A tapered parallel plate waveguide for frequency up-conversion of terahertz radiation.

A tapered parallel plate waveguide was developed for frequency up-conversion experiments in the terahertz (THz) region by flash ionization. The element at the plasma-source-wave interaction area determines the conversion efficiency. It causes THz pulses to converge to a narrow plate separation, which is smaller than the wavelength. The waveguide exhibited good performance for transmitting p-polarized THz pulses in a 50 µm separation, making it suitable for flash ionization experiments.

Efficacy of folinic acid in preventing oral mucositis in allogeneic hematopoietic stem cell transplant patients receiving MTX as prophylaxis for GVHD.

As the safety of folinic acid administration and its efficacy for reducing the toxicity of MTX remain controversial, we assessed the effect of folinic acid administration after MTX treatment for GVHD prophylaxis on the incidence of oral mucositis and acute GVHD. We retrospectively analyzed data for 118 patients who had undergone allogeneic hematopoietic SCT and had received MTX for GVHD prophylaxis. Multivariate analysis showed that systemic folinic acid administration significantly reduced the incidence of severe oral mucositis (odds ratio (OR)=0.13, 95% confidence interval (CI) 0.04-0.73, P=0.014). There was also a tendency for a lower incidence of severe oral mucositis in patients who received folinic acid mouthwash (OR=0.39, 95%CI 0.15-1.00, P=0.051). No significant difference was observed in the incidence of acute GVHD between patients who received systemic folinic acid administration and those who did not (P=0.88). Systemic folinic acid administration and mouthwash appear to be useful for reducing the incidence of severe oral mucositis in patients who have received allogeneic hematopoietic SCT using MTX as GVHD prophylaxis.

p73 is not mutated in meningiomas as determined with a functional yeast assay but p73 expression increases with tumor grade.

The p53 gene is normally wild type in meningiomas. Since all three members of the p53 gene family recognize the same DNA sequence, tumors containing wild type p53 could decrease transactivation of p53 target genes by mutating either p63 or p73. In meningiomas the most likely target is p73, because loss of heterozygosity of the chromosomal band containing p73 is the commonest genetic lesion in these tumors. To screen p73 for mutations we have developed a functional assay which tests the ability of p73 to activate transcription from a p53-responsive promoter in yeast. The assay correctly identified p73 mutants with mutations equivalent to hotspot mutations in p53, demonstrating that the assay can detect transcriptionally inactive p73. No mutations in p73 were identified in meningiomas. p73 RNA level was higher in more advanced tumors, but there was no correlation between the expression level of p73 and p21, a known p53 target gene. The yeast assay was also used to measure the intrinsic sensitivity of the p73 protein to mutagenesis. Like p53, p73 is exceptionally easy to inactivate as a transcription factor by point mutation. Taken together, these results indicate that p53 and p73 serve very different functions in tumors.

Sympathetic predominance in young male white-collar workers with mild to moderate hypertension.

In order to investigate the difference in diurnal variation of autonomic control between young male mild to moderate hypertensive and normotensive white-collar workers, we accessed heart rate variability with ambulatory blood pressure for over 24 hours including work and sleep periods. Both mean systolic and diastolic blood pressures (SBP and DBP) were significantly (p<0.01) higher in hypertensives (HT, n=11) than normotensives (NT, n=11) in all phases, whereas heart rate was not different between them in any phase. Blood pressures during work period (on-duty) were significantly higher than those during non-work period (off-duty) in both NT and HT. High frequency (HF; 0.15-0.40 Hz) power showed a circadian pattern in phase with the sleep-wake cycle. Both HF and low frequency (LF; 0.05-0.15 Hz) powers in HT were significantly lower than that in NT during the sleep period (p<0.05). LF/HF ratio showed a circadian pattern that was almost opposite of the pattern in HF power. Twenty-four-hour average LF/HF in HT was higher than that in NT (p<0.05). Especially during on-duty, LF/HF in HT was significantly higher than in NT (p<0.05). The increase of SBP and LF/HF from off-duty period to on-duty period (deltaSBP and deltaLF/HF) showed significant positive correlation (r=0.67, P<0.05) in HT group, suggesting that the SBP elevation during on-duty in HT may be coupled with sympathetic activation. These results suggest that HT showed significantly reduced parasympathetic control during sleep and an increased sympathetic activity especially during on-duty as compared with NT. Our findings imply that persistence of sympathodominant states together with reduced vagal activity in young mild to moderate hypertensive workers may be related to future cardiovascular disorders.

