Unusual Accumulation Research Articles

Unusual Accumulation of Demethylspheroidene in Anaerobic-Phototrophic Growth of crtA-Deleted Mutants of Rhodovulum sulfidophilum

Rhodovulum sulfidophilum produces carotenoids in the spheroidene pathway. Spheroidene monooxygenase, CrtA, catalyzes the conversion of spheroidene to spheroidenone. crtA-deleted mutants of R. sulfidophilum did not produce spheroidenone and demethylspheroidenone. In these mutants, the ratio of demethylspheroidene to spheroidene increased with exposure to light. One mutant exhibiting a spheroidene-predominant phenotype did not grow under anaerobic-light conditions and was devoid of bacteriochlorophyll a, even under semiaerobic-light conditions There was no difference in the growth of the mutants under aerobic-dark conditions. These data suggest that demethylspheroidene is important for photosynthesis in R. sulfidophilum.

Splenic Accumulation of Tc-99m HMDP in a Patient With Severe Alcoholic Cirrhosis of the Liver

The authors report unusual splenic accumulation of Tc-99m hydroxymethylene diphosphonate (HMDP) on bone scintigraphy of a patient with alcoholic cirrhosis of the liver. Laboratory studies showed hemolytic anemia and hemochromatosis resulting from alcohol abuse, both of which are thought to be related to the diffuse splenic uptake observed. When diffuse splenic accumulation by Tc-99m HMDP is seen, the existence of alcoholic hepatopathy might be considered.

Histopathological changes in the eyes in systemic lupus erythematosus: an electron microscope and immunohistochemical study.

This paper reports the histopathological findings in the eyes of a 26-year-old female patient diagnosed with systemic lupus erythematosus (SLE) with peripheral neuropathy. The patient had no significant ocular problems. She died of pneumonia after two years of suffering. The eyeballs were procured at autopsy and the retina, choroid and optic nerve processed for light and electron microscopy, and immunohistochemistry for immunoglobulin G (IgG), glial fibrillary acidic protein (GFAP), calbindin and parvalbumin. Histologically, there was haemorrhaging in the retinal nerve fibre layer. Ultrastructurally, the axons of this layer were swollen, and contained an unusual accumulation of microtubules and smooth endoplasmic reticulum. There were degenerative changes in the pericytes and smooth muscle cells of blood vessels. The capillary lumen was partially obliterated, and contained IgG, which was also detected throughout the choroid and wall of choroidal arterioles. The latter and Bruch's membrane showed fibrin deposits. The optic nerve showed infiltrated mononuclear cells near the degenerated axons, these axons lacked immunoreactivity to calbindin and parvalbumin. Compared to the control, the connective tissue sheaths of the central retinal vessels possessed a vast number of proliferated fibroblast cells, and trichrome staining showed transmural vessel scarring. Dense GFAP immunoreactivity was observed surrounding the vessel wall. These pathological changes are due to impaired blood circulation caused by haemorrhaging and vasculitis, and vessel occlusion by fibrin. The nature of the changes observed tends to indicate that a regular, thorough ophthalmic examination should be conducted even in the absence of significant ocular symptoms in SLE.

ABX-EGF monotherapy in patients (pts) with metastatic colorectal cancer (mCRC): An updated analysis

