Biomedical subjects
Publications and source records attributed to J S Yu.
Two childhood cases of intracranial melanotic neuroectodermal tumor, one benign and one malignant, are presented. The clinical, radiologic, and pathologic features of these cases are described and the relevant literature is reviewed.
The activating factor of ATP.Mg-dependent protein phosphatase (FA) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates, FA could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with a Km value of 0.4 microM, and tau proteins to 4 moles of phosphates per mole of proteins with a Km value of about 3 microM. When using microtubules as substrates, FA could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca+2/phospholipid-dependent protein kinase, the FA-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced by FA. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed that FA could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca+2/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due to FA. Taken together, the results provide initial evidence that the ATP.Mg-dependent protein phosphatase activating factor (FA) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.
We report a child in whom dihydropteridine reductase deficiency was diagnosed prenatally because of an affected sibling and who was treated from birth with apparent good response. This family has been reported before (Firgaira et al., 1980, 1981, 1983; Lipson et al., 1984; Cotton et al., 1986; Dahl et al., 1988). The parents are Lebanese Muslims who are first cousins, and their first and third children, both male, are well. The third child was diagnosed as normal in utero (Firgaira et al., 1983). The second child, the subject of an early report (Lipson et al., 1984), has dihydropteridine reductase deficiency. The dihydropteridine reductase is nonfunctional due to an amino acid insertion (Howells et al., 1990).
In order to better characterize the molecular events that accompany lesion development in multiple sclerosis (MS), we studied the accumulation of RNA specific to the nuclear proto-oncogenes c-fos and c-myb in post mortem white matter brain tissue. RNA was prepared from plaque and periplaque regions of 6 different MS brains, from "normal" white matter regions of 3 MS brains and from 6 normal control samples. Quantitation of specific RNA corresponding to each proto-oncogene was performed by Northern blot hybridization and by scanning densitometry. Results indicate a 2-fold increase in c-fos RNA in MS white matter, compared to control tissue. No c-myb signal was identified in any sample. In situ hybridization studies confirmed the selective upregulation of c-fos RNA levels in MS tissue, and suggested that glial cells and not inflammatory cells were responsible for the enhanced c-fos signal. These results suggest that persistent glial cell activation is present within chronic MS lesions irrespective of whether the lesions are active (e.g., inflammatory) or inactive.
Two forms of type-1 protein phosphatase activating factor (FA) termed FA1 and FA2 have been identified in plasma membranes of pig brain. FA1 is spontaneously active and trypsin-labile whereas FA2 is inactive and trypsin-resistant. Phospholipid reconstitution studies further indicate that the FA activity in the neutral phospholipids-reconstituted complex is spontaneously active and trypsin-labile whereas the FA activity in the acidic phospholipids-reconstituted complex is trypsin-resistant and inactive. The results indicate that inactive FA2 may have its catalytic domain interacted with negatively-charged phospholipids in brain membranes. This provides initial evidence for the regulation of protein kinase FA (a transmembrane signal of insulin and epidermal growth factor) in the central nervous system.
Protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase) has been characterized to exist in two forms in the purified brain myelin. One form of kinase FA is spontaneously active and trypsin-labile, whereas the other form of kinase FA is inactive and trypsin-resistant, suggesting a different membrane topography with active FA exposed on the outer face of the myelin membrane and inactive FA buried within the myelin membrane. When myelin was solubilized in 1% Triton X-100, all kinase FA became active and trypsin-labile. Phospholipid reconstitution studies further indicated that when kinase FA was reconstituted in acidic phospholipids, such as phosphatidylinositol and phosphatidylserine, the enzyme activity was inhibited in a dose-dependent manner, suggesting that kinase FA interacts with acidic phospholipids which inhibit its activity. Furthermore, when myelin was incubated with exogenous phospholipase C, the inactive/trypsin-resistant FA could be converted to the active/trypsin-labile FA in a time- and dose-dependent manner. Taken together, it is concluded that membrane phospholipids play an important role in modulating the activity of kinase FA in the brain myelin. It is suggested that phospholipase C may mediate the activation-sequestration of inactive/trypsin-resistant kinase FA in the brain myelin through the phospholipase C-catalyzed degradation of acidic membrane phospholipids. The activation-sequestration of protein kinase FA may represent one mode of control modulating the activity of kinase FA in the central nervous system myelin.
Exposure of rat adipocytes to physiological concentrations of insulin resulted in a time- and concentration-dependent activation-translocation of kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase) in plasma membranes and the subsequent activation of ATP.Mg-dependent protein phosphatase in the cytosol. The insulin-induced activation of membrane-associated kinase FA and cytosolic ATP.Mg-dependent protein phosphatase occurred very rapidly, reaching the maximal activity levels within 3 min. Moreover, the insulin effect is transient; the insulin-stimulated FA activity in membranes and ATP.Mg-dependent phosphatase activity in the cytosol returned to control levels within 30 min. It is concluded that insulin may induce the activation of kinase FA in membranes and thereby promotes the activation of ATP.Mg-dependent multifunctional protein phosphatase in the cytosol of rat adipocytes in order to mediate some of its intracellular effects through the dephosphorylation reactions. The release of factor FA from plasma membranes may represent one of the transmembrane signalling mechanisms for insulin actions.
A Mn2+/phospholipid-dependent protein phosphatase has been identified and characterized from brain membranes. The phosphatase contains three subunits with molecular weights of 64,000, 54,000, and 35,000 in a 1:1:1 molar ratio. On gel filtration, the enzyme has an apparent molecular weight of approximately 180,000. The phosphatase was active on many substrates, including p-nitrophenyl phosphate, phosphotyrosine, phosphothreonine, phosphorylase a, myelin basic protein, histones, type 1 phosphatase inhibitor-2, microtubule tau protein, and synapsin I. To dephosphorylate phosphoproteins, the phosphatase was dependent on such acidic phospholipids as phosphatidylinositol and phosphatidylserine but not on neutral phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The phospholipid-mediated activation of the phosphatase was time and dose dependent and could be reversed by Triton X-100 or gel filtration. Kinetic study further indicates that phospholipid was able to increase the Vmax of the phosphatase but had no effect on the Km value for substrates, suggesting a direct interaction of phospholipids with the phosphatase. Conversely, in order to dephosphorylate phosphoamino acids such as phosphotyrosine and phosphothreonine, this phosphatase was entirely dependent on Mn2+. Phospholipids had no effect on the dephosphorylation of phosphoamino acids, whereas Mn2+ had no effect on the dephosphorylation of phosphoproteins. It is concluded that this Mn2+/phospholipid-dependent membrane phosphatase has two distinct activation mechanisms. The enzyme requires Mn2+ to dephosphorylate micromolecules, whereas acidic phospholipids are needed to dephosphorylate macromolecules. This suggests that Mn2+ and phospholipids may play a role in regulating the substrate specificity of this multisubstrate membrane phosphatase.
A cyclic AMP and calcium-independent protein kinase has been identified and purified from pig brain to near homogeneity. This independent protein kinase was isolated in an inactive form, and activation required ATP and Mg2+. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme contains 1 subunit with a molecular mass of about 36 kDa. Although there was no significant phosphorylation of phosphorylase, phosphorylase b kinase, casein, phosvitin, and protamine, this kinase was found to be very active toward myelin basic protein and histones H1, 2A, and 2B. Trypsinolysis completely destroyed the kinase activity, indicating that this is not a protease-activated protein kinase. More interesting, this cAMP and calcium-independent protein kinase can be regulated by its state of phosphorylation. In its non-phosphorylated state, the kinase was essentially inactive but could be fully activated when the enzyme was phosphorylated up to a 1:1 molar ratio. Conversely, partial dephosphorylation of the phosphorylated enzyme was associated with a time-dependent decrease in the kinase activity and a loss of 32P. All the results taken together point out that this kinase is distinguished from all the reported protein kinases and may represent a previously undiscovered protein kinase. The results also provide initial evidence that a cascade activation mechanism may possibly be involved in the regulation of a protein kinase activity which is independent of cAMP and calcium.
Although the activating factor FA of the type-1 protein phosphatase has long been recognized as a cytosolic enzyme involved in the regulation of cell metabolism and nervous functions, strong indications have been obtained that FA is in fact a membrane-bound protein kinase in most mammalian tissues. For instance, direct treatment of the tissue extracts including brain, liver, cardiac, smooth and skeletal muscles with 1% Triton X-100 can cause several fold stimulation of the FA activity. Moreover, at least 50% of the FA can be detected in the particulate fractions of the extracts. Chromatography of the extracts in the presence and absence of Triton X-100 further demonstrate these results. The data can now explain the reason why most people can not isolate reasonable amount of FA from mammalian tissues. It is recommended that Triton X-100 should be used for purification of FA from most mammalian tissue extracts. The results also suggest that most previous studies on the action of FA involved in the regulation of cell functions should be re-evaluated and the membrane-associated FA should be taken into consideration.
Direct treatment of brain myelin with freezing/thawing in 0.2 M 2-mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P-labeled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X-100, this endogenous latent phosphatase activity was further stimulated 2.5-fold. Diethylaminoethyl-cellulose and Sephadex G-200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M 2-mercaptoethanol and with a molecular weight of 350,000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP X Mg-dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high-molecular-weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.
In addition to handling urgent emergency calls for advice about major poisonings, the NSW Poisons Information Centre handles calls from the general community about poisonings of a much more minor nature. Although these poisonings may be perceived by the callers to be significant, the only action required is either reassurance that there is, in fact, no problem or simple treatment that does not require further intervention. The study demonstrates that callers in this category would have sought professional advice elsewhere had the Centre not existed and the subsequent cost effectiveness of the Centre's role in taking these calls.
Two inactive/latent protein phosphatases termed LP-1 (Mr 260,000) and LP-2 (Mr 350,000) were identified and purified from pig brain. Examination of molecular structures indicated that LP-1 has three subunits with molecular weights of 69,000, 55,000, and 34,000, respectively, whereas LP-2 contains only one subunit, with molecular weight of 49,000. When using phosphorylase a as a substrate, LP-1 was completely inactive and could be dramatically activated by freezing and thawing in 0.2 M 2-mercaptoethanol, whereas LP-2 contained some basal activity but could also be stimulated 40-fold by the same treatment. Kinetic analysis further indicated that both LP-1 and LP-2 enzymes dephosphorylate histone 2A, myelin basic protein, and phosphorylase a at a rather comparable rate, but the dephosphorylation of histone 2A and myelin basic protein seems to be spontaneously active. This, together with the results that trypsinolysis could specifically knock off phosphorylase phosphatase activity but caused no effect on the associated myelin basic protein/histone phosphatase activities, supports the notion that a two-site mechanism may possibly be involved in the regulation of substrate specificity of LP-1 and LP-2 enzymes in the central nervous system.
Paediatric Casualty has evolved into a blend of true Accident and Emergency and Primary Care Services. With the impending redevelopment of the Accident and Emergency Department at the Camperdown Children's Hospital a survey of families utilising the service was conducted to assess their perceptions of the services provided. It emerged that although many families travelled long distances to the Hospital and had to contend with long waits, they perceived the trip as worthwhile with most finding the quality of care satisfactory (95%) and friendly (90%), coming away with a good understanding of their child's problem (94%), the investigations performed and management prescribed (97%), and arrangements made for follow up (89%). A large percentage (51%) came to the hospital seeking a second opinion for which they had already consulted another agency. Nineteen percent were directly referred, with the remainder seeking a second opinion on their own initiative. This high referral rate necessitates a staffing formula that provides appropriate expertise and supervision. As the demand on hospitals to provide primary care facilities is likely to remain, recommendations are made for improvement in the services provided as well as making more use in forward planning of available expertise and supervision.
Four cases of tyrosinaemia type II (Richner-Hanhart syndrome) are reported. This syndrome consists of corneal erosions, palmar and plantar hyperkeratoses, and sometimes mental retardation. Presentation with photophobia and dendritic corneal ulceration or circumscribed palmoplantar keratoderma should alert the physician to the possible diagnosis of tyrosinaemia II. Early diagnosis is important, as the clinical picture can be modified by dietary restriction.
Generalized arterial calcification in infancy is a rare disorder in which death usually occurs in infancy, the diagnosis generally being made at autopsy. Three patients are reported. The diagnosis was made during life in two, enabling new information to be collected. Cardiac catheterization in one provided evidence suggesting stiffness of pulmonary and systemic arterial walls. Another is a long-term survivor with spontaneous regression of calcification. The third case, diagnosed at necropsy, was associated with endocardial fibroelastosis.
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