Effect of alendronate on bone metabolic indices and bone mineral density in patients treated with high-dose glucocorticoid: a prospective study.

SUMMARY: This prospective study, in the very early phase after initiation of glucocorticoid (GC) treatment, showed that alendronate was effective in suppressing accelerated bone resorption and subsequent decrease in bone mineral density (BMD) at the lumbar spine of patients with high-dose GC treatment. INTRODUCTION: How bisphosphonates affect bone metabolism and BMD of patients with high-dose GC in the early phase, especially within 1 month is unclear. METHODS: We examined the prospective effects of daily 5 mg alendronate on bone metabolism and BMD in 20 patients with high-dose GC (at least 40 mg prednisolone/day) and compared them to 34 high-dose GC-treated patients without alendronate. RESULTS: Serum levels of calcium decreased at day 28 in the alendronate group. Urinary calcium excretion significantly increased after day 7 in both groups. The increase in serum parathyroid hormone (PTH) level at day 7 in the control group was not observed in the alendronate group, but PTH levels increased at day 28 and month 3 in the alendronate group. As for the bone turnover markers, the serum osteocalcin level decreased in both alendronate and control groups, but serum bone-type alkaline phosphatase levels did not show significant changes. Although the urinary type I collagen cross-linked N-telopeptide (NTX) level showed significant increases on days 7 and 28 in the control group; such early increases in urinary NTX were not observed in the alendronate group. Thereafter, the urinary NTX levels fell slowly in the alendronate group significantly. BMD at the lumbar spine significantly decreased from month 1 in the control group, whereas in the alendronate group, BMD at the lumbar spine maintained almost the same level at all time points observed. CONCLUSION: Alendronate was effective in suppressing bone resorption and subsequent BMD decrease at the lumbar spine in patients with high-dose GC treatment.

Substrate recognition of isopeptidase: specific cleavage of the epsilon-(alpha-glycyl)lysine linkage in ubiquitin-protein conjugates.

The structural chromatin protein A24 (uH2A) is a conjugate of histone H2A and a non-histone protein, ubiquitin. Eukaryotic cells contain an enzyme, generically termed isopeptidase, which can cleave A24 stoichiometrically into H2A and ubiquitin in vitro. Isopeptidase, free of proteinase activity, has been partially purified from calf thymus by ion-exchange chromatography, gel filtration and affinity chromatography, and analyzed for its substate specificity. There are three major types of isopeptide bonds besides the epsilon-(alpha-glycyl)lysine bond between H2A and ubiquitin; namely, the disulfide bridge, the aldol and aldimide bonds and the epsilon-(gamma-glutamyl)lysine crosslink. Under conditions where A24 was completely cleaved into H2A and ubiquitin, none of these naturally occurring isopeptide bonds was cleaved by isopeptidase. Furthermore, the bonds formed in vitro by transglutaminase reaction between casein and putrescine, through the gamma-NH2 of glutamine residue and the NH2 of putrescine, were not cleaved by the enzyme. The enzyme also failed to cleave the glycyl-lysyl and other orthodox peptide linkages within proteins. Among various proteins examined, the substrates for isopeptidase reaction were confined to conjugates between ubiquitin and other proteins, formed through epsilon-(alpha-glycyl)lysine bonds. Since ubiquitin released by isopeptidase is re-usable for an ATP-dependent conjugation with other proteins, its carboxyl terminal -Gly-Gly-COOH most likely is preserved intact, and is not blocked. These results suggest that isopeptidase specifically recognizes and cleaves the epsilon-(alpha-glycyl)lysine bond. A possible biological significance of this enzyme is discussed.

Glycogen storage disease type IIIa: first report of a causative missense mutation (G1448R) of the glycogen debranching enzyme gene found in a homozygous patient.