3511 Background: The epidermal growth factor receptor (EGFr) is a transmembrane tyrosine kinase expressed in mCRC. ABX-EGF is a high-affinity, fully human IgG2 monoclonal antibody directed against EGFr. Methods: This phase 2 study assessed the efficacy and safety of ABX-EGF monotherapy in pts with mCRC who previously failed therapy with a fluoropyrimidine and irinotecan or oxaliplatin or both. Tumors were tested for EGFr expression by immunohistochemistry in all pts screened; cohort A enrolled pts with EGFr expression of 2+ or 3+ in >=10% of tumor cells and cohort B enrolled pts with <10% 2+ or 3+, but 1+, 2+ or 3+ in >=10% of tumor cells. Pts received 2.5 mg/kg ABX-EGF weekly IV over 1 hr. The primary endpoint was response (RECIST) at week 8 with responses confirmed >=4 weeks later. Pharmacokinetic (PK) samples were collected before and after dose 8 of each cycle. Enrollment is complete, with treatment ongoing. Results: 148 pts (82 men/66 women; ECOG status 0 [42 pts] or 1 [106 pts]) were enrolled in cohort A (n=105) and cohort B (n=43). Of 148 pts with evaluations after 8 weeks, 15 (10.1%; 95%CI: 5.8%, 16.2%) had confirmed partial responses (12 cohort A, 3 cohort B) and 54 (36.5%) had stable disease (39 cohort A, 15 cohort B) by investigator assessment. The most frequent adverse event (AE) was skin rash (> 90%) [3.4% grade 3]. Other grade 3/4 AEs in >3 pts were fatigue (9 pts) and anemia (4 pts). One pt had a grade 3 infusion-related reaction considered possibly related to ABX-EGF and was premedicated for further treatment. PK appeared to reach steady state after 8 doses, and no unusual accumulation of ABX-EGF was observed for up to 4 cycles. Conclusions: This updated interim analysis suggests that ABX-EGF is well-tolerated, with encouraging single-agent antitumor activity in mCRC pts failing standard chemotherapy. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen Amgen Amgen Amgen Amgen

Unusual accumulation of S-methylmethionine in aerobic-etiolated and in anoxic rice seedlings: An 1H-NMR study

An unknown signal at 2.93 ppm in 1H-NMR spectra of rice, Oryza sativa, was assigned to the methyl groups of sulphur-methylmethionine (SMM), thereby devising a new method for the determination of this compound. Rice seedlings growing aerobically in the dark and in the light engaged for the synthesis of SMM an amount of Met corresponding to 23 and 8%, respectively, of the total seed reserves of this amino acid. In etiolated shoots, SMM reached 1.2 micromol g(-1) fresh weight, an unusually high level in vegetative tissues of wild-type plants. This is compared to a value of 0.4 micromol g(-1) fresh weight in green tissues. A decreased demand for Met during growth caused the higher accumulation of SMM in etiolated, rather than green, tissues. At the same time, dark seedlings were endowed with a readily utilizable and translocable alternative form of Met, as shown by retrieval of SMM from the coleoptile. The importance of methyl group storage in SMM is shown by comparison with choline and choline phosphate pools.

Coating selection for ‘Delicious’ and other apples

Abstract The gas permeabilities of shellac and several experimental coating formulations, including candelilla wax and shellac-carnauba were measured. These coatings, selected to have a wide range of gas permeabilities, were applied to freshly harvested and 5-month commercially stored ‘Delicious’, ‘Fuji’, ‘Braeburn’ and ‘Granny Smith’ apples. The coated apples were monitored during storage of 2 or 4 weeks at 20 °C for changes in internal gases level, and for quality parameters (surface gloss, weight loss, flesh firmness, Brix, titratable acidity and ethanol content). The shellac coating resulted in maximum fruit gloss, lowest internal O 2 , highest CO 2 , and least loss of flesh firmness for all of the varieties. The ‘Granny Smith’ with shellac had low internal O 2 ( 2 (25 kPa). This excessive modification of internal gas induced an abrupt rise of the respiratory quotient, prodigious accumulation of ethanol in both ‘Braeburn’ and ‘Granny Smith’, and flesh browning at the blossom end of ‘Braeburn’. In addition the shellac coating gave an unusual accumulation of ethanol in freshly harvested and 5 month-stored ‘Fuji’. Candelilla and carnauba-shellac coatings maintained more optimal internal O 2 and CO 2 and better quality for ‘Fuji’, ‘Braeburn’ and ‘Granny Smith’ apples, although even these coatings may present too much of a gas barrier for ‘Granny Smith’. Therefore, this research recommends the best coatings as shellac for ‘Delicious’, and carnauba-shellac for ‘Braeburn’ or ‘Fuji’. Best for ‘Granny Smith’ would seem to be a high-permeability wax similar to polyethylene, which has not been approved for apple by FDA. In general, the gas permeabilities of the coatings were useful as an indicator of differences in coating barrier properties, but did not account for differences in pore blockage.

