[The clinical characteristics and two different treatment outcomes of 321 patients with hypopharyngeal squamous cell carcinoma].

Objective:To investigate the clinical characteristics, two different treatment outcomes and prognostic factors of hypopharyngeal carcinoma. Method:The life table method was used to calculate the overall survival rates, Log-rank test was used to compare the overall survival rates between the two groups．The Cox proportional hazard model was used to perform the multivariate analysis to confirm independent treatment modalities as prognostic factors. Result:Among the 321 patients, 197 patients received surgery combine with radiotherapy or concurrent chemoradiotherapy treatment（S+R/CRT） and 124 patients received radiotherapy or concurrent chemoradiotherapy treatment(R/CRT). For 321 patients, the 1,3,5year overall survival rates were 75.87%,49.39%,41.38% and the median survival time was 37.65 months. The difference in throat retention ratio between the radiotherapy or concurrent chemoradiotherapy treatment(41.94%) and surgery combine with radiotherapy or concurrent chemoradiotherapy treatment（11.17%) was statistically significant (P<0.01).Univariate analysis showed that clinical stage of tumor, T stage, N stage, M stage and two different treatment modalities have impact on survival prognosis. Cox regression multivariate analysis showed that T stage, N stage, two different treatment modalities were independent risk factors of prognosis. Conclusion:The overall prognosis of hypopharyngeal carcinoma was poor and dismal. Hypopharyngeal carcinoma is characterized by high degree of malignancy, difficult to be found early, prone to recurrence and metastasis after operation, large trauma and poor prognosis. Comprehensive examination should be conducted to define the stage of tumor and choose the rational treatment plan before treatment. Surgery combine with radiotherapy or chemotherapy treatment modality is still the main treatment strategy for advanced-stage hypopharyngeal carcinoma..

Transparent flexible thermoelectric material based on non-toxic earth-abundant p-type copper iodide thin film.

Thermoelectric devices that are flexible and optically transparent hold unique promise for future electronics. However, development of invisible thermoelectric elements is hindered by the lack of p-type transparent thermoelectric materials. Here we present the superior room-temperature thermoelectric performance of p-type transparent copper iodide (CuI) thin films. Large Seebeck coefficients and power factors of the obtained CuI thin films are analysed based on a single-band model. The low-thermal conductivity of the CuI films is attributed to a combined effect of the heavy element iodine and strong phonon scattering. Accordingly, we achieve a large thermoelectric figure of merit of ZT=0.21 at 300 K for the CuI films, which is three orders of magnitude higher compared with state-of-the-art p-type transparent materials. A transparent and flexible CuI-based thermoelectric element is demonstrated. Our findings open a path for multifunctional technologies combing transparent electronics, flexible electronics and thermoelectricity.

Human bone marrow-derived adult stem cells for post-myocardial infarction cardiac repair: current status and future directions.

Stem cell-based cell therapy has emerged as a potentially therapeutic option for patients with acute myocardial infarction (AMI) and heart failure. With the completion of a number of trials using bone marrow (BM)-derived adult stem cells, critical examination of the overall clinical benefits, limitations and potential side effects of this revolutionary treatment will pave the way for future clinical research. At present, clinical trials have been conducted almost exclusively using BM stem cells. The primary endpoints of these trials are mainly safety and feasibility, with secondary endpoints in the efficacy of post-myocardial infarction (MI) cardiac repair. Intervention with BM-derived cells was mainly carried out by endogenously-mobilised BM cells with granulocyte-colony stimulating factor, and more frequently, by intracoronary infusion or direct intramyocardial injection of autologous BM cells. While these studies have been proven safe and feasible without notable side effects, mixed outcomes in terms of clinical benefits have been reported. The major clinical benefits observed are improved cardiac contractile function and suppressed left ventricular negative remodelling, including reduced infarct size and improved cardiac perfusion of infarct zone. Moderate and transient clinical benefits have been mostly observed in studies with intracoronary infusion or direct intramyocardial injection of BM cells. These effects are widely considered to be indirect effects of implanted cells in association with paracrine factors, cell fusion, passive ventricular remodelling, or the responses of endogenous cardiac stem cells. In contrast, evidence of cardiac regeneration characterised by differentiation of implanted stem cells into cardiomyocytes and other cardiac cell lineages, is weak or lacking. To elucidate a clear risk-benefit of this exciting therapy, future studies on the mechanisms of cardiac cell therapy will need to focus on confirming the ideal cell types in relation to dosage and timing for post-MI cardiac repair, developing more effective cell delivery techniques, and devising innovative cell tracking modalities that could unveil the fates of implanted cells such as survival, engraftment and functionality.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100 percent; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6 percent, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83 percent, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7 percent ethanol (62 vs 62 percent, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62 percent, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development.

