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Although lifestyle interventions have shown promise in oncology and for cancer survivorship, their potential to improve outcomes in allogeneic hematopoietic cell transplantation (allo-HCT) and chronic graft-versus-host disease (cGVHD) patients remains to be fully explored. Given the high rates of cardiovascular disease, metabolic syndrome, and secondary malignancy in this patient population, lifestyle modifications can serve as a vital frontline defense against chronic diseases. Current research has illuminated the potential supportive role of lifestyle interventions in the solid cancer patient population, which is encouraging future lifestyle medicine research for patients with hematologic malignancies and allo-HCT recipients. Recent studies have indicated the pernicious effects of poor lifestyle choices on the course of cGVHD development and survival. The intersection between certain pillars of lifestyle medicine (ie, nutrition and exercise) and allo-HCT patient outcomes has been more well documented than that of other pillars (ie, social relationships and spirituality). Ongoing randomized trials studying the effects of exercise and nutrition on clinical outcomes in cGVHD and allo-HCT patients may provide important future evidence of the role of lifestyle medicine in this patient population. In this review, we describe the current landscape of lifestyle medicine in allo-HCT and cGVHD, its potential, and propose ways to further develop this evolving field of medicine., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: Incidence and mortality rates of uterine cancer are increasing and, obesity, which is also rising, has been associated with uterine cancer development and mortality. A recent study found that poor sleep quality is common among endometrial cancer survivors and those with obesity had more sleep disturbances than those having normal weight. However, it is unclear if higher levels of obesity (Class III, BMI ≥ 40 kg/m 2 ), which are rising rapidly, are differentially associated with sleep as well as depression and quality of life in endometrial cancer survivors., Methods: We evaluated sleep, depression, and quality of life in 100 Stage I endometrial cancer survivors with obesity seeking weight loss enrolled in a lifestyle intervention (NCT01870947) at baseline., Results: The average age was 60 years and mean BMI was 42.1 kg/m 2 with 58% having a BMI ≥ 40 kg/m 2 . Most survivors (72.3%) had poor sleep quality and most (71.2%) reported sleeping < 7 h/night. Survivors with class III compared with class I obesity had significantly more sleep disturbances and daytime dysfunction; and, those with poor sleep had higher depression and lower quality of life. Survivors with a BMI ≥ 50 kg/m 2 (~ 25%) had the highest levels of depression and lowest physical and emotional well-being., Conclusions: Our results reveal that endometrial cancer survivors with class III compared with class I obesity have poorer sleep quality, higher depression, and lower quality of life. Given the rising rates of obesity and uterine cancer mortality, interventions to combat both obesity and poor sleep are needed.

Objective: To perform a randomized trial to determine whether there is cardiovascular disease (CVD) risk reduction from a plant-based (PB), no-added-fat diet and the American Heart Association (AHA) diet in children., Study Design: A 4-week (April 20, 2013 to May 18, 2013), prospective randomized trial was undertaken in a large, Midwestern hospital system's predominantly middle class outpatient pediatric practices. Thirty children (9-18 years of age) parent pairs with a last recorded child body mass index >95th percentile and child cholesterol >169 mg/dL were randomized to PB or AHA with weekly 2-hour classes of nutrition education., Results: Children on PB had 9 and children on AHA had 4 statistically significant (P < .05) beneficial changes from baseline (mean decreases): body mass index z-score(PB) (-0.14), systolic blood pressure(PB) (-6.43 mm Hg), total cholesterol(PB) (-22.5 mg/dL), low-density lipoprotein(PB) (-13.14 mg/dL), high-sensitivity C-reactive protein(PB) (-2.09 mg/L), insulin(PB) (-5.42 uU/mL), myeloperoxidase(PB/AHA) (-75.34/69.23 pmol/L), mid-arm circumference(PB/AHA) (-2.02/-1.55 cm), weight(PB/AHA) (-3.05/-1.14 kg), and waist circumference(AHA) (-2.96 cm). Adults on PB and AHA had 7 and 2, respectively, statistically significant (P < .05) beneficial changes. The significant change favoring AHA was a 1% difference in children's waist circumference. Difficulty shopping for food for the PB was the only statistically significant acceptability barrier., Conclusions: PB and the AHA in both children and adults demonstrated potentially beneficial changes from baseline in risk factors for CVD. Future larger, long-term randomized trials with easily accessible PB foods will further define the role of the PB in preventing CVD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: Plant-based nutrition achieved coronary artery disease (CAD) arrest and reversal in a small study. However, there was skepticism that this approach could succeed in a larger group of patients. The purpose of our follow-up study was to define the degree of adherence and outcomes of 198 consecutive patient volunteers who received counseling to convert from a usual diet to plant-based nutrition., Methods: We followed 198 consecutive patients counseled in plant-based nutrition. These patients with established cardiovascular disease (CVD) were interested in transitioning to plant-based nutrition as an adjunct to usual cardiovascular care. We considered participants adherent if they eliminated dairy, fish, and meat, and added oil., Results: Of the 198 patients with CVD, 177 (89%) were adherent. Major cardiac events judged to be recurrent disease totaled one stroke in the adherent cardiovascular participants—a recurrent event rate of .6%, significantly less than reported by other studies of plant-based nutrition therapy. Thirteen of 21 (62%) nonadherent participants experienced adverse events., Conclusion: Most of the volunteer patients with CVD responded to intensive counseling, and those who sustained plant-based nutrition for a mean of 3.7 years experienced a low rate of subsequent cardiac events. This dietary approach to treatment deserves a wider test to see if adherence can be sustained in broader populations. Plant-based nutrition has the potential for a large effect on the CVD epidemic.

