A BstUI PCR/RFLP marker in an intron of the canine fibrillin 1 gene.
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Animals↗
Biomedical subjects
M L Katz
Publications and source records attributed to M L Katz.
Length polymorphism in a CT-rich microsatellite in an intron of the canine tyrosinase-related protein-2 gene.
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Animals↗
A BseRI PCR/RFLP in an intron of the canine phenol sulfotransferase gene.
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Alleles↗
Modulation of nerve growth factor internalization by direct interaction between p75 and TrkA receptors.
While the central role played by TrkA in nerve growth factor (NGF) signalling has been established by dissecting its signal transduction pathways, insight into the mechanism of action of p75LNR, the low-affinity neurotrophin receptor, has only recently been achieved. The relative contribution of p75LNR and TrkA to the constitution of high-affinity receptors for NGF and, similarly, with TrkB an TrkC to the formation of those for other neurotrophins, is presently under debate. Some form of collaboration in mediating neurotrophin activities has been observed between the Trk and p75LNR receptors, but only recent indirect evidence indicates a molecular interaction. In the present work, we have ectopically coexpressed p75LNR and TrkA in Sf9 insect cells by using baculovirus vectors, and show a direct association between the two NGF receptors. In addition, we show that the intracellular and extracellular domains of both receptors contribute to this interaction. Finally, we demonstrate that NGF becomes endocytosed in TrkA-expressing cells but not in p75LNR-expressing cells, and that such function can be modulated by the presence of the intracellular domain of p75LNR receptor.
Animals↗
Late-infantile ceroid-lipofuscinosis: lysine methylation of mitochondrial ATP synthase subunit c from lysosomal storage bodies.
Late-infantile ceroid-lipofuscinosis is a fatal autosomal recessively inherited disease characterized by massive accumulations of lysosomal storage bodies in many tissues. A major constituent of the storage bodies is the subunit c protein of mitochondrial ATP synthase. Juvenile ceroid-lipofuscinosis, a disease that is similar to but genetically distinct from the late-infantile disorder, also involves lysosomal accumulation of the subunit c protein. In the juvenile disease, the stored form of the protein contains an epsilon-N-trimethyllysine (TML) residue at position 43. Analyses were performed to determine whether subunit c protein stored in the late-infantile disease is also trimethylated at lysine residue 43. Amino acid composition analysis of the subunit c protein stored in brains from subjects with the late-infantile disease indicated that one of the two lysine residues in the protein is trimethylated. Data from molecular mass analysis of the protein was consistent with the presence of three methyl groups not present in the unmodified protein. The TML in the storage body subunit c protein was found by amino acid sequence analysis to occur exclusively at residue 43. The lysine at this position in the stored protein was completely methylated. Recent studies suggest that the subunit c protein from normal mitochondria may also have the same amino acid modification. Thus, it appears that specific methylation of lysine residue 43 of mitochondrial ATP synthase subunit c is probably a normal post-translational modification, and that the lysosomal storage of this protein in late-infantile, as well as in juvenile ceroid-lipofuscinosis, does not result from a defect in its methylation.
Cerebral Cortex↗
Immunochemical localization of the Batten disease (CLN3) protein in retina.