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca(2)+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca(2)+]i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 microg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca(2)+]i oscillations, completion of meiosis and parthenogenetic development.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte.

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Capsaicin and nonivamide as novel skin permeation enhancers for indomethacin.

The study was conducted in vitro to investigate the changes of indomethacin transdermal permeation pretreated by capsaicin and nonivamide, two compounds chemically similar to Azone. The combined effect of low frequency ultrasound (20 kHz) and enhancers on the indomethacin permeation was also evaluated. The experimental data demonstrated that capsaicin and nonivamide significantly enhanced the flux of indomethacin across nude mouse skin. Enhancement effects of both analogues were very similar and depended predominantly on the concentration tested. Histological examination coupled with visual scores indicated the safety of capsaicin and nonivamide on skin structure. Simultaneous application of ultrasound and enhancers significantly increased skin permeation of indomethacin compared with either ultrasound or enhancers alone. Better effect was obtained by the combination with capsaicin than nonivamide.

[Significance of the unbalanced expression of Th1/Th2 type cytokines in human glioma].

OBJECTIVE: To study the significance of the unbalanced expression of Th1/Th2 type cytokines in human glioma. METHODS: The gene expressions of Th1/Th2 type cytokines in 62 specimens of human glioma tissues, 4 glioma cell lines, peripheral blood mononuclear cell (PBMC) of 15 glioma patients, 5 specimens of normal adult brain tissue and 5 brain meningioma tissues were detected by semiquantitative reverse transcription polymerase chain reaction. IFN-gamma and IL-2 represent Th1 type cytokines. IL-4, IL-6, IL-10 and IL-13 represent Th2 type cytokines. RESULTS: There were obviously predominant expression of Th2 type cytokines in glioma cell lines (P < 0.01) and specimens of human glioma tissues (P < 0.01). The tendency of distinct expression of Th2 type cytokines in PBMC was also existent. There wasn't obvious discrepancy of the expression of two type cytokines in normal adult brain tissues and meningioma tissues. CONCLUSIONS: It is likely that the switching of Th1/Th2 type cytokines in gliomas as predominant expression of Th2 type cytokine genes is related to the origination of gliomas and the evasion of glioma cells from immune surveillance.

[Influence of adjustment of balance of Th1/Th2 type cytokines on proliferation of glioma cells].

OBJECTIVE: To study the influence of adjustment of balance of Th1/Th2 by external cytokines on proliferation of glioma cells. METHODS: The gene expressions of Th1/Th2 type cytokines in C6, 9L, U251 and SHG44 glioma cells were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). After the cells were induced with IFN-gamma + IL-4 McAb and IL-4 + IFN-gamma McAb respectively, we isolated the total RNA to proceed RT-PCR again. The evaluation of cell proliferation was proceeded by MTT assay method. RESULTS: There was obviously predominant expression of Th2 type cytokines in glioma cell lines (P < 0.01). The expression intensity of IFN-gamma was improved in IFN-gamma + IL-4 McAb groups and Th2 type cytokines were enhanced in IL-4 + IFN-gamma McAb groups. IFN-gamma and IL-4 McAb could cause the switch from Th2 to Th1, and could remarkably inhibit the proliferation of glioma cells in a dose-dependent way (P < 0.01). On the other hand, IL-4 and IFN-gamma McAb could strengthen the switch of Th2, and might stimulate the glioma cell growth, also in a dose-dependent way (P < 0.01). CONCLUSIONS: There is a Th2 preponderance in glioma cells. IFN-gamma and IL-4 McAb could regulate the switch from Th2 to Th0 or Th1, and inhibit the proliferation of glioma cells.

