agents, including MMS, MNNG, and TMZ, with changes in the IC50 of 6.0, 4.5, and 2.5 fold respectively. CONCLUSIONS: These data establish transient MPG overexpression as a potential therapeutic approach for increasing HOS cellular sensitivity to DNA damage agent chemotherapy.

Adenoviridae↗

[Individualized chemotherapy based on drug sensitivity and resistance assay and MGMT protein expression for patients with malignant glioma--analysis of 42 cases].

BACKGROUND & OBJECTIVE: Malignant glioma cells are resistant to most chemotherapeutic agents. Nitrosourea and temozolomide (TMZ) are main agents for treating malignant glioma. Resistance of malignant glioma to these agents is frequently associated with high levels of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). This study was to evaluate the efficacy of individualized chemotherapy, according to chemotherapy sensitivity and resistance assays (CSRAs) and MGMT expression pattern, on malignant glioma, and observe the adverse events. METHODS: The pathologically confirmed malignant glioma patients, treated by operation at Cancer Center of Sun Yat-sen University from Dec. 2001 to Feb. 2006, were enrolled. The fresh tumor tissues obtained during operation were immediately sent for CSRAs using MTT assay. The expression of MGMT protein was detected by immunohistochemistry. After radiotherapy,the patients received chemotherapy according to the results of CSRAs and MGMT expression. The patients were evaluated for response to chemotherapy according to WHO criteria, and for toxicity according to National Cancer Institute (NCI) criteria. RESULTS: Forty-two patients were evaluated for response to chemotherapy. Seven patients received 2 chemotherapy regimens consecutively, therefore, overall 49 cases were evaluable. Of the 49 cases, 6 (12%) achieved complete remission (CR), 10 (20%) achieved partial remission (PR), 20 (41%) had stable disease (SD), and 13 (27%) had progressive disease (PD). The objective response rate (CR and PR) was 33%, and the disease control rate (CR, PR, and SD) was 73%. Hematologic toxicities were the main adverse events observed in this study, included grade IV anemia (1%), grade III-IV leukopenia (28%), and grade III-IV thrombocytopenia (8%). Non-hematologic toxicities mainly included nausea/vomit, fatigue, and alopecia. CONCLUSION: Individualized chemotherapy based on in vitro CSRAs and MGMT expression for patients with malignant glioma could improve overall response rate.

Adolescent↗

[In vitro studies on omeprazole metabolism in rat liver microsomes].

The metabolism of omeprazole to its two major metabolites, hydroxyomeprazole (OH-OPZ) and omeprazole sulfone (OPZ-SFN) was studied in rat liver microsomes by a reversed phase HPLC assay. The formation of metabolites of OPZ depended on incubation time, substrate concentration, microsomal protein concentration, and was found to be optimal at pH 7.4. The Vmax and Km of OPZ hydroxylation in the rat liver microsomal preparation were 2033 nmol/(min.mg protein) and 46.8 mumol.L-1 respectively. The maximum rate of formation of OPZ-SFN (Vmax) was 187.9 nmol/(min.mg protein), with a Km value of 120.7 mumol.L-1 in rat liver microsome. Moreover, the effects of 7 drugs on OPZ metabolism were tested. The results showed that mephenytoin, benzodiazepines (DZ, NDZ, TMZ, FNZ, NZ) and papaverine caused inhibition of OPZ metabolism, among them papaverine was the only fairly strong inhibitor.

Animals↗

[Trimetazidine therapy in atrio-cochlear disturbances of vascular origin].

Therapeutic effects of TMZ treatment were evaluated in 31 patients with atrio-cochlear disorders caused by an insufficiency of cerebral circulation. It was found that the results of vestibular or cochlear disorders remission was dependent on the time interval between the onset of symptoms and the beginning of trimetazidine therapy. The best results were also found in patients suffering from atrio-cochlear disorders lasting not longer than two years and due to arterial hypertension or cerebral basilar artery insufficiency.

Cerebrovascular Disorders↗

Is epinephrine-induced platelet aggregation autoregulated by its metabolic degradation products in vivo?

BACKGROUND: Catecholamines play an important role in platelet activation and aggregation, epinephrine being the most potent one. Catecholamines are substantially increased during stress, exercise or smoking and could result in clinically important platelet activation if their action was not rapidly regulated. In the present study the possible fast regulation of epinephrine-induced platelet aggregation by its metabolic degradation products is investigated. MATERIALS AND METHODS: Human platelet rich plasma (hPRP) and washed rabbit platelets(wRP) were used for the study. The platelets irreversible aggregation induced by epinephrine and ADP were monitored by an aggregometer prior to and after the addition of the catecholamines degradation products metanephrine, 3-methoxy-4-hydroxy-phenyl-glycole-aldehyde (MHPGA),3-methoxy-4-hydroxy-phenyl-mandelic acid (VMA) 3,4-dihydroxy phenyl glycole (DHPG),3,4-Dihydroxy-phenyl-glycole-aldehyde (DHPGA) and the trimethoxy-phenyl-methyl-piperazine(TMP), a known free radical scavenger and calcium antagonist. Linoleic acid-lipoxygenase reaction, in vitro was monitored in the presence and absence of VMA. RESULTS: Metabolic degradation products possessing a methoxy group at position 3 of the phenolic ring markedly inhibited epinephrine and ADP-induced platelet aggregation at microM concentrations. The most potent inhibitor of both agonists was metahephrine, followed by MHPGA and VMA. TMZ also inhibited platelet aggregation at concentrations similar to VMA. Dihydroxy-phenyl compounds failed to induce any inhibition. None of the substances tested induced any aggregatory effect even at high concentrations (1 mM). VMA significantly inhibited linoleic acid-lipoxygenase reaction at 0.1 microM. CONCLUSIONS: Results indicate that catecholamines' degradation products possessing methoxy (-OCH3) groups can rapidly inhibit in vitro and ex vivo epinephrine-induced platelet aggregation. The inhibitory effects of methoxy phenolic derivatives on epinephrine-induced platelet aggregation may possibly be attributed to their free radical scavenging properties. There is substantial evidence to conclude that an internal rapid autoregulation of epinephrine-induced platelet aggregation, caused by its metabolic degradation products, takes place in vivo.

Animals↗

Absorption of sodium diclofenac after ocular administration in rabbit.

Sodium diclofenac (DCF, CAS 1507-79-6) is a non-steroidal anti-inflammatory drug whose activity is mainly due to an inhibitory effect on the synthesis of the prostaglandins. At present, a tendency towards its topical use in the treatment of the inflammatory state of the anterior segment of the eye as an alternative to the steroid drugs is observed. The pharmacokinetics of DCF was studied in rabbits by assessing the ocular and systemic absorption of DCF after administering DCF eye drops. The chromatographic methods available were not sensitive enough to quantify the extremely low drug concentrations which appeared in the biological fluids after ocular treatment. For this reason, the concentrations of DCF in plasma and aqueous humor were evaluated by a coupled liquid chromatography/gas chromatography (LC/GC) method. DCF was absorbed well through the cornea with the aqueous humor concentration peak being 757.8 ng/ml at 120 min. This good ocular absorption of DCF was confirmed by the concentrations observed in plasma. The presence of tramazoline (TMZ, CAS 74195-73-6) in the eye drops increases the levels of DCF in aqueous humor.

Absorption↗