Docosahexaenoic acid (DHA), a long-chain omega-3 polyunsaturated fatty acid, has been used to treat a range of different conditions, including periodontal disease (PD) and inflammatory bowel disease (IBD). That DHA helps with these oral and gastrointestinal diseases in which inflammation and bacterial dysbiosis play key roles, raises the question of whether DHA may assist in the prevention or treatment of other inflammatory conditions, such as the metabolic syndrome, which have also been linked with inflammation and alterations in normal host microbial populations. Here we review established and investigated associations between DHA, PD, and IBD. We conclude that by beneficially altering cytokine production and macrophage recruitment, the composition of intestinal microbiota and intestinal integrity, lipopolysaccharide- and adipose-induced inflammation, and insulin signaling, DHA may be a key tool in the prevention of metabolic syndrome.

Numerous studies reveal the cardiovascular benefits of consuming dietary fiber and, especially, cereal fiber. Cereal fiber is associated with cardiovascular risk reduction through multiple mechanisms and consuming a variety of cereal fiber sources offers health benefits specific to the source. Certain cereal fibers have been studied more extensively than others and provide greater support for their incorporation into a healthful diet. β-glucan from oats or barley, or a combination of whole oats and barley, and soluble fiber from psyllium reduces the risk of coronary heart disease; inulin-type fructans added to foods and beverages may modestly decrease serum triacylglycerols; arabinoxylan and resistant starch may improve glycemic control. Individuals with low cereal fiber intake should increase their intake of whole grains in order to receive the benefits of whole grains in addition to fiber. For those adjusting to the texture and palatability of whole grains, turning to added-fiber products rich in β-glucan and psyllium may allow them to reach their fiber goals without increasing caloric intake.

Object: This study was undertaken to test a hypothesis that meningiomas of the midline skull base and spine are predominantly of the meningothelial histological subtype., Methods: The cases of 794 consecutive patients who underwent resection for meningioma at the Cleveland Clinic between January 1991 and March 2004 were reviewed retrospectively. The authors analyzed the relationship between the tumors' histological subtypes and sites of origin in the 731 patients from this group who harbored tumors that were determined to be benign histologically (World Health Organization Grade I). Meningothelial meningiomas (MMs) accounted for 63.5% (464/731) of the Grade I tumors. The incidence of MM according to the site of origin was as follows: 84.9% (186/219) in the midline skull base, 58.3% (35/60) in the lateral skull base, 48.5% (183/377) in a non-skull base location, and 80% (60/75) in spinal locations. The incidence of MM in the midline skull base and spinal locations were significantly higher than in non-skull base or lateral skull base locations., Conclusions: Meningiomas of the midline neuraxis are predominantly meningotheliomas. Analysis of the increasingly available data on genetic and topographic characteristics of MMs suggests that they may represent a unique entity, contrary to the prevailing belief that all benign meningiomas are identical tumors.