Replicating adenoviruses that target tumors with constitutive activation of the wnt signaling pathway.

Despite important advances in understanding the molecular basis of cancer, few treatments have been devised which rationally target known causal oncogenic defects. Selectively replicating viruses have a major advantage over nonreplicating viruses to target these defects because the therapeutic effect of the injected virus is augmented by virus produced within the tumor. To permit rational targeting of colon tumors, we have developed replicating adenoviruses that express the viral E1B and E2 genes from promoters controlled by the Tcf4 transcription factor. Tcf4 is constitutively activated by mutations in the adenomatous polyposis coli and beta-catenin genes in virtually all colon tumors and is constitutively repressed by Groucho and CtBP in normal tissue. The Tcf-E2 and Tcf-E1B promoters are active in many, but not all, cell lines with activation of the wnt pathway. Viruses with Tcf regulation of E2 expression replicate normally in SW480 colon cancer cells but show a 50- to 100-fold decrease in replication in H1299 lung cancer cells and WI38 normal fibroblasts. Activation of wnt signaling by transduction of a stable beta-catenin mutant into normal fibroblasts renders the cells permissive for virus replication. Insertion of Tcf4 sites in the E1B promoter has only small effects on replication in vitro but significantly reduces the inflammatory response in a rodent lung model in vivo. Replicating adenoviruses with Tcf regulation of both E1B and E2 transcription are potentially useful for the treatment of liver metastases from colorectal tumors, but additional changes will be required to produce a virus that can be used to treat all colon tumors.

Distinct prognostic values of p53 mutations and loss of estrogen receptor and their cumulative effect in primary breast cancers.

A total of 76 primary breast cancers were screened for p53 mutations using the yeast p53 functional assay, and the mutations were determined by DNA sequencing. Clonal mutations of p53 were detected in 30 tumors (39%). Immunohistochemical staining for nuclear p53 accumulation performed on the yeast assay-positive cases clearly differentiated missense mutations in the DNA binding domain (contact mutant; 17 cases) as positive stain and nonsense-type mutations or missense mutations that may affect 3D-structure of p53 protein (structural mutant; 13 cases) as negative stain. Enzyme immunoassay revealed loss of estrogen receptor in 36 tumors (50%). Prognostic values of p53 mutation and loss of estrogen receptor were evaluated after a median follow-up period of 44 months. p53 mutations were associated with a short overall survival (log rank test, p = 0.0319), whereas it was not related to disease-free (recurrence-free) survival. Contact mutants were associated with slightly shorter survival compared with structural mutants. Inversely, loss of estrogen receptor was associated with early recurrence (p = 0.0461) but not with short overall survival. The patients with tumors harboring both p53 mutation and loss of estrogen receptor had the poorest outcome (p = 0.0019 and 0.0075 for overall and disease-free survivals, respectively), suggesting independent and additive effects of the 2 factors. The independent role of the 2 factors was confirmed by a multivariate analysis using the Cox proportional hazard model stratified according to clinical tumor stages. Although preliminary, due to the small number of patients studied and the relatively short follow-up time, our results suggest that p53 mutations and loss of estrogen receptor cooperatively affect the prognosis of primary breast cancer patients.

Dominant-negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme.

