Plant toxin ß-ODAP activates integrin ß1 and focal adhesion: A critical pathway to cause neurolathyrism.

Neurolathyrism is a unique neurodegeneration disease caused by ß-N-oxalyl-L-α, ß- diaminopropionic (ß-ODAP) present in grass pea seed (Lathyrus stativus L.) and its pathogenetic mechanism is unclear. This issue has become a critical restriction to take full advantage of drought-tolerant grass pea as an elite germplasm resource under climate change. We found that, in a human glioma cell line, ß-ODAP treatment decreased mitochondrial membrane potential, leading to outside release and overfall of Ca2+ from mitochondria to cellular matrix. Increased Ca2+ in cellular matrix activated the pathway of ECM, and brought about the overexpression of ß1 integrin on cytomembrane surface and the phosphorylation of focal adhesion kinase (FAK). The formation of high concentration of FA units on the cell microfilaments further induced overexpression of paxillin, and then inhibited cytoskeleton polymerization. This phenomenon turned to cause serious cell microfilaments distortion and ultimately cytoskeleton collapse. We also conducted qRT-PCR verification on RNA-sequence data using 8 randomly chosen genes of pathway enrichment, and confirmed that the data was statistically reliable. For the first time, we proposed a relatively complete signal pathway to neurolathyrism. This work would help open a new window to cure neurolathyrism, and fully utilize grass pea germplasm resource under climate change.

Bone fracture toughness and strength correlate with collagen cross-link maturity in a dose-controlled lathyrism mouse model.

Collagen cross-linking is altered in many diseases of bone, and enzymatic collagen cross-links are important to bone quality, as evidenced by losses of strength after lysyl oxidase inhibition (lathyrism). We hypothesized that cross-links also contribute directly to bone fracture toughness. A mouse model of lathyrism using subcutaneous injection of up to 500 mg/kg ß-aminopropionitrile (BAPN) was developed and characterized (60 animals across 4 dosage groups). Three weeks of 150 or 350 mg/kg BAPN treatment in young, growing mice significantly reduced cortical bone fracture toughness, strength, and pyridinoline cross-link content. Ratios reflecting relative cross-link maturity were positive regressors of fracture toughness (HP/[DHLNL + HLNL] r(2) = 0.208, p < 0.05; [HP + LP]/[DHNL + HLNL] r(2) = 0.196, p < 0.1), whereas quantities of mature pyridinoline cross-links were significant positive regressors of tissue strength (lysyl pyridinoline r(2) = 0.159, p = 0.014; hydroxylysyl pyridinoline r(2) = 0.112, p < 0.05). Immature and pyrrole cross-links, which were not significantly reduced by BAPN, did not correlate with mechanical properties. The effect of BAPN treatment on mechanical properties was dose specific, with the greatest impact found at the intermediate (350 mg/kg) dose. Calcein labeling was used to define locations of new bone formation, allowing for the identification of regions of normally cross-linked (preexisting) and BAPN-treated (newly formed, cross-link-deficient) bone. Raman spectroscopy revealed spatial differences attributable to relative tissue age and effects of cross-link inhibition. Newly deposited tissues had lower mineral/matrix, carbonate/phosphate, and Amide I cross-link (matrix maturity) ratios compared with preexisting tissues. BAPN treatment did not affect mineral measures but significantly increased the cross-link (matrix maturity) ratio compared with newly formed control tissue. Our study reveals that spatially localized effects of short-term BAPN cross-link inhibition can alter the whole-bone collagen cross-link profile to a measureable degree, and this cross-link profile correlates with bone fracture toughness and strength. Thus, cross-link profile perturbations associated with bone disease may provide insight into bone mechanical quality and fracture risk.

New insights into the mechanism of neurolathyrism: L-ß-ODAP triggers [Ca2+]i accumulation and cell death in primary motor neurons through transient receptor potential channels and metabotropic glutamate receptors.