Objective: 5-Lipoxygenase (5-LO) oxidizes arachidonic acid into proinflammatory eicosanoids that may promote tumorigenesis. In this study, we investigated whether 5-LO is expressed in human astrocytomas and what effect its expression may have on patient outcome., Methods: Increased 5-LO messenger ribonucleic acid and protein expression was detected by the polymerase chain reaction and antibody-based approaches, respectively, in surgical astrocytoma specimens and established glioblastoma multiforme cell lines compared with primary cell culture from the human white matter., Results: Immunohistochemical analysis revealed predominantly nuclear 5-LO staining in 44 of 49 glioblastoma multiforme samples (90%), 8 of 10 (80%) anaplastic astrocytomas samples, and 3 of 13 (23%) low-grade astrocytoma samples analyzed. Double-staining experiments with anti-CD-68 (macrophage/microglial marker) and anti-5-LO antibodies suggest that both CD-68-positive and CD-68-negative tumor cells express 5-LO protein. Staining of 5-LO was significantly more frequent in high-grade than in low-grade tumors (P = 0.001). Patients whose tumors expressed 5-LO were significantly older, had lower preoperative Karnofsky performance scores and shorter survival than patients whose tumors did not express 5-LO. After adjusting for pathological diagnosis and age, respectively, neither Karnofsky performance score nor survival were significantly associated with 5-LO staining., Conclusion: These data indicate that 5-LO is overexpressed in high-grade astrocytomas and supports the idea that eicosanoids may play a role in tumorigenesis of these brain tumors.

Acetyl-11-keto-beta-boswellic acid (AKBA) is a naturally occurring pentacyclic triterpene isolated from the gum resin exudate of the tree Boswellia serrata (frankincense). Because pentacyclic triterpenes have antiproliferative and cytotoxic effects against different tumor types, we investigated whether AKBA would act in a similar fashion on primary human meningioma cell cultures. Primary cell cultures were established from surgically removed meningioma specimens. The number of viable cells in the absence/presence of AKBA was determined by the non-radioactive cell proliferation assay. The activation status of the proliferative cell marker, extracellular signal-regulated kinase-1 and -2 (Erk-1 and Erk-2) was determined by immunoblotting with the antibody that recognizes the activated form of these proteins. Treatment of meningioma cells by AKBA revealed a potent cytotoxic activity with half-maximal inhibitory concentrations in the range of 2 - 8 microM. At low micromolar concentrations, AKBA rapidly and potently inhibited the phosphorylation of Erk-1/2 and impaired the motility of meningioma cells stimulated with platelet-derived growth factor BB. The cytotoxic action of AKBA on meningioma cells may be mediated, at least in part, by the inhibition of the Erk signal transduction pathway. Because of the central role the Erk pathway plays in signal transduction and tumorigenesis, further investigation into the potential clinical use for AKBA and related boswellic acids is warranted.

Acetyl-11-keto-beta-boswellic acid (AKBA) is a naturally occurring pentacyclic triterpene isolated from the gum resin exudate from the stem of the tree Boswellia serrata (frankincense). AKBA has been recently identified as a novel, orally active, non-redox and non-competitive 5-lipoxygenase inhibitor that also inhibits topisomerase I and II in vitro. Because natural pentacyclic triterpenes have an antiproliferative effect against different tumor types, we investigated the effects of AKBA on the proliferation of 11 primary cell cultures established from human surgical specimens of meningiomas, common central nervous system tumors. Treatment of meningioma cells by AKBA revealed a potent cytotoxic activity with half-maximal inhibitory concentrations in the range of 2-8 microM. At similar, physiologically achievable concentrations, AKBA rapidly (within minutes) and potently inhibited the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk-1 and Erk-2) in meningioma cells stimulated with platelet-derived growth factor BB. High expression level of 5-LO was detected in primary meningioma cells and surgical specimens by immunoblotting analysis, suggesting the possible role of 5-LO in meningioma tumorigenesis. Considering the critical importance of the Erk-1/2 signal transduction pathway not only in meningiomas but in other human neoplasms, the interruption of signaling through this evolutionarily conserved pathway might be one of the mechanisms by which AKBA induces suppression of proliferation and apoptosis of different tumor types.

