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Background and objective Alexander disease is a slowly progressive CNS disorder that most commonly occurs in children. Until recently, the diagnosis could only be established by the histologic finding of Rosenthal fibers in brain specimens. Mutations in the glial fibrillary acidic protein (GFAP) gene have now been shown in a number of biopsy- or autopsy-proven patients with Alexander disease. A prospective study on patients suspected to have Alexander disease was conducted to determine the extent to which clinical and MRI criteria could accurately diagnose affected individuals, using GFAP gene sequencing as the confirmatory assay. Methods Patients who showed MRI white matter abnormalities consistent with Alexander disease, unremarkable family history, normal karyotype, and normal metabolic screening were included in this study. Genomic DNA from patients was screened for mutations in the entire coding region, including the exon-intron boundaries, of the GFAP gene. Results Twelve of 13 patients (approximately 90%) were found to have mutations in GFAP. Seven of those 12 patients presented in infancy with seizures and megalencephaly. Five were juvenile-onset patients with more variable symptoms. Two patients in the latter group were asymptomatic or minimally affected at the time of their initial MRI scan. The mutations were distributed throughout the gene, and all involved sporadic single amino acid heterozygous changes that changed the charge of the mutant protein. Four of the nine changes were novel mutations. Conclusions In symptomatic and asymptomatic patients with a predominantly frontal leukoencephalopathy by MRI, GFAP gene mutation analysis should be included in the initial diagnostic evaluation process for Alexander disease.

Objective: To characterize Alexander disease (AxD) phenotypes and determine correlations with age at onset (AAO) and genetic mutation. AxD is an astrogliopathy usually characterized on MRI by leukodystrophy and caused by glial fibrillary acidic protein ( GFAP ) mutations. Methods: We present 30 new cases of AxD and reviewed 185 previously reported cases. We conducted Wilcoxon rank sum tests to identify variables scaling with AAO, survival analysis to identify predictors of mortality, and χ 2 tests to assess the effects of common GFAP mutations. Finally, we performed latent class analysis (LCA) to statistically define AxD subtypes. Results: LCA identified 2 classes of AxD. Type I is characterized by early onset, seizures, macrocephaly, motor delay, encephalopathy, failure to thrive, paroxysmal deterioration, and typical MRI features. Type II is characterized by later onset, autonomic dysfunction, ocular movement abnormalities, bulbar symptoms, and atypical MRI features. Survival analysis predicted a nearly 2-fold increase in mortality among patients with type I AxD relative to those with type II. R79 and R239 GFAP mutations were most common (16.6% and 20.3% of all cases, respectively). These common mutations predicted distinct clinical outcomes, with R239 predicting the most aggressive course. Conclusions: AAO and the GFAP mutation site are important clinical predictors in AxD, with clear correlations to defined patterns of phenotypic expression. We propose revised AxD subtypes, type I and type II, based on analysis of statistically defined patient groups.

We have postulated that steroidogenesis activator polypeptide (SAP) is a product of glucose-regulated protein-78 (grp78) proteolysis on the basis of a number of considerations, including a striking sequence similarity between the carboxyl-terminal region of grp78 and SAP. Since ACTH stimulates the rapid intracellular accumulation of SAP, experiments were conducted to determine whether ACTH might also regulate levels of grp78 mRNA and/or protein. Using a grp78 cDNA probe, Northern analysis of total RNA isolated from hypophysectomized or dexamethasone-suppressed rats revealed that neither treatment had a measurable influence on steady-state levels of grp78 mRNA over a 4-day period. Moreover, immunoblotting with an antiserum directed against a shared grp78/SAP sequence failed to detect a significant change in the grp78 content of adrenal homogenates from dexamethasone-suppressed rats as compared with untreated controls. On the other hand, grp78 in cultured rat adrenocortical cells fell to 50% of that in time-zero controls after 72 h in the absence of ACTH, whereas inclusion of ACTH in the medium blocked this decline. We conclude that while adrenocortical grp78 may be under some measure of trophic ACTH control, the rapid fluctuations reported for SAP are not likely to be driven by large changes in the size of the grp78 pool.