Neurolathyrism is a motor neuron (MN) disease caused by ß-N-oxalyl-L-α,ß-diaminopropionic acid (L-ß-ODAP), an AMPA receptor agonist. L-ß-ODAP caused a prolonged rise of intracellular Ca(2+) ([Ca(2+)]i) in rat spinal cord MNs, and the [Ca(2+)]i accumulation was inversely proportional to the MN's life span. The [Ca(2+)]i rise induced by L-ß-ODAP or (S)-AMPA was antagonized completely by NBQX, an AMPA-receptor blocker. However, blocking the L-type Ca(2+) channel with nifedipine significantly lowered [Ca(2+)]i induced by (S)-AMPA, but not that by L-ß-ODAP. Tetrodotoxin completely extinguished the [Ca(2+)]i rise induced by (S)-AMPA or kainic acid, whereas that induced by L-ß-ODAP was only attenuated by 65.6±6% indicating the prominent involvement of voltage-independent Ca(2+) entry. The tetrodotoxin-resistant [Ca(2+)]i induced by L-ß-ODAP was blocked by 2-APB, Gd(3+), La(3+), 1-(ß-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF-96365) and flufenamic acid, which all are blockers of the transient receptor potential (TRP) channels. Blockers of group I metabotropic glutamate receptors (mGluR I), 7-(hydroxyiminocyclopropan[b]chromen-1α-carboxylate ethyl ester (CPCCPEt) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) also lowered the [Ca(2+)]i rise by L-ß-ODAP. MN cell death induced by L-ß-ODAP was prolonged significantly with SKF-96365 as well as NBQX. The results show the involvement of TRPs and mGluR I in L-ß-ODAP-induced MN toxicity through prolonged [Ca(2+)]i mobilization, a unique characteristic of this neurotoxin.

Role of glutamate and nitric oxide in onset of motor neuron degeneration in neurolathyrism.

Neurolathyrism is associated with a complex pattern of alterations in the glutamatergic system of the cortical motor region of brain. It is a neurological disorder consorted with excessive consumption of Lathyrus sativus (Grass pea), comprising large amounts of the neurotoxin, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ODAP). ODAP being a potent agonist of ionotropic glutamate receptors enhances their activity and also blocks the astrocytic glutamate/cystine transporters, abutting the neurons. This leads to the sustained increase in the concentration of Glutamate in the synapse which triggers excitotoxicity. L. sativus also contains high levels of arginine and homoarginine which are natural substrates of nitric oxide production, when NO levels increases, it forms peroxynitrite radicals which cause irreparable damage to mitochondria and cellular macromolecules leading to motor neuron degeneration. This review brings together all the molecular events reported so far, emphasizing on the possible role of glutamate and nitric oxide mediated cell death.

The ratio 1660/1690 cm(-1) measured by infrared microspectroscopy is not specific of enzymatic collagen cross-links in bone tissue.

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm(-1) area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm(-1) by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm(-1) by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm(-1) area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm(-1) ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm(-1) was unmodified. In conclusion, the 1660/1690 cm(-1) is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process.

Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral.

In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and ß-APN treated animals were fed additionally with ß-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2-L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that ß-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p<0.05). Further, compression tests revealed a significant negative impact of ß-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, ß-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence of ß-APN, despite a similar mineral content. In conclusion the results emphasize the pivotal role of collagen cross-links in the determination of bone quality and mechanical integrity. However, in this rat animal model of lathyrism, the coupled alterations of tissue structural properties make it difficult to weigh the contribution of the anatomically confined material changes to the overall mechanical performance of whole bone. Interestingly, the collagen cross-link ratio in bone forming areas had the same profile as seen in actively bone forming trabecular surfaces in human iliac crest biopsies of osteoporotic patients.

[Altered collagene characteristics and lysyl oxidase activity in lathyrism].

Experiments were carried out on rats with lathyrism, which was induced by adding semicarbazide (0.075%) into drinking water for 45 days. The data obtained show a 30% reduction in the body weight and an increase in.organ weight coefficients. Semicarbazide intake led to the pelvic limb paralysis, scoliosis, bone tissue degradation, cartilage growth, 46% decrease of the calcium level in the femur. It has been detected essential structural changes in extracellular matrix based on the collagen cross-links reduction. The activity of lysyl oxidase, a key enzyme for the collagen development, showed 5-fold decrease in the aorta tissues. The level of formaldehyde, a nonenzymic cross-links developer, has been measured in the liver tissue by the aldehyde trap (5,5-dimethyleyclohexane-1,3-dione) administration and then fluorimetric determination of formaldimedone. Under semicarbazide load, the formaldehyde level in the liver tissue was reduced by 47%. Therefore, semicarbazide influences not only the enzymic development of aldehyde groups in collagen, but the level of other aldehydes, which can cause cross-links. This experimental model of lathyrism is appropriate for investigation of the lysyl oxidase inhibitors effect on extracellular matrix.