Object: The neurofibromatosis Type 2 (NF2) gene is the only tumor suppressor gene that has been clearly implicated in the development of benign meningiomas. Interestingly, previous data obtained by the authors indicate that reduced NF2 protein expression seldom occurs in meningothelial meningiomas, the most common histological type of meningioma. The goal of the current study was to explore further the hypothesis of NF2 gene-independent tumorigenesis of meningothelial meningiomas., Methods: The authors performed a mutational analysis of all 17 exons of the NF2 gene by using single-stranded conformational polymorphism (SSCP). In addition, expression levels of the NF2 protein and mu-calpain, a protease suggested to inactivate the NF2 protein, were determined by immunoblotting analysis of 27 meningiomas (20 meningothelial and seven nonmeningothelial). Mutations of the NF2 gene were found in only one (5%) of 20 meningothelial meningiomas and three (43%) of seven nonmeningothelial tumors (Fisher's exact test, p = 0.042). The levels of NF2 protein were severely reduced in six (28.5%) of 21 meningothelial meningiomas, in contrast to six (86%) of seven nonmeningothelial meningiomas (Fisher's exact test, p = 0.023). Activation of IL-calpain did not correlate with the status of NF2 protein expression in the meningiomas analyzed, demonstrating that mu-calpain activation does not account for the loss of NF2 protein in meningiomas with apparently normal NF2 genes., Conclusions: These results clearly demonstrate that NF2 gene mutations and decreased NF2 protein expression rarely occur in meningothelial meningiomas compared with other histological types of meningiomas. The clinical behavior of meningothelial meningiomas, however, is similar to that of other benign meningiomas. It is likely, therefore, that the tumorigenesis of meningothelial meningiomas is the result of deleterious alterations of genes that have final phenotypical effects similar to inactivation of the NF2 gene.

Neurofibromin and p120 GTPase activating protein (p120 GAP) down-regulate the activity of cellular Ras proteins. How the activity of these two proteins is controlled is not yet clear. In this study, we analyzed the effects of eight nutritionally relevant fatty acids on GTPase stimulatory activity of full-length neurofibromin and p120 GAP. The fatty acids tested were: saturated stearic acid, monounsaturated oleic acid, three omega-6 and three omega-3 polyunsaturated fatty acids. The analysis was performed by Ras immunoprecipitation GTPase assay. The full-length p120 GAP expressed in insect Sf9 cells and the immunoaffinity purified full-length neurofibromin were used. Neurofibromin was readily inhibited by stearic and oleic acid, but p120 GAP was not inhibited even at high concentrations (> 80 microM). Neurofibromin was also inhibited by low concentrations of all the polyunsaturated fatty acids tested (IC50 of 6 to 16 microM). p120 GAP was 2-3 fold less sensitive to inhibition by these fatty acids. The GTPase stimulatory activity of neurofibromin was also inhibited by arachidonic and oleic acid in the presence of a lipid mixture representing the major lipid components of the cell membrane. Chimeric proteins of neurofibromin and p120 GAP were used to determine that differential sensitivity to fatty acid inhibition maps to the catalytic domain of the proteins. These results indicated that fatty acids can modulate the GTPase function of the c-Ha-Ras protein by inhibiting the GTPase stimulatory activity of two Ras regulators, full-length neurofibromin and p120 GAP, at physiologically relevant concentrations in vitro.

Mutations in neurofibromatosis type 1 target the gene coding for neurofibromin. While neurofibromin is able to accelerate the rate of GTP hydrolysis by cellular Ras proteins, its biological function is not well understood. To gain information regarding its function, the intracellular localization of neurofibromin was analyzed in cultured cell lines using polyclonal antisera raised against four neurofibromin-specific peptides, three from the carboxyl terminus and one from the amino terminus. In methanol-fixed cells distinct rod-like structures distributed throughout the cytoplasm were recognized by the antisera. Similar structures were seen with each antiserum, including affinity-purified antibodies, and in each of the cultured cell lines tested. Similar structures were seen in paraformaldehyde-fixed cells. Double staining experiments showed that these structures colocalize with mitochondria, but not with actin, beta-tubulin, or endoplasmic reticulum. When actin or tubulin structures within the cell were disrupted by separate antimitotic drugs, these stained structures retained their shape. Neurofibromin association with mitochondria was confirmed biochemically when highly purified mitochondrial fractions from bovine heart tissue were shown in Western analysis to contain neurofibromin. This association might be helpful in predicting identification of some of the cellular proteins with which neurofibromin interacts.

