Genetic studies of human apolipoproteins. III. Polymorphism of apolipoprotein C-II

Using a simple and rapid one-dimensional isoelectric focusing technique followed by immunoblotting, we have detected genetic polymorphism of human apolipoprotein C-II (APO C-II) in normal unfractionated plasma samples of individuals of black ancestry. Two common autosomal codominantly expressed alleles, designated APO C-II*1 and APO C-II*2, at the APO C-II structural locus have been observed with frequencies of 0.975 and 0.025 in US blacks and 0.943 and 0.049 in Nigerian blacks. In addition, the gene product of a rare allele designated APO C-II*3 was observed in a single Nigerian black. Apart from a single example of an APO C-II 2-1 phenotype in plasma samples from 187 whites, which was electrophoretically identical to the 2-1 phenotype observed in blacks, it appears that APO C-II*2 is a unique black marker of potential importance in anthropogenetic and atherosclerosis studies.     

Genetic studies of human apolipoproteins. IV. Structural heterogeneity of apolipoprotein H (beta 2-glycoprotein I).

Human beta 2-glycoprotein I has recently been identified as a component of several human plasma lipoprotein fractions and therefore termed as apolipoprotein H. Its metabolic function in lipid metabolism is not known with certainty, though it may be involved in very-low-density-lipoprotein metabolism. Previously, inherited quantitative variation in beta 2-glycoprotein I has been suggested in man. In this investigation, we document the evidence of genetically determined structural polymorphism of apolipoprotein H or beta 2-glycoprotein I by using thin-layer polyacrylamide isoelectric focusing gels followed by immunological identification by double antibody staining. The apolipoprotein H structural locus is characterized by the occurrence of three common alleles in U.S. whites and blacks. The frequency distributions of the three alleles designated APO H1, APO H2, and APO H3 are .059, .882, and .059 in whites and .017, .902, and .068 in blacks, respectively. In addition, the gene product of a fourth allele, APO H4, has been observed at polymorphic frequency in black individuals and may represent a black marker variant. Family data confirm the hypothesis of four alleles at a single APO H gene locus with an autosomal codominant pattern of inheritance.     

Genetic studies of human apolipoproteins

Summary Apolipoprotein H (APO H) has recently been identified as a structural component of chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Although the precise metabolic function of APO H in lipid metabolism is not certain, it has been suggested that APO H may be involved in triglyceride (TG) metabolism. In addition to the previously described quantitative polymorphism, we have recently detected a common qualitative polymorphism at the APO H structural locus. To test the role of APO H genetic variation in determining lipoprotein and lipid levels, we have estimated the allelic effects of APO H variation on TG, VLDL, LDL, HDL, HDL3, and total cholesterol on 356 Nigerian blacks(189 males, 167 females). While no significant effect of phenotype was observed on lipoprotein levels, the effect of interaction between phenotype and gender was significant. Therefore, data on males and females were analyzed separately using analysis of variance after adjusting for age and body mass index. Logarithmic transformation of pertinent variables was done to bring the distribution of the variables closer to normality. A statistically significant effect of phenotype was observed on triglyceride levels in females only (P<0.05). Further analysis of this phenotypic effect revealed that it is due to the impact of the APO H ^* 3 allele, which raises triglycerides by 9.92 mg/dl as compared to the common allele, APO H ^* 2. These findings are in accordance with the postulated role of APO H in triglyceride metabolism. On the basis of its sex-specific effect, we propose a hypothesis that may explain the combined influence of the quantitative and qualitative polymorphisms at the APO H locus on triglyceride levels in females.     