[Differentiation of natural killer cells into two functional subsets: NKh1 and NKh2].

OBJECTIVE: To verify the presence of functional subsets of natural killer cells based on the cytokine production. METHODS: NK cells were purified and cultured in complete RPMI1640 medium in the presence of either IFN gamma + anti-IL-4(classical Th1 polarization) or IL-4 + anti-IFN gamma (classical Th2 polarization) for three days, and then were collected and detected for type I/type II cytokines by RT-PCR method. RESULTS: NK cells were purified from 15 healthy donors, over 70% purity of NK cells were determined by flow cytometry. NK cells in peripheral blood expressed high level of type I cytokines, mainly IFN gamma, but low level of type II cytokines such as IL-10 and IL-13, IL-4 was not produced by NK cells. Cells cultured in IFN gamma + anti-IL-4 condition exhibited significantly increased level of IFN gamma, unchanged IL-2, and decreased type II cytokines. Cells grew in IL-4 + anti-IFN gamma condition exhibited increased IL-10 and IL-13, and decreased IFN gamma expressions. CONCLUSIONS: Based on the cytokine production, NK cells may be divided into two functional subsets in the same manner as that of T lymphocytes(e.g. Th1/Th2): NKh1 and NKh2. The biological characterization and phenotypic marker are under investigate.

X-linked adrenoleukodystrophy: genes, mutations, and phenotypes.

X-linked adrenoleukodystrophy (X-ALD) is a complex and perplexing neurodegenerative disorder. The metabolic abnormality, elevated levels of very long-chain fatty acids in tissues and plasma, and the biochemical defect, reduced peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity, are ubiquitous features of the disease. However, clinical manifestations are highly variable with regard to time of onset, site of initial pathology and rate of progression. In addition, the abnormal gene in X-ALD is not the gene for VLCS. Rather, it encodes a peroxisomal membrane protein with homology to the ATP-binding cassette (ABC) transmembrane transporter superfamily of proteins. The X-ALD protein (ALDP) is closely related to three other peroxisomal membrane ABC proteins. In this report we summarize all known X-ALD mutations and establish the lack of an X-ALD genotype/phenotype correlation. We compare the evolutionary relationships among peroxisomal ABC proteins, demonstrate that ALDP forms homodimers with itself and heterodimers with other peroxisomal ABC proteins and present cDNA complementation studies suggesting that the peroxisomal ABC proteins have overlapping functions. We also establish that there are at least two peroxisomal VLCS activities, one that is ALDP dependent and one that is ALDP independent. Finally, we discuss variable expression of the peroxisomal ABC proteins and ALDP independent VLCS in relation to the variable clinical presentations of X-ALD.

Gene redundancy and pharmacological gene therapy: implications for X-linked adrenoleukodystrophy.

As more functional redundancy in mammalian cells is discovered, enhanced expression of genes involved in alternative pathways may become an effective form of gene therapy. X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder with impaired very-long-chain fatty acid metabolism. The X-ALD gene encodes a peroxisomal membrane protein (ALDP) that is part of a small family of related peroxisomal membrane proteins. We show that 4-phenylbutyrate treatment of cells from both X-ALD patients and X-ALD knockout mice results in decreased levels of and increased beta-oxidation of very-long-chain fatty acids; increased expression of the peroxisomal protein ALDRP; and induction of peroxisome proliferation. We also demonstrate that ALDP and ALDRP are functionally related, by ALDRP cDNA complementation of X-ALD fibroblasts. Finally, we demonstrate the in vivo efficacy of dietary 4-phenylbutyrate treatment through its production of a substantial reduction of very-long-chain fatty acid levels in the brain and adrenal glands of X-ALD mice.

Hydroxyethyl starch solution attenuates blood-brain barrier disruption caused by intracarotid injection of hyperosmolar mannitol in rats.