The effector binding domain and the switch II region of c-Ha-Ras are necessary for p120GAP-stimulated GTP hydrolysis. We report a third region of c-Ha-Ras located within the alpha 3 helix (amino acids 101-103) which is also required for efficient p120GAP, but not neurofibromin-mediated hydrolysis. This highly conserved region of the Ras protein was investigated using an insertion-deletion mutant (Ras-100LIR104) originally characterized by Willumsen et al. (Willumsen, B. M., Adari, H., Zhang, K., Papageorge, A. G., Stone, J. C., McCormick, F., and Lowy, D. R (1989) in The Guanine Nucleotide Binding Proteins; Common Structural and Functional Properties (Bosch, L., Kraal, B., and Parmeggiani, A., eds) pp. 165-178, Plenum Press, New York). The 100LIR104 substitution did not alter the intrinsic hydrolytic rate of the protein. The p120GAP-stimulated hydrolysis of Ras-100LIR104, however, was decreased by 2-3-fold compared to wild type Ras. This decrease in p120GAP-stimulated hydrolysis was not due to its inability to physically associate with Ras-100LIR104. GTP (as determined by competitive binding assays). Surprisingly, neurofibromin-stimulated GTP hydrolysis was unaltered by the mutation. Finally, no differences were observed in the ability of either the p120GAP catalytic domain or the neurofibromin GRD to accelerate Ras-100LIR104 GTPase activity, indicating that the amino-terminal noncatalytic GAP region is critical for p120GAP-stimulated GTP hydrolysis. This is the first report of a Ras mutation which differentiates between p120GAP and neurofibromin activity.

To determine the organization of the DP region in the Mbc of anthropoid primates, we constructed contig maps from cosmid clones of the chimpanzee and orangutan, representatives of the infraorder Catarrhini, as well as of the cotton-top tamarin, a representative of the infraorder Platyrrhini. We found the maps to be remarkably similar to each other and to the previously published map of the human DP region. In each of the four species, the DP region consists of four loci arranged in the same order (DPB2 . . . DPA2 . . . DPB1 . . . DPA1) and in the same transcriptional orientation (tail-to-tail). The regions in the four species are of approximately the same length and many of the restriction sites are shared between species. The inserts of most Alu elements, of a ribosomal protein pseudogene, and of an IgC epsilon-like pseudogene are found in corresponding positions in all four species. The data indicate that the human-type organization of the DP region was established before the divergence of the Catarrhini and Platyrrhini lines more than 37 million years ago and that it has remained principally intact since that time. This conservation of the DP region is in striking contrast to the evolutionary instability of certain other Mbc regions, in particular those occupied by the DRB or C4 and CYP21 loci. We interpret the stability of the DP region as an indication that the region is being phased out functionally.

Neurofibromin, the product of the neurofibromatosis type 1 gene, was found to form a stable complex with immobilized p21c-Ha-ras-GMP-PNP (a non-hydrolyzable GTP analog). This complex, detectable as early as 30 min after addition of crude brain extract, is extremely stable, with less than 50% dissociating after 5 h at 4 degrees C. We interpret this to suggest that the dissociation of full-length neurofibromin from p21c-Ha-ras-GMP-PNP is tightly linked to the hydrolysis of GTP to GDP. Failure to remove a significant proportion of the bound neurofibromin in the presence of EDTA and GDP implies that the binding of neurofibromin to p21c-Ha-ras-GMP-PNP results in the ras protein becoming resistant to guanine nucleotide exchange. Under conditions in which neurofibromin quantitatively binds to p21c-Ha-ras-GMP-PNP, we were unable to detect a complex between p21c-Ha-ras and GAP (GTPase-activating protein). The failure to detect GAP binding to immobilized p21c-Ha-ras-GMP-PNP cannot be explained by the known differences in affinities of the GAP-related domain of neurofibromin and GAP for p21c-Ha-ras-GTP. GAP is, however, able to interact biochemically with immobilized p21c-Ha-ras, suggesting a difference in the interaction between GAP and neurofibromin with p21c-Ha-ras-GMP-PNP.

The beta chain-encoding (B) class II genes of the primate major histocompatibility complex belong to several families. The DRB family of class II genes is distinguished by the occurrence of haplotype polymorphism--the existence of multiple chromosomal forms differing in length, gene number, and gene combinations, each form occurring at an appreciable frequency in the population. Some of the haplotypes, or fragments thereof, are shared by humans, chimpanzees, and gorillas. In an effort to follow the DRB haplotype polymorphism further back in time, we constructed DRB contig maps of the two chromosomes present in the orangutan cell line CP81. Two types of genes were found in the two haplotypes, Popy-DRB5 and Popy-DRB1*03, the former occurring in two copies and one gene fragment in each haplotype, so that the CP81 cell line contains four complete DRB5 genes and two DRB5 fragments altogether. Since the four genes are more closely related to one another than they are to other DRB5 genes, they must have arisen from a single ancestral copy by multiple duplications. At the same time, however, the two CP81 haplotypes differ considerably in their restriction enzyme sites and in the presence of Alu elements at different positions, indicating that they have been separated for a length of time that exceeds the lifespan of a primate species. Moreover, a segment of about 100 kilobase pairs is shared between the orangutan CP81-1 and the human HLA-DR2 haplotype. These findings indicate that part of the haplotype polymorphism may have persisted for more than 13 million years, which is the estimated time of human-orangutan divergence.