Previous experiments have suggested that some mutant forms of p53 are able to inactivate the endogenous wild-type p53 protein in a dominant-negative fashion. However, it remains unknown whether tumors with such dominant-negative (transdominant) p53 mutants have a biological significance that is different from that of recessive p53 mutants. In this study, we examined the dominant-negative potential of various p53 mutants using a yeast-based assay in which both wild-type and mutant p53 were efficiently expressed. We tested a total of 106 p53 mutants, which were identified in brain tumors, glioblastoma multiforme-derived cell lines, breast cancers, or premalignant lesions and squamous cell carcinomas of oral epithelium or were otherwise created by mutagenesis. In agreement with the previous studies, our results demonstrated that transdominant mutations affected amino acid residues that are essential for the stabilization of the DNA-binding surface in the p53 core domain and for the direct interaction of p53 with its DNA-binding sequence. Among 40 patients with sporadic glioblastomas, the average age at diagnosis was significantly younger in the patients with tumors harboring dominant-negative mutations (30.4 +/- 14.7 years, n = 7) than it was in those with recessive mutations (55.2 +/- 18.6 years, n = 9, P < 0.012) and in those without mutations (54.7 +/- 17.1 years, n = 24, P < 0.003). Our data suggest that dominant-negative p53 mutants accelerate development and/or growth of glioblastoma anlagen.

Heart rate monitoring as a field method for estimating energy expenditure as evaluated by the doubly labeled water method.

Energy expenditure (TEE) determined by the doubly labeled water technique (DLW) in 10 adult subjects (5 males and 5 females) was used to evaluate the energy expenditure estimated from 24 h heart rate monitoring records (HR method) for 2 d randomly sampled during 2 weeks of DLW study. Individual data on HR and oxygen consumption, obtained during a step test (resting conditions, and up and down at 3 or 4 stepping rates) and postabsorptive conditions (resting metabolic rate: RMR), were used to calculate three types of calibration regression line, i.e., straight-linear regression (EE-HR: A for TEEhr-A), log-linear regression (lnEE-HR: B for TEEhr-B), and two-linear regression (flex-HR method: C for TEEhr-C). When the 24 h HR records were applied, these calibration regressions provided three estimates of TEE (mean +/- SD kcal/24 h): TEEhr-A (3,059 +/- 1,246 in all subjects), TEEhr-B (2,472 +/- 843), and TEEhr-C (2,759 +/- 1,228). Mean TEE determined by the DLW method (TEEdlw) was 2,544 +/- 378 kcal/24 h. Although no mean values estimated by HR methods were significantly different statistically from the mean value of TEEdlw, the variances of the estimates (e.g., SD) by the HR method were much greater than that of TEEdlw (between twofold and threefold). TEEhr-B estimated by lnEE-HR regression provided the smallest differences from that of TEEdlw (mean difference of -3.1% with a range of -35.1(-)+36.6%). From these observations, the following conclusions were made: 1) The estimates of TEE by HR are useful as a group mean, but interpretation of the individual TEE estimates requires caution because of great deviations from the reference values. 2) Among the calibration methods tested, the log-linear calibration regression (lnEE on HR) gives the best estimates of TEE by the HR method and is recommended for use in future studies.

Hematocrit correlates with blood pressure in young male office workers.

High hematocrit (Ht) level has been reported to be a correlating factor of hypertension in aged people, but has not been examined in younger generation. To investigate the association between Ht and blood pressure (BP) in healthy young workers, statistical analysis was performed for 646 male employees, using cross-sectional health-check data. Ht was positively correlated with systolic blood pressure (SBP) and with diastolic blood pressure (DBP) by Pearson's simple correlation analysis. Multiple regression analysis for SBP and DBP was conducted by stepwise procedure, using Ht, age, body mass index (BMI), and drinking and smoking habits as independent variables. It was revealed that Ht was a significant independent variable for DBP (p < 0.001), as well as age and BMI, but not for SBP. These findings suggest that increased Ht is an important variable for assessing risk for cardiovascular disorders, especially diastolic hypertension, in young male office workers.

Characteristics of mutations in the p53 gene of oral squamous-cell carcinomas associated with betel-quid chewing in Sri Lanka.