Several different mutations in the glycogen-debranching enzyme gene AGL have been found in patients with glycogen storage disease type III (GSD III) to date, but no missense mutations have been reported for GSD III, only nonsense, splicing, and deletion/insertion lesions. Here we describe a novel G1448R missense mutation in a Japanese GSD IIIa patient from a consanguineous family. Sequence analysis of cDNA from the patient' liver specimen revealed two separate nucleotide changes: a G-to-A transition at nucleotide 3737 in exon 26 (3737G>A) and a G-to-C transversion at nucleotide 4742 in exon 33 (4742G>C), both of which result in substitution of glycine by arginine (G1115R and G1448R). Because homo-zygotes for G1115R were found in healthy controls, G1115R seems to be a polymorphism. Restriction fragment length polymorphism analysis with Bsa JI showed that the patient was homozygous for G1448R and that none of the normal controls had the mutation. This missense mutation is located at a putative glycogen-binding site that is indispensable for enzyme activity. Thus, G1448R is likely to be the causative mutation in this patient. This is the first report of a missense mutation associated with GSD III.

Loss of serotonin-containing neurons in the raphe of patients with myotonic dystrophy: a quantitative immunohistochemical study and relation to hypersomnia.

Hypersomnia occurs frequently in patients with myotonic dystrophy (MyD). We performed a quantitative immunohistochemical study of serotonin (5-HT)-containing neurons linked to hypersomnia in the dorsal raphe nucleus (DRN) and the superior central nucleus (SCN) in 8 patients with MyD, 5 of whom showed hypersomnia, and in 12 age-matched controls. The densities of 5-HT neurons in the DRN and the SCN were significantly lower in MyD patients with hypersomnia than in MyD patients without hypersomnia and controls. These data suggest that the loss of 5-HT neurons of the DRN and the SCN is associated with the presence of hypersomnia in MyD.

Loss of catecholaminergic neurons in the medullary reticular formation in myotonic dystrophy.

OBJECTIVE: To clarify the possible relation between the extent of involvement of catecholaminergic neurons and the presence of alveolar hypoventilation in patients with myotonic dystrophy (MyD). BACKGROUND: Respiratory insufficiency has been reported frequently in MyD patients. Recent data support the hypothesis that this respiratory failure results from a primary dysfunction of the CNS. METHODS: The authors performed a quantitative immunoreactive study of tyrosine hydroxylase immunoreactive (TH+) neurons linked to hypoventilation in the dorsal central medullary nucleus (DCMN), the ventral central medullary nucleus (VCMN), and the subtrigeminal medullary nucleus (SMN)--where the autonomic respiratory center is thought to be located--in eight MyD patients and in 10 age-matched control subjects. Alveolar hypoventilation of the central type was present in three of the MyD patients but not in the remaining MyD patients or the control subjects. RESULTS: The densities of TH+ neurons of the DCMN, the VCMN, and the SMN in MyD patients with hypoventilation were significantly lower than in those without hypoventilation (p < 0.02, p < 0.01, and p < 0.01, respectively) and control subjects (p < 0.01, p < 0.01, and p < 0.01, respectively). CONCLUSIONS: These data suggest that the loss of TH+ neurons of the DCMN, the VCMN, and the SMN is associated with the presence of hypoventilation in MyD and may be an important feature of MyD.

Neuronal loss in the medullary reticular formation in myotonic dystrophy: a clinicopathological study.

Respiratory insufficiency occurs frequently in patients with myotonic dystrophy (MyD). We have performed a quantitative study of neurons linked to respiratory function in the dorsal central medullary nucleus (DCMN), the ventral central medullary nucleus (VCMN), and the subtrigeminal medullary nucleus (SMN) in seven patients with MyD and eight age-matched controls. Alveolar hypoventilation of the central type occurred in three of the MyD patients but not in the remaining MyD patients or controls. The densities of neurons of the DCMN, the VCMN, and the SMN in MyD patients with hypoventilation were significantly lower than in MyD without hypoventilation and controls. These data suggest the neuronal loss of the DCMN, VCMN, and SMN is associated with the presence of hypoventilation in MyD and may be an important feature of MyD.

Decreased expression of full-length mRNA for cBCD541 does not correlate with spinal muscular atrophy phenotype severity.