The DRB region of the human major histocompatibility complex displays length polymorphism: Five major haplotypes differing in the number and type of genes they contain have been identified, each at appreciable frequency. In an attempt to determine whether this haplotype polymorphism, like the allelic polymorphism, predates the divergence of humans from great apes, we have worked out the organization of the DRB region of the chimpanzee Hugo using a combination of chromosome walking, pulsed-field gel electrophoresis, and sequencing. Hugo is a DRB homozygote whose single DRB haplotype is some 440 kilobases (kb) long and contains five genes. At least one and possibly two of these are pseudogenes, while three are presumably active genes. The genes are designated DRB*A0201, DRB2*0101, DRB3*0201, DRB6*0105, and DRB5*0301, and are arranged in this order on the chromosome. The DRB2 and DRB3 genes are separated by approximately 250 kb of sequence that does not seem to contain any additional DRB genes. The DRB*A0201 gene is related to the DRB1 gene of the human DR2 haplotype; the DRB2*0101 and DRB3*0201 genes are related to the DRB2 and DRB3 genes of the human DR3 haplotype, respectively; the DRB6*0105 and DRB5*0301 genes are related to the DRBVI and DRB5 genes of the human DR2 haplotype, respectively. Thus the Hugo haplotype appears to correspond to the entire human DR2 haplotype, into which a region representing a portion of the human DR3 haplotype has been inserted. Since other chimpanzees have their DRB regions organized in different ways, we conclude that, first, the chimpanzee DRB region, like the human DRB region, displays length polymorphism; second, some chimpanzee DRB haplotypes are longer than the longest known human DRB haplotypes; third, in some chimpanzee haplotypes at least, the DRB genes occur in combinations different from those of the human haplotypes; fourth, and most importantly, certain DRB gene combinations have been conserved in the evolution of chimpanzees and humans from their common ancestors. These data thus provide evidence that not only allelic but also haplotype polymorphism can be passed on from one species to another in a given evolutionary lineage.

Fifty-five single-copy DNA probes were isolated from the library LL06NS01, which was constructed from a complete HindIII digest of a flow-sorted human chromosome 6. Because chromosomes from a human x Chinese hamster somatic cell hybrid were used as the starting material for the flow-sorting, the library could be expected to contain some contaminating Chinese hamster DNA as well as DNA from human chromosomes other than 6. Thirty-seven of the 55 probes, however, were shown to map to human chromosome 6 by Southern blot hybridization with DNA from a panel of somatic cell hybrids. Eleven of the probes were mapped further by in situ hybridization. Four probes were localized to the short arm of chromosome 6, six to the long arm, and one to the centromeric region.

We prepared a cosmid library from chimpanzee DNA and screened it with a mouse probe specific for the complement component 4 (C4)-encoding gene. We isolated 29 clones and constructed restriction maps for 20 of these. The clones could be arranged into two overlapping clusters covering the entire C4 region of both chromosomes in this particular heterozygous chimpanzee. The region is about 100 kilobases (kb) long and contains two C4 and two CYP21 genes, the latter coding for the enzyme 21-hydroxylase. Using oligonucleotide probes we identified the genes as corresponding to human C4A, C4B, CYP21 and CYP21P genes. The last gene apparently contains an 8-base pair (bp) deletion (as does the corresponding human gene), which renders it a pseudogene. The genes are arranged in the order C4A...CYP21P...C4B...CYP21. Each of the two C4 genes is 16 kb long and thus corresponds to the short version of the human C4 genes. We suggest that the duplication of the basic C4-CYP21 unit that generated the standard arrangement of the human C4-CYP21 region occurred before the separation of the evolutionary lineages leading to humans and chimpanzees (i.e., more than five million years ago). We suggest further that the original form of the C4 gene was of the long variety and was generated by the insertion of a 6.8-kb element into one of the C4 introns. The element was subsequently excised in the ancestors of the chimpanzees and in at least one lineage of the human C4B gene. We speculate that the presence of the 6.8-kb insert in the human C4A and some C4B genes might largely be responsible for the great instability of this chromosomal region which leads to frequent duplications and deletions, some of which cause 21-hydroxylase deficiency.