This study was performed to investigate whether a fraction of hydroxyethyl starch macromolecules, prepared from pentastarch and known as "Hes-Pz," with molecular weights of 100,000-1,000,000, protects against blood-brain barrier (BBB) disruption due to intracarotid injection of hyperosmolar mannitol. Rats were anesthetized with isoflurane, and retrograde catheterization of a unilateral eternal carotid artery was performed. Except for the Control group (n = 8), hemodilution was performed using lactated Ringer's solution LR group, n = 7), 6% hetastarch (HES group, n = 7), or 6% HES-Pz (HES-Pz group, n = 8) to reduce the hematocrit to about 23%. The BBB transfer coefficient (Ki) of 14C-alpha-aminoisobutyric acid was determined after a unilateral intracarotid injection of 25% mannitol. Blood pressure and hematocrit were similar in all groups. In the control group, Ki was increased significantly in the ipsilateral cortex (IC) where mannitol was injected (16.3 +/- 6.1 vs 4.1 +/- 1.4 microL.min-1) when compared with the contralateral cortex (CC). Ki was similar in the CC in all four groups. The Ki in the IC was significantly lower in the HES-Pz(6.4 +/- 3.5 microL.g-1.min-1) than in the Control, HES, or LR group (16.3 +/- 6.1, 19.0 +/- 12.9, 17.9 +/- 10.8 microL.g-1.min-1, respectively). Our data suggest that HES-Pz significantly attenuates disruption of the BBB caused by an injection of hyperosmolar mannitol.

Cerebral microregional oxygen balance during chronic versus acute hypertension in middle cerebral artery occluded rats.

This study was performed to compare microregional 0(2) supply and consumption balance in spontaneously hypertensive rats (SHR), normotensive Wistar Kyoto rats (WKY), and in phenylephrine-induced acutely hypertensive WKY (WKY + ph) rats. Under isoflurane anesthesia, a middle cerebral artery (MCA) of SHR (n = 7) and WKY (n = 14) rats was occluded. Seven of the WKY rats were infused with phenylephrine (WKY + ph) to keep the mean arterial pressure (MAP) at the same level as that of the SHR. In all animals, 1 h after MCA occlusion, regional cerebral blood flow (rCBF) was determined using an autoradiographic technique, and microregional arterial and venous 02 saturations were determined using microspectrophotometry. MAP was 76 +/- 4 (SD), 136 +/- 15, and 132 +/- 12 mm Hg for the WKY, WKY + ph, and SHR groups, respectively. All variables describing regional O2 balance and rCBF were similar between the SHR and the WKY groups in the ischemic cortex as well as in the contralateral cortex. With phenylephrine infusion, rCBF of both the ischemic cortex and the contralateral cortex were increased in the WKY group. The average 02 supply-to-consumption ratio in the ischemic cortex was higher in the WKY + ph than in the WKY or SHR group. In the ischemic cortex, heterogeneity of venous 02 saturation (SvO2), expressed as a coefficient of variation (CV = 100 X SD/mean), was significantly lower in the WKY + ph (18.3 +/- 2.4) group than in the SHR (30.5 +/- 11.8) or in the WKY (31.3 +/- 9.0) group. The number of veins with low 02 saturation (SvO2 < 40%) in the ischemic cortex was significantly lower in the WKY + ph than in the SHR or in the WKY group. Our data suggest that in chronically hypertensive animals, cerebrovascular adaptations enable the microregional 02 balance in focal ischemia to be maintained at a level similar to that of normotensive animals. However, in normotensive animals with focal cerebral ischemia, an acute increase of MAP improves microregional O2 balance.

Increased blood-brain permeability with hyperosmolar mannitol increases cerebral O2 consumption and O2 supply/consumption heterogeneity.