This experiment safety assurance package (ESAP) is a revision of the last mixed uranium and plutonium oxide (MOX) ESAP issued in June 2002). The purpose of this revision is to provide a basis to continue irradiation up to 52 GWd/MT burnup [as predicted by MCNP (Monte Carlo N-Particle) transport code The last ESAP provided basis for irradiation, at a linear heat generation rate (LHGR) no greater than 9 kW/ft, of the highest burnup capsule assembly to 50 GWd/MT. This ESAP extends the basis for irradiation, at a LHGR no greater than 5 kW/ft, of the highest burnup capsule assembly from 50 to 52 GWd/MT.

A sensitive, optimized assay for serum AMP deaminase (EC 3.5.4.6) is presented, which is based on a colorimetric indophenol determination of ammonia liberated during the enzyme reaction. An average enzyme activity in serum of 2.7 U/liter (n = 36, range 0-6.2) for healthy adults was established. Both the enzyme and colorimetric reactions were optimized to give an assay sensitivity of 0.2 U/liter of serum, a 98-99% analytical recovery in the normal serum enzyme concentration range, and a CV of 7.9% in-run and 10.6% between-run. This optimization included studies on specific buffer, pH, ionic strength, and potential activators. The Km for the enzyme in serum was determinated to be 1.4 X 10(-3) mol/liter, which agrees well with the value reported for human skeletal muscle enzyme. Above-normal serum AMP deaminase activity may be present in various myopathies and could be useful in detecting carriers of the X-linked dystrophies.

The behavior of steroidogenesis activator polypeptide (SAP), a recently described modulator of cholesterol side-chain cleavage activity (Pedersen, R. C., and Brownie, A. C. (1987) Science 236, 188-190), was investigated in rat adrenocortical cells using a specific radioimmunoassay. In response to a maximal dose of adrenocorticotropic hormone (ACTH) (1 nM) or of 8-Br-cAMP (1 mM), an increase in intracellular SAP begins rapidly (less than 1 min) and reaches half-maximal and maximal levels (16-fold greater than basal) at 3 and 15 min, respectively. A plateau at this maximal concentration of SAP is then maintained. The levels of intracellular SAP content and of corticosterone output exhibit a similar dose-dependent response to ACTH (EC50 = 25 and 30 pM, respectively). Treatment of ACTH-stimulated cells with cycloheximide reverses the rise in SAP (t1/2 congruent to 5-7 min). In vivo the SAP content of adrenals from quiescent rats is concordant with the circadian rhythm of the pituitary-adrenal axis; at the apex (1800 h), adrenal SAP is 13-fold higher than at the nadir (0800 h), paralleling 2- and 7-fold variations in cholesterol side-chain cleavage activity and serum corticosterone levels, respectively. At both time points, SAP levels rise in response to stress. Of the rat tissues examined, only the major steroid-forming organs (adrenal cortex and gonads) had significant levels of immunoreactive, cAMP-responsive SAP, while cAMP-unresponsive immunoreactivity was also detectable in the thymus, spleen, and brain. Considered together with the biological activity previously demonstrated for SAP in vitro, these data are consistent with its role as a cAMP-dependent, cycloheximide-sensitive modulator of steroid biosynthesis.

The behavior of steroidogenesis activator polypeptide (SAP), a recently described modulator of cholesterol side-chain cleavage activity (Pedersen, R. C., and Brownie, A. C. (1987) Science 236, 188-190), was investigated in rat adrenocortical cells using a specific radioimmunoassay. In response to a maximal dose of adrenocorticotropic hormone (ACTH) (1 nM) or of 8-Br-cAMP (1 mM), an increase in intracellular SAP begins rapidly (less than 1 min) and reaches half-maximal and maximal levels (16-fold greater than basal) at 3 and 15 min, respectively. A plateau at this maximal concentration of SAP is then maintained. The levels of intracellular SAP content and of corticosterone output exhibit a similar dose-dependent response to ACTH (EC50 = 25 and 30 pM, respectively). Treatment of ACTH-stimulated cells with cycloheximide reverses the rise in SAP (t1/2 congruent to 5-7 min). In vivo the SAP content of adrenals from quiescent rats is concordant with the circadian rhythm of the pituitary-adrenal axis; at the apex (1800 h), adrenal SAP is 13-fold higher than at the nadir (0800 h), paralleling 2- and 7-fold variations in cholesterol side-chain cleavage activity and serum corticosterone levels, respectively. At both time points, SAP levels rise in response to stress. Of the rat tissues examined, only the major steroid-forming organs (adrenal cortex and gonads) had significant levels of immunoreactive, cAMP-responsive SAP, while cAMP-unresponsive immunoreactivity was also detectable in the thymus, spleen, and brain. Considered together with the biological activity previously demonstrated for SAP in vitro, these data are consistent with its role as a cAMP-dependent, cycloheximide-sensitive modulator of steroid biosynthesis.