PURPOSE: Batten disease, also known as juvenile ceroid-lipofuscinosis and CLN3, is an autosomal recessively inherited disorder that results in blindness due to retinal degeneration. The CLN3 gene has been identified, but the function of the protein that this gene encodes is unknown. Experiments were conducted to determine where the CLN3 protein is localized in the mouse retina. Localization should provide a clue in evaluating potential functions of this protein. METHODS: Using oligonucleotide primers based on the reported human CLN3 cDNA sequence, the mouse cDNA nucleotide sequence was determined from products of the reverse transcriptase-polymerase chain reaction and 3' rapid amplification of cDNA ends. A synthetic 20-amino-acid peptide corresponding to an internal hydrophilic region of the predicted amino acid sequence of the mouse CLN3 protein was used to immunize rabbits. The resulting antiserum was used in immunoblot analysis of mouse retina homogenates and in electron microscopic immunocytochemical labeling of mouse retina sections. RESULTS: The peptide antibody labeled a single protein band of approximately 50 kDa on immunoblots of mouse retina homogenates. No labeling was detected with homogenates from human retinas. The antibody specifically labeled mitochondria of Müller cells and inner retinal neurons. Little labeling was observed in mitochondria of the photoreceptor cells. Mitochondria of other cell types, including the retinal pigment epithelium and choroidal cells, were not labeled. CONCLUSIONS: The retinal CLN3 protein appears to be localized almost exclusively in the mitochondria, but was detected only in certain cell types. Batten disease is characterized by massive lysosomal accumulations of a small inner mitochondrial membrane protein (subunit c of ATP synthase). The mitochondrial localization of the CLN3 protein suggests that it may play a role in the normal processing of subunit c.
Amino Acid Sequence↗
Formation of lipofuscin-like fluorophores by reaction of retinal with photoreceptor outer segments and liposomes.
During the aging process the retinal pigment epithelium (RPE) accumulates autofluorescent lysosomal storage bodies (lipofuscin). Data from previous studies led to the hypothesis that at least one of the fluorescent components of RPE lipofuscin is formed by reaction of vitamin A aldehyde with phosphatidylethanolamine (PE) in the photoreceptor outer segments. Experiments were performed to test this hypothesis. All-trans retinaldehyde was incubated with isolated bovine photoreceptor outer segments and with synthetic liposomes. Liposomes were made with two different lipid compositions. One type of liposome consisted of a mixture of lipids, including phosphatidylcholine (PC), none of which contained a primary amine. The other liposome type was identical in composition accept that some of the PC was replaced with an equimolar amount of phosphatidylethanolamine (PE). After incubation of the samples, aliquots were examined with fluorescence microscopy to assess whether any lipofuscin-like fluorescence had developed. Lipids were extracted from additional aliquots of the samples and analyzed with thin layer chromatography. Photoreceptor outer segments incubated with retinaldehyde developed an intense golden yellow fluorescent emission when illuminated with 395-440 nm light. Similar fluorescence developed in the liposomes containing PE, whereas the liposomes lacking PE or any other primary amine did not develop any detectable fluorescence. The development of fluorescence in the samples in situ correlated with the appearance of an orange colored component in the lipid extracts that displayed a weak red emission upon ultraviolet light illumination. Incorporation of this component into liposomes resulted in the appearance of a golden yellow fluorescent emission. The results of these experiments suggest that retinal, generated during visual pigment bleaching can react with PE in the photoreceptor outer segments to form a fluorophore, a derivative of which subsequently accumulates in RPE lipofuscin. An RPE lipofuscin fluorophore was previously shown to be identical to a reaction product of retinal and ethanolamine. This fluorophore is probably derived from the reaction product of outer segment PE and retinal.
Animals↗
Decreased plasma carnitine and trimethyl-L-lysine levels associated with lysosomal accumulation of a trimethyl-L-lysine containing protein in Batten disease.
Batten disease, or juvenile neuronal ceroid-lipofuscinosis, is an autosomal-recessive hereditary disorder that leads to blindness, severe neurological degeneration, and premature death. The disease is characterized by massive accumulation of lysosomal storage bodies in most tissues. A significant constituent of the storage material is a protein that appears to be almost identical to a small hydrophobic inner mitochondrial membrane protein, subunit c of ATP synthase. The protein isolated from the storage bodies contains an epsilon-N-trimethyl-L-lysine (TML) residue at amino acid position 43. The presence of TML in the stored protein suggests that one of the lysine residues in subunit c is normally trimethylated, and this trimethylation may act as a signal to initiate degradation of the protein. Free TML produced by the degradation of TML-containing proteins is the first intermediate in the carnitine biosynthetic pathway. It is possible that trimethylated subunit c is a major source of the free TML used in carnitine biosynthesis. If this is the case, one would predict that the genetic defect resulting in the accumulation of TML containing subunit c would also reduce systemic levels of free TML and carnitine. To evaluate this possibility, plasma TML and carnitine levels were measured in affected human subjects, heterozygous carriers, and normal controls. Both TML and carnitine levels were significantly depressed in the affected individuals. This suggests that subunit c is normally a major source of TML for carnitine biosynthesis. In Batten disease, failure to degrade the TML-containing form of subunit c is probably responsible for the reduction in plasma TML and carnitine levels.
Adult↗
Glycation mediated crosslinking between alpha-crystallin and MP26 in intact lens membranes.
With advancing age, progressive crosslinking occurs between lens crystallin proteins and other lenticular components. This crosslinking may be involved in the development of senile cataracts. Experiments were conducted to determine whether non-enzymatic glycation could be involved in the crosslinking between lens alpha-crystallin and MP26, an abundant lens fiber cell membrane intrinsic protein. In vitro crosslinking of alpha-crystallin and MP26 of bovine lens membranes was observed in presence of two degradation products of ascorbic acid (ASA), dehydroascorbic acid (DHA) and threose. Alkali-washed bovine lens membranes, isolated after glycation with DHA and threose, contained both alpha-crystallin and MP26, as determined by immunoblot and double immunocytochemical labeling studies. In contrast, membranes incubated without these glycating compounds contained only MP26. SDS-PAGE analysis of [125I] alpha-crystallin incubated with lens membranes in the presence of threose showed a higher amount of radioactivity in high molecular weight aggregates than in the aggregates produced when alpha-crystallin and threose were incubated without membranes. A slot-blot immunoassay of alkali-washed human lens membranes showed a higher amount of covalently bound alpha-crystallin in aged, cataractous or diabetic lens membranes than was present in lens membranes from young normal donors. Based on the in vitro results, we hypothesize that non-enzymatic glycation is one of the vivo mechanisms in the crosslinking of alpha-crystallin to lens membrane proteins, such as MP26. This crosslinking may contribute significantly to the development of age-related and diabetic cataracts.
Aging↗
Thrombolytic therapy for acute deep venous thrombosis: how much is enough?
Twenty-eight patients treated with thrombolytic therapy for acute deep venous thrombosis were monitored prospectively with non-invasive testing every 12-24h during treatment to evaluate thrombus response and whether duration of therapy was appropriate. Some 75% (21 of 28) of patients demonstrated improvement with lytic therapy with 36% (10 of 28) demonstrating complete lysis; 95% of responders (20 of 21) initiated lysis within 24h. Some 33% (7 of 21) of all responders and 64% (7 of 11) of those having partial lysis had treatment terminated during thrombus resolution but before maximal lysis. Non-invasive testing indicated that thrombolytic therapy for acute deep venous thrombosis is frequently terminated before maximal lysis of the thrombus. Monitoring thrombus response with venous duplex imaging should be part of the treatment strategy of deep venous thrombosis if thrombolytic therapy is used. This approach should increase efficacy and potentially reduce complications of thrombolytic therapy for acute deep venous thrombosis.
Acute Disease↗
Venous outflow of the leg: anatomy and physiologic mechanism of the plantar venous plexus.
PURPOSE: Mechanisms of venous outflow from the leg and foot have not been clearly defined. The purpose of this study was to evaluate the anatomy and physiologic mechanism of the plantar venous plexus and its impact on venous drainage from the tibial veins. METHODS: Fifty phlebograms that contained complete foot and calf films were reviewed. On lateral films, the number of veins in the plantar venous plexus and its tibial outflow tract were counted. The length and diameter of the longest vein in the plantar venous system and the length of the foot arch were measured. The ratio of the length of the plantar venous plexus to the arch length was calculated. The presence or absence of valves within the plexus was recorded. Plantar venous plexus outflow was evaluated by an duplex ultrasonographic scan of the posterior tibial, anterior tibial, and peroneal veins during intermittent external pneumatic compression of the plantar surface of the foot. RESULTS: The plantar venous plexus was composed of one to four large veins (mean, 2.7 veins) within the plantar aspect of the foot. The diameter of these veins was 4.0 +/- 1.2 mm. The veins coursed diagonally from a lateral position in the forefoot to a medial position at the level of the ankle, spanning 75% of the foot arch. Prominent valves were recognized within the plantar veins in 22 of 50 patients. The plexus coalesced into an outflow tract of one to four veins (mean, 2.5 veins) that flowed exclusively into the posterior tibial venous system. Small accessory veins that drained the plantar surface of the forefoot flowed into either the posterior tibial or peroneal veins. This pattern of selective drainage of the plantar venous plexus was confirmed by duplex imaging. Mechanical compression of the plantar venous plexus produced a mean peak velocity in the posterior tibial veins of 123 +/- 71 cm/sec, in the anterior tibial veins of 24 +/- 14 cm/sec, and in the peroneal veins of 29 +/- 26 cm/sec. CONCLUSIONS: The plantar venous plexus is composed of multiple large-diameter veins that span the arch of the foot. Compression of the plantar venous plexus, such as that which occurs during ambulation, is capable of significantly increasing flow through the posterior tibial venous system into the popliteal vein. Its function may be integral to venous outflow from the calf and priming of the more proximal calf muscle pump.
Child↗
Vitamin A incorporation into lipofuscin-like inclusions in the retinal pigment epithelium.
Intravitreal injection of the protease inhibitor leupeptin causes a rapid accumulation of lipofuscin-like autofluorescent inclusions in the retinal pigment epithelium (RPE) of the eye. In vitamin A-deprived animals, similar inclusions form in response to leupeptin treatment, but they do not become autofluorescent. Because vitamin A is necessary to the development of fluorescence, it appears likely that retinoids are directly incorporated into the inclusions. Experiments were conducted to determine whether this is the case. Rats were reared on a diet containing retinoic acid as the only retinoid. Retinoic acid cannot be utilized in visual transduction by the retina. When the eyes had been over 90% depleted of visual cycle retinoids, the animals were given a single intramuscular injection of 3H-all-trans retinol. After 7 days, when visual cycle retinoids had returned to an average of almost 70% of normal, the animals were given an intravitreal injection of leupeptin in each eye. At either 1 day or 7 days after the leupeptin treatment, some of the animals were dark-adapted for at least 12 h. The eyes were enucleated and fixed under dim red light. A region of each retina just superior to the optic nerve head was examined with electron microscopic autoradiography. At both one day and 7 days after the leupeptin treatment, the radiolabel in the RPE was primarily associated with the leupeptin induced inclusion bodies. Label was also present in the photoreceptor outer segments. The localization of vitamin A to the leupeptin-induced inclusions in the RPE strongly suggests that vitamin A is covalently bound to outer segment proteins that have been phagocytosed by the RPE but remain undegraded due to protease inhibition. This bound vitamin A is probably responsible for the autofluorescence of the leupeptin-induced inclusions. Vitamin A is not likely to be bound through a Schiff base linkage, since retinal-Schiff base compounds do not exhibit lipofuscin-like fluorescence.
Animals↗
Mitochondrial ATP synthase subunit c stored in hereditary ceroid-lipofuscinosis contains trimethyl-lysine.