L-ß-N-oxalyl-α,ß-diaminopropionic acid toxicity in motor neurons.

The excitatory amino acid L-ß-N-oxalyl-α,ß-diaminopropionic acid (L-ß-ODAP) in Lathyrus sativus L. is proposed as the causative agent of the neurodegenerative disease neurolathyrism. We investigated the effect of L-ß-ODAP on [Ca2+]i handling, redox homeostasis, and cell death in rat spinal motor neurons. L-ß-ODAP and L-glutamate triggered [Ca2+]i transients, which were inhibited by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor blockers; 2,3-dioxo-6-nitro-1,2,3, 4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide and 1-naphthyl acetylspermine, the latter specifically blocking Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. In addition, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, and to a lesser extent 1-naphthyl acetylspermine, protected the neurons against cell death induced by L-ß-ODAP or L-glutamate. Methionine and cysteine were also protective against neuronal cell death. We conclude that deregulation of [Ca2+]i homeostasis and oxidative stress contribute to motor neuron cell death in neurolathyrism.

In search of a new therapeutic target for the treatment of genetically triggered thoracic aortic aneurysms and cardiovascular conditions: insights from human and animal lathyrism.

Lathyrism, or the effect of certain plants on connective tissue disruption, particularly involving the vascular and skeletal systems, especially aortic dissection/rupture, has been well documented in animals. Its impact on the pathogenesis of connective tissue diseases in humans is still unclear. An extensive review of the scientific literature from the 1800s until the present time was performed to examine the common pathways between animal and human lathyrism and genetically triggered thoracic aortic aneurysms and cardiovascular conditions in humans, with special focus on the identification of potential therapeutic targets. Search areas covered the following subjects: lathyrism/spontaneous aortic dissection/rupture in animals; beta-aminopropionitrile, semicarbazide and their effects; collagen and elastin synthesis and cross-linking; genetic and molecular biology characteristics of the genetically triggered thoracic aortic conditions. Search results demonstrate Marfan syndrome as a model for the genetically triggered thoracic aortic aneurysms, has been linked to mutations of the fibrillin-1 gene, via transforming growth factor beta-1. Several other conditions do not share this mutation. Inhibition of semicarbazide-sensitive amine oxidase [vascular adhesion protein-1 (VAP-1)] in animals has been shown to result in aortic dilatation due to disruption of elastin cross-linking. Significantly low activity of this enzyme was identified in annulo-aortic ectasia; a condition similar to Marfan syndrome. In conclusion, the precise molecular and genetic pathways responsible for the clinical findings in Marfan syndrome and related conditions remain unclear. Observational and experimental findings relating to the vascular and systemic effects of molecular pathways implicated in the phenomenon of animal and human lathyrism suggest that VAP-1 seems to be involved in the molecular and developmental pathways of the genetically triggered thoracic aortic diseases and thus could be a potential therapeutic target for these conditions.

Downregulation of glutaredoxin but not glutathione loss leads to mitochondrial dysfunction in female mice CNS: implications in excitotoxicity.