Genetic polymorphism of human plasma apolipoprotein A-IV is due to nucleotide substitutions in the apolipoprotein A-IV gene

Genetic polymorphism of human plasma apolipoprotein A-IV has been detected by isoelectric focusing techniques followed by immunoblotting. The molecular basis for this apoA-IV polymorphism has been elucidated. Analysis of the protein coding sequences of the apoA-IV alleles 1 and 2 revealed a single G to T substitution in the apoA-IV-2 allele. The point mutation, occurring in a region highly conserved among the mouse, rat, and human A-IV apolipoproteins, converts the glutamine at position 360 of the mature protein to a histidine. This amino acid substitution adds one positive charge unit to the apoA-IV-1 isoprotein (pI 4.97) thus creating the more basic apoA-IV-2 isoprotein (pI 5.02). Computer analysis of the apoA-IV-2 allele revealed that the single G to T substitution results in the loss of a BbvI and a Fnu4HI restriction enzyme site and in the formation of a new restriction site for the enzyme SfaNI. Protein primary and secondary structure predictions were largely unaffected by this amino acid exchange. These results on the structure of the apoA-IV-1 and apoA-IV-2 alleles suggest that the three other rare isoproteins (apoA-IV-0, apoA-IV-3, and apoA-IV-4) are also due to nucleotide and subsequent amino acid substitutions in the apoA-IV sequence.     

Genetic studies of human apolipoproteins. XVII: Population genetics of apolipoprotein polymorphisms in American Samoa

Variation in human apolipoprotein genes is a major source of phenotypic differences in human lipid metabolism. Data regarding genetic variation at apolipoprotein loci in various populations are only beginning to accumulate, and they suggest that different populations vary widely in distribution of apolipoprotein alleles. Using isoelectric focusing-immunoblotting techniques, we screened 67 serum samples from self-identified Samoan residents of American Samoa to investigate structural variation at six apolipoprotein loci: A-I, A-II, A-IV, C-II, E, and H. The APO A-I, A-II, and C-II loci were found to be monomorphic by isoelectrical focusing. In Samoans, the common three-allele polymorphism was observed for APO E, with no striking differences in frequencies from Caucasian populations. The three common alleles of the APO H locus also were identified; however, frequencies of the less common alleles (APO H*I and APO H*3) were different from those observed elsewhere for Caucasians.     

Genetic studies of human apolipoproteins. V. A novel rapid procedure to screen apolipoprotein E polymorphism

A simple and new method has been developed to detect apolipoprotein E polymorphism directly from plasma or serum without prior ultracentrifugation and delipidation. The method combines the use of dialyzed plasma or serum samples with or without neuraminidase treatment followed by monodimensional isoelectric focusing in simple or 3 M urea gels at a constant low power and progressively increasing voltage over a 3-hr period, and finally protein blotting to a nitrocellulose membrane. Apolipoprotein E phenotypes are identified immunologically using a double antibody reaction, the primary antibody being a monospecific, polyclonal goat anti-apolipoprotein E, and the secondary antibody being a rabbit anti-goat IgG conjugated with alkaline phosphatase. The method was employed to screen apolipoprotein E polymorphism in two white populations in the United States. The frequency values are comparable to those reported previously by other investigators using conventional detection methods. The procedure is simple, accurate, suitable for large scale epidemiologic, clinical, and genetic studies.     

Genetic studies of human apolipoproteins. XV. An overview of IEF immunoblotting methods to screen apolipoprotein polymorphisms

Genetically determined structural variation in the gene products of various apolipoproteins plays a significant role in modulating lipid levels in the population at large. However, due to tedious and cumbersome experimental problems involved, the detailed characterization of this genetic variation has been limited. The recent development of simple and sensitive isoelectric focusing and immunoblotting methods has circumvented these technically associated problems to a large extent, and this has allowed us to expand the genetic data on various apolipoproteins to previously uncharacterized populations. We have reviewed here these isoelectric focusing and immunoblotting methods. A comprehensive listing of allele frequencies has also been given for the polymorphic apolipoproteins.     

Genetic studies of human apolipoproteins. VII. Population distribution of polymorphisms of apolipoproteins A-I, A-II, A-IV, C-II, E, and H in Nigeria.

Familial porphyria cutanea tarda (PCT) results from a generalized deficiency of uroporphyrinogen decarboxylase (URO-D) activity. The molecular defect responsible for this disorder has not been characterized. To determine whether decreased levels of URO-D mRNA are responsible for subnormal URO-D activity, steady-state levels of URO-D mRNA in lymphoblastoid cells were determined. Northern blots were hybridized with a URO-D cDNA probe and quantified by densitometry. No difference in the levels of URO-D mRNA was detected between affected individuals and their normal relatives. Thus, the deficiency of URO-D activity in two familial PCT pedigrees characterized here does not arise from a deficiency of URO-D mRNA.     

Genetic studies of human apolipoproteins. XX. Genetic polymorphism of apolipoprotein J and its impact on quantitative lipid traits in normolipidemic subjects.

Apolipoprotein J (apo J) is a newly identified member of a growing family of proteins associated with various lipoprotein particles. Apo J is a glycoprotein which exists in the plasma associated with high-density lipoprotein subfractions which also contain apo A-I and cholesteryl ester transfer protein (CETP). We have investigated the possible existence of genetic polymorphism at the apo J structural locus and have evaluated its role in lipid metabolism. By employing isoelectric focusing and immunoblotting techniques, we have screened plasma or serum samples from six population groups: U.S. whites, Amerindians, Eskimos, New Guineans, U.S. blacks, and Nigerian blacks. Apo J revealed a common two-allele polymorphism only in populations with African ancestry and was found to be monomorphic in all other population groups tested. The genetic basis of the two alleles designated--APO J*1 and APO J*2, at a single structural locus, apo J-- was confirmed in a large number of segregating families. In the U.S. blacks, the frequencies of the APO J*1 and APO J*2 alleles were .76 and .24, respectively, and in the Nigerian blacks these values were .72 and .28, respectively. In addition, a single example of a rare allele designated APO J*3 was also encountered in the U.S. black sample. In Nigerian blacks, the apo J polymorphism's impact on seven quantitative lipid traits--total cholesterol, LDL-cholesterol, HDL-cholesterol, HDL3-cholesterol, HDL2-cholesterol, VLDL-cholesterol, and triglycerides--was investigated. No significant impact of the apo J polymorphism was observed for any of these lipid traits.     

Structure of human apolipoprotein A-IV: a distinct domain architecture among exchangeable apolipoproteins with potential functional implications

Apolipoprotein A-IV (apoA-IV) is an exchangeable apolipoprotein that shares many functional similarities with related apolipoproteins such as apoE and apoA-I but has also been implicated as a circulating satiety factor. However, despite the fact that it contains many predicted amphipathic alpha-helical domains, relatively little is known about its tertiary structure. We hypothesized that apoA-IV exhibits a characteristic functional domain organization that has been proposed to define apoE and apoA-I. To test this, we created truncation mutants in a bacterial system that deleted amino acids from either the N- or C-terminal ends of human apoA-IV. We found that apoA-IV was less stable than apoA-I but was more highly organized in terms of its cooperativity of unfolding. Deletion of the extreme N and C termini of apoA-IV did not significantly affect the cooperativity of unfolding, but deletions past amino acid 333 on the C terminus or amino acid 61 on the N terminus had major destabilizing effects. Functionally, apoA-IV was less efficient than apoA-I at clearing multilamellar phospholipid liposomes and promoting ATP-binding cassette transporter A1-mediated cholesterol efflux. However, deletion of a C-terminal region of apoA-IV, which is devoid of predicted amphipathic alpha helices (amino acids 333-376) stimulated both of these activities dramatically. We conclude that the amphipathic alpha helices in apoA-IV form a single, large domain that may be similar to the N-terminal helical bundle domains of apoA-I and apoE but that apoA-IV lacks the C-terminal lipid-binding and cholesterol efflux-promoting domain present in these apolipoproteins. In fact, the C terminus of apoA-IV appears to reduce the ability of apoA-IV to interact with lipids and promote cholesterol efflux. This indicates that, although apoA-IV may have evolved from gene duplication events of ancestral apolipoproteins and shares the basic amphipathic helical building blocks, the overall localization of functional domains within the sequence is quite different from apoA-I and apoE.     

Distribution of apolipoprotein A-IV in human plasma

Human apoA-IV was purified from delipidated urinary chylomicrons. Monospecific antibodies were raised in rabbits and used to develop a double antibody radioimmunoassay (RIA). Displacement of 125I-labeled apoA-IV by plasma or purified chylomicron apoA-IV resulted in parallel displacement curves, indicating that apoA-IV from both sources share common antigenic determinants. The apoA-IV level in plasma from normal healthy fasting male subjects (n = 5) was 37.4 +/- 4.0 mg/dl, while fat-feeding increased the level to 49.1 +/- 7.9 mg/dl (P less than 0.05) at 4 hr. The apoA-IV level in plasma from abetalipoproteinemic fasting subjects was 13.7 +/- 3.1 mg/dl (n = 5). Plasma from a single fasting Tangier subject showed a reduced apoA-IV level of 21.1 mg/dl. The distribution of apoA-IV in fasting and postprandial plasma was determined by 6% agarose gel chromatography. Fifteen to 25% of plasma apoA-IV eluted in the region of plasma high density lipoprotein (HDL), with the remainder eluting in subsequent column fractions. In abetalipoproteinemic plasma this HDL fraction is reduced and lacks apoA-IV, suggesting that at least some of the apoA-IV on these particles is normally derived from triglyceride-rich lipoproteins. Lipemic plasma from a fat-fed subject showed a small rise (3%) in chylomicron-associated apoA-IV. Gel-filtered HDL and subsequent apoA-IV-containing fractions were subjected to 4-30% polyacrylamide gradient gel electrophoresis (4/30 GGE), and apoA-IV was identified by immunolocalization following transfer of proteins to nitrocellulose paper. In normal plasma apoA-IV was localized throughout all HDL fractions. In addition, normal plasma contained apoA-IV localized in a small particle (diameter 7.8-8.0 nm). This particle also contained apoA-I and lipid. A markedly elevated saturated to unsaturated cholesteryl ester ratio was present in gel-filtered plasma fractions containing small HDL, suggesting an intracellular origin of these particles. In abetalipoproteinemic plasma apoA-IV was absent from all HDL fractions except for the small HDL particles, suggesting that they are not derived from the surface of triglyceride-rich particles. All plasmas contained free apoA-IV. In contrast to gel-filtered plasma, lipoprotein subfractions of fasted normal plasma prepared in the ultracentrifuge primarily contained apoA-IV in the d greater than 1.26 g/ml fraction, suggesting an artifactual redistribution of the apolipoprotein during centrifugation. Overall, these data suggest that apoA-IV secretion into plasma is increased with fat feeding, and that apoA-IV normally exists as both a free apolipoprotein and in association with HDL particles.     

The primary structure of human apolipoprotein A-IV

Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded.     

Genetic studies of human apolipoproteins. XI. The effect of the apolipoprotein C-II polymorphism on lipoprotein levels in Nigerian blacks

The human apolipoprotein C-II locus exhibits genetically determined structural polymorphism in United States and African blacks. In the present study, we have investigated the effect of the apoC-II polymorphism on quantitative serum levels of total cholesterol, total high density lipoprotein (HDL) cholesterol, cholesterol in high density lipoprotein subfractions, low density lipoprotein (LDL) cholesterol, and triglycerides (TG) in a sample of 368 unrelated Nigerian blacks. The frequencies of the APOC-II*1 and APOC-II*2 alleles in the samples were 0.947 and 0.053, respectively. In males, the effect of the APOC-II*2 allele was to lower the total serum cholesterol and LDL-cholesterol levels by 13.28 mg/dl and 10.55 mg/dl, respectively, relative to the common allele, APOC-II*1. In females, the effect was to lower total plasma cholesterol by 4.49 mg/dl and LDL-cholesterol by 3.21 mg/dl. The effect of apoC-II on quantitative lipoprotein levels is shown to be independent of variation at the linked apoE locus, but the products of the two loci interact in determining overall quantitative phenotypes.     

Accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve. Identification of apolipoprotein D, apolipoprotein A-IV, apolipoprotein E, and apolipoprotein A-I

In this report, we have identified two apolipoproteins (apo), apoD and apoA-IV, that, together with the previously identified apoA-I and apoE, accumulate in the regenerating peripheral nerve. These four apolipoproteins were identified in regenerating rat sciatic nerves by their molecular weights, their isoelectric points, and their recognition by specific antibodies. Antibodies were also used to document the changing concentrations of these apolipoproteins in homogenates of regenerating sciatic nerves collected 1 day to 6 weeks after a denervating crush injury. By 3 weeks after injury, at their peak accumulation, apoA-IV and apoA-I had increased 14- and 26-fold, respectively, relative to their concentrations in the normal nerve. Apolipoproteins D and E, in contrast, increased over 500- and 250-fold, respectively, by 3 weeks. These same apolipoproteins also accumulated in the regenerating sciatic nerves of two other species, the rabbit and the marmoset monkey. Immunocytochemistry showed that apoD was produced by astrocytes and oligodendrocytes in the normal central nervous system, and by neurolemmal or fibroblastic cells in the normal peripheral nervous system. Metabolic labeling of both apoD and apoE by [35S]methionine during an in vitro incubation of regenerating rat sciatic nerve segments confirmed that these apolipoproteins are synthesized by the nerve. Neither apoA-IV nor apoA-I was metabolically labeled, however, suggesting that they enter the nerve from the plasma. The results from this study provide evidence that several different apolipoproteins from various sources may play a role in lipid transport within neural tissues.     

Genetic studies of human apolipoproteins. XVIII. Apolipoprotein polymorphisms in Australian Aborigines

A tribal aboriginal community, the Mowanjum, from the Kimberley region in Western Australia has been screened to determine the extent of genetic variation in the products of genes coding for apolipoproteins, which are intimately involved in lipid metabolism. Of the seven systems tested, APOE and APOH revealed common structural variations, but their distribution patterns are significantly different from those found in European populations. Australian Aborigines were found to be unique because they have no APOE*2 and APOH*3 alleles and have strikingly high frequencies of the APOE*4 (26%) and APOH*1 (13%) alleles. The contrast in variation observed at these apolipoprotein loci between Australian Aborigines and Europeans not only makes these loci useful genetic markers in biologic anthropology studies but also provides a unique opportunity to investigate the role of genetic-environment interaction in determining interpopulation differences in cardiovascular disease risk factors.     

Conformational properties of human and rat apolipoprotein A-IV

Apolipoprotein A-IV has been isolated from four sources: human and rat lymph and plasma. Conformational properties of the rat and human apoA-IV in solution and denaturation changes induced by guanidine hydrochloride (Gnd X HCl) were studied using circular dichroic and fluorescence spectroscopy, and analytical sedimentation equilibrium ultracentrifugation. We have shown that both rat and human apoA-IV have similar secondary structure with negative maxima in the circular dichroic spectra at 222 nm and 207 nm. Furthermore, we have found no significant difference in the alpha-helical content of the apoA-IV from rat plasma (33%), rat lymph (37%), human plasma (35%), or human lymph (35%). Our denaturation studies with Gnd X HCl demonstrated reversibility and the fact that each apoA-IV had a tendency to self-associate in solution and the self-association could be disrupted by low concentrations of Gnd X HCl (less than or equal to 0.4 M). Unfolding of the secondary structure of each apoA-IV occurred at higher concentrations of Gnd X HCl (midpoint less than or equal to 1.0 M). The apparent free energy of denaturation of the four apoA-IV proteins calculated from changes in the circular dichroic spectra upon addition of increasing concentrations of Gnd X HCl varied in a range from 3.0 to 4.2 kcal/mol. The fluorescence experiments revealed that apoA-IV from all sources had a maximum fluorescence emission at 342.5 nm, which shifted to the red region upon addition of increasing concentrations of Gnd X HCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic studies of human apolipoproteins. X. The effect of the apolipoprotein E polymorphism on quantitative levels of lipoproteins in Nigerian blacks.

Human apolipoprotein E exhibits genetic polymorphism in all populations examined to date. By isoelectric focusing and immunoblotting, three common alleles have been demonstrated in 365 unrelated Nigerian blacks. Furthermore, the APO E genetic polymorphism's effect on quantitative levels of lipids and lipoproteins has been determined. The respective frequencies of the APO E*2, APO E*3, and APO E*4 alleles are .027, .677, and .296. The effect of APO E polymorphism is significant only on total cholesterol and low-density lipoprotein cholesterol. The average excesses of the APO E*2 allele are to lower total cholesterol and low-density lipoprotein cholesterol by 9.19 mg/dl and 11.11 mg/dl, respectively. The average excesses of the APO E*4 allele are to increase total cholesterol and low-density lipoprotein cholesterol by 5.64 mg/dl and 6.18 mg/dl, respectively. On the basis of the differences in (a) the distribution of APO E allele frequencies between the Nigerians and other populations and (b) dietary lipids, we propose a model that shows that lipid metabolism is influenced by the combined effects of the APO E polymorphism and environmental factors.     