The subunit c protein of mitochondrial ATP synthase accumulates in lysosomal storage bodies of numerous tissues in human subjects with certain forms of ceroid-lipofuscinosis, a degenerative hereditary disease. Subunit c appears to constitute a major fraction of the total storage-body protein. Lysosomal accumulation of subunit c has also been reported in putative animal models (dogs, sheep and mice) for ceroid-lipofuscinosis. In humans with the juvenile form of the disease, hydrolysates of total storage-body protein have been found to contain significant amounts of epsilon-N-trimethyl-lysine (TML). TML is also abundant in storage-body protein hydrolysates from affected dogs and sheep. These findings suggested that one or both of the two lysine residues of subunit c might be methylated in the stored form of the protein. The normal subunit c protein from mitochondria does not appear to be methylated. In a putative canine model for human juvenile ceroid-lipofuscinosis, residue 43 of the storage-body subunit c was previously found to be TML. In the present study, subunit c was isolated from the storage bodies of humans with juvenile ceroid-lipofuscinosis, and from sheep and mice with apparently analogous diseases. In all three species, partial amino acid sequence analysis of the stored subunit c indicated that the protein contained TML at residue 43. These findings strongly suggest that specific methylation of lysine residue 43 of mitochondrial ATP synthase plays a central role in the lysosomal storage of this protein.
Adolescent↗
Canine hereditary ceroid-lipofuscinosis: evidence for a defect in the carnitine biosynthetic pathway.
The ceroid-lipofuscinoses are a group of autosomal-recessive hereditary lysosomal storage diseases that have been characterized in humans and other mammalian species. In a canine model for the juvenile form of the human disease, a major constituent of the storage bodies is the subunit c protein of mitochondrial ATP synthase that contains an epsilon-N-trimethyllysine (TML) residue. TML is a precursor in carnitine biosynthesis. To determine whether accumulation of the TML-containing protein could result from a defect in the carnitine biosynthetic pathway, plasma carnitine and trimethyllysine levels were measured in homozygous affected, heterozygous carriers, and in normal dogs. When compared top normal animals, mean carnitine levels were reduced by 67% in affected and 50% in carrier dogs. Mean plasma TML levels were elevated almost 50% above control levels in the carriers, but were decreased by approximately 25% in the affected animals. The changes in plasma carnitine and TML levels in the carriers are consistent with the possibility that the disease involves a defect in the carnitine biosynthetic pathway. Secondary effects of the disease process may account for the apparently contradictory decrease in plasma TML levels in affected animals.
Animals↗
A noninvasive screening test to detect "critical" deep venous reflux.
PURPOSE: The venous filling index (VFI) was introduced as a noninvasive measure of venous valvular reflux. Because it is quantitative, identifying increasingly severe reflux should be possible. The purpose of this study is to evaluate the sensitivity and predictive value of the VFI as a predictor of phlebographically demonstrated "critical" venous reflux. METHODS: Thirty-one limbs with suspected venous insufficiency underwent both descending phlebography and air plethysmography. Nine limbs had deep venous reflux ending above the knee on descending phlebography (group 1), and 22 limbs had "critical" deep reflux to the below-knee level (group 2). The VFI, ejection fraction, and residual volume fraction were calculated, and a tourniquet was used to distinguish superficial from deep venous reflux. RESULTS: Three of six group 2 limbs with a VFI less than 7 had obliteration of their iliac veins. Tourniquet application improved the VFI in 13 limbs, all of which showed either phlebographic or venous duplex evidence of greater saphenous incompetence. CONCLUSIONS: A VFI greater than 7 showed a 73% sensitivity and 100% positive predictive value of identifying "critical venous reflux." The VFI may underestimate the degree of reflux in patients with proximal venous obstruction. Improvement of venous hemodynamics with tourniquet application suggests that venous reflux might be improved in some patients by correcting superficial venous insufficiency.
Adult↗
Is transcranial Doppler a worthwhile addition to screening tests for cerebrovascular disease?