Spinal muscular atrophy (SMA) is characterized by degeneration of spinal cord anterior horn cells and muscular atrophy and has three phenotypes based on clinical severity and age of onset. One of the responsible genes for SMA is the survival motor neuron (SMN) gene, which is homozygously absent or interrupted in more than 90% of SMA patients. The cBCD541 (BCD) gene is a highly homologous copy of the SMN gene, which has a single synonymous transition in the coding region and may compensate for the loss of the SMN gene. To evaluate the effects of the BCD gene expression on the phenotypes of SMA, we examined lymphocyte mRNA from 9 SMA patients lacking the SMN gene, 10 asymptomatic parents, and 15 control subjects. We amplified mRNA fragments containing exon 7 of the SMN or BCD genes using reverse transcription-polymerase chain reaction since the transcript lacking exon 7 encodes a putative protein with a different C-terminal end. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript as an internal control, and the relative expression level of the SMN or BCD gene was shown as the ratio of SMN or BCD transcript to GAPDH transcript (S/G ratio). The mean S/G ratios of the patients were significantly lower than that of the parents and controls. However, among the patients examined in this study, there was no relationship between the S/G ratios and phenotypes of SMA. The results showed that the BCD gene expression was not related to the phenotypes of SMA. Furthermore, there was an overlap between the S/G ratios in patients and controls. As our discrimination study showed that the S/G ratio reflected the expression of the BCD transcripts in patients and the SMN transcripts in controls, this finding suggested that the BCD gene expression per se does not compensate for the loss of the SMN gene.

Synthesis and structure-activity relationships of 2-substituted D-tryptophan-containing peptidic endothelin receptor antagonists: importance of the C-2 substituent of the D-tryptophan residue for endothelin A and B receptor subtype selectivity.

Continuing studies on modifications of potent cyclic pentapeptide endothelin (ET) receptor antagonists, represented by BQ-123, and potent linear tripeptide derivative ET receptor antagonists, represented by BQ-788, are described herein. The introduction of D-tryptophan analogues with C-2 substituents in these peptidic ET antagonists resulted in potent ET receptor antagonists with various ETA/ETB subtype selectivity. Combined ETA/ETB receptor antagonists were found in both cyclic pentapeptide and linear tripeptide series with 2-halo- and 2-methyl-D-tryptophans. In contrast, compounds with 2-cyano-D-tryptophan were ETB receptor-selective antagonists. The C-2 substituent of the D-tryptophanyl residue appeared to be very important for the discrimination of ETA/ETB subtype selectivity of the antagonists. The potent ET receptor antagonists with various ETA/ETB subtype selectivity synthesized in this study may be useful tools for elucidating the physiological and pathophysiological roles of ET and ET receptors.

Nuclear ribonucleoprotein particles in the cellular slime mold Dictyostelium discoideum.

The nuclear ribonucleoprotein (RNP) particles containing rapidly labeled RNA were isolated from interphase cells of the cellular slime mold Dictyostelium discoideum and characterized. The size of the isolated RNP particles was small (10S to 50S) in comparison with that of nuclear RNP particles found in higher eukaryotes. These small RNP particles do not seem to be artifacts due to degradation during the preparation of nuclear extracts. The rapidly labeled RNA of the nuclear RNP particles was heterogeneous in size and a considerable amount contained polyadenylic acid sequences. Synthesis of RNA in the nuclear RNP particles was resistant to a relatively high concentration of actinomycin D. The protein component of the RNP particle consists of at least four proteins with molecular weights of 80,000, 66,000, 60,000, and 42,000. Thus it is suggested that almost all of the nuclear RNP particles containing rapidly labeled RNA in interphase cells are RNP complexes consisting of Heterogeneous nuclear RNA and several protein species.

Competitive inhibition of hexokinase isoenzymes by mercurials.

A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by p-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by p-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.

Somatosensory evoked potentials in patients with hypocalcaemia after parathyroidectomy.

The effects of hypocalcaemia on somatosensory evoked potentials (SEPs) were studied in five patients after parathyroidectomy. Despite normal latencies the mean value of amplitudes of the SEPs in hypocalcaemic patients was greater than that in normocalcaemic subjects. Recovery functions of the SEPs showed a significant decrease in hypocalcaemic patients at interstimulus intervals of about 10 ms compared with those in normocalcaemic patients and in normal volunteers. Recovery functions appear to be a valid indicator of synaptic efficacy, especially for evaluation of the reduction in conduction efficacy of the central nervous system in hypocalcaemia.

Clinical features and skewed X-chromosome inactivation in female carriers of X-linked recessive spinal and bulbar muscular atrophy.

In X-linked recessive disorders, a few female gene carriers become symptomatic. Recent evidence implicates skewed X-chromosome inactivation in such female carriers. We studied the clinical features of eight female gene carriers of X-linked recessive spinal and bulbar muscular atrophy (SBMA), and evaluated the relationship between phenotype and genotype from the viewpoint of X-chromosome inactivation. Seven of eight cases were symptomatic, showing mild muscle weakness, frequent muscle cramps, slight elevation of the serum creatinine kinase level, or neurogenic changes on the electromyogram. Only one carrier was asymptomatic clinically. For the estimation of X-chromosome inactivation, the methylation status of the androgen receptor (AR) gene was determined by polymerase chain reaction-based assay. Highly skewed inactivation of the affected AR gene was found in the asymptomatic carrier, while symptomatic carriers had a random or lower inactivation pattern of the affected AR gene. These findings suggest that most female carriers of SBMA show some clinical abnormalities, and highly skewed inactivation of the affected X-chromosome seems to closely relate with escape of the manifestation in female carriers of SBMA.

Acute autonomic and sensory neuropathy: a case report.

A female patient with acute autonomic and sensory neuropathy is described. Urinary disturbance developed rapidly and was followed by orthostatic syncope, absence of lacrimation, salivation and sweating, and sensory impairment. Muscle strength had been consistently normal despite diffuse muscular atrophy. Marked decrease in the number of small myelinated and unmyelinated fibres was revealed in biopsied sural nerve. Eighteen months after the onset, her autonomic symptoms have partially improved.

Insulin-like growth factor 1 inhibits glucocorticoid-induced glutamine synthetase activity in cultured L6 rat skeletal muscle cells.

We previously demonstrated the preventive effect of insulin-like growth factor 1 (IGF-1) on steroid myopathy in rats. However, the mechanism by which IGF-1 inhibits steroid myopathy remains unclear. Recent studies have revealed that glutamine synthetase (GS) is induced by glucocorticoid and may be related to the development of steroid myopathy. In this study, we examined whether IGF-1 affected steroid-induced enhancement of GS activity in L6 rat skeletal muscle cells. Dexamethasone (10(-6) M) significantly increased GS activity in L6 cells (P < 0.01). IGF-1 dose-dependently inhibited dexamethasone-induced GS activity. Addition of IGF-1 (750 ng/ml) decreased GS activity to approximately 50% of that with dexamethasone alone (P < 0.01). These results suggest that a decrease in GS activity may be involved in the preventive effect of IGF-1 on steroid myopathy.

Calcitonin gene-related peptide enhances spontaneous acetylcholine release from the rat motor nerve terminal.

The effect of synthetic rat calcitonin gene-related peptide (rCGRP) on neuromuscular transmission was examined in a superfused rat phrenic nerve-diaphragm preparation using an intracellular microelectrode technique. The superfusion with rCGRP (10(-8) to 2 x 10(-7) M) caused significant increases in the frequency of miniature endplate potentials (MEPPs). It, however, had no effect on the resting membrane potential, amplitude of MEPPs, or acetylcholine quantum size and content. Enhancement of spontaneous acetylcholine release from the motor nerve terminal by rCGRP was demonstrated.

Neuropathological changes of the brain in myotonic dystrophy--some new observations.

Brain autopsy materials from 2 patients with myotonic dystrophy (MyD) were studied. The results obtained in these 2 cases were quite similar. Besides thalamic inclusion bodies and minor abnormalities in gyral architecture with a disordered cortical cellular arrangement, some new observations have been made. First, no more than one intracytoplasmic inclusion body per cell was present in the cerebral cortex, the thalamus, the caudate nucleus and the putamen; this inclusion body was oval or elongated with smooth, sharply defined contours and was usually located at the periphery of the cell. Second, irregular intracytoplasmic inclusion bodies, often multiple and not surrounded by a halo, were found at the periphery or within accumulations of neuromelanin granules in the pigmented cells of the substantia nigra. All the bodies described above stained highly eosinophilic with hematoxylin-eosin and the ultrastructure of the bodies in the thalamus and the substantia nigra was almost the same; these bodies were composed of stacks of alternating parallel, light and dark rectilinear profiles oriented perpendicularly to the longitudinal axis of the bodies. Third, Marinesco bodies were observed with a very high frequency in the pigmented cells of the substantia nigra.