A non-ACTH aldosterone-stimulating factor(s) has been implicated in the pathogenesis of idiopathic hyperaldosteronism (IHA). Although this factor has not been fully characterized, some evidence suggests that it may be related to a pro-gamma-melanotropin (pro-gamma-MSH), derived from the NH2-terminal region of pro-opiomelanocortin. In the present study, plasma immunoreactive (IR-) gamma-MSH levels at 0800 h in patients with IHA were evaluated (90 +/- 17 fmol/ml; range: 13-173 fmol/ml) and found to be significantly higher (P less than 0.05) than those in subjects with aldosterone-producing adenomas (33 +/- 8 fmol/ml), essential hypertension (33 +/- 6 fmol/ml), and normotensive controls (19 +/- 2 fmol/ml). Seven of nine IHA subjects had circulating IR-gamma-MSH levels above the normal range (greater than 35 fmol/ml). In plasmas sampled at 1200 h, IR-gamma-MSH was significantly higher in patients with IHA (95 +/- 26 fmol/ml) and adenomas (63 +/- 23 fmol/ml), as compared with essential hypertensives (31 +/- 6 fmol/ml) and normotensives (19 +/- 3 fmol/ml). Mean plasma IR-ACTH, plasma cortisol, and urinary cortisol levels did not differ significantly between any of these groups. In order to evaluate the effect of a pro-gamma-MSH in vitro, adrenal adenoma tissue was obtained from two patients, one with elevated IR-gamma-MSH (61 fmol/ml) and a second with low IR-gamma-MSH (12 fmol/ml). Aldosterone secretion by dispersed adenoma cells from the former, but not the latter, underwent a fourfold dose-dependent (10(-14)-10(-9) M) increase in response to human Lys-gamma 3-MSH. These data suggest that a pro-gamma-MSH may be implicated as a pathogenic factor in a subset of patients with primary aldosteronism, particularly among those differentially diagnosed as having IHA.

A sensitive, optimized assay for serum AMP deaminase (EC 3.5.4.6) is presented, which is based on a colorimetric indophenol determination of ammonia liberated during the enzyme reaction. An average enzyme activity in serum of 2.7 U/liter (n = 36, range 0-6.2) for healthy adults was established. Both the enzyme and colorimetric reactions were optimized to give an assay sensitivity of 0.2 U/liter of serum, a 98-99% analytical recovery in the normal serum enzyme concentration range, and a CV of 7.9% in-run and 10.6% between-run. This optimization included studies on specific buffer, pH, ionic strength, and potential activators. The Km for the enzyme in serum was determinated to be 1.4 X 10(-3) mol/liter, which agrees well with the value reported for human skeletal muscle enzyme. Above-normal serum AMP deaminase activity may be present in various myopathies and could be useful in detecting carriers of the X-linked dystrophies.

Pro-gamma-melanotropins (pro-gamma-MSH) are implicated in the control of aldosterone secretion by the normal adrenal cortex and in idiopathic hyperaldosteronism. In contrast with other melanotropins (alpha- and beta-MSH), the pro-gamma-MSH increase aldosterone secretion by normal adrenals only in the presence of adrenocorticotrophic hormone (ACTH). However, in aldosteronoma cells this stimulatory effect of the pro-gamma-MSH is not ACTH-dependent. The mechanism of pro-gamma-MSH action on aldosterone biosynthesis may involve activation of cholesteryl ester hydrolase in zona glomerulosa, resulting in an increased provision of cholesterol for steroidogenesis.