Oxidative stress, excitotoxicity and mitochondrial dysfunction play synergistic roles in neurodegeneration. Maintenance of thiol homeostasis is important for normal mitochondrial function and dysregulation of protein thiol homeostasis by oxidative stress leads to mitochondrial dysfunction and neurodegeneration. We examined the critical roles played by the antioxidant, non-protein thiol, glutathione and related enzyme, glutaredoxin in maintaining mitochondrial function during excitotoxicity caused by beta-N-oxalyl amino-L-alanine (L-BOAA), the causative factor of neurolathyrism, a motor neuron disease involving the pyramidal system. L-BOAA causes loss of GSH and inhibition of mitochondrial complex I in lumbosacral cord of male mice through oxidation of thiol groups, while female mice are resistant. Reducing GSH levels in female mice CNS by pretreatment with diethyl maleate or L-propargyl glycine did not result in inhibition of complex I activity, unlike male mice. Further, treatment of female mice depleted of GSH with L-BOAA did not induce inhibition of complex I indicating that GSH levels were not critical for maintaining complex I activity in female mice unlike their male counterpart. Glutaredoxin, a thiol disulfide oxidoreductase helps maintain redox status of proteins and downregulation of glutaredoxin results in loss of mitochondrial complex I activity. Female mice express higher levels of glutaredoxin in certain CNS regions and downregulation of glutaredoxin using antisense oligonucleotides sensitizes them to L-BOAA toxicity seen as mitochondrial complex I loss. Ovariectomy downregulates glutaredoxin and renders female mice vulnerable to L-BOAA toxicity as evidenced by activation of AP1, loss of GSH and complex I activity indicating the important role of glutaredoxin in neuroprotection. Estrogen protects against mitochondrial dysfunction caused by excitotoxicity by maintaining cellular redox status through higher constitutive expression of glutaredoxin in the CNS. Therapeutic interventions designed to upregulate glutaredoxin may offer neuroprotection against excitotoxicity in motor neurons.

Periodontal & systemic bone changes in rats with experimental lathyrism.

BACKGROUND & OBJECTIVE: It is not clear how lathyrism affects the systemic bone metabolism. We therefore undertook a study to observe periodontal and systemic bone changes by performing radiological, metabolic, and bone densitometric evaluations in rats with experimental lathyrism. METHODS: A total of 30 rats were used. Experimental lathyrism was induced by once daily subcutaneous administration of beta-aminopropionitrile (beta-APN), at a dose of 5 mg beta-APN/0.4 ml per 100 g of body weight for 40 days. After 40 days, vertebral bone mineral density was analyzed by means of dual energy X-ray absorbtiometry in both groups. Blood was drawn by cardiac puncture and the animals were decapitated. Serum calcium levels were measured. Right mandibles were removed and radiographs were obtained. Alveolar bone level was determined in the radiographs. RESULTS: In all lathyritic rats, alveolar bone level was pathologically decreased with visible resorption. Vertebral bone mineral density values of lathyritic rats did not differ significantly from those of the control group. Compared to controls, there was a statistically significant decrease in serum calcium levels in the lathyritic group (P<0.001). INTERPRETATION & CONCLUSION: Significant alveolar bone resorption without alterations in vertebral bone mineral density indicated that lathyrogen administration for 40 days presumably has not caused systemic demineralization. This model could be used for studying the role of local and systemic agents on periodontal alveolar bone resorption.

Impact of the gliotoxin L-serine-O-sulphate on cellular metabolism in cultured rat astrocytes.

L-serine-O-sulphate is a member of a group of amino acids collectively called gliotoxins and is a substrate for the high affinity sodium-dependent glutamate transporters. Previous studies have shown that it is toxic to primary cultures of astrocytes but the mode of toxicity is unknown. The current study demonstrates that L-serine-O-sulphate, at a sub-toxic concentration (400 microM), causes significant disruption to glucose and alanine metabolism in cultures of rat cortical astrocytes. More specifically, using (13)C NMR spectroscopy a significant reduction in labelled end products from [1-(13)C]glucose and [3-(13)C]alanine was found in the presence of L-serine-O-sulphate. Additionally, using [2-(13)C]glycine a 27% reduction in de novo glutathione synthesis was observed in the presence of the gliotoxin. Incubation of the cells with L-serine-O-sulphate reduced the activity of alanine and aspartate aminotransferase by 53% and 67%, respectively. Collectively these results show that the gliotoxin, L-serine-O-sulphate, causes major disruptions to metabolic pathways in primary cultures of astrocytes.

Determination of systemically & locally induced periodontal defects in rats.

BACKGROUND & OBJECTIVE: The role of lathyrogens on bone metabolism is unclear, therefore we undertook this study to observe periodontal and systemic alterations in experimental lathyrism in rat and compare these changes to that observed in the locally induced periodontitis group. METHODS: A total of 45 male Wistar rats were equally divided in the lathyritic group (group 1), ligature-induced periodontitis group (group 2), and healthy controls (group 3). Experimental lathyrism was induced by once daily subcutaneous administration of beta-aminoproprionitrile (beta-APN), at a dose of 5 mg/0.4 ml per 100 g of body weight for 40 days. Ligature-induced periodontitis was created by tying silk ligatures on the necks of mandibular molars. After 40 days, blood samples were obtained and the animals were decapitated. Radiographic observations, extraction tests, histologic evaluations were performed, and serum ALP activity and gingival tissue IL-1beta levels were measured. RESULTS: Significant alveolar bone resorption around the mandibular molar teeth (P<0.001); lower extraction force levels (P<0.001); higher numbers of lymphocytes and macrophages (P<0.01) (both in connective tissue and epithelium at the dentogingival junction); decreased ALP activity (P<0.001); and increased gingival tissue IL-1beta levels (P<0.001) were observed in groups 1 and 2, compared to those in group 3. ALP activity was higher in group 1 than in group 2 rats (P<0.05). INTERPRETATION & CONCLUSION: Similar radiographical and histopathological findings and comparable increases in gingival tissue IL-1beta levels both in groups 1 and 2 showed that in addition to resorption of alveolar bone, chronic inflammation of periodontium also occurred both in the lathyritic rats as well as in ligature-induced periodontitis group rats.

Neurolathyrism: mitochondrial dysfunction in excitotoxicity mediated by L-beta-oxalyl aminoalanine.

beta-N-Oxalyl amino-L-alanine (L-BOAA); synonym beta-N-oxalyl-alpha,beta-diaminopropionic acid (beta-ODAP) is a naturally occurring non-protein amino acid present in the chickling pea from the plant Lathyrus sativus grown in drought prone areas. Ingestion of L-BOAA as a staple diet results in a progressive neurodegenerative condition, neurolathyrism, a form of motor neuron disease which affects the upper motor neurons and anterior horn cells of the lumbar spinal cord. L-BOAA is an excitatory acid and acts as an agonist at the AMPA receptor. One of the primary effects of L-BOAA toxicity is the inhibition of mitochondrial complex I selectively in the motor cortex and lumbar spinal cord. Recent evidence has suggested that the mitochondrial dysfunction is a consequence of oxidation protein thiol groups as a result of generation of reactive oxygen species. Mitochondrial complex I is highly to vulnerable to inactivation through oxidation of vital sulfhydryl groups. Thiol antioxidants such as alpha-liopic acid offer a method of protecting mitochondrial function. A common mechanism involving oxidation of protein thiol groups may underlie neurodegeneration occurring through mitochondrial dysfunction induced by excitatory amino acid.

Effects of lathyritic drugs and lathyritic demineralized bone matrix on induced and sustained osteogenesis.

Demineralized bone matrix was implanted in normal and lathyritic rats. At 2 weeks, the bone that formed in the lathyritic animals had an elevated alkaline phosphatase activity and a reduced calcium content compared with the controls. Four weeks after implantation, these biochemical parameters were reversed, with a decrease in alkaline phosphatase activity and an increase in calcium content to control levels. The histology of the recovered implants revealed new bone formation. Lathyritic demineralized bone matrix was prepared from bones of rats fed beta-aminopropionitrile for 2 weeks (2-week BAPN-DBM) or 4 weeks (4-week BAPN-DBM), and was implanted in normal rats. Two weeks after implantation, both preparations of lathyritic demineralized bone matrix demonstrated early bone formation, although alkaline phosphatase activity and calcium content were reduced. By 4 weeks after implantation, no biochemical or histological evidence of bone formation remained at the site of the 4-week BAPN-DBM implants; continued but reduced bone formation was observed at the site of the 2-week BAPN-DBM implants. Reconstitution of inactivated normal demineralized bone matrix with the guanidine-soluble extracts restored the osteoinductive capacity. However, reconstitution of inactivated lathyritic demineralized bone matrix (4-week BAPN-DBM) failed to restore the osteoinductive capacity. These results indicate that the degree of crosslinking of the collagen matrix that acts as a carrier for osteoinductive proteins plays a key role in inducing and sustaining osteogenesis.

From soil to brain: zinc deficiency increases the neurotoxicity of Lathyrus sativus and may affect the susceptibility for the motorneurone disease neurolathyrism.

Zinc deficiency and oversupply of iron to the roots of grass pea (Lathyrus sativus) induce increases in the content of the neurotoxin beta-L-ODAP (3-oxalyl-L-2,3-diaminopropanoic acid) in the ripe seeds. The transport of zinc to the shoots is enhanced by the addition of beta-L-ODAP. The neurotoxin of L. sativus is proposed to function as a carrier molecule for zinc ions. Soils, depleted in micronutrients from flooding by monsoon rains (Indian subcontinent) or otherwise poor in available zinc and with high iron content (Ethiopian vertisols), may be responsible for higher incidence of human lathyrism, one of the oldest neurotoxic diseases known to man. A role for brain zinc deficiency in the susceptibility for lathyrism is postulated.

Tyrosine-rich acidic matrix protein (TRAMP) accelerates collagen fibril formation in vitro.

Tyrosine-rich acidic matrix protein (TRAMP) is a recently discovered protein that co-purifies with porcine skin lysyl oxidase and is equivalent to the M(r) 22,000 extracellular matrix protein from bovine skin that co-purifies with dermatan sulfate proteoglycans (Cronshaw, A. D., MacBeath, J. R. E., Shackleton, D. R., Collins, J. F., Fothergill-Gilmore, L. A., and Hulmes, D. J. S. (1993) Matrix 13, 255-266; Neame, P. J., Choi, H. U., and Rosenberg, L. C. (1989) J. Biol. Chem. 264, 5474-5479). The effect of TRAMP on collagen fibril formation was studied in vitro by reconstitution of fibrils from lathyritic rat skin collagen I. Fibril formation was initiated by the warm start procedure, in which acidic collagen solutions and double strength neutral buffer, both preincubated separately at 34 degrees C, were mixed. When monitored by turbidimetry, TRAMP was found to accelerate collagen fibril formation. Acceleration occurred at sub-stoichiometric molar ratios of TRAMP collagen, and the presence of TRAMP stabilized the fibrils against low temperature dissociation. It was confirmed by centrifugation that the amount of fibrillar collagen formed in the presence of TRAMP was greater than in its absence. By SDS-polyacrylamide gel electrophoresis and scanning densitometry, binding of TRAMP to collagen was detected that approached saturation with a molar ratio of TRAMP to collagen of approximately 1:2. Fibrils formed in the presence of TRAMP were normal when observed by electron microscopy, although fibril diameters were smaller than the controls. TRAMP was found to partially reverse the inhibitory effects of urea and increased ionic strength on the kinetics of fibril formation, although inhibition by glucose was unaffected. TRAMP also accelerated the assembly of pepsin-treated collagen, where the non-helical, telopeptide regions were partially removed. Acceleration of collagen fibril formation by TRAMP is discussed in the light of the known effects of other extracellular matrix components on this process.

Studies on neurolathyrism.

Neurolathyrism is a neurological condition seen among people who eat the seeds of Lathyrus sativus (LS) as a principal source of food energy for 2 months or more. It is characterized by severe muscular rigidity and paralysis of the lower limbs. beta-N-Oxalyl-L-alpha,beta-diaminopropionic acid is the principal toxin found in the seed. No experimental animal model for neurolathyrism could be produced by feeding either the seeds or the toxin, although the condition has been known for centuries. We discovered that experimental neurolathyrism could be produced in guinea pigs and primates that needed an external supply of ascorbic acid by making them subclinically deficient in ascorbic acid and feeding them the seeds of LS or extracts thereof. Autoclaving the seeds of LS with lime removes the toxin.

Lathyrism and the half-life of collagen.

Maturation of collagen in the periodontal ligament has often been designated as the cause of tooth eruption. The fact that collagen has a high turnover rate provided some sort of proof of this hypothesis. It is well known that lathyrogens decrease eruption rate. Experiments were performed, using Wistar rats, which looked into the relationship between collagen turnover and lathyrism. The collagen turnover was established in teeth not erupting after the administration of lathyrogens. These results were compared to those obtained after having stopped eruption by the placement of a goldcrown on the lower incisors. It was found that lathyrism, and therefore collagen maturation, did not influence turnover rate. Thus the supposition, that collagen is responsible for tooth eruption, becomes doubtful.