Inborn errors of metabolism discovered in Asian department of pediatrics and mental retardation research center.

To heighten the effectiveness of chemical diagnosis for inborn errors of metabolism (IEM) using urease pretreatment and GC-MS analysis, a sample collection and transportation method was contrived. The resulting "filter paper set" allows simple urine collection and transportation, and enables anyone from anywhere to receive the GC-MS analysis without the limitations of place or time. Using filter paper sets, high-risk screening of undiagnosed children or mentally retarded children with unknown cause was conducted in cooperation with hospitals and universities in several Asian countries. During 8 months 203 patients from China and India were analyzed and 20 cases of IEM were chemically diagnosed. These diagnoses greatly contributed to the treatment of children with intractable diseases who lived in Asian countries where analytical techniques and facilities for IEM were not sufficient.

Asparagine-proline sequence within membrane-spanning segment of SREBP triggers intramembrane cleavage by site-2 protease.

The NH(2)-terminal domains of membrane-bound sterol regulatory element-binding proteins (SREBPs) are released into the cytosol by regulated intramembrane proteolysis, after which they enter the nucleus to activate genes encoding lipid biosynthetic enzymes. Intramembrane proteolysis is catalyzed by Site-2 protease (S2P), a hydrophobic zinc metalloprotease that cleaves SREBPs at a membrane-embedded leucine-cysteine bond. In the current study, we use domain-swapping methods to localize the residues within the SREBP-2 membrane-spanning segment that are required for cleavage by S2P. The studies reveal a requirement for an asparagine-proline sequence in the middle third of the transmembrane segment. We propose a model in which the asparagine-proline sequence serves as an NH(2)-terminal cap for a portion of the transmembrane alpha-helix of SREBP, allowing the remainder of the alpha-helix to unwind partially to expose the peptide bond for cleavage by S2P.

ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs.

ATF6 is a membrane-bound transcription factor that activates genes in the endoplasmic reticulum (ER) stress response. When unfolded proteins accumulate in the ER, ATF6 is cleaved to release its cytoplasmic domain, which enters the nucleus. Here, we show that ATF6 is processed by Site-1 protease (S1P) and Site-2 protease (S2P), the enzymes that process SREBPs in response to cholesterol deprivation. ATF6 processing was blocked completely in cells lacking S2P and partially in cells lacking S1P. ATF6 processing required the RxxL and asparagine/proline motifs, known requirements for S1P and S2P processing, respectively. Cells lacking S2P failed to induce GRP78, an ATF6 target, in response to ER stress. ATF6 processing did not require SCAP, which is essential for SREBP processing. We conclude that S1P and S2P are required for the ER stress response as well as for lipid synthesis.

Second-site cleavage in sterol regulatory element-binding protein occurs at transmembrane junction as determined by cysteine panning.

In response to sterol deprivation, two sequential proteolytic cleavages release the NH2-terminal fragments of sterol regulatory element-binding proteins (SREBPs) from cell membranes. The fragments translocate to the nucleus where they activate genes involved in cholesterol and fatty acid metabolism. The SREBPs are bound to membranes in a hairpin fashion. The NH2-terminal and COOH-terminal domains face the cytoplasm, separated by two membrane spanning segments and a short lumenal loop. The first cleavage occurs at Site-1 in the lumenal loop. The NH2-terminal fragment is then released by cleavage at Site-2, which is believed to lie within the first transmembrane segment. Here, we use a novel cysteine panning method to identify the second cleavage site (Site-2) in human SREBP-2 as the Leu484-Cys485 bond that lies at the junction between the cytoplasmic NH2-terminal fragment and the first transmembrane segment. We transfected cells with cDNAs encoding fusion proteins with single cysteine residues at positions to the NH2-terminal and COOH-terminal sides of cysteine 485. The NH2-terminal fragments were tested for susceptibility to modification with Nalpha-(3-maleimidylpropionyl)biocytin, which attaches a biotin group to cysteine sulfhydryls. Cysteines to the NH2-terminal side of cysteine 485 were retained on the NH2-terminal fragment, but cysteines to the COOH-terminal side of leucine 484 were lost. Leucine 484 is three residues to the COOH-terminal side of the tetrapeptide Asp-Arg-Ser-Arg, which immediately precedes the first transmembrane segment and is required for Site-2 cleavage.

Evaluation of BR-16 A (Mentat) in cognitive and behavioural dysfunction of mentally retarded children--a placebo-controlled study.

It is important to control abnormal behaviour and hyperactivity, and improve cognition in mentally retarded children (MRC), which would help in their education, training and subsequent rehabilitation. Recently it has become known that amongst other side-effects, protracted use of anti-convulsant medication induces cognitive and behavioural dysfunction, which is a major problem in mentally retarded epileptics. In a placebo-controlled study, we confirmed the efficacy of a herbal preparation, BR-16A (Mentat) in controlling such behavioural and cognitive deficits in 40 mentally retarded children. The efficacy of this remedy was further evaluated in 19 MRCs with epilepsy. Twelve patients had generalised seizure, 4 with partial and 3 with mixed seizure pattern was continued. Inspite of the usual antiepileptic treatment, the frequency of seizures ranged from 1 to 7 attacks in periods from 1 week to 1 year. With active drug Br-16A, it was possible to note a reduction in seizure frequency. Patients with higher frequency responded better. There was no further increase in the dosage of antiepileptic drugs. There was significant control of other abnormal behaviour as shown by reduction in rating score on the Children's Behavioural Inventory test. BR-16A was effective in controlling abnormal behaviour, especially hyperactivity and incongruous behaviour in mentally retarded children with and without epilepsy.

Brain tumours in childhood in Bombay: I: Histopathology showing changing patterns; II: Tissue culture with light and electronmicroscopy, stressing ingestion & degradation of bacteria by glial cells in vitro.

The pathological pattern of 86 brain 'tumours' in childhood during the years 1981-85 (out of a total of 586 for all ages), showed a higher proportion of neoplasms and a much lower of tuberculomas compared to the preceding three decades. A large number of histologically unusual cases was revealed. Through tissue culture of brain tumours we carried out morphological, histochemical and fine structural study of the tumour cells in vitro. The abundant presence of lysosomal acid phosphatase, in outgrowing cells, correlated with the detection of lysosomal dense bodies and vacuoles in araldite sections, by light and electronmicroscopy. In view of the phagocytic propensity of schwann cells for M. leprae as the important factor in leprous neuritis, TC preparations of gliomas, (in addition to acoustic schwannomas and meningiomas), were inoculated with two mycobacteria, M. scrofulaceum and the ICRC bacillus. There was a pronounced intracytoplasmic uptake, i.e. endocytosis, of acid-fast bacilli by the growing cells of these tumours. This was confirmed by electronmicroscopy which showed intact and degrading bacilli in various stages, in such cells of a typical cerebral astrocytoma used as an illustrative case in this paper. Ingestion and Digestion appear to be an inherent property of growing tumour cells in vitro. Fine structural examination of in vitro growth of an unusual subependymal giant cell astrocytoma, not inoculated with bacilli, served as a control. Cells of both tumours showed copious autophagic activity and cytoskeletal features of developing microtubules and filaments.

Effect of prolonged anticonvulsant medication in epileptic patients: serum lipids, vitamins B6, B12, and folic acid, proteins, and fine structure of liver.

Twenty-seven epileptic patients, most from low socioeconomic groups and aged 15-54 years, were studied for effects of prolonged anticonvulsant medication. They had received the usual doses of phenobarbitone and diphenylhydantoin (PHT) regularly for 3-32 years, with control of seizures, and had not taken any B-vitamins in the year before investigation. Besides reduced serum and cerebrospinal fluid (CSF) folate levels, significantly increased levels of total vitamin B6 in CSF and serum and of vitamin B12 in serum were found in patients as compared with normal healthy subjects. The bone marrow was normoblastic, and significant elevation of serum triglycerides and/or cholesterol was observed in patients. The total protein level was only slightly reduced as compared with that of controls, but there was significant increase in beta-lipoprotein fraction on gel electrophoresis. Plasma proteins concerned with vitamins and lipid transport showed no remarkable change, and no abnormal protein was detected. Although there was no clinical hepatic involvement, liver biopsy performed in 9 of 27 patients revealed fine structural changes in hepatocytes suggestive of varying degrees of drug-induced changes. A ramifying network of short, smooth, endoplasmic cisternae with depleted rough endoplasmic reticulum (RER), distended sinusoids with Kupffer cells, dark shrunken hepatocytes with reduced mitochondria, and increased lipofuscin were observed. This suggested an adaptive response of the liver, a reversible change, possibly related to the increased serum lipids in the same patients.

Fine structure of cellular and vascular reaction in brain tuberculomas: a model for phagocytosis in the CNS.

The fine structure of cells of the mononuclear phagocyte series (MPS) and a few other cells with phagocytic capacity, has been critically evaluated, mainly from an electronmicroscopic examination of the reactive border zone of 11 human brain tuberculomas, which provide ideal material for the study of macrophages. Most of them appeared to be blood monocyte-derived epithelioid cells of various forms and stages. The cytoplasm of these cells showed either more rough ER representing protein synthesising activity; or more frequently, phagosomes, phagolysosomes, dense bodies or empty vacuoles, representing various stages of ingestion and digestion of necrotic material. Often such material, which was more or less osmiophilic, was seen abundantly between the cells. These actively phagocytic cells occasionally undergoing, mitosis, are referred to as "epithelioid macrophages" and were morphologically similar to the "activated microglia" described in other conditions. They also showed a tendency to be closely adjacent to each other and occasionally fuse to form giant cells. There were also a number of lymphocytes and plasma cells. The latter showed various stages of active and granular or depleted and distended rough ER tubules, phagocytic activity and tendency to fuse. Expected vasculitis and small vessel necrosis formed part of this granulomatous reaction. Constituents of oedematous or necrosed brain tissue were seen immediately around the reactive zone of these tuberculomas, the most frequent being reactive astrocytes, many of which showed membrane-bound vacuoles. It is conceivable that the excessive pleomorphic cellular, vascular and necrotic reaction in these brain tuberculomas could have resulted from a delayed type of hypersensitivity to a very small quentity of antigenic tuberculoprotein, which probably initiates the chain of immunologic responses.

Ultrastructural basis of the vasculopathy in and around brain tuberculomas. Possible significance of altered basement membrane.

The fine structure of small blood vessels in and around ten brain tuberculomas was examined. In the peripheral reactive zone of the tuberculomas, examination of 1-mu-thick survey sections established the chronic inflammatory process and the vasculitis characterized by infiltration of the vasomurium (vessel wall) by large and small mononuclear cells. This reaction was typical of chronic epithelioid cell granuloma. Electron microscopic examination of the reactive zone confirmed the vascular proliferation and vasculitis, the venule being the most frequently involved type of blood vessel. It showed the infiltrating cells to lie amidst osmiophilic, concentrically proliferated basement membrane laminae, which formed the greater part of the thickened vessel wall, generally surrounding the endothelial cells directly, the pericytes having disappeared. This appearance, together with the results of Gomori's reticulin stain on paraffin sections, suggested that the altered basement membrane material was reticulin. The possibility is discussed that the altered basement membrane material could be antigenic and that it might be responsible for perpetuating the necrotic vascular and perivascular reaction in tuberculous meningitis and tuberculomas. The above change in the basement membrane was not encountered in the blood vessels of the surrounding edematous brain. The endothelial cells and tight junctions were relatively well-preserved. Intact arterioles could be recognized even in severely edematous brain tissue. At both sites the fine structure of the blood vessels was typical of that expected in the central nervous system. Fenestrated vessels were not seen. The perivascular astrocytic end-feet were destroyed in the reactive zone and either distended or ruptured in the overtly edematous brain tissue also. In the central caseous part of the tuberculoma, there were few blood vessels, and they were in a state of advanced necrosis, but ghost outlines of proliferated basement membrane could be seen.