PURPOSE: Carotid duplex imaging has become the standard diagnostic evaluation for patients with suspected cerebrovascular disease. Transcranial Doppler ultrasonography expands the noninvasive diagnostic capabilities to the intracranial circulation. The purpose of this study was to evaluate the results of routine transcranial Doppler studies on patients referred for noninvasive cerebrovascular evaluation. METHODS: A total of 670 patients had routine transcranial Doppler examinations as part of their noninvasive cerebrovascular evaluation. Patients were categorized clinically and according to their severity of extracranial internal carotid artery stenosis (< 50%, 50% to 79%, 80% to 99%, occlusion). Transcranial Doppler examinations were classified as normal or abnormal (intracranial stenosis, collateral pathway, > 30% velocity difference normal or abnormal (intracranial stenosis, collateral pathway, > 30% velocity difference between sides, flow reversal, and velocities +/- SD from normal). RESULTS: Forty-eight percent of the patients were women, and 52% were men. The average age was 65.5 years. Fifty-four percent of the patients were white, 42% were black, 3% were Hispanic, and 1% were other. Forty-eight percent presented with hemispheric symptoms, 34% had no symptoms, and 18% had nonhemispheric symptoms. Forty-five percent (304 of 670) had an interpretable transcranial Doppler examination. The ability to insonate the basal cerebral arteries through the temporal bone was significantly reduced in women (p < 0.0001), black patients (p < 0.0001), and older patients (p < 0.0001). The results of forty-four percent of interpretable examinations were normal, 19% demonstrated side-to-side velocity differences, 13% showed collateral pathways, 11% showed velocities +/- 2 SD, 10% showed an intracranial stenosis, and 4% showed reversed flow pattern. Although 56% of the patients had notable findings, no patient had their diagnostic or therapeutic plan altered by the transcranial Doppler results. CONCLUSION: Less than 50% of the patients referred for first-time cerebrovascular examination had access for an interpretable transcranial Doppler examination. Though the number of positive findings is reasonably high, no material impact on diagnostic or treatment plans was seen in the patients in this series. These results indicate that selection criteria for examination of the intracranial arteries should be refined and that transcranial Doppler scanning should not be incorporated as part of the "routine" noninvasive cerebrovascular examination.
Aged↗
Air plethysmography: a clinical review.
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Air↗
Lysine methylation of mitochondrial ATP synthase subunit c stored in tissues of dogs with hereditary ceroid lipofuscinosis.
Certain forms of ceroid lipofuscinosis, a hereditary degenerative disease, are characterized by accumulation of large amounts of subunit c of mitochondrial ATP synthase in lysosomal storage bodies of numerous tissues. The subunit c protein appears to constitute a major fraction of the total storage body protein. In previous studies it was demonstrated that hydrolysates of total storage body protein from affected humans and sheep contain significant amounts of epsilon-N-trimethyllysine (TML). This finding suggested that one or both of the two lysine residues of subunit c might be methylated in the stored form of the protein. The normal subunit c protein from mitochondria does not appear to be methylated. Using a putative canine model for the juvenile form of ceroid lipofuscinosis, analyses were conducted to determine whether lysosomal storage of subunit c was accompanied by lysine methylation of this protein. In affected dogs, as in humans and sheep with hereditary ceroid lipofuscinosis, the storage bodies were found to contain large amounts of subunit c protein, as indicated by polyacrylamide gel electrophoresis and partial amino acid sequence analysis. The subunit c protein partially purified from isolated storage bodies was found to contain lysine and TML in an almost equimolar ratio. Normal subunit c contains 2 lysine residues, one at position 7 and the other at position 43. Removal of the first 7 residues of the partially purified protein through sequential Edman degradation resulted in a dramatic increase in the TML to lysine ratio in the residual protein. This suggests that lysine residue 43 is methylated. Confirmation that residue 43 of the stored protein is TML was obtained by amino acid sequence analysis after cleavage of the protein with trypsin. This finding strongly suggests that specific methylation of lysine residue 43 of mitochondrial ATP synthase plays a central role in the lysosomal storage of this protein.
Amino Acid Sequence↗