Oral squamous-cell carcinoma (SCC) is the most common neoplasm in Sri Lanka, accounting for approximately 30% of all cancers in males. Epidemiologic evidence indicates that there is an unequivocal relationship between betel chewing and oral carcinogenesis, suggesting that there may be specific genetic targets of betel-quid ingredients. The p53 gene has been indicated to be a tumor-suppressor gene that is found in mutated form in common human cancers; however, there are few reports about "carcinogen-specific" p53 mutation. Because of this background, primary resected specimens from 23 oral SCCs, 7 leukoplakias and 2 oral submucous fibrosis were collected from oral SCC patients in Sri Lanka and were used for p53 mutation analysis. Exons 5 through 8 of the p53 gene were examined by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. Mutations in the p53 gene were frequent (10/23) in oral SCC specimens from Sri Lanka. Moreover, the mutations clustered significantly in exon 5 (7/10) of the p53 gene, and small deletions and inclusions other than point mutations were observed. These results indicate that 1) betel-quid chewing may cause specific genetic changes, including mutation in the p53 gene; 2) mutations in the p53 gene are not rare events in SCC patients who are betel-quid chewers, which contrasts with other reports; 3) exon 5 of the p53 gene could be one of the specific targets for some betel-quid ingredients; and 4) betel-quid chewing may be a critical environmental factor in the development of oral SCC.

[Role of school lunch in primary school education: a trial analysis of school teachers' views using an open-ended questionnaire].

We tried to analyze synthetically teachers' view points associated with health education and roles of school lunch in primary education. For this purpose, a survey using an open-ended questionnaire consisting of eight items relating to health education in the school curriculum was carried out in 100 teachers of ten public primary schools. Subjects were asked to describe their view regarding the following eight items: 1) health and physical guidance education, 2) school lunch guidance education, 3) pupils' attitude toward their own health and nutrition, 4) health education, 5) role of school lunch in education, 6) future subjects of health education, 7) class room lesson related to school lunch, 8) guidance in case of pupil with unbalanced dieting and food avoidance. Subjects described their own opinions on an open-ended questionnaire response sheet. Keywords in individual descriptions were selected, rearranged and classified into categories according to their own meanings, and each of the selected keywords were used as the dummy variable. To assess individual opinions synthetically, a principal component analysis was then applied to the variables collected through the teachers' descriptions, and four factors were extracted. The results were as follows. 1) Four factors obtained from the repeated principal component analysis were summarized as; roles of health education and school lunch program (the first principal component), cooperation with nurse-teachers and those in charge of lunch service (the second principal component), time allocation for health education in home-room activity and lunch time (the third principal component) and contents of health education and school lunch guidance and their future plan (the fourth principal component). 2) Teachers regarded the role of school lunch in primary education as providing daily supply of nutrients, teaching of table manners and building up friendships with classmates, health education and food and nutrition education, and developing food preferences through eating lunch together with classmates. 3) Significant positive correlation was observed between "the teachers' opinion about the role of school lunch of providing opportunity to learn good behavior for food preferences through eating lunch together with classmates" and the first principal component "roles of health education and school lunch program" (r = 0.39, p < 0.01). The variable "the role of school lunch is health education and food and nutrition education" showed positive correlation with the principle component "cooperation with nurse-teachers and those in charge of lunch service" (r = 0.27, p < 0.01). Interesting relationships obtained were that teachers with longer educational experience tended to place importance in health education and food and nutrition education as the role of school lunch, and that male teachers regarded the roles of school lunch more importantly for future education in primary education than female teachers did.

High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay.

To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT-PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR-SSCP) of exon 5-8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR-SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.

[Detectability and diagnostic criteria of p53 gene mutations in human oral squamous cell carcinoma using yeast functional assay].

