Effect of Oxygen Free Radicals on Corneal Collagen

Corneal collagen was labeled in vivo by injection of ¹⁴C-proline into the anterior chamber of rabbit eyes. The isolated corneal collagen was incubated in iron-free phosphate buffered saline (pH 7.4) containing I mM axorbate and 0.1 mM CuSO₄ for either 1 hour or 3 hours at 37°. Addition of 2 volumes of 8 M urea-I mM dithiothreitol and heating for 1 min at 100° solubilized virtually all of the collagen in the control incubations but left a significant amount of insoluble collagen in specimens exposed to the hydroxyl radical generating system. This residue amounted to 19% and 38% of the initial radioactivity in samples incubated for 1 h and 3 h, respectively. The chromatographic profiles (gel filtration on CL-4B) of the soluble fraction showed an increase in both aggregation and degradation products of collagen in the 1 h incubation mixture, whereas after 3 h there was an increase only in degradation products. These observations suggest that additional crosslinking of the soluble collagen aggregates observed at 1 h may be responsible for their subsequent disappearance at 3 h, with concomitant increase of the insoluble fraction. Collagen degradation by OH may play a role in corneal ulceration, whereas hydroxyl radical-mediated crosslinking is consistent with age-dependent increases in insoluble collagen.     

Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7

Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75–80 °C and its half life was 30 min at 70 °C. The K_m and V_max were calculated to be, respectively, 0.33 mM and 188 μM min⁻¹ mg⁻¹ with p-nitrophenyl palmitate (pNPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well.     

In vivo labeling studies on the biosynthesis and degradation of collagen in experimental myocardial infarction.

The biosynthesis and degradation of myocardial collagen was studied in myocardium infarcted rats after a single intraperitoneal injection of 3H-proline. The incorporation of tritiated proline into collagen as 3H-hydroxyproline was regarded as a measure of collagen synthesis. The total content as well as total activity of hydroxyproline were measured in the whole homogenate, neutral salt soluble fraction, insoluble fraction and in urine collected at different time intervals and specific activities were calculated. Both collagen anabolism and catabolism were found to be affected in infarcted rat hearts. Degradation of existing collagen at the earlier stages of myocardial infarction and a simultaneous new collagen synthesis and deposition as insoluble form later took place.     

An in vitro study on the role of metal catalyzed oxidation in glyeation and crosslinking of collagen

The present investigation was carried out to understand the effect of metal catalyzed oxidation on glycation and crosslinking of collagen. Tail tendons obtained from rats weighing 200-225 g were incubated with glucose (250 mM) and increasing concentrations of copper ions (5, 25, 50 and 100 M) under physiological conditions of temperature and pH. Early glycation, crosslinking and late glycation (fluorescence) of collagen samples were analyzed periodically. Early glycation was estimated by phenol sulfuric acid method, and the crosslinking was assessed by pepsin and cyanogen bromide digestion. A concentrationdependent effect of metal ions on the rate of glycation and crosslinking of collagen was observed. Tendon collagen incubated with glucose and 100 M copper ions showed 80% reduction in pepsin digestion within seven days, indicating extensive crosslinking, whereas collagen incubated with glucose alone for the same period showed only 7% reduction. The presence of metal ions in the incubation medium accelerated the development of Maillard reaction fluorescence on collagen, and the increase was dependent on the concentration of metal ions used. The metal chelator Diethylene triamine penta-acetate significantly prevented the increase in collagen crosslinking by glucose and copper ions. Free radical scavengers benzoate and mannitol effectively prevented the increased crosslinking and browning of collagen by glucose. The results indicate that the metal catalyzed oxidation reactions play a major role in the crosslinking of collagen by glucose. It is also suggested that the prevention of increased oxidative stress in diabetes may prevent the accelerated advanced glycation and crosslinking of collagen.     

Synthesis and degradation of collagens in skin of healthy and protein-malnourished rats in vivo, studied by 18O2 labelling.

To explore the effects of growth retardation, caused by restricted protein intake, on collagen turnover in the whole skin, Sprague-Dawley rats (n = 20) were labelled with 18O2 and fed on either an adequate (18%) or a low (3%) lactalbumin diet. Skin biopsies were obtained at intervals during the following 6 months. Independent groups of animals (n = 186) were used to determine the size of the 0.5 M-acetic acid-soluble and -insoluble collagen pools in the entire skin of healthy and malnourished rats. Collagen was estimated by measurement of hydroxyproline. Soluble-collagen synthesis rates were equivalent to 99 +/- 8 mumol of hydroxyproline/day in healthy animals and 11 +/- 2 mumol/day in malnourished rats. Insoluble-collagen synthesis rates were 32 and 5 mumol of hydroxyproline/day in the healthy and protein-depleted rats respectively. The degradation of soluble collagen amounted to 37 +/- 8 and 6 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Efflux of collagen from the soluble collagen, defined as the sum of the rate of soluble collagen that is degraded plus that which matures into insoluble collagen, was 70 +/- 8 and 11 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Insoluble collagen was not degraded in either group. The fraction of soluble collagen leaving the pool that was converted into insoluble collagen was 0.46 in both diet groups. It is concluded that the turnover of soluble collagen is markedly decreased with malnutrition, but degradation and conversion into insoluble collagen account for the same proportions of efflux from the soluble-collagen pool as in rapidly growing rats.     

Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 1802 labelling.

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.     

Synergistic Effects of Cu(II) and Dimethylammonium 2,4-Dichlorophenoxyacetate (U46 D Fluid) on PM2 DNA and Mechanism of Dna Damage

Dimethylammonium 2,4-dichlorophenoxyacetate (2,4-D · DMA) induced strand breaks in PM2 DNA when incubated with CuCl₂, whereas 2,4-D · DMA alone or CuCl₂ alone did not show any or only a negligible effect. The formation of single strand breaks increased linearly with time and concentration of 2,4-D · DMA. Neocuproine, a specific Cu(I) chelator totally prevented strand break formation. So did catalase (up to 100mM 2,4-D · DMA), but DMSO had only a small protective effect. 2,4-Dichlorophenol, CO₂ and formaldehyde were detected as reaction products of 2,4-D and CuCl₂. From these results a redox reaction of Cu(II) and 2,4-D is proposed, which could explain the DNA damaging properties of CuCl₂/2,4-D · DMA.     

Procedural improvements for in vitro production of viable uterine stage caprine embryos

Efforts to improve proportions of caprine immature oocytes developing into viable uterine-stage embryos in vitro involved study of 1924 oocytes in experiments designed to examine influences of fertilization media, sperm incubation temperatures, sperm treatment procedures, different protein supplementations, and different insemination intervals. Oocytecumulus complexes (OCCs) were matured during 27 h in TCM-199 supplemented with 20% FBS, 100 μg LH ml⁻¹, 0.5 μg FSH ml⁻¹, and 1 μg Estradiol-17-β ml⁻¹at 38.5 °C in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere. Freshly collected sperm were washed and incubated at either 22 °C or 38.5 °C for 5 h and then treated with either 0.1 μM calcium ionophore A23187 for 1 min, or with 7.35 mM calcium lactate in the presence of oocytes during the insemination interval, or with 100 μg heparin +2 mM caffeine ml⁻¹ for 15 min. The interval for insemination was experimentally varied i.e. 14 or 24 h. Results showed that: (a) when used as a fertilization medium mDM supported more blastocyst development than TALP (10.5% vs. 0%, P < 0.05); (b) incubation temperatures of 22 °C or 38.5 °C prepared goat spermatozoa equally for capacitation in mDM containing 20% FBS; (c) when oocytes were inseminated with sperm incubated in mDM with 20% FBS and capacitated with calcium lactate more embryos reached the blastocyst stage (P < 0.05) than after incubation in the same conditions but after sperm capacitation with heparin, and A23187 (31.8% vs. 24.2% and 10.2%, respectively; (d) a 24 h insemination interval was not superior to 14 h when sperm were incubated with either 20% FBS or 6 mg BSA ml⁻¹ and capacitated with calcium lactate (P > 0.05). Three morulae resulting from the best conditions in this work (FBS, calcium lactate, 14 h insemination) were transferred into the uterine horn ipsilateral to the corpus luteum of a recipient and two normal female kids were born after normal gestation. This is the first report in which it has been possible to consistently take caprine development to the blastocyst stage in vitro, and to obtain offspring following uterine transfer. Methodology reported here should facilitate implementation of new reproductive and genetic strategies in goat breeding.     

Quantitative and metabolic changes of hepatic collagens in rats after carbon tetrachloride poisoning

1. The collagen hydroxyproline in rat liver was composed of 3.5% neutral-soluble collagen, 4.9% acid-soluble collagen and 91.6% insoluble collagen. In labelling studies with [(14)C]proline in vitro, the specific radioactivities of neutral-soluble, acid-soluble and insoluble collagens in rat liver were found to be 233000, 69000 and 830d.p.m./mumol of hydroxyproline respectively after 1h. 2. During subacute carbon tetrachloride poisoning the hepatic content of insoluble collagen markedly increased, whereas those of soluble collagens did not change. During recovery from subacute poisoning hepatic contents of soluble collagens were markedly decreased. 3. After 8 weeks of carbon tetrachloride poisoning the specific radioactivities of hepatic soluble collagens increased, while that of insoluble collagen decreased. During recovery from subacute poisoning, the specific radioactivities of soluble collagens decreased to the normal range and that of insoluble collagen further decreased. 4. Hepatic collagenolytic activity solubilizing insoluble collagen, which differs from mammalian collagenase, decreased under the conditions of the subacute poisoning and also during recovery from subacute poisoning.     

In vitro degradation behavior of microspheres based on cross-linked dextran

The aim of this study was to investigate the in vitro degradation of hydroxyl ethyl methacrylated dextran (dex-HEMA) microspheres. Dextran microspheres were incubated in phosphate buffer pH 7.4 at 37 degrees C, and the dry mass, mechanical strength, and chemical composition of the microspheres were monitored in time. The amount and nature of the formed degradation products were established for microspheres with different cross-link densities by FT-IR (Fourier transformed infrared spectroscopy), NMR, mass spectrometry, SEC analysis, and XPS (X-ray photoelectron microscopy). The dex-HEMA microspheres DS 12 (degree of HEMA substitution; the number of HEMA groups per 100 glucose units) incubated at pH 7.4 and 37 degrees C showed a continuous mass loss, leaving after 6 months a residue of about 10% (w/w) of water-insoluble products. NMR, mass spectrometry, and SEC showed that the water-soluble degradation products consisted of dextran, low molecular weight pHEMA (M(n) approximately 15 kg/mol), and small amounts of unreacted HEMA and HEMA-DMAP (intermediate reaction product of the Baylis-Hillman reaction of HEMA with DMAP (4-dimethyl aminopyridine)). Microscopy revealed that the water-insoluble residue consisted of particles with shape and size similar to that of nondegraded microspheres. However, these particles had lost their mechanical strength as evidenced from micromanipulation experiments. FT-IR and XPS (X-ray photoelectron microscopy) revealed that these particles consisted of pHEMA, of which a small fraction was soluble in methanol (M(n) ranging between 27 and 82 kg/mol). The insoluble material likely consisted of lightly cross-linked pHEMA. In conclusion, in vitro degradation of dex-HEMA microspheres results in the formation of water-soluble degradation products (mainly dextran), leaving a small water-insoluble residue mainly consisting of pHEMA.     

Nitrate assimilation and nitrogen circulation in Austrian pine

Nitrate uptake, reduction and translocation were examined in 5-week-old Austrian pines ( Pinus nigra Arnold var. nigricans Host.) during exposure to 5 m M NaNO₃. The rate of nitrate uptake was linear during the 7 h light period. ¹⁵N-NO₃⁻ was detected in all parts of the pine except in the needles. By the 7th hour, 43% of the absorbed nitrate had been reduced, and this increased to 64% by the 24th hour. The major part of the total reduction occurred in the roots at this growth stage. Accumulation of ¹⁵N in reduced soluble and insoluble fractions was more prevalent in roots than in shoots. In the needles, the translocated nitrogen was mainly incorporated into the insoluble fraction. It is likely that most of the nitrogen from nitrate was transported from the roots to the aerial organs as organic nitrogen; however part of the upward nitrogen flux took place as nitrate ions.
An experiment in which an exposure for 24 h to 5 m M Na¹⁵NO₃ was followed by 13 days exposure to Na¹⁴NO₃ (pulse chase experiment) revealed a half time of about 1 day for depletion of root nitrate. A large part of this depletion was due to the loss of ¹⁵N-NO₃⁻ to the nutrient solution. The remaining pool of ¹⁵N-nitrate was partitioned between a metabolically inactive and an active pool. During the chase period, the simultaneous decrease of ¹⁵N-incorporation in the soluble N fraction and increase in the insoluble N fraction in different pine parts, particularly in the needles, suggested that protein synthesis occurred mostly in young tissues of the shoot and was the major sink of the newly absorbed ¹⁵N-NO₃.     

Protective Effects of Soluble Collagen during Ultraviolet-A Crosslinking on Enzyme-Mediated Corneal Ectatic Models

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.     

Increase in crosslinking of nonenzymatically glycosylated collagen induced by products of lipid peroxidation

The increase in crosslinking in normal and nonenzymatically glycosylated rat tail tendon collagen after treatment with decomposing lipid hydroperoxides was assessed by measuring the breaking time of tendons immersed in 7 M urea under a 3 g weight at 40 degrees C (thermal rupture time). The incubation of tendons in 200 mM glucose for 43 h at 40 degrees C increased thermal rupture times from 5.15 to 26.38 min, (P less than 0.001) with no significant corresponding increase in tendons incubated in buffer alone. After incubation of the glycosylated tendons in the presence of peroxidized linoleic/arachidonic acid vesicles for about 20 h, their thermal rupture time increased to 3360 min (P less than 0.001). The rupture time for normal tendons after the same treatment was 206 min. These apparent crosslinking increases cannot be fully accounted for by reactions involving malondialdehyde, as incubation of both glycosylated and normal tendons in enzymatically produced malondialdehyde resulted in a modest two- to threefold increase in thermal rupture time.     

Metal composition and solubility determine lung toxicity induced by residual oil fly ash collected from different sites within a power plant

Residual oil fly ash (ROFA) is a particulate pollutant comprised of soluble and insoluble metals and is produced by the combustion of fossil fuels. The objective was to examine the pulmonary responses to chemically distinct ROFA samples collected from either a precipitator or air heater within the same power plant. The collected ROFA samples were suspended in saline (total sample), incubated for 24 h at 37 degrees C, centrifuged, separated into soluble and insoluble fractions, and the metal composition was determined. In addition, electron spin resonance (ESR) was used to detect short-lived free radical intermediates produced by the ROFA samples and the different fractions. On day 0, Male Sprague-Dawley rats were intratracheally instilled with saline (vehicle control) or the ROFA samples (1 mg/100 g body wt). At day 1, bronchoalveolar lavage was performed, and lung inflammation was assessed. On day 3, additional rats that had been treated with ROFA were intratracheally inoculated with 5 x 10(5) Listeria monocytogenes, and pulmonary bacterial clearance was measured at days 6, 8, and 10. The precipitator ROFA was found to be more soluble and acidic with a significantly greater mass of each metal compared with the air heater ROFA. A prominent hydroxyl radical signal was measured for the total and soluble precipitator ROFA after the addition of H2O2, whereas the air heater ROFA and its fractions did not produce a signal. Precipitator ROFA induced a greater inflammatory response than air heater ROFA illustrated by a significant elevation in lung neutrophils. In addition, pulmonary clearance of L. monocytogenes was greatly diminished in the rats treated with the soluble and total precipitator ROFA samples. None of the air heater ROFA samples had an effect on lung bacterial clearance. In conclusion, precipitator ROFA, particularly the soluble fraction, generated a metal-dependent hydroxyl radical as measured by ESR and was shown to cause more inflammation and result in reduced lung defense against infection compared with air heater ROFA. These results are most likely due to differences in metal composition and solubility of the ROFA samples.     

Acetylation of wheat straw hemicellulose B in a new non-aqueous swelling system

The acetylation of wheat straw hemicellulose B was carried out in a homogeneous N,N-dimethylformamide and lithium chloride system with acetic anhydride using 4-dimethylaminopyridine as a catalyst. The degree of substitution of hemicellulose B acetates ranged between 0.59 and 1.25 as a function of experimental conditions. Under an optimum condition (85°C, 60 h), approximately 75% of the free hydroxyl groups in native hemicellulose B were acetylated. The molecular weight measurements (31,890–34,090 g mol⁻¹) showed a controllable degradation of hemicellulose B chains during the reactions at temperature 60–85°C and duration of 2–60 h. It was found that the thermal stability of the products was increased by chemical modification.     

Inhibition of collagen glycation and crosslinking in vitro by methanolic extracts of Finger millet (Eleusine coracana) and Kodo millet (Paspalum scrobiculatum)

The present investigation was carried out to study the effects of methanolic extracts of Finger millet (Eleusine coracana) and Kodo millet (Paspalum scrobiculatum) on glycation and crosslinking of collagen. Tail tendons obtained from rats weighing 200-225 g were incubated with glucose (50 mM) and 3 mg of extracts of the above millets in methanol under physiological conditions of temperature and pH for 10 days. Early glycation was estimated by phenol-sulfuric acid method and the crosslinking was assessed by pepsin digestion, cyanogen bromide peptide map and viscosity measurements. Tendon collagen incubated with glucose (50 mM) showed 65% solubility on pepsin treatment; poor resolution of bands in the cyanogen bromide peptide map, and intrinsic viscosity of 0.84 dl/g. The collagen incubated with Finger millet and Kodo millet extracts inhibited glycation; 89% and 92% solubility in pepsin; good resolution of bands in the cyanogen bromide peptide map and intrinsic viscosity of 0.46 and 0.58 dl/g respectively. The study implicates the potential usefulness of the above millets in protection against glycation and crosslinking of collagen.     

Difference of molecular association in two types of curdlan gel

The molecular association in a curdlan gel formed by neutralizing an alkaline solution of curdlan with carbon dioxide was compared with those in gels obtained by heating aqueous suspensions of curdlan at various temperatures.

The neutralized and 60°C-set preparations were soluble in 0·01 sodium hydroxide, whereas preparations set at above 90°C were soluble only in concentrations of sodium hydroxide above 1 . The absorption of Aniline blue or Congo red to the preparations decreased with an increase in the temperature of heat treatment and the adsorption to a gel heated at 120°C for 4 h was about 30% of that for the unheated neutralized gel. Seventy-three per cent of the heated preparation was resistant to treatment with 32% sulfuric acid at 32°C for 30 days, whereas none of the neutralized gel was resistant. An electron micrograph of the resistant part of the curdlan showed that it had a pseudocrystalline form. X-ray studies showed a much higher crystalline structure in the resistant part than in the preparation without heat treatment. The X-ray patterns were almost the same for preparations treated with 32% sulfuric acid or (1 → 3)-β-glucanase.     

Effect of beta-D-xylosides on collagen synthesis in cultured fibroblasts.

Cultured normal human skin fibroblasts were incubated with [¹⁴C]proline in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose. Formation of non-dialyzable hydroxyproline was used as a measure of collagen synthesis. Although total [¹⁴C]proline incorporation was similar in the two cultures, [¹⁴C]hydroxyproline formation was significantly decreased in the β-xyloside-treated cultures. Increasing the period of incubation increased the radioactivity of the insoluble collagen fraction in untreated fibroblasts, however, in β-xyloside-treated cultures no such increase was observed. In contrast to the decreased production of collagen, growth of cells in the presence of the β-xyloside induced the synthesis of high levels of soluble glycosaminoglycans as measured by ³⁵SO₄ incorporation into isolated polysaccharide.     

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g^-1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8-9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a K_m of 6.6 mM for H₂O₂ and V_max of 707 mM H₂O₂ min^-1 g^-1 wet wt. cells, and showed saturation kinetics at 50 mM H₂O₂. The cells were entrapped in calcium alginate and used for H₂O₂ degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.     

Malnutrition increases insoluble-to-soluble tubulin ratio and in vitro incorporation of 32ATP in rat cerebral cortex.

Wistar rats were fed a normal protein (25% casein) or an isoenergetic low protein (8% casein) diet from the day of birth to weaning on day 21. Litters were killed at weaning and cerebral cortex was removed. Tubulin was prepared by centrifugation at 100,000 g, 4°C, as described by Shelansky et al. [Proc. Natn. Acad. Sci. U.S.A. 70, 765–768 (1973)]. Cold-insoluble tubulin was recovered in the pellet (Pl) fraction and cold-soluble tubulin in the supernatant (Sl) fraction. Alpha and beta tubulin were quantified by electrophoretic and immunological methods in both fractions. Our results indicated that malnutrition enhanced the ratio of cold-insoluble-tubulin-to-cold-soluble-tubulin. Furthermore malnutrition induced an increased in vitro incorporation of ³²P into both soluble and insoluble tubulins. Although tubulin phosphorylation has been related to tubulin stability properties, we cannot unequivocally ascribe the increased insoluble/soluble tubulin ratio with malnutrition to increased in vitro incorporation of ³²P.