This study was performed to evaluate whether increasing the permeability of the blood-brain barrier by unilateral intracarotid injection of hyperosmolar mannitol would alter O2 consumption and the O2 supply/consumption balance in the ipsilateral cortex. Rats were anesthetized with 1.4% isoflurane using mechanical ventilation. Retrograde catheterization of a unilateral external carotid artery was performed to administer 25% mannitol at a rate of 0.25 ml/kg/s for 30 s. The blood-brain barrier transfer coefficient (K(i) of 14C-alpha aminoisobutyric acid was measured in one group (N = 7) after administering mannitol. Regional cerebral blood flow (rCBF), regional arterial and venous O2 saturation and O2 consumption were measured in another group using a 14C-iodoantipyrine autoradiographic technique and microspectrophotometry (N = 7). Vital signs were similar before and after administering mannitol. K(i) was significantly higher in the ipsilateral cortex (IC) (22.3 +/- 8.4 microliters/g/min) than in the contralateral cortex (CC) (4.4 +/-1.1). rCBF was similar between the IC (105 +/- 21 ml/g/min) and the CC (93 +/- 20). Venous O2 saturation was lower in the IC (43 +/- 7%) than in the CC (55 +/- 4%). The coefficient of variation (100 x SD/mean) of venous O2 saturation was significantly elevated in the IC (32.3) compared with the CC (18.2), indicating increased heterogeneity of O2 supply/consumption balance. O2 consumption was higher in the IC (9.6 +/- 3.0 ml O2/100 g/min) than in the CC (6.7 +/- 1.5). Our data suggested that increasing permeability of the blood-brain barrier increased cerebral O2 consumption and the heterogeneity of local O2 supply/consumption balance.

Cyclic GMP-phosphodiesterase inhibition does not alter cerebral oxygen consumption.

The effect of zaprinast, a cyclic guanosine monophosphate inhibitor, on the level of cyclic GMP and cerebral O2 consumption was determined. Anesthetized male Long-Evans rats were divided into a control group (n = 15) and a zaprinast treated group (n = 15). Vehicle was applied topically to the left cortex and 3*10-3 M zaprinast was applied to the right cortex. A saline treated control group was also studied. Regional cerebral blood flow was determined by [14C]-iodoantipyrine and regional 0(2) extraction was determined by microspectrophotometry. The level of cyclic GMP was measured by radioimmunoassay. There were no hemodynamic or blood gas differences between groups. The level of cyclic GMP was not significantly different between the right and left cerebral cortex of the control group (17.0 + or - 4.3 and 17.7 + or - 4.6 pmol/g). In the zaprinast treated group, there was a significant (46%) increase in the level of cyclic GMP in the zaprinast treated cortex (20.5 + or - 8.1) in comparison to the vehicle treated cortex (14.0 + or - 5.7). Zaprinast did not significantly alter cerebral blood flow. There were no significant differences in regional 0(2) extraction. The 0(2) consumption of the zaprinast treated cortex (8.0 + or - 3.3 ml O(2)*min(-1)*100 g(-1)) was not different from that of the vehicle ) treated cortex (7.0 + or - 2.9) or those of the control group. Thus, our data indicated that the increased level of cyclic GMP had no significant effect on cerebral oxygen consumption.

Altered expression of ALDP in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is a neurodegenerative disorder with variable phenotypic expression that is characterized by elevated plasma and tissue levels of very long-chain fatty acids. However, the product of the gene defective in ALD (ALDP) is a membrane transporter of the ATP-binding cassette family of proteins and is not related to enzymes known to activate or oxidize fatty acids. We generated an antibody that specifically recognizes the C-terminal 18 amino acids of ALDP and can detect ALDP by indirect immunofluorescence. To better understand the mechanism by which mutations in ALDP lead to disease, we used this antibody to examine the subcellular distribution and relative abundance of ALDP in skin fibroblasts from normal individuals and ALD patients. Punctate immunoreactive material typical of fibroblast peroxisomes was observed in cells from seven normal controls and eight non-ALD patients. Of 35 ALD patients tested, 17 had the childhood-onset cerebral form of the disease, 13 had the milder adult phenotype adrenomyeloneuropathy, 3 had adrenal insufficiency only, and 2 were affected fetuses. More than two-thirds (69%) of all patients studied showed no punctate immunoreactive material. There was no correlation between the immunofluorescence pattern and clinical phenotype. We determined the mutation in the ALD gene in 15 of these patients. Patients with either a deletion or frameshift mutation lacked ALDP immunoreactivity, as expected. Four of 11 patients with missense mutations were also immunonegative, indicating that these mutations affected the stability or localization of ALDP. In the seven immunopositive patients with missense mutations, correlation of the location and nature of the amino acid substitution may provide new insights into the function of this peroxisomal membrane protein. Furthermore, the study of female relatives of immunonegative ALD probands may aid in the assessment of heterozygote status.