Cadmium and zinc adsorption maxima of geochemically anomalous soils (Oxisols) in Jamaica

Soil samples were collected from a Miocene limestone area of Jamaica (Manchester Parish) where unusual accumulations of Cd and other metals have been described previously. The source of the metals is natural (geological). The soils are aluminous Oxisols and, geochemically, are closely similar to local karst bauxite deposits. For comparison a karst bauxite sample was collected from Alabama (USA) and an Ultisol sample from South Carolina (USA). All the Jamaican soils were in the pH range neutral to slightly alkaline and CaCO3 contents ranged from 1.3 to 23.1%. Mean total Cd = 102.5 mg/kg (range 13.6-191.8 mg/kg) and mean Zn = 362.6 (range = 125.8-683.3) mg/kg. These values are higher than world averages. The mean readily exchangeable Cd was 0.05 (range 0.01-0.15) mmol/kg and for Zn mean = 0.02 (range 0.01-0.02) mmol/kg. Adsorption data were obtained experimentally and modelled using the Langmuir isotherm. For the Manchester soils the mean Cd adsorption maximum was 9.15 (range 2.26-32.0) mmol/kg; the values were higher than the karst bauxite sample (0.08 mmol/kg) or the Ultisol (0.08 mmol/kg). Reliable Zn isotherms were not obtained for all soils; for three Manchester soils the mean Zn sorption maximum was 2.99 mmol/kg compared with 3.13 mmol/kg for Cd in the same three soils. Mean Al and Fe values are 38.7% Al2O3 and 17.7% Fe2O3 compared with the Ultisol (14.5% Al2O3 11.3% Fe2O3) and the bauxite (52.6% Al2O3, 0.7%Fe2O3). Interpretation of the major element values and the known mineralogy of the soils implies that the high adsorption maxima of the Manchester soils can best be explained by their calcareous nature. It is concluded that the Manchester soils have ample adsorption capacity to trap any incoming Cd or Zn solutes.

Lipid metabolome-wide effects of the PPARγ agonist rosiglitazone

Successful therapy for chronic diseases must normalize a targeted aspect of metabolism without disrupting the regulation of other metabolic pathways essential for maintaining health. Use of a limited number of single molecule surrogates for disease, or biomarkers, to monitor the efficacy of a therapy may fail to predict undesirable side effects. In this study, a comprehensive metabolomic assessment of lipid metabolites was employed to determine the specific effects of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist rosiglitazone on structural lipid metabolism in a new mouse model of Type 2 diabetes. Dietary supplementation with rosiglitazone (200 mg/kg diet) suppressed Type 2 diabetes in obese (NZO x NON)F1 male mice, but chronic treatment markedly exacerbated hepatic steatosis. The metabolomic data revealed that rosiglitazone i) induced hypolipidemia (by dysregulating liver-plasma lipid exchange), ii) induced de novo fatty acid synthesis, iii) decreased the biosynthesis of lipids within the peroxisome, iv) substantially altered free fatty acid and cardiolipin metabolism in heart, and v) elicited an unusual accumulation of polyunsaturated fatty acids within adipose tissue. These observations suggest that the phenotypes induced by rosiglitazone are mediated by multiple tissue-specific metabolic variables. Because many of the effects of rosiglitazone on tissue metabolism were reflected in the plasma lipid metabolome, metabolomics has excellent potential for developing clinical assessments of metabolic response to drug therapy.

Cis-4-methylsphingosine phosphate induces apoptosis in neuroblastoma cells by opposite effects on p38 and ERK mitogen-activated protein kinases.

Intracellular phosphorylation of cis-4-methylsphingosine was previously shown to result in a metabolically stable compound that accumulates in Swiss 3T3 fibroblasts and mimics the mitogenic effect induced by the short-lived sphingosine metabolite, sphingosine-1-phosphate. In the present study incubation of neuroblastoma B104 cells with cis-4-methylsphingosine (10 microM) also resulted in an intracellular accumulation of its phosphorylated derivative that was, however, associated with the concentration-dependent induction of apoptosis, not observed after treatment with 10 microM of sphingosine-1-phosphate or sphingosine, respectively. In B104 cells, cis-4-methylsphingosine stimulated p38 mitogen-activated protein kinase (p38 MAPK) and simultaneously inhibited extracellular signal-regulated kinase (ERK), whereas sphingosine and sphingosine-1-phosphate only stimulated p38 MAPK without suppression of ERK. Inhibition of cis-4-methylsphingosine phosphorylation reduced both, apoptosis and concurrent regulation of mitogen-activated protein kinases (MAPKs), suggesting that the unusual accumulation of the phosphorylated sphingoid base was responsible for the biological effects. Furthermore, inhibition of p38 MAPK prevented cis-4-methylsphingosine-induced apoptosis, while suppression of the ERK pathway in the presence of sphingosine or sphingosine-1-phosphate resulted in apoptosis, indicating that the simultaneous opposite regulation of the two MAPKs was required for the induction of apoptosis.

Chemical forms of metallothionein and cellular oxidative stress in human hepatocellular carcinoma: A comparative study with the hepatitis of LEC rat

AbstractMetallothionein (MT) has been described as a cell‐protective protein that binds several heavy metals. In mammals, MT plays an important physiological role that includes detoxifying heavy metals, accumulating essential trace elements in cells to maintain homeostasis, and scavenging reactive oxygen species or free radicals. In contrast, the generation of hydroxyl radical (·OH) has been reported in the reaction of hydrogen peroxide and copper (Cu)‐MT separated from the livers of Long–Evans Cinnamon (LEC) rats, an animal model of human hepatic injury with unusual accumulation of Cu‐MT in the liver. On the other hand, Cu has been reported to accumulate in human hepatocellular carcinoma (HCC). Thus, we investigated whether the induction of MT, accumulation of Cu, and enhancement of oxidative stress are relevant to the development of human HCC, similarly to the observations in the liver of LEC rats. Although the kinetic behaviors of metal and MT levels were different in the livers of humans and LEC rats, Cu‐MT induction in the tumor portion of human HCC tissues was suggested to occur similarly to that in the liver of LEC rats. In addition, it was found that oxidative stress in terms of the level of thiobarbituric acid‐reactive substances was enhanced in the tumor portion of human HCC, corresponding well with increasing in the age of LEC rats, which developed jaundice and hepatitis. On the basis of these results, we conclude that the induction of Cu‐MT in human HCC might be related to the hepatic oxidative stress, which in turn develops hepatic injury involving carcinoma. J. Trace Elem. Exp. Med. 15:31–45, 2002. ©2002 Wiley‐Liss, Inc.

Late Quaternary variations in calcium carbonate preservation of deep-sea sediments in the northern Cape Basin: results from a multiproxy approach

In this study, we test various parameters in deep-sea sediments (bulk sediment parameters and changes in microfossil abundances and preservation character) which are generally accepted as indicators of calcium carbonate dissolution. We investigate sediment material from station GeoB 1710-3 in the northern Cape Basin (eastern South Atlantic), 280 km away from the Namibian coast, well outside today’s coastal upwelling. As northern Benguela upwelling cells were displaced westward and periodically preceded the core location during the past 245 kyr (Volbers et al., submitted), GeoB 1710-3 sediments reflect these changes in upwelling productivity. Results of the most commonly used calcium carbonate dissolution proxies do not only monitor dissolution within these calcareous sediments but also reflect changes in upwelling intensity. Accordingly, these conventional proxy parameters misrepresent, to some extent, the extent of calcium carbonate dissolution. These results were verified by an independent dissolution proxy, the Globigerina bulloides dissolution index (BDX′) (Volbers and Henrich, submitted). The BDX′ is based on scanning electronic microscope ultrastructural investigation of planktonic foraminiferal tests and indicates persistent good carbonate preservation throughout the past 245 kyr, with the exception of one pronounced dissolution event at early oxygen isotopic stage (OIS) 6. The early OIS 6 is characterized by calcium carbonate contents, sand contents, and planktonic foraminiferal concentrations all at their lowest levels for the last 245 kyr. At the same time, the ratio of radiolarian to planktonic foraminiferal abundances and the ratio of benthic to planktonic foraminiferal tests are strongly increased, as are the rain ratio, the fragmentation index, and the BDX′. The sedimentary calcite lysocline rose above the core position and GeoB 1710-3 sediments were heavily altered, as attested to by the unusual accumulation of pellets, aggregates, sponge spicules, radiolaria, benthic foraminifera, and planktonic foraminiferal assemblages. Solely the early OIS 6 dissolution event altered the coarse fraction intensely, and is therefore reflected by all conventional calcium carbonate preservation proxies and the BDX′. We attribute the more than 1000 m rise of the sedimentary calcite lysocline to the combination of two processes: (a) a prominent change in the deep-water mass distribution within the South Atlantic and (b) intense degradation of organic material within the sediment (preserved as maximum total organic carbon content) creating microenvironments favorable for calcium carbonate dissolution.

Inositol phosphates from barley low-phytate grain mutants analysed by metal-dye detection HPLC and NMR

Inositol phosphates from barley low-phytate grain mutants and their parent variety were analysed by metal-dye detection HPLC and NMR. Compound assignment was carried out by comparison of retention times using a chemical hydrolysate of phytate [Ins(1,2,3,4,5,6)P6] as a reference. Co-inciding retention times indicated the presence of phytate, D/L-Ins(1,2,3,4,5)P5, Ins(1,2,3,4,6)P5, D/L-(1,2,4,5,6)P5, D/L-(1,2,3,4)P4, D/L-Ins(1,2,5,6)P4 and D/L-Ins(1,4,5,6)P4 in PLP1B mutants as well as the parent variety. In grain extracts from mutant lines PLP1A, PLP2A and PLP3A unusual accumulations of D/L-Ins(1,3,4,5)P4 were observed whereas phytate and the above-mentioned inositol phosphates were present in relatively small amounts. Assignment of D/L-Ins(1,3,4,5)P4 was corroborated by precise co-chromatography with a commercial Ins(1,3,4,5)P4 standard and by NMR spectroscopy. Analysis of inositol phosphates during grain development revealed accumulation of phytate and D/L-Ins(1,3,4,5)P4, which suggested the tetrakisphosphate compound to be an intermediate of phytate synthesis. This assumption was strengthened further by phytate degradation assays showing that D/L-Ins(1,3,4,5)P4 did not belong to the spectrum of degradation products generated by endogenous phytase activity. Metabolic scenarios leading to accumulation of D/L-Ins(1,3,4,5)P4 in barley low-phytate mutants are discussed.

Inositol phosphates from barley low-phytate grain mutants analysed by metal-dye detection HPLC and NMR.

Inositol phosphates from barley low-phytate grain mutants and their parent variety were analysed by metal-dye detection HPLC and NMR. Compound assignment was carried out by comparison of retention times using a chemical hydrolysate of phytate [Ins(1,2,3,4,5,6)P(6)] as a reference. Co-inciding retention times indicated the presence of phytate, D/L-Ins(1,2,3,4,5)P(5), Ins(1,2,3,4,6)P(5), D/L-(1,2,4,5,6)P(5), D/L-(1,2,3,4)P(4), D/L-Ins(1,2,5,6)P(4) and D/L-Ins(1,4,5,6)P(4) in PLP1B mutants as well as the parent variety. In grain extracts from mutant lines PLP1A, PLP2A and PLP3A unusual accumulations of D/L-Ins(1,3,4,5)P(4) were observed whereas phytate and the above-mentioned inositol phosphates were present in relatively small amounts. Assignment of D/L-Ins(1,3,4,5)P(4) was corroborated by precise co-chromatography with a commercial Ins(1,3,4,5)P(4) standard and by NMR spectroscopy. Analysis of inositol phosphates during grain development revealed accumulation of phytate and D/L-Ins(1,3,4,5)P(4), which suggested the tetrakisphosphate compound to be an intermediate of phytate synthesis. This assumption was strengthened further by phytate degradation assays showing that D/L-Ins(1,3,4,5)P(4) did not belong to the spectrum of degradation products generated by endogenous phytase activity. Metabolic scenarios leading to accumulation of D/L-Ins(1,3,4,5)P(4) in barley low-phytate mutants are discussed.