A novel missense mutation in the early growth response 2 gene associated with late-onset Charcot--Marie--Tooth disease type 1.

A novel mutation (Arg381Cys) in the second zinc-finger domain of early growth response 2 (EGR2) was identified in a late-onset Charcot--Marie--Tooth disease type 1 (CMT1) patient. This patient had initial symptoms of numbness and weakness in the leg at age 59, and a median nerve motor conduction velocity of 27 m/s. A sural nerve biopsy showed a severe loss of myelinated fibers with numerous onion bulbs. This is the first report of the EGR2 mutation presenting a late onset of CMT1 phenotype. Its mutation was a different amino acid substitution at codon 381 (Arg381His) which demonstrated congenital hypomyelinating neuropathy or early-onset CMT1. This report suggests that the EGR2 mutation represents divergent phenotypes at codon 381, which may be a mutation hotspot.

Immunohistochemical study of intracytoplasmic inclusion bodies of the thalamus in myotonic dystrophy.

Intracytoplasmic inclusion bodies of the thalamus in eight patients with myotonic dystrophy (MyD) were studied immunohistochemically. The intracytoplasmic inclusion bodies of the thalamus (thalamic inclusions, TIs) were strongly immunostained with anti-ubiquitin antibody (Ab) and some of them were mildly stained with anti-microtubule associated protein 1 (MAP 1) and anti-MAP 2 antibodies. However, TIs did not react with any of the following: anti-neurofilament protein Ab, anti-tau Ab, anti-paired helical filament Ab, anti-tubulin Abs (alpha and beta), anti-neuron-specific enolase Ab, anti-glial fibrillary acidic protein Ab, anti-synaptophysin Ab, anti-myelin basic protein Ab, anti-actin Ab and anti-phosphorylated epitope of neurofilaments Ab. Thus, our study demonstrates the unique immunohistochemistry of TIs in MyD which differentiates them from other intracytoplasmic inclusions in various neurodegenerative disorders.

Intracytoplasmic inclusion bodies of the substantia nigra in myotonic dystrophy. Immunohistochemical observations.

We recently reported a significantly higher incidence of intracytoplasmic inclusion bodies (IIBs) of the substantia nigra in patients with myotonic dystrophy (MyD) than in age-matched controls. The changes are, per se, not specific, since a small percentage of disease and normal controls also showed similar inclusions. To elucidate the pathological significance of the inclusion in MyD, we studied immunohistochemical characteristics of IIBs of the substantia nigra in eight patients with MyD. Many IIBs showed moderately intense immunoreactivity for ubiquitin, microtubule-associated protein (MAP) 1 and MAP 2. However, the IIBs did not react with any of the following: anti-neurofilament protein antibodies (Abs) (68, 160 and 200 kDa), anti-neuron-specific enolase antibody (Ab), anti-tau Ab, anti-tubulin Abs (alpha and beta), anti-paired helical filament Ab, anti-actin Ab, anti-phosphorylated epitope of neurofilaments Ab, anti-synaptophysin Ab, anti-myelin basic protein Ab, anti-actin Ab and anti-glial fibrillary acidic protein Ab. Our results suggest that IIBs of the substantia nigra in MyD are related to an alteration of neuronal cytoskeleton metabolism affecting microtubular proteins in conjunction with activation of ubiquitin proteolytic systems.

Alteration in amino acids in motor neurons of the spinal cord in amyotrophic lateral sclerosis.

Little is known concerning the changes of amino acid composition in different regions of the spinal cord in patients with amyotrophic lateral sclerosis (ALS). We performed quantitative amino acid analyses in the posterior funiculus, the lateral corticospinal tract, and the anterior horn of cervical enlargement of the spinal cord from seven ALS patients, and the results were compared with those of seven patients with other neurologic diseases (control A) and seven patients without neurologic diseases (control B). The levels of collagen-associated amino acids, hydroxyproline, proline, glycine, and hydroxylysine, were markedly lower in the lateral corticospinal tract and the anterior horn of ALS patients than in controls A and B. The contents of the acidic amino acids glutamate and aspartate were also significantly decreased in the lateral corticospinal tract and the anterior horn of ALS patients as compared with those of controls A and B. These data suggest that decreased contents of collagen-associated amino acids and excitatory amino acids are related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS.