In vivo acylation of proteolipid protein and DM-20 in myelin and myelin subfractions of developing rat brain: Immunoblot identification of acylated PLP and DM-20

The acylation of proteolipid protein (PLP) was examined in myelin and myelin subfractions from rat brain during the active period of myelination. Proteolipid protein and DM-20 in myelin and myelin subfractions were readily acylated in developing rat brain 22 hours after intracerebral injection of [3H]palmitic acid. No differences in the relative specific activity of PLP in myelin from 9-, 15-, and 30-day-old rat brains was observed; however, the relative specific activity of PLP in the heavy myelin subfraction tended to be higher than that in the light myelin subfraction. The acylation of PLP was confirmed by fluorography of immuno-stained cellulose nitrate sheets, clearly establishing that the acylated protein is in fact the oligodendroglial cell- and myelin-specific protein, PLP. Since PLP is acylated in the 9-day-old animal, when little compact myelin is present, it is possible that the acylation of PLP is a prerequisite for the incorporation of this protein into the myelin membrane.     

Purification and immunohistochemical localization of rat brain myelin proteolipid protein.

Abstract— A homogeneous preparation of proteolipid protein (PLP) from rat brain myelin was isolated by preparative gel electrophoresis in sodium dodecyl sulfate and chemically characterized. The results of amino acid and N-terminal amino acid analyses are reported. The same preparation of myelin PLP was used to produce specific precipitating antibodies. Rabbit and goat antisera to myelin PLP each gave a single precipitin line with purified PLP dissolved in Triton X-100. Under identical conditions, no precipitation was observed with antiserum to myelin basic protein or with control serum. Immunofluorescence localization employing antiserum to PLP demonstrated bright specific fluorescence restricted to the myelin sheaths of axons in all anatomical areas of the rat brain examined. Neuronal cell bodies and their dendrites were completely negative with respect to the presence of proteolipid protein. PLP could not be localized in the cell bodies or fibrous processes in any of the glial elements in the adult rat brain. However, myelin PLP was clearly visible in the cytoplasm and processes of actively myelinating oligodendrocytes in the corpus callosum in the brains of 10-day-old rats.     

Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein

The 3200 and 1600 nucleotide mRNAs encoding rat brain proteolipid protein (PLP), the major protein component of central nervous system myelin, are heterogeneous at their 5' ends, differ in their 3' polyadenylation sites, and are transcribed from a single gene. The mRNAs, which first appear postnatally, encode identical 277 amino acid proteins that are 99% identical to the bovine protein sequence. Thus, PLP has been highly conserved during mammalian evolution. A single amino-terminal methionine is removed post-translationally, indicating that PLP does not require a signal peptide sequence for insertion into the myelin membrane. Mouse and monkey utilize the 3200 but not the 1600 nucleotide mRNA, suggesting that there is no functional necessity for two sizes of rat PLP mRNAs.     

Rapid metabolism of fatty acids covalently bound to myelin proteolipid protein

Proteolipid protein (PLP), the major protein of central nervous system myelin, contains approximately 2 mol of covalently bound fatty acids. In this study, the in vivo turnover rate of the acyl chains bound to PLP was determined in 40-day-old rats after a single intracranial injection of [3H]palmitic acid. The apparent half-life of total fatty acids bound to PLP was approximately 7 days. After correction for acyl chain interconversion, the half-life of palmitate bound to PLP was only 3 days. This turnover rate is much more rapid than that of the protein moiety calculated under the same experimental conditions (t1/2 = 1 month). Additional evidence for the dynamic metabolism of acyl groups was provided by experiments in brain tissue slices which showed that acylation of PLP occurs in adult animals as well as during active myelination. Acylation of endogenous PLP in purified myelin and its subfractions was also studied during rat brain development using either [3H]palmitoyl-CoA or [3H]palmitic acid plus ATP and CoA. Labeling of endogenous PLP with [3H]palmitoyl-CoA was observed as early as 10 days postnatal and continued at the same rate throughout development. When [3H]palmitic acid was used as precursor in the presence of both ATP and CoA, esterification of myelin PLP occurred rapidly in adult animals, indicating that both nonacylated PLP and acyl-CoA ligase are present in myelin. Finally, pulse-chase experiments in a cell-free system showed that PLP-bound fatty acids turn over with a half-life shorter than 10 min. These observations are consistent with the concept that acylation of myelin PLP is a dynamic process involved mainly in myelin maintenance and function.     

In vitro synthesis of myelin proteolipid protein from rat brain using a total homogenate system

In vitro synthesis of myelin proteolipid protein (PLP) was explored at different ages using rat brain total homogenates, incubated for 30 min with [³H]glycine. Total proteolipids, extracted from the incubated samples, were separated by SDSPAGE and the radioactivity was measured in the band corresponding to myelin PLP. The incorporation into PLP in relation to the incorporation into brain total proteins increased from 0.04% at 10 days of age to 0.63% at 20 days, and declined slowly thereafter. Time course experiments were carried out using brain homogenates obtained from rats of 20 days of age (i.e. at the period of maximal synthesis of PLP). Labeled PLP molecules were already found at 2.5 min of incubation and the incorporation of the label into this protein, relative to the incorporation into total proteins, did not vary throughout the entire incubation time (30 min). Pulsechase experiments using a similar system and adding cycloheximide at different incubation times showed that the appearance of label into mature PLP was immediately blocked by the inhibitor of protein synthesis. These data suggest that PLP is synthesized as such and not as a pre-protein which is subsequently processed to render mature PLP.     

Effects of monensin on posttranslational processing of myelin proteins

Rat brain slices were incubated with [3H]palmitic acid and [14C]glycine to label the lipid and protein moieties, respectively, of myelin proteolipid protein (PLP). The effects of monensin on posttranslational processing of proteins were examined by measuring the appearance of [14C]glycine- and [3H]palmitate-labeled proteins in myelin and myelin-like fractions. At 0.01 and 0.10 microM, monensin did not appreciably affect total lipid or protein synthesis; higher concentrations caused increased inhibition. Monensin at 0.10 microM markedly decreased the appearance of [14C]glycine-labeled PLP in myelin, but had little effect on the 14C basic proteins or the incorporation of [3H]palmitic acid into total or myelin PLP. The same relative effect was apparent at higher monensin concentrations. In the myelin-like fraction, monensin at 0.10 microM also depressed entry of [14C]glycine into protein comigrating with PLP, and again had no effect on incorporation of [3H]palmitic acid. In addition, monensin increased the [3H]palmitate label associated with two high-molecular-weight proteins in the myelin-like fraction with no concomitant increase in [14C]glycine label.     

Cell-free acylation of rat brain myelin proteolipid protein and DM-20.

Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective labelling of 'PLP' [proteolipid protein; Folch & Lees (1951) J. Biol. Chem. 191, 807-817] and 'DM-20' [Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J. Neurochem. 19, 2083-2089] which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography. These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin. Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively. Incubation of myelin with [3H]palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively. The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids. The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of [3H]palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with [3H]palmitic acid and [14C]palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin. We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin. Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.     

Acylation of Rat Brain Myelin Proteolipid Protein with Different Fatty Acids

The acylation of rat brain proteolipid protein (PLP) with tritiated palmitic, oleic, and myristic acids was studied in vivo and in vitro and compared with the acylation of lipids. Twenty-four hours after intracranial injection of [3H]myristic acid, only 16% of the PLP-bound label appeared as myristic acid, with 66% as palmitic, 9% as stearic, and 6% as oleic acid, whereas greater than 63% of the label in total or myelin phospholipid was in the form of myristic acid. In contrast, after labelling with [3H]palmitic or oleic acids, 75% and 86%, respectively, of the radioactivity in PLP remained in the original form. When brain tissue slices were incubated for short periods of time, the incorporation of palmitic and oleic acids into PLP exceeded that of myristic acid by a factor of 8. In both systems and with all precursors studied, the label associated with PLP was shown to be in ester linkage. The results suggest a preferential acylation of PLP with palmitic and oleic acids as compared with myristic acid. This is consistent with the fatty acid composition of the isolated PLP.     

Characterization of myelin proteolipid mRNAs in normal and jimpy mice.

A clone specific for the rat myelin proteolipid protein (PLP) was isolated from a cDNA library made in pUC18 from 17-day-old rat brain stem mRNA. This clone corresponded to the carboxyl-terminal third of the PLP-coding region. The clone was used to identify PLP-specific mRNAs in mouse brain and to establish the time course of PLP mRNA expression during mouse brain development. Three PLP-specific mRNAs were seen, approximately 1,500, 2,400, and 3,200 bases in length, of which the largest was the most abundant. During brain development, the maximal period of PLP mRNA expression was from 14 to 25 days of age, and this was a similar time course to that for myelin basic protein mRNA expression. When the jimpy mouse, an X-linked dysmyelination mutant, was studied for PLP mRNA expression, low levels of PLP mRNA were seen which were approximately 5% of wild-type levels at 20 days of age. When jimpy brain RNA was analyzed by Northern blotting, the PLP-specific mRNA was shown to be 100 to 200 bases shorter than the wild-type PLP-specific mRNA. This size difference was seen in the two major PLP mRNAs, and it did not result from a loss of polyadenylation of these mRNAs.     

Experimental allergic encephalomyelitis mediated by murine encephalitogenic T cell lines specific for myelin proteolipid apoprotein

T cell lines specific for bovine myelin proteolipid apoprotein (PLP) were established from SJL/J mice. The line cells bore surface phenotypes of T helper/inducer cells (Lyt-1+, Lyt-2-, L3T4+) and responded well to bovine, rat, and guinea pig PLP but not to myelin basic protein. One line responded to major PLP, and another responded to both major PLP and DM-20, which are the two major intrinsic membrane proteins of the central nervous system (CNS) myelin. Intraperitoneal inoculation of 4 to 30 X 10(6) PLP-activated line cells followed by injection of pertussis vaccine induced acute inflammatory disease of the CNS, with typical clinical signs of EAE mostly in a week in recipient mice that had been treated with low-dose irradiation. Almost all animals recovered completely, and two of the 12 animals relapsed 42 or 75 days after inoculation. The lesions were restricted to the CNS and were characterized by perivascular and parenchymal infiltration of inflammatory cells, fibrin deposit, and demyelination. In the severe lesions, axons were also damaged. These observations suggest that PLP is a definite encephalitogen, and PLP-sensitized effector T cells induce inflammatory demyelination in the CNS.     

Characterization of proteolipid protein fatty acylesterase from rat brain myelin.

A protein fatty acylesterase activity that catalyzes the removal of fatty acid from exogenous proteolipid protein (PLP) has been demonstrated in isolated rat brain myelin. Optimum enzyme activity for the deacylation of PLP was obtained in 0.5% Triton X-100, 1 mM dithiothreitol at pH 7.0 and at 37 degrees C. Other detergents (octyl beta-D-glucoside, Nonidet P-40, and Tween 20) have little or no effect, whereas deacylation was completely abolished by 0.1% sodium dodecyl sulfate or boiling the membrane fraction for 5 min prior to incubation. Under optimal conditions, the rate of deacylation was linear up to 20 min, and the apparent Km for bovine [3H]palmitoyl-PLP was 18 microM. The myelin-associated PLP fatty acylesterase has no apparent requirements for divalent cations (Ca2+, Mg2+, Mn2+), and chelators such as EDTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, and 1,10-phenantroline have little or no effect on enzyme activity. Sulfhydryl and histidine residues are needed for full enzyme activity, whereas the "active serine"-directed inhibitor phenylmethylsulfonyl fluoride has no effect. The myelin-associated protein fatty acylesterase was present throughout brain development and in all myelin subfractions, in agreement with the dynamic metabolism of PLP-bound fatty acids. Enzyme activity was also present in sciatic nerve, brain cortex, and heart whereas liver was devoid of activity. Several esterases, including phospholipase A2, glyoxalase II, and acetylcholinesterase, did not remove fatty acid from PLP. Myelin basic protein, palmitoyl-CoA hydrolase, and myelin-associated nonspecific esterase were also ruled out as the PLP fatty acylesterase. Thus, all data seem to indicate that this enzyme is different from esterases of the lipid metabolism. Finally, stimulation of protein phosphorylation with Ca2+, but not with cyclic-AMP, inhibited PLP deacylation, suggesting that the myelin-associated protein fatty acylesterase activity is regulated by endogenous Ca(2+)-dependent protein kinases.     

Developmental expression of the myelin gene MOBP in the rat nervous system

The myelin-associated/oligodendrocyte basic proteins (MOBPs) are recently discovered constituents of myelin and are small, cytoplasmic, and highly basic proteins exclusively expressed postnatally by oligodendrocytes. Due to a clustering of positively charged amino acids observed in the most abundant MOBP isoform similar to myelin basic protein (MBP) and P0, it was speculated that MOBP could function in myelin sheath compaction. The present report strongly supports this view. A direct comparison of MBP and proteolipid protein (PLP) gene expression with that of MOBP by in situ hybridization revealed a very similar regional distribution. It was found that MOBP expression was abundant in the rat CNS at postnatal day 15 (P 15) but is restricted to densely myelinated regions. In contrast to MBP and PLP, expression of MOBP was undetectable in the peripheral nervous system during the entire development. Interestingly, MOBP mRNA was localized in oligodendrocyte processes even at early postnatal stages and throughout development. MOBP showed a very specific timing of expression: in spinal cord and brain, MOBP gene expression occurred significantly later (2–3 days) than that of MBP and PLP, but slightly earlier than myelin oligodendrocyte glycoprotein gene expression. MOBP proteins appeared in spinal cord and brain stem also after MBP protein, suggesting that the MOBPs functionally act after the structural myelin proteins MBP and PLP. Our findings imply a function of MOBP during the late steps of myelin formation, presumably at the initiation of sheath compaction.     

Study of the expression of myelin proteolipid protein (lipophilin) using a cloned complementary DNA.

We have prepared a lambda gt10 cDNA library with the mRNA isolated from fetal calf brains which were actively myelinating. Using two oligonucleotides made according to the known amino acid sequence of myelin proteolipid protein (PLP or lipophilin), we have isolated several cDNA clones for this major intrinsic membrane protein of myelin. One of these clones, designated as pLP1, is found to contain 444 bp of coding sequence for the C-terminal half of PLP and 486 bp of 3' untranslated sequence. Using pLP1 as a hybridization probe, we have studied the regulation of PLP at the mRNA level during rat brain development. Our results show that the relative amounts of mRNA for PLP and that for the major extrinsic protein of the myelin membrane, myelin basic protein, increase coordinately during the course of myelination in the brain.     

Major Central Nervous System Myelin Glycoprotein of the African Lungfish (Protopterus dolloi) Cross-Reacts with Myelin Proteolipid Protein Antibodies, Indicating a Close Phylogenetic Relationship with Amphibians

CNS myelin was isolated from the spinal cord of the African lungfish Protopterus dolloi. Its proteins consisted of (1) two basic proteins (16,000 and 18,500 apparent Mr) that reacted with anti-human CNS myelin basic protein antibodies and (2) a major protein (29,000 apparent Mr) that stained with concanavalin A-horseradish peroxidase and bound to anti-rat CNS myelin proteolipid protein (PLP) antibodies. This dominant 29,000 Mr protein showed no reaction with antibodies against the major bovine PNS myelin glycoprotein P0. Following treatment with endoglycosidase F the 29,000 Mr protein was reduced in size to a 26,000 apparent Mr component that no longer bound concanavalin A but retained the anti-PLP reactivity. These results agree with a concanavalin A-binding oligosaccharide linked through asparagine to a protein backbone of PLP homology. The major 29,000 Mr lungfish CNS myelin protein was therefore termed g-PLP (glycosylated proteolipid protein). This is the first report demonstrating the occurrence of a PLP-cross-reactive protein in CNS myelin of a fish. It attests to the close phylogenetic relationship of lungfishes to amphibians. Amphibians were previously recognized as the oldest class bearing PLP in its CNS myelin.     

Conserved Fatty Acid Composition of Proteolipid Protein During Brain Development and in Myelin Subfractions

Myelin proteolipid protein (PLP) is modified after translation by the attachment of long-chain fatty acids to several cysteine residues. In this study, the amount and pattern of fatty acids covalently bound to rat PLP were determined during brain development and in myelin subfractions. For this purpose, PLP was isolated by gel-filtration chromatography in organic solvents, subjected to alkaline methanolysis, and the released fatty acid methyl esters were analyzed by gas-liquid chromatography. At all ages examined, PLP had the same amount of covalently-bound fatty acids (3–4% w/w) and palmitate, oleate and stearate were always the major acyl chains. In contrast to myelin lipids, the fatty acid composition of PLP showed only minor changes between 15-days and 90-days of age. The amount and pattern of fatty acids bound to PLP prepared from three myelin subfractions were also indistinguishable. The conservation of a characteristic PLP-fatty acid make-up during brain development and in various myelin compartments suggests that this post-translational modification is essential for the normal functioning of the protein.     

Studies on the Wolfgram High Molecular Weight CNS Myelin Proteins: Relationship to 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase

Evidence is presented that the major protein components of the high molecular weight CNS myelin proteins designated as the Wolfgram protein doublet (W1 and W2) contain the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP). CNP is a basic hydrophobic protein containing about 830 to 840 amino acid residues. When electrophoresed on SDS polyacrylamide gels, CNP appears as a protein doublet, separated by a molecular weight difference of about 2500-3000 in bovine, human, rat, guinea pig, and rabbit. A similar protein doublet has been identified as the Wolfgram proteins W2 and W1 in myelin and in the chloroform-methanol-insoluble pellet obtained from myelin. Moreover, the relative Coomassie blue staining intensity of the CNP2 plus CNP1 protein doublet among the species examined was remarkably similar to that observed for electrophoresed myelin and chloroform-methanol-insoluble pellet derived from myelin. Antisera raised against purified bovine CNP recognized the W1 and W2 proteins isolated from bovine and human brain. The amino acid composition of pure bovine CNP is presented and compared with the compositions of several rat and bovine Wolfgram proteins obtained by other investigators. Our electrophoretic, compositional, and immunological data support the contention that the enzyme CNP is a major component of the Wolfgram protein doublet.     

Calcium-Stimulated Proteolysis in Myelin: Evidence for a Ca2+-Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS

Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.     

Fatty Acid Acylation of Rat Brain Myelin Proteolipid Protein In Vitro: Identification of the Lipid Donor

The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.     

Emergence of three myelin proteins in oligodendrocytes cultured without neurons

Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat brain and optic nerve to allow us to analyze whether two transmembranous myelin proteins, myelin-associated glycoprotein (MAG) and proteolipid protein (PLP), were expressed together with myelin basic protein (MBP) in defined medium with low serum and in the absence of neurons. Using double label immunofluorescence, we investigated when and where these three myelin proteins appeared in cells expressing galactocerebroside (GC), a specific marker for the oligodendrocyte membrane. We found that a proportion of oligodendrocytes derived from brain and optic nerve invariably express MBP, MAG, and PLP about a week after the emergence of GC, which occurs around birth. In brain-derived oligodendrocytes, MBP and MAG first emerge between the fifth and the seventh day after birth, followed by PLP 1 to 2 d later. All three proteins were confined to the cell body at that time, although an extensive network of GC positive processes had already developed. Each protein shows a specific cytoplasmic localization: diffuse for MBP, mostly perinuclear for MAG, and particulate for PLP. Interestingly, MAG, which may be involved in glial-axon interactions, is the first myelin protein detected in the processes at approximately 10 d after birth. MBP and PLP are only seen in these locations after 15 d. All GC-positive cells express the three myelin proteins by day 19. Simultaneously, numerous membrane and myelin whorls accumulate along the oligodendrocyte surface. The sequential emergence, cytoplasmic location, and peak of expression of these three myelin proteins in vitro follow a pattern similar to that described in vivo and, therefore, are independent of continuous neuronal influences. Such cultures provide a convenient system to study factors regulating expression of myelin proteins.     

Regional studies of myelin proteins in human brain and spinal cord

The myelin specific proteins, myelin basic protein (MBP) and myelin proteolipid protein (PLP) were quantitated by radioimmunoassay (RIA) and the activity of the enzyme 23-cyclic 3 phosphohydrolase (CNP) measured, in 27 regions of normal brain and spinal cord. Varying regional concentrations for each protein and regional variations for protein ratios were noted, supporting the concept of a varying chemical composition for myelin throughout the central nervous system (CNS). Variation was also noted among myelin subfractions from a single region. Regions with special sensitivity to the multiple sclerosis process had relatively lower proportions of CNP in several, but not all cases.