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Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.

N-terminally truncated amyloid-beta (Abeta) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbetaN3(pE)-40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Abeta accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Abeta1-40, Abeta1-42, AbetaN3(pE)-40 and AbetaN3(pE)-42. Our data show that fibre morphology of Abeta peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AbetaN3(pE)-40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbetaN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbetaN3(pE)-40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while full-length peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance beta-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.

The nuclear matrix-intermediate filament complex (NM-IF) is a protein scaffold which spans the whole cell, and several lines of evidence suggest that this structural frame represents also a functional unit, which could be involved in the epigenetic control of cancer development. Here we report the characterization by high resolution two-dimensional gel electrophoresis and Western blot analysis of the NM-IF complex isolated from prostate cancer (PCa); tumor-associated proteins were identified by comparing the electrophoretic patterns with those of normal human prostate (NHP). Extensive changes in the expression of both the NM and IF proteins occur; they are, however, related in a different way to tumor progression. Poorly differentiated PCa (Gleason score 8-9) shows a strong down regulation of several constitutive cytokeratins (CKs 8, 18, and 19); their expression significantly (P < 0.05) decreases with respect to both NHP and benign prostatic hyperplasia (BPH) and, more interestingly, also with respect to moderately (Gleason score 6-7) and well (Gleason score 4-5) differentiated tumors. Moreover, we have identified a tumor-associated species which is present in all of the tumors examined, systematically absent in NHP and occurs only in a few samples of BPH; this polypeptide, of M(r) 48,000 and pI 6.0, represent a proteolytic fragment of CK8. At variance with these continuing alterations in the expression, the NM proteins undergo stepwise changes correlating with the level of differentiation. The development of less differentiated tumors is characterized by the appearance of several new proteins and by the decrease in the expression of others. Six proteins were found to be expressed with a frequency equal to one in poorly differentiated tumor, namely in all the samples of tumor examined, while in moderately and well differentiated tumors the frequency is less than one, and decreases with increasing the level of differentiation. When tumors of increasing Gleason score are compared with NHP a dramatic increase in the complexity of the protein patterns is observed, indicating that tumor dedifferentiation results in a considerable increase in the phenotypic diversity. These results suggest that tumor progression can be characterized using an appropriate subset of tumor-associated NM proteins., (Copyright 2000 Wiley-Liss, Inc.)

Using differential scanning calorimetry in combination with pulsed field gel electrophoresis, we relate here the changes in the thermal profile of rat liver nuclei induced by very mild digestion of chromatin by endogenous nuclease with the chain length distribution of the DNA fragments. The enthalpy of the endotherm at 106 degrees C, which reflects the denaturation of the heterochromatic domains, decreases dramatically after the induction of a very small number of double-strand breaks per chromosome; the thermal transition disappears when the loops have undergone on average one DNA chain scission event. Quantitative analysis of the experimental data shows that the loop behaves like a topologically isolated domain. Also discussed is the process of heterochromatin formation, which occurs according to an all-or-none mechanism. In the presence of spermine, a strong condensation agent, only the loops that have undergone one break are able to refold, in confirmation of the extremely cooperative nature of the transition. Furthermore, our results suggest a relationship between the states that give rise to the endotherms at 90 degrees C and 106 degrees C and the morphologies referred to as class II and class III in a previous physicochemical study of the folding of chromatin fragments (Widom, 1986. J. Mol. Biol. 190:411-424) and support the view that the overall process of condensation follows a sequential (two-step) pathway.

Chromatin condensation and DNA cleavage at internucleosomal sites have been recognized early as hallmarks of apoptosis, and it has been suggested that extensive DNA chain scission could directly result in the formation of dense chromatin bodies. Here we have shown that no causal relationship exists between DNA degradation and chromatin condensation in glucocorticoid-induced thymocyte apoptosis. The chromatin rearrangement occurred independent of as well as prior to DNA cleavage and involved a specific conformational change at the nucleosome level. In the early stages of the process, the core particles appeared to be tightly packed face-to-face in smooth 11-nm filaments that progressively folded to generate a closely woven network. The network finally collapsed, producing dense apoptotic bodies. Since trypsin digestion relaxed condensed chromatin and histone H4 underwent appreciable deacetylation in the apoptotic cell, we suggest that changes in the DNA-histone interactions represented a major modulating factor of condensation.

We have characterized the changes in composition of the nuclear matrix-intermediate filament complex (NM-IF) isolated from prostate cancer (PCa), compared with benign prostatic hyperplasia (BPH). We prepared the NM-IF from ten patients undergoing radical retropubic prostectomy; the benign hyperplastic tissue was obtained from the prostate lobe contralateral to the cancer zone. Several quantitative and qualitative changes have been identified. Three new proteins of molecular weight 48, 47 and 29 kDa and isoelectric point 6.0, 4.9 and 6.4, respectively, were detected in PCa, referred to here as P8, P5 and NM-1, P8 was present in all ten of the tumors examined, P5 was expressed in 9/10 PCa; conversely, they were present in only one and two BPH, respectively; NM-1 was found in eight tumors out of nine and never in BPH. These proteins are expressed in moderately differentiated malignant cells, suggesting that the proteins of the NM-IF complex can be interesting biomarkers for prostate cancer. Immunoblot analysis shows that P8 and P5 proteins belong to the IF superfamily. This observation, taken together with previous data obtained by our and other groups, suggests that the characterization of NM-IF protein changes could also shed light on mechanistic aspects of cancer progression.

In a previous paper (Barboro et al., 1993, Biophys. J. 65, 1690-1699) we have shown that cancer development in the resistant hepatocyte model of Solt and Farber is characterized by the progressive unfolding of the higher-order structure of chromatin. A possible functional role of decondensation phenomena in cell transformation cannot be ruled out. Genetic activation involves the relaxation of the superstructure of chromatin, which may be, at least in part, modulated by its interaction with the nuclear matrix. Moreover, recent observations suggest that gene expression can be stimulated by alterations in the organization of the cytoskeleton. Therefore, we have characterized the changes in composition that the nuclear matrix-intermediate filament complex undergoes during the evolution of rat hepatocyte nodules. Dramatic changes in the expression of both the nuclear matrix and intermediate filament proteins occur during transformation; they are, however, related in a different way to the stages of carcinogenesis. Several new nuclear matrix proteins appear in early nodules, isolated 9 weeks after initiation. The subsequent evolution of persistent nodules is also characterized by discrete changes in the composition. Thus, the new synthesis of nuclear matrix proteins reflects the emergence of successive cellular populations, in line with the recent finding that a subset of components of the nuclear matrix is cell type-specific. In contrast, intermediate filament proteins undergo continuing changes. A new keratin with apparent molecular weight of 39 kDa, analogous to human keratin 19, appears in early nodules, and its expression steadily increases up to the 32nd week from initiation; at the same time, the amount of the proteolytic fragments of keratins A and D increases sharply. These findings suggest that the inappropriate expression of keratin 19 may be involved in the epigenetic activation of new cellular programs, through the rearrangement of the cytoskeleton which in turn may perturb nuclear matrix function.

In a series of related papers, we have recently presented the results of a thermodynamic approach to the conformational transitions of bulk chromatin induced in vitro by different structure-perturbing agents, such as the intercalating dye ethidium bromide or the ionic strength. In all these studies, we took advantage of the capability of differential scanning calorimetry to detect the changes in the melting behavior of the structural domains of chromatin (the linker and the core particle) associated with the order-disorder transitions. This technique also revealed that the higher-order structure undergoes a catastrophic decondensation process in the course of the transformation of rat hepatocytes as well as of cultured cells. Therefore, several questions arose concerning the biological function (if any) of the changes in the degree of condensation of bulk chromatin, as well as the mechanism of transition and the nature of the modulating agents. In this paper, we report a thermodynamic analysis of the reconstitution of H1-depleted calf thymus chromatin with the purpose of establishing (1) the binding mode of H1 and (2) the energetics and cooperativity of the transition from the unfolded to the condensed state. When H1 is progressively extracted from calf thymus nuclei by high-salt treatment, the endotherm at 107 degrees C, characteristic of the core particles interacting within condensed domains, converts into the thermal transition at 90 degrees C, resulting from the denaturation of noninteracting core particles. Binding of H1 fully restores the thermal profile of native chromatin. Analysis of H1 association shows that binding occurs at independent sites with KA = (3.67 +/- 0.60) x 10(4) M-1 and each site comprising 180 +/- 10 bp. The experimental dependence of the fraction of condensed chromatin on R, the moles of bound H1 per nucleosome mole, was compared with a simple thermodynamic model for the conformational change. This analysis yields a value of -5 kcal per nucleosome mole for the interaction free energy of nucleosomes within the ordered state. The process of condensation, is not, however, a highly cooperative (all-or-none) one, as expected from a consideration of the solenoidal model for the 30 nm fiber. Rather, nucleation of the helical state involves the face-to-face interaction between consecutive core particles, and the growth is largely determined by the mergence and rearrangement of neighboring clusters of helically arrayed nucleosomes.

In chronic renal failure hypertriglyceridemia is a well-known complication that persists during hemodialysis treatment: it may be one of the major risk factor for cardiovascular death in these patients. We studied the effects of two lipid lowering drugs; simvastatin (20 mg/day for six months) and probucol (500 mg/day for six months), on lipid profile in 12 hemodialysis patients. Simvastatin therapy reduced plasma total cholesterol by 26% (p > 0.002), LDL-cholesterol by 36% (p > 0.002) and triglycerides by 28% (p > 0.05): plasma HDL-cholesterol and apo-A were raised, but not significantly. Probucol therapy decreased plasma triglycerides by 38% (p > 0.05), total cholesterol by 15% and LDL-cholesterol by 19%: plasma HDL-cholesterol and apo-A were decreased but not significantly. No side effects occurred with either drug. These data suggest that in hemodialysis patients both simvastatin and probucol profoundly affect the lipid profile, as well as the triglyceride levels.

Chronic renal failure is characterized by abnormalities in glucose metabolism. In fact there are present a normal fasting plasma glucose level )or mild hyperglycemia) in the presence of hyperinsulinemia, blunted decrease in the plasma glucose concentration in response to exogenous insulin administration, and diminished effect of intravenous insulin on glucose uptake in forearm perfusion studies. The glucose intolerance is not the result of reduced insulin secretion, or circulating insulin antagonists, and does not correlate with the coexisting metabolic acidosis. Glucose intolerance exists because the peripheral insulin-sensitive tissue (muscle, adipose tissue, liver) of the patients with chronic renal failure are insulin resistant. However there are two subgroups of uremic patients with regard to glucose tolerance: about half of uremic patients can augment their insulin secretion sufficiently to maintain normal glucose tolerance despite glucose intolerance. In the other half, insulin secretion following glucose loads is not different from normal values, so that glucose intolerance results. The cause of the peripheral insulin resistance remain unclear. Besides deranged renal function can result in the development of hypoglycemia. The most important predisposing mechanism to hypoglycemia is diminished glucose availability due to substrate limitation; the second important mechanism (alcohol, insulin, propranolol, etc.). Finally, in chronic renal failure persistent hyperinsulinemia can contribute hyperlipemia and to high incidence of cardiovascular disease.

Using differential scanning calorimetry and complementary ultrastructural observations, we have characterized the status of chromatin during the transformation of rat hepatocytes in the resistant hepatocyte model of Solt and Farber (1976. Nature (Lond.). 263:701-703). Differential scanning calorimetry affords a measure of the degree of condensation of chromatin in situ and has therefore been used in this work for the purpose of establishing the nature of the structural changes associated with the emergence of successive cellular populations. Since the resistant hepatocyte model generates a series of synchronous phenotypic changes, it was possible to determine unambiguously the content of heterochromatin at each step of the process. The higher-order structure undergoes a partial relaxation in early developing nodules, isolated 16 weeks after initiation; the thermal transition at 90 degrees C, which is characteristic of noninteracting core particles, increases with respect to control hepatocytes. Dramatic changes occur in persistent (46-week) nodules. The 90 degrees C endotherm dominates the thermogram, while the transition at 107 degrees C, corresponding to the denaturation of the core particle packaged within the heterochromatic domains, disappears. The complete loss of the higher-order structure at this stage of transformation has been further verified by ultrastructural observations on thin nuclear sections. Ten-nm filaments, having a beaded appearance, are scattered throughout the nucleoplasm and clearly result from the decondensation of 30-nm-thick fibers. This catastrophic relaxation process cannot be related to an effective increase in gene activity. Rather, our observations suggest that during transformation chromatin is in a state of high transcriptional competence associated with the alert of general cellular programs. This view is consistent with the finding that in persistent nodules the DNA is extensively hypomethylated with respect to normal liver.

The occurrence of malnutrition in patients with chronic renal failure has been well documented, mostly in patients undergoing regular dialysis: these patients often suffer from protein-calorie malnutrition, but more seldom there are disturbances to be due to deficit of 1,25 dihydroxyvitamin D3, folic acid, vitamin B6, iron, zinc and L-carnitine, mostly in patients who do not get adequate supplementation. There are several causes for protein-calorie malnutrition in chronic renal insufficiency. These include uremia per se, altered hormonal milieu, abnormal amino acid metabolism due to uremia or to loss of metabolically active renal tissue. Dialysis treatment improves some of these abnormalities, but introduces others. On the other hand, it is well established that morbidity and mortality in chronic renal failure are influenced by their nutritional status: therefore it is important to know the factors and the mechanisms that cause malnutrition for its prevention, recognition and correction.

The efficacy, tolerability and safety of simvastatin in the treatment of hyperlipemia in uremic patients undergoing hemodialysis were evaluated in 6 patients; a further 6 patients were treated with placebo and represented the control group. All patients treated completed the study. No clinical or laboratory side-effects were noted during the entire period of observation. Simvastatin caused a significant 26% reduction in total cholesterol, a 36% reduction in LDL cholesterol and a 28% reduction in triglycerides; HDL cholesterol and Apolipoprotein A increased by 19% and 12% respectively.

Carnitine plays an important role in regulating the flow of energetic substrates and their balance through cellular membranes. Usually in chronic renal failure, increased plasma levels of carnitine are found. In early hemodialysis a slow decrease of the muscle concentrations of carnitine is found, followed by a decrease of plasma concentrations long after substitutive treatment. Several signs and symptoms are attributable to carnitine deficiency. It is reported that these changes regress with suitable supply of intravenous carnitine after dialysis end for 15-30 days, and subsequently with chronic administration in the dialysis fluid.

We present a detailed thermodynamic investigation of the conformational transitions of chromatin in calf thymus nuclei. Differential scanning calorimetry was used as the leading method, in combination with infrared spectroscopy, electron microscopy, and techniques for the molecular characterization of chromatin components. The conformational transitions were induced by changes in the counterion concentration. In this way, it was possible to discriminate between the interactions responsible for the folding of the higher order structure and for the coiling of nucleosomal DNA. Our experiments confirm that the denaturation of nuclear chromatin at physiological ionic strength occurs at the level of discrete structural domains, the linker and the core particle, and we were able to rule out that the actual denaturation pattern might be determined by dissociation of the nucleohistone complex and successive migration of free histones toward native regions, as recently suggested. The sequence of the denaturation events is (1) the conformational change of the histone complement at 66 degrees C, (2) the unstacking of the linker DNA at 74 degrees C, and (3) the unstacking of the core particle DNA, that can be observed either at 90 or at 107 degrees C, depending on the degree of condensation of chromatin. Nuclear chromatin unfolds in low-salt buffers, and can be refolded by increasing the ionic strength, in accordance with the well-known behavior of short fragments. The process is athermal, therefore showing that the stability of the higher order structure depends on electrostatic interactions. The transition between the folded conformation and the unfolded one proceeds through an intermediate condensation state, revealed by an endotherm at 101 degrees C. The analysis of the thermodynamic parameters of denaturation of the polynucleosomal chain demonstrates that the wrapping of the DNA around the histone octamer involves a large energy change. The most striking observation concerns the linker segment, which melts a few degrees below the peak temperature of naked DNA. This finding is in line with previous thermal denaturation investigations on isolated chromatin at low ionic strength, and suggests that a progressive destabilization of the linker occurs in the course of the salt-induced coiling of DNA in the nucleosome.

Non-destructive electron microscopy of native chromatin from rat liver nuclei reveals that the 30 nm fibre is formed of four 11 nm nucleofilaments, arranged in a coiled-coil (or rope-like) conformation. At low ionic strength, native fibres show an alternating pattern of compact and unwound regions. Freeze-etching experiments carried out on the same nuclei are compatible with the existence of periodic attachments of the fibres to the nuclear envelope near the pores in a regular, drapery-like fashion. For the first time, computer image analysis has been applied to electron micrographs of giant chromatin fibres and a few essential geometrical parameters characterizing the conformation of the higher-order structures have been determined. No significant difference has been found between calf thymus and rat liver chromatin.

DNA chain scission, induced both in vitro and in vivo by various agents, is an event of great biological relevance. The damage is currently evaluated by empirical membrane separation techniques; the results are quite reproducible and the sensitivity higher than 1 single strand break per 10(9) Daltons. We outline a simple theory of the filtration of coiled macrosolutes, having a random size distribution, through porous membranes, considered as being in quasi-steady flow. The basic transport equation Jj = cj (1 - sigma)Jv is solved by considering that the value of sigma j, the reflection coefficient of component j, (1 less than or equal to j less than or equal to N), is given by (1 - KjRj), where Kj is the partition constant between pore and solution, a function of the conformational entropy loss of the coil, and Rj accounts for the frictional force experienced by a particle moving along the pore. The problem of evaluating the volume Vs filled up with solute has been approached according to a simplified theory of the excluded volume for flexible polymers; the result is Vs = sigma nj4/3 pi(rGj)3 where rGj is the jth radius of gyration. The solution of the resulting set of N differential equations gives nj, the number of molecules of component j remaining on the filter, as a function of the elution volume V. The theory demonstrates that the process is governed by the average dimensions of the coil, so affording a universal calibration of filter elution methods, in excellent agreement with the experiments.

Different forms of renin have been purified from bull kidney by combined gel filtration, affinity chromatography and ion-exchange chromatography. The specific activity of the enzyme was determined by a biochemical method of synthetic substrate and by radioimmunoassay on both synthetic and natural substrates; molecular characterization was carried out by molecular weight determinations, polyacrylamide gel electrophoresis, isoelectric focusing, amino acid analysis and optical rotatory dispersion. Three forms (renin C, D, E) are distinct on the basis of amino acid composition and chromatographic behavior, while possessing the same molecular weight, and displaying only minor differences in specific activity, alpha-helix content and isoelectric point; the occurrence of a group of renin isoenzymes may be suggested. Another form (A) has a lower specific activity and a higher molecular weight (57 000) compared with C, D and E and further differs markedly in chromatographic behavior, amino acid composition, alpha-helix content and isoelectric point, as well as in substrate specificity; it may be regarded as a pseudorenin. The fifth form (B) possesses the highest specific activity and does not correspond to a single molecular form; the presence of two components of different molecular weight (27 000 and 46 000 respectively) has been established both by polyacrylamide gel electrophoresis and isoelectric focusing.

Extremely large domains of the genome of resting cells (calf thymus) have been visualized in the electron microscope by combining mild extraction procedures with a non-artifactual method of mounting the sample (the phospholipid monolayer technique). The observed chromatin strands, free from distortion, reach contour lengths up to 60 micrometers. After lysis of the nuclei, four classes of fibres may be identified on the basis of their diameters (30, 24, 18 and 11 nm, respectively). The morphology of giant chromatin strands is strikingly regular; long trains of equally sized, arc-shaped segments are observed, their length being, in many cases, multiples of a fixed value. The inflection points delimiting contiguous segments are often associated with laminar fragments of the nuclear envelope or, less frequently, linked to fibrillar elements. It appears that higher-order structures of chromatin in resting cells conform, to a large extent, to a so called 'drapery-like' mode, according to which a continuous strand runs between contiguous anchorage sites placed on the nuclear envelope. Because of the presence of regularly spaced inflection points, this organization is much more ordered than expected. Spontaneous unwinding of the fibres at low ionic strength, limited nuclease digestion, and relaxation in the presence of ethidium bromide, have been used as probes of the conformation. All these experiments rule out its identification with a single-strand helix. The final ordered state is attained by folding the basic 11 nm strand and by winding up this configuration on itself. This leads to a coiled-coil or 'rope-like' model. The 11 nm strand is 'punctuated' by sharp kinks. Roughly, it may be assimilated to a chain of semirigid, freely joined elements. As a consequence, local flexibility is greatly enhanced, so allowing the assembly mode described.

A light scattering method, together with complementary electron microscopy observations, was designed to investigate the self-assembly of fibrin. Calcium-free monomer was used, and clot reconstitution was carried out in solvents corresponding to limit interaction energies of the protein with the medium. The self-assembly process, under physiological conditions, conforms to the following sequence of events. 1) A fast polymerization step leading to linear aggregates. 2) Fiber growth; at this stage onset of the network occurs. 3) Gelation (clot formation). Bound calcium was found to be structurally required for gelation. Its removal results in the formation of thick fibers, which are unable to clot. Evidence is reported favouring our previous hypothesis (Conio et al., 1976) on the onset of the network: branching of linear aggregates is a prerequisite for clotting. The occurrence of a crystallization process, which overlaps to fiber growth, is demonstrated in this paper for the first time. Its dependence on solvent-protein interactions is analyzed. Our results suggest that fibrin monomer is to some extent a flexible molecule. Both flexibility and crystallization may play a functional role in the clotting process in vivo.

Differential scanning calorimetry of chromatin isolated from rat liver cells revealed three discrete thermal transitions whose temperatures and melting enthalpies depend on ionic strength in the range 0 to 600 millimolar NaCl. Intact nuclei showed a fourth thermal transition at a lower temperature and different melting enthalpies for the other three transitions still present at temperatures similar to those obtained in isolated chromatin. The data are discussed in terms of the tertiary, quaternary, and quinternary structures of chromatin DNA.

A good deal of information on the thermodynamic properties of chromatin was derived in the last few years from optical melting experiments. The structural domains of the polynucleosomal chain, the linker, and the core particle denature as independent units. The differential scanning calorimetry profile of isolated chromatin is made up of three endotherms, at approximately 74, 90, and 107 degrees C, having an almost Gaussian shape. Previous work on this matter, however, was mainly concerned with the dependence of the transition enthalpy on external parameters, such as the ionic strength, or with the melting of nuclei from different sources. In this paper we report the structural assignment of the transitions of rat liver nuclei, observed at 58, 66, 75, 92, and 107 degrees C. They are representative of the quiescent state of the cell. The strategy adopted in this work builds on the method developed for the investigation of complex biological macromolecules. The heat absorption profile of the nucleus was related to the denaturation of isolated nuclear components; electron microscopy and electrophoretic techniques were used for their morphological and molecular characterization. The digestion of chromatin by endogenous nuclease mimics perfectly the decondensation of the higher order structure and represented the source of several misinterpretations. This point was carefully examined in order to define unambiguously the thermal profile of native nuclei. The low-temperature transitions, centered around 58 and 66 degrees C, arise from the melting of scaffolding structures and of the proteins associated with heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 250 WORDS)