In yeast functional assay (YF assay), a newly developed screening system for p53 mutation, wild-type p53 gives white yeast colonies and transcriptionally inactive mutant p53 gives red colonies. In the present study, the author applied YF assay to the detection of p53 mutations in human oral squamous cell carcinoma (SCC). Total RNA was extracted from samples and YF assay was performed. Four SCC cell lines (SAS, HSC-2, HSC-3 and Ca9-22) known to have p53 mutations all gave 100% red colonies, whereas nine oral non-tumor tissues gave 2.9-10% (average 5.2 +/- 2.7%) red colonies. Furthermore, a rat hepatoma cell line, WHp53, which had been transfected with human wild-type p53 expression vector, presented 7.8% red colonies. Thus the functional assays of tissues or cells containing only wild-type p53 give 3-10% red colonies as a background. To assess the detectability of p53 mutations, YF assay was performed on mixtures of wild-type and mutant p53 PCR products at serial ratios. The result showed that the mutation was detectable if 6% population of transcriptionally inactive mutant p53 mRNA were present in the total p53 mRNA. Twenty-two clinical samples of human oral SCC were then tested by YF assay. Fourteen out of 22 cases gave more than 20% red colonies. In these 14 cases, clonal p53 mutations with deletion, nonsense mutation or missense mutation were identified. In a case which gave 17% red colonies, identical p53 mutation was found in 2 out of 6 independent red colonies. However, no identical mutations were found in the cases giving 13, 9 and 8% red colonies. Based on these results, the author proposes that 20% of red colonies is the minimal value for the diagnosis of p53 mutation in YF assay under PCR conditions using Pfu polymerase and hot start method.

Hepatocyte growth factor and c-met expression in Long-Evans Cinnamon rats with spontaneous hepatitis and hepatoma.

The Long-Evans Cinnamon (LEC) rat is characterized by the spontaneous onset of acute and chronic hepatitis, followed by occurrence of liver cancer, and is thus able to provide a unique experimental model for human genetical liver disease, Wilson's disease. Hepatocyte growth factor (HGF) is a potent hepatotrophic factor in liver regeneration, and its expression is up-regulated in response to liver injuries. We found that the plasma HGF level in LEC rats rose markedly during the fulminant hepatitis phase, fell during the phase of chronic/cholangiofibrosis, and fluctuated during the hepatoma phase. Immunohistological staining of the liver revealed that the number of HGF-positive cells increased remarkably during the fulminant hepatitis phase, and that many of these cells were localized at the portal triads. Fewer HGF-positive cells were observed during the phase of chronic hepatitis. The surface of the hepatocellular carcinoma (HCC) cells and the cytoplasm of the nonepithelial cells in cancerous liver tissues were HGF-positive. The HGF-messenger RNA (mRNA) level in the liver rose in the fulminant hepatitis phase, fell in the chronic hepatitis phase, and was intermediate or high during the hepatoma phase. The expression of c-met mRNA was strong in the tissues of LEC rats with fulminant hepatitis and, especially, in the cholangiofibrosis tissues. c-met mRNA was also detected in HCCs. These results suggest that the HGF-c-met system may play an important role in the regeneration of hepatocytes as well as in the development of HCC in paracrine or autocrine mechanisms.

Energy expenditure determined by the doubly labeled water method in Bolivian Aymara living in a high altitude agropastoral community.

Using the doubly labeled water method (DLW), we determined total energy expenditure (TEE) under free-living conditions in 23 rural Bolivian Aymara (males and females aged 4-65), natives of a small, high-altitude (4000-4100 m), rural agropastoral community in the Andes mountains. In the adults (18-65 y of age), mean TEEs for males and females were 11.1 +/- 1.8 MJ/24 h (range: 9.3-14.1) and 9.8 +/- 0.9 MJ/24 h (8.8-11.3). Non basal energy expenditure expressed as TEE relative to basal energy expenditure (TEE:RMR) and as the difference between TEE and RMR per unit of weight [(TEE-RMR)/wt] showed no significant sex differences. TEE:RMR in the adults (2.00 +/- 0.21) was significantly greater (P < 0.05) than that of adolescents and children (1.67 +/- 0.25), but the mean (TEE-RMR)/wt values were similar in children, adolescents, and adults. Significantly high RMR:FFM values in children and adolescents, reflecting a curvilinear relation of RMR and FFM, have enlarged the differences in TEE:RMR by age groups. When compared with other DLW studies for free-living nonobese adults, the RMR of adult Aymara subjects normalized by the ratio method (RMR:FFM) and by the regression-based method (RMR adjusted with FFM as the covariate) was not significantly different from that observed in subjects living in low altitudes. As compared to FAO/WHO/UNU (1985) recommendations, activity levels were classified as heavy for the adult females and moderate-heavy for the adult males. Energy requirements for maintaining everyday tasks in the Andean people are much higher than expected from the previous studies on food consumption.

Selenium levels and glutathione peroxidase activities in blood in an andean high-altitude population.

Selenium (Se) levels in blood (whole blood, erythrocytes and serum) and blood glutathione peroxidase (GSH-Px) activity were investigated in people living at high altitude in Bolivia (4,000 m above sea level). These parameters were compared to those of people living at low altitude (300 m above sea level). The Se levels in whole blood of the high-altitude subjects did not differ significantly from those of the low-altitude subjects. However, the Se levels in erythrocytes were significantly lower in the high-altitude subjects than in the low-altitude subjects, whereas serum Se levels were higher in the high-altitude subjects than in the low-altitude subjects. GSH-Px activity (Unit/g Hb) was significantly lower in the high-altitude subjects than in the low-altitude subjects. The mean corpuscular Hb concentration (MCHC), an indicator of the age of erythrocytes, in the high-altitude subjects was significantly higher than in the low-altitude subjects. These results show that the GSH-Px activity in the blood of the high-altitude subjects is relatively low. This may be due to their aged erythrocytes and/or to relatively low Se intake in the high-altitude population compared with low-altitude population.

Differing distribution of hepatocyte growth factor-positive cells in the liver of LEC rats with acute hepatitis, chronic hepatitis and hepatoma.

Using anti-rat hepatocyte growth factor (HGF) antibody, we investigated the distribution of HGF-positive cells in the liver tissues of LEC rats at various phases of liver diseases. During the phase of fulminant hepatitis, HGF-positive cells increased remarkably, and many of them were localized at the portal triads; these cells were identified from their shape as non-epithelial cells. A reduced number of HGF-positive cells was observed during the phase of chronic hepatitis, while no HGF-positive cells were seen in the tissue of cholangiofibrosis. During the phase of carcinoma, staining revealed that both the hepatocellular carcinoma cells and the non-epithelial cells in cancerous liver tissue were HGF-positive. These results suggest that, in LEC rats, HGF may play an important role in the regeneration of hepatocytes as well as in the development of hepatocellular carcinoma.

Geographical variation in urinary mercury concentrations among populations living in highland and lowland Bolivia.

Mercury (Hg) concentrations were determined in spot urine samples of populations living in the highland and lowland areas of Bolivia. The mean levels of the Hg (U-Hg) were 0.43 and 0.34 micrograms Hg/g creatinine in lowlanders and in highlanders, respectively, which did not suggest existence of high-level exposure to Hg in these populations. In both highlanders and lowlanders, there was a marked difference in levels of U-Hg among different populations. Several lines of evidence suggested that local dietary patterns resulted in the observed difference in U-Hg among different populations. Several lines of evidence suggested that local dietary patterns resulted in the observed difference in U-Hg levels, although the responsible food items could not be specified. In most of the populations examined, females showed higher U-Hg levels than males. Other factors possibly related to the observed geographical differences are discussed.

Physical growth of Aymara children in a herding community of the Bolivian Altiplano.

In a small agropastoral Aymara community called Wariscata in the Andean Altiplano of Bolivia, anthropometric measurements were made in 1988. In comparison with those of published data for the other rural and urban Andean populations (Aymara, Quechua and Mestizo at high and low altitudes), the Aymara children of Wariscata were taller and heavier than other rural high altitude native children, but similar in height to urban high altitude children. This is possibly due to secular change of growth accompanied with nutritional improvement that has taken place in recent years. Chest width and depth had similar values to those in other Aymara children. But, Aymara children in Wariscata of both sexes had smaller chest dimensions (depth and width) than those of Quechua children. However, these ethnic differences in chest dimensions were not reflected in the adult Aymara and Quechua, suggesting different process of chest growth in Aymara and Quechua populations.