Binding of human apolipoprotein A-IV to human hepatocellular plasma membranes

We have investigated the binding of human apolipoprotein A-IV (apo A-IV) to human hepatocellular plasma membranes. Addition of increasing concentrations of radiolabeled apo A-IV to hepatic plasma membranes, in the presence and absence of a 25-fold excess of unlabeled apo A-IV, revealed saturation binding to the membranes with a KD of 154 nM and a binding maximum of 1.6 ng/microgram of membrane protein. The binding was temperature-insensitive, partially calcium-dependent, abolished when apo A-IV was denatured by guanidine hydrochloride or when the membranes were treated with Pronase and decreased when apo A-IV was incorporated into phospholipid/cholesterol proteoliposomes. In displacement studies using purified apolipoproteins and isolated lipoproteins, only unlabeled apo A-IV, apo A-I and high-density lipoproteins effectively competed with radiolabeled apo A-IV for membrane binding sites. We conclude that human apo A-IV exhibits high-affinity binding to isolated human hepatocellular plasma membranes which is saturable, reversible and specific.     

Lipoprotein affinity of human apolipoprotein A-IV during cholesterol esterification

We have studied the lipoprotein distribution of human apo A-IV during cholesterol esterification by the action of endogenous lecithin-cholesterol acyltransferase. Using immunologic and radiotracer techniques at 4 degrees C, apo A-IV was found in two discrete monomeric and dimeric populations, unassociated with plasma lipoproteins. With incubation at 37 degrees C, apo A-IV initially associated with the high density lipoprotein-3 fraction, but thereafter dissociated from its surface, and reappeared as unbound protein and in association with a complex in the low density lipoprotein size range. Inclusion of LCAT inhibitors in the incubations abolished these changes. We conclude that the changes in lipoprotein distribution of human apo A-IV closely parallel the formation and exchange of plasma cholesteryl esters.     

Structural properties and lipid binding of human apolipoprotein A-IV

The in vivo affinity of human apolipoprotein A-IV (apo-A-IV) for plasma lipoproteins is considerably less than that of other apolipoproteins. We have therefore studied its spectroscopic properties and its association with model chylomicrons to investigate its structural characteristics and to define their influence upon its affinity for lipids. Fluorescence emission spectra of apo-A-IV in dilute aqueous solution revealed that its single tryptophan residue resides in a pH-sensitive hydrophobic domain, which is maximally protected from iodide quenching at pH 7.5. Denaturation of apo-A-IV by guanidine hydrochloride caused a multiphasic fluorescence emission red shift, with an unusual enhancement of quantum yield. Circular dichroism spectroscopy of apo-A-IV demonstrated negative ellipticity maxima at 210 and 222 nm, consistent with 54% alpha-helical structure. The alpha-helicity of apo-A-IV as measured by [theta]222 was also pH-sensitive and displayed a distinctive decrease between pH 7.0 and 8.0. Apo-A-IV was exquisitely sensitive to denaturation by guanidine hydrochloride, and its estimated free energy of stabilization in aqueous solution was near zero. Apo-A-IV bound to the surface of Sf greater than 400 particles of a phospholipid-triglyceride emulsion in a noncooperative, concentration-dependent manner. The affinity of apo-A-IV for these model chylomicrons was influenced by changes in pH or addition of guanidine hydrochloride in a manner which correlated well with the structural changes observed under similar conditions. We conclude that human apolipoprotein A-IV possesses several biophysical properties characteristic of the better studied plasma apolipoproteins, yet, apo-A-IV appears to be marginally stable in aqueous solution and its structural characteristics and lipid binding properties are particularly sensitive to environment.