Chromatographic identification of a biochemical alteration in the aqueous humour of megalophthalmic Black Moor goldfish

Purpose: To observe if any biochemical abnormalities exist between the eye of megalophthalmic and non-megalophthalmic goldfish by high-performance liquid chromatography (HPLC). Method: Aqueous humour and sera from megalophthalmic and non-megalophthalmic goldfish were subjected to HPLC and monitored by photodiode array detection (Waters, MA, USA). Results: An unusual accumulation of a compound with a UV absorption maximum at 290 nm was observed in the aqueous humour of megalophthalmic eye. This compound was also present in the sera of both normal goldfish and one of its megalophthalmic mutant. However, it was significantly elevated in the aqueous humour of the megalophthalmic eye only. This compound concentration was very high in the eye of small fish and its concentration increased only slightly with the expansion of the eye in larger fish. Conclusions: The presence of this compound in the serum and aqueous humour indicates a specific systemic metabolic variation in Black Moor goldfish not seen other animal species we had studied (humans, bovine, chick, rabbits and rats). The marked elevation of this compound in the megalophthalmic eye indicates a possible association of this compound with the metabolic variation accounting for the expansion of the eye in megalophthalmic goldfish.

Plant growth inhibitory activity of L-canavanine and its mode of action.

L-Canavanine is a nonprotein amino acid contained in jack bean [Canavalia ensiformis (L.) DC] and shows a plant inhibitory effect. The inhibitory effect was determined by an immersion test and a microdrop test that employed rice seedlings. L-Canavanine inhibited elongation of the second leaf sheath of rice seedlings more than other natural bioactive substances, such as salicylic acid and cinnamic acid. The modified microdrop test revealed that the mode of action of L-canavanine had no relation to gibberellin synthesis. In the microdrop test, the inhibitory effect of L-canavanine was decreased by simultaneous addition of L-arginine, an analog of L-canavanine. Free amino acid analysis of rice shoots clearly showed that L-canavanine induced an unusual accumulation of L-arginine. However, accumulation of L-arginine did not cause the inhibitory effect on plant growth. These results suggest that the mechanism of inhibition of L-canavanine is closely related to the inhibition of arginine metabolism.

Reduced latency but no increased brain tumor penetrance in mice with astrocyte specific expression of a human p53 mutant.

p53-germline mutations located in the core DNA-binding domain have been associated with a more dominant tumor penetrance especially for breast cancer and brain tumors. We previously reported an unusual accumulation of CNS tumors associated with a unique p53 germline mutation, Y236delta (deletion of codon 236). To test whether this tissue-specific tumor predisposition reflects a gain-of-function activity of Y236delta, we generated transgenic mice expressing Y236delta in astrocytes using the regulatory elements of the glial fibrillary acidic protein (GFAP) gene. After transplacental exposure to N-ethyl-N-nitrosourea (25 mg/kg BW) brain tumors developed in 18% (7/39) of GFAP-Y236delta transgenic p53-/- mice, while in p53+/- mice the incidence was 28% (11/40) (P>0.3). However, the mean tumor latency for GFAP-Y236delta/p53+/- mice was significantly shorter than for p53+/- mice, with 19.9 weeks vs 31.6 weeks (P=0.039), respectively. Taken together, cell specific expression of Y236delta results in an acceleration of tumor progression but does not confer a higher tumor penetrance. Conceivably, the transdominant effect of Y236delta provided a growth advantage early in the progression of neoplastic cells, since the endogenous p53 wild-type allele was lost in all brain tumors independent of the genotype. This reflects well observations from human astrocytic neoplasms with p53 mutations.

Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation.

BackgroundThe relationship between cell shape, proliferation, and extracellular matrix (ECM) production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D) culture.ResultsThree experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1) Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2) Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3) Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth.ConclusionsThe cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

Decreased Elastin Deposition and High Proliferation of Fibroblasts from Costello Syndrome Are Related to Functional Deficiency in the 67-kD Elastin-Binding Protein

Costello syndrome is characterized by mental retardation, loose skin, coarse face, skeletal deformations, cardiomyopathy, and predisposition to numerous malignancies. The genetic origin of Costello syndrome has not yet been defined. Using immunohistochemistry and metabolic labeling with [3H]-valine, we have established that cultured skin fibroblasts obtained from patients with Costello syndrome did not assemble elastic fibers, despite an adequate synthesis of tropoelastin and normal deposition of the microfibrillar scaffold. We found that impaired production of elastic fibers by these fibroblasts is associated with a functional deficiency of the 67-kD elastin-binding protein (EBP), which is normally required to chaperone tropoelastin through the secretory pathways and to its extracellular assembly. Metabolic pulse labeling of the 67-kD EBP with radioactive serine and further chase of this tracer indicated that both normal fibroblasts and fibroblasts from patients with Costello syndrome initially synthesized comparable amounts of this protein; however, the fibroblasts from Costello syndrome patients quickly lost it into the conditioned media. Because the normal association between EBP and tropoelastin can be disrupted on contact with galactosugar-bearing moieties, and the fibroblasts from patients with Costello syndrome revealed an unusual accumulation of chondroitin sulfate-bearing proteoglycans (CD44 and biglycan), we postulate that a chondroitin sulfate may be responsible for shedding EBP from Costello cells and in turn for their impaired elastogenesis. This was further supported by the fact that exposure to chondroitinase ABC, an enzyme capable of chondroitin sulfate degradation, restored normal production of elastic fibers by fibroblasts from patients with Costello syndrome. We also present evidence that loss of EBP from fibroblasts of Costello syndrome patients is associated with an unusually high rate of cellular proliferation.

Activation of Chick Tendon Lysyl Oxidase in Response to Dietary Copper

Lysyl oxidase (EC 1.4.3.13), a cuproenzyme, can account for 10–30% of the copper present in connective tissue. Herein, we assess the extent to which tissue copper concentrations and lysyl oxidase activity are related because the functional activity of lysyl oxidase and the copper content of chick tendon are both related to dietary copper intake. Chicks (1-d old) were fed diets (basal copper concentration, 0.4 μg/g diet) to which copper was added from 0 to 16 μg/g diet. Liver and plasma copper levels tended to normalize in chickens that consumed from 1 to 4 μg copper/g of diet, whereas tendon copper concentrations suggested an unusual accumulation of copper in chickens that consumed 16 μg copper/g diet. The molecular weight of lysyl oxidase was also estimated using matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS). A novel aspect of these measurements was estimation of protein mass directly from the surface of chick tendons and aortae. Whether copper deficiency (0 added copper) or copper supplementation (16 μg copper/g of diet) caused changes in the molecular weight of protein(s) in tendon corresponding to lysyl oxidase was addressed. The average molecular weight of the peak corresponding to lysyl oxidase in tendon and aorta from copper-deficient birds was 28,386 Da ± 86, whereas the average molecular weight of corresponding protein in tendon from copper-supplemented birds was 28,639 Da ± 122. We propose that the shift in molecular weight is due in part to copper binding and the formation of lysyl tyrosyl quinone, the cofactor at the active site of lysyl oxidase.

Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.

Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin.