A heterozygous protein-truncating RFX6 variant in a family with childhood-onset, pregnancy-associated and adult-onset diabetes.

BACKGROUND: Recently, heterozygous RFX6 mutations including p.Arg377Ter were identified in individuals with maturity-onset diabetes of the young (MODY). Clinical analysis of 36 individuals suggested that RFX6 mutation-induced MODY is characterized by low penetrance and relatively late onset. However, given the small number of previous reports and the limited clinical information of each case, further studies are necessary to clarify the phenotypic characteristics of RFX6 mutations. CASE REPORT: We identified a previously reported p.Arg377Ter variant of RFX6 in a three-generation family with diabetes. The variant was detected through mutation screening for 30 diabetes-associated genes. The variant was not found in public databases and was predicted to encode a truncated protein or undergo nonsense-mediated mRNA decay. The proband showed glycosuria from 8 years of age and was diagnosed with MODY at 10 years of age, before the onset of puberty. She received basal and bolus insulin injection as initial therapy. The proband's mother exhibited glycosuria at 26 years of age when she conceived the first child. The mother was treated with insulin, oral hypoglycaemic drugs and diet. The proband and her mother were negative for islet cell autoantibodies. The maternal grandmother showed glycosuria around 50 years of age and was treated with oral hypoglycaemic drugs alone. CONCLUSION: This study provides supporting evidence for the causal relationship between heterozygous RFX6 mutations and MODY. Furthermore, our results indicate that phenotypic consequences of RFX6 mutations are highly variable even within a single family, and possibly include childhood-onset and pregnancy-associated non-autoimmune diabetes.

Low prevalence of maternal microchimerism in peripheral blood of Japanese children with type 1 diabetes.

AIM: To clarify the prevalence and degree of maternal microchimerism in Japanese children with type 1 diabetes, as well as its effect on phenotypic variation. METHODS: We studied 153 Japanese children with type 1 diabetes, including 124 children positive for ß-cell autoantibodies, and their 71 unaffected siblings. The number of circulating microchimeric cells per 105 host cells was estimated by the use of quantitative-polymerase chain reaction targeting non-transmitted maternal human leukocyte antigen alleles. The results were compared to previous data from white European people. Phenotypic comparison was performed between maternal microchimerism carriers and non-carriers with diabetes. RESULTS: Maternal microchimerism was detected in 15% of children with autoantibody-positive type 1 diabetes, 28% of children with autoantibody-negative type 1 diabetes, and 16% of unaffected siblings. There were no differences in the prevalence or levels of maternal microchimerism among the three groups or between the children with type 1 diabetes and their unaffected siblings. Furthermore, maternal microchimerism carriers and non-carriers exhibited similar phenotypes. CONCLUSIONS: Maternal microchimerism appears to be less common in Japanese children with type 1 diabetes than in white European people. Our data indicate that maternal microchimerism is unlikely to be a major trigger or a phenotypic determinant of type 1 diabetes in Japanese children and that the biological significance of maternal microchimerism in type 1 diabetes may differ among ethnic groups.

Aneuploid rescue precedes X-chromosome inactivation and increases the incidence of its skewness by reducing the size of the embryonic progenitor cell pool.

STUDY QUESTION: Do monosomy rescue (MR) and trisomy rescue (TR) in preimplantation human embryos affect other developmental processes, such as X-chromosome inactivation (XCI)? SUMMARY ANSWER: Aneuploid rescue precedes XCI and increases the incidence of XCI skewness by reducing the size of the embryonic progenitor cell pools. WHAT IS KNOWN ALREADY: More than half of preimplantation human embryos harbor aneuploid cells, some of which can be spontaneously corrected through MR or TR. XCI in females is an indispensable process, which is predicted to start at the early-blastocyst phase. STUDY DESIGN, SIZE, DURATION: We examined the frequency of XCI skewness in young females who carried full uniparental disomy (UPD) resulting from MR or TR/gamete complementation (GC). The results were statistically analyzed using a theoretical model in which XCI involves various numbers of embryonic progenitor cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 39 children and young adults ascertained by imprinting disorders. XCI ratios were determined by DNA methylation analysis of a polymorphic locus in the androgen receptor gene. We used Bayesian approach to assess the probability of the occurrence of extreme XCI skewness in the MR and TR/GC groups using a theoretical model of 1-12 cell pools. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 12 of 39 individuals (31%) showed skewed XCI. Extreme skewness was observed in 3 of 15 MR cases (20%) and 1 of 24 TR/GC cases (4.2%). Statistical analysis indicated that XCI in the MR group was likely to have occurred when the blastocyst contained three or four euploid embryonic progenitor cells. The estimated size of the embryonic progenitor cell pools was approximately one-third or one-fourth of the predicted size of normal embryos. The TR/GC group likely had a larger pool size at the onset of XCI, although the results remained inconclusive. LIMITATIONS, REASONS FOR CAUTION: This is an observational study and needs to be validated by experimental analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study provides evidence that the onset of XCI is determined by an intrinsic clock, irrespectively of the number of embryonic progenitor cells. Our findings can also be applied to individuals without UPD or imprinting disorders. This study provides a clue to understand chromosomal and cellular dynamics in the first few days of human development, their effects on XCI skewing and the possible implications for the expression of X-linked diseases in females. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Grants-in-aid for Scientific Research on Innovative Areas (17H06428) and for Scientific Research (B) (17H03616) from Japan Society for the Promotion of Science (JSPS), and grants from Japan Agency for Medical Research and Development (AMED) (18ek0109266h0002 and 18ek0109278h0002), National Center for Child Health and Development and Takeda Science Foundation. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Protein-altering variants of PTPN2 in childhood-onset Type 1A diabetes.

AIM: To examine the contribution of PTPN2 coding variants to the risk of childhood-onset Type 1A diabetes. METHODS: PTPN2 mutation analysis was carried out for 169 unrelated Japanese people with childhood-onset Type 1A diabetes. We searched for coding variants that were absent or extremely rare in the general population and were scored as damaging by multiple in silico programs. We performed mRNA analysis and three-dimensional structural prediction of the detected variants, when possible. We also examined possible physical links between these variants and previously reported risk SNPs as well as clinical information from variant-positive children. RESULTS: One frameshift variant (p.Q286Yfs*24) and two probably damaging missense substitutions (p.C232W and p.R350Q) were identified in one child each. Of these, p.Q286Yfs*24 and p.C232W were hitherto unreported, while p.R350Q accounted for 2/121,122 alleles of the exome datasets. The p.Q286Yfs*24 variant did not encode stable mRNA, and p.C232W appeared to affect the structure of the tyrosine-protein phosphatase domain. The three variants were physically unrelated to known risk SNPs. The variant-positive children manifested Type 1A diabetes without additional clinical features and invariably carried risk human leukocyte antigen alleles. CONCLUSIONS: The results provide the first indication that PTPN2 variants contribute to the risk of Type 1A diabetes, independently of known risk SNPs. PTPN2 coding variants possibly induce non-specific Type 1A diabetes phenotypes in individuals with human leukocyte antigen-mediated disease susceptibility. Our findings warrant further validation.

Catastrophic cellular events leading to complex chromosomal rearrangements in the germline.

Although complex chromosomal rearrangements were thought to reflect the accumulation of DNA damage over time, recent studies have shown that such rearrangements frequently arise from 'all-at-once' catastrophic cellular events. These events, designated chromothripsis, chromoanasynthesis, and chromoanagenesis, were first documented in the cancer genome and subsequently observed in the germline. These events likely result from micronucleus-mediated chromosomal shattering and subsequent random reassembly of DNA fragments, although several other mechanisms have also been proposed. Typically, only one or a few chromosomes of paternal origin are affected per event. These events can produce intrachromosomal deletions, duplications, inversions, and translocations, as well as interchromosomal translocations. Germline complex rearrangements of autosomes often result in developmental delay and dysmorphic features, whereas X chromosomal rearrangements are usually associated with relatively mild clinical manifestations. The concept of these catastrophic events provides novel insights into the etiology of human genomic disorders. This review introduces the molecular characteristics and phenotypic outcomes of catastrophic cellular events in the germline.

FUT2 non-secretor status is associated with Type 1 diabetes susceptibility in Japanese children.

AIM: To examine the contribution of the FUT2 gene and ABO blood type to the development of Type 1 diabetes in Japanese children. METHODS: We analysed FUT2 variants and ABO genotypes in a total of 531 Japanese children diagnosed with Type 1 diabetes and 448 control subjects. The possible association of FUT2 variants and ABO genotypes with the onset of Type 1 diabetes was statistically examined. RESULTS: The se2 genotype (c.385A>T) of the FUT2 gene was found to confer susceptibility to Type 1A diabetes in a recessive effects model [odds ratio for se2/se2, 1.68 (95% CI 1.20-2.35); corrected P value = 0.0075]. CONCLUSIONS: The FUT2 gene contributed to the development of Type 1 diabetes in the present cohort of Japanese children.

Risk assessment of medically assisted reproduction and advanced maternal ages in the development of Prader-Willi syndrome due to UPD(15)mat.

Recent studies have suggested that disomic oocyte-mediated uniparental disomy 15 (UPD(15)mat) is increased in patients with Prader-Willi syndrome (PWS) born after medically assisted reproduction (MAR). However, it remains unknown whether the increase is primarily due to MAR procedure itself or advanced maternal childbearing ages as a predisposing factor for the disomic oocyte production. To examine this matter, we studied 122 naturally conceived PWS patients (PWS-NC group) and 13 MAR-conceived patients (PWS-MAR group). The relative frequency of disomic oocyte-mediated UPD(15)mat was significantly higher in PWS-MAR group than in PWS-NC group (7/13 vs 20/122, p = 0.0045), and the maternal childbearing ages were significantly higher in PWS-MAR group than in PWS-NC group [median (range), 38 (26-45) vs 30 (19-42), p = 0.0015]. However, the logistic regression analysis revealed no significant association between the occurrence of disomic oocyte-mediated UPD(15)mat and MAR, after adjusting for childbearing age (p = 0.25). Consistent with this, while the frequency of assisted reproductive technology (ART)-conceived livebirths was higher in the PWS patients than in the Japanese general population (6.4% vs 1.1%, p = 0.00018), the distribution of childbearing ages was significantly skewed to the increased ages in the PWS patients (p < 2.2 × 10(-16) ). These results argue against a positive association of MAR procedure itself with the development of UPD(15)mat.

Variants associated with autoimmune Type 1 diabetes in Japanese children: implications for age-specific effects of cis-regulatory haplotypes at 17q12-q21.

AIMS: The aim of this study was to clarify the significance of previously reported susceptibility variants in the development of autoimmune Type 1 diabetes in non-white children. Tested variants included rs2290400, which has been linked to Type 1 diabetes only in one study on white people. Haplotypes at 17q12-q21 encompassing rs2290400 are known to determine the susceptibility of early-onset asthma by affecting the expression of flanking genes. METHODS: We genotyped 63 variants in 428 Japanese people with childhood-onset autoimmune Type 1 diabetes and 457 individuals without diabetes. Possible association between variants and age at diabetes onset was examined using age-specific quantitative trait locus analysis and ordered-subset regression analysis. RESULTS: Ten variants, including rs2290400 in GSDMB, were more frequent among the people with Type 1 diabetes than those without diabetes. Of these, rs689 in INS and rs231775 in CTLA4 yielded particularly high odds ratios of 5.58 (corrected P value 0.001; 95% CI 2.15-14.47) and 1.64 (corrected P value 5.3 × 10-5 ; 95% CI 1.34-2.01), respectively. Age-specific effects on diabetes susceptibility were suggested for rs2290400; heterozygosity of the risk alleles was associated with relatively early onset of diabetes, and the allele was linked to the phenotype exclusively in the subgroup of age at onset ≤ 5.0 years. CONCLUSIONS: The results indicate that rs2290400 in GSDMB and polymorphisms in INS and CTLA4 are associated with the risk of Type 1 diabetes in Japanese children. Importantly, cis-regulatory haplotypes at 17q12-q21 encompassing rs2290400 probably determine the risk of autoimmune Type 1 diabetes predominantly in early childhood.

Molecular basis of non-syndromic hypospadias: systematic mutation screening and genome-wide copy-number analysis of 62 patients.

STUDY QUESTION: What percentage of cases with non-syndromic hypospadias can be ascribed to mutations in known causative/candidate/susceptibility genes or submicroscopic copy-number variations (CNVs) in the genome? SUMMARY ANSWER: Monogenic and digenic mutations in known causative genes and cryptic CNVs account for >10% of cases with non-syndromic hypospadias. While known susceptibility polymorphisms appear to play a minor role in the development of this condition, further studies are required to validate this observation. WHAT IS KNOWN ALREADY: Fifteen causative, three candidate, and 14 susceptible genes, and a few submicroscopic CNVs have been implicated in non-syndromic hypospadias. STUDY DESIGN, SIZE, DURATION: Systematic mutation screening and genome-wide copy-number analysis of 62 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study group consisted of 57 Japanese and five Vietnamese patients with non-syndromic hypospadias. Systematic mutation screening was performed for 25 known causative/candidate/susceptibility genes using a next-generation sequencer. Functional consequences of nucleotide alterations were assessed by in silico assays. The frequencies of polymorphisms in the patient group were compared with those in the male general population. CNVs were analyzed by array-based comparative genomic hybridization and characterized by fluorescence in situ hybridization. MAIN RESULTS AND THE ROLE OF CHANCE: Seven of 62 patients with anterior or posterior hypospadias carried putative pathogenic mutations, such as hemizygous mutations in AR, a heterozygous mutation in BNC2, and homozygous mutations in SRD5A2 and HSD3B2. Two of the seven patients had mutations in multiple genes. We did not find any rare polymorphisms that were abundant specifically in the patient group. One patient carried mosaic dicentric Y chromosome. LIMITATIONS, REASONS FOR CAUTION: The patient group consisted solely of Japanese and Vietnamese individuals and clinical and hormonal information of the patients remained rather fragmentary. In addition, mutation analysis focused on protein-altering substitutions. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide evidence that pathogenic mutations can underlie both mild and severe hypospadias and that HSD3B2 mutations cause non-syndromic hypospadias as a sole clinical manifestation. Most importantly, this is the first report documenting possible oligogenicity of non-syndromic hypospadias. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology; by the Grant-in-Aid from the Japan Society for the Promotion of Science; by the Grants from the Ministry of Health, Labour and Welfare, from the National Center for Child Health and Development and from the Takeda Foundation. The authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs.

Pseudoautosomal deletions encompassing a novel homeobox gene cause growth failure in idiopathic short stature and Turner syndrome.

Growth retardation resulting in short stature is a major concern for parents and due to its great variety of causes, a complex diagnostic challenge for clinicians. A major locus involved in linear growth has been implicated within the pseudoautosomal region (PAR1) of the human sex chromosomes. We have determined an interval of 170 kb of DNA within PAR1 which was deleted in 36 individuals with short stature and different rearrangements on Xp22 or Yp11.3. This deletion was not detected in any of the relatives with normal stature or in a further 30 individuals with rearrangements on Xp22 or Yp11.3 with normal height. We have isolated a homeobox-containing gene (SHOX) from this region, which has at least two alternatively spliced forms, encoding proteins with different patterns of expression. We also identified one functionally significant SHOX mutation by screening 91 individuals with idiopathic short stature. Our data suggest an involvement of SHOX in idiopathic growth retardation and in the short stature phenotype of Turner syndrome patients.

An improved method for the isolation of dense storage granules from human platelets.

Pretreatment of human platelets with the metabolic inhibitors rotenone and 2-deoxyglucose, before French press homogenization, has led to the isolation of dense storage granules in an overall yield of about 20%. The concentrations of serotonin, ATP and ADP were estimated in the dense granules. Serotonin was 40--60-fold enriched in the dense granules compared to the platelet homogenate. Stored ATP and ADP were also 40-fold enriched in the dense granules compared to the estimated storage nucleotide pool in intact platelets. The ATP to ADP ratio in the isolated dense granules was 0.68-0.70, the same as the ratio of the secreted ATP and ADP. In platelets prelabeled with [3H]adenine, the specific radioactivities of the ATP and ADP in the isolated dense granules and of the secreted ATP and ADP were both negligible, whereas the estimated specific radioactivity of the metabolically active ATP and ADP was 2,000 cpm/nmol. These results confirm that the ATP and ADP in the isolated dense granules are the same as the secreted ATP and ADP in terms of metabolic inactivity and their ATP to ADP ratios.

Next-generation sequencing for patients with non-obstructive azoospermia: implications for significant roles of monogenic/oligogenic mutations.

Azoospermia affects up to 1% of adult men. Non-obstructive azoospermia is a multifactorial disorder whose molecular basis remains largely unknown. To date, mutations in several genes and multiple submicroscopic copy-number variations (CNVs) have been identified in patients with non-obstructive azoospermia. The aim of this study was to clarify the contribution of nucleotide substitutions in known causative genes and submicroscopic CNVs in the genome to the development of non-obstructive azoospermia. To this end, we conducted sequence analysis of 25 known disease-associated genes using next-generation sequencing and genome-wide copy-number analysis using array-based comparative genomic hybridization. We studied 40 Japanese patients with idiopathic non-obstructive azoospermia. Functional significance of molecular alterations was assessed by in silico analyses. As a result, we identified four putative pathogenic mutations, four rare polymorphisms possibly associated with disease risk, and four probable neutral variants in 10 patients. These sequence alterations included a heterozygous splice site mutation in SOHLH1 and a hemizygous missense substitution in TEX11, which have been reported as causes of non-obstructive azoospermia. Copy-number analysis detected five X chromosomal or autosomal CNVs of unknown clinical significance, in addition to one known pathogenic Y chromosomal microduplication. Five patients carried multiple molecular alterations. The results indicate that monogenic and oligogenic mutations, including those in SOHLH1 and TEX11, account for more than 10% of cases of idiopathic non-obstructive azoospermia. Furthermore, this study suggests possible contributions of substitutions in various genes as well as submicroscopic CNVs on the X chromosome and autosomes to non-obstructive azoospermia, which require further validation.

Studies on catalase compartmentation in digitonin-treated rat hepatocytes.

The cellular compartmentation of catalase was studied in digitonin-permeabilized rat hepatocytes. A biphasic dose-response curve was observed for the unmasking of catalase activity by digitonin in latency studies. About 40-60% of the total catalase activity was seen in the range of 5-200 microM digitonin compared to 13% free activity in control preparations without digitonin. The free catalase activity began to increase again above 300 micron digitonin, and all latency was lost around 500 microM and above. These results indicate that there exists in rat hepatocytes catalase with two levels of crypticity to digitonin, only one of which was seen in a mixed organelle preparation containing peroxisomes.

Adenine nucleotide metabolism of blood platelets. IX. Time course of secretion and changes in energy metabolism in thrombin-treated platelets.

Changes in the energy metabolism of washed human platelets were compared with the kinetics of secretion induced by thrombin (5 units/ml). A 50% decrease in the level of metabolic ATP (3H-labelled), which was essentially complete in 30s, was matched in rate by adenine nucleotide secretion from storage in dense granules. Incubation of platelets with antimycin before thrombin addition increased the rate of fall in metabolic ATP, but did not affect the rate of adenine nucleotide secretion. beta-N-Acetylglucosaminidase secretion, which was slower than adenine nucleotide secretion in control platelets, was noticeably inhibited by antimycin, confirming previous reports that different regulatory mechanisms exist for dense and alpha-granule secretion. The rates of rephosphorylation of metabolic ADP to ATP via glycolysis and oxidative phosphorylation were estimated by measuring lactate production and O2 consumption in resting and thrombin-stimulated platelets and compared to the level of metabolic ATP (9-10 nmol/mg of platelet protein in the resting state). The rate of ATP production was stimulated at least two fold from 12 nmol to 24 nmol/min/mg within seconds of thrombin addition. This increased rate was maintained over the observed period of 5 min although the level of metabolic ATP had decreased to 4-5 nmol/mg within 30 s; the turnover of the remaining metabolic ATP thus increased four fold over the resting state although the actual stimulation of energy production was only two fold.

Thrombin-induced oxygen consumption, malonyldialdehyde formation and serotonin secretion in human platelets.

The relationship of a thrombin-induced burst in O2 consumption to lipid peroxidation in washed human platelets was investigated by measuring malonyldialdehyde, a by-product of endoperoxide degradation in platelets. The ratio of O2 consumed by malonyldialdehyde produced was approximately 7:1. Acetylsalicylate blocked the formation of malonyldialdehyde completely and partially inhibited the O2 burst induced by thrombin. 5,8,11,14-Eicosatetraynoic acid inhibited the O2 burst as well as the malonyldialdehyde formation completely. The release of [14C]serotonin was not affected by either inhibitor.

The mechanism of lack of hypocholesterolemic effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in rats.

In order to clarify the reason why pravastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, did not show hypocholesterolemic effects in rats, the changes of various parameters affecting the serum cholesterol levels by pravastatin were determined in rats and rabbits, as a comparison. In rabbits, pravastatin administration at 50 mg/kg for 14 days decreased serum and liver cholesterol by 40% and 8%, respectively. The hepatic LDL receptor activity was increased 1.7-fold, and VLDL cholesterol secretion was decreased. Cholesterol 7 alpha-hydroxylase activity was not changed. In contrast, in rats, serum cholesterol was increased by 14% at 50 mg/kg and 27% at 250 mg/kg for 7 days, respectively. At 250 mg/kg, liver cholesterol was significantly increased by 11%. Under these conditions, neither the hepatic LDL receptor activity nor cholesterol 7 alpha-hydroxylase was changed, and VLDL cholesterol secretion was increased. At 250 mg/kg, net cholesterol synthesis in rat liver was increased after 7 days of consecutive administration. These results imply that in rats, stimulated net cholesterol synthesis caused the increase of liver cholesterol followed by the increase of VLDL cholesterol secretion, and resulted in the raise of plasma cholesterol. Although hepatic HMG-CoA reductase was induced almost the same fold in both animals at 50 mg/kg, the induced HMG-CoA reductase activity in rats might overcome the inhibitory capability of pravastatin, resulting in an increase of net cholesterol synthesis, but not in rabbits. This over response to pravastatin in rats might cause the lack of hypocholesterolemic effects of this drug.

Subcellular fractionation of pig platelets.

Subcellular components were obtained from pig platelets, disrupted by means of a French press and separated into 4 primary fractions. The granule fraction (10 000 g) was subjected to a sucrose gradient fractionation. Primary fractions and the granule subfractions were studied electron microscopically and biochemically by following the distribution of markers of membranes, lysosomes or alpha-granules, mitochondria and dense granules. With this technique of platelet homogenization, 80% of the serotonin and 93% of the beta-N-acetylglucosaminidase were found to be particulate. In the gradient, mitochondria were sharply banded in a fraction (density 1.16--1.17) having a specific activity 10--100 times higher than the other fractions of the gradient. Serotonin-containing granules were found in a pellet of density greater than 1.27 and contained 60% of the serotonin and adenine nucleotides of the granule fraction. The lysosome markers that were monitored, acid phosphatase and beta-N-acetylglucosaminidase, exhibited different distribution patterns. Acid phosphatase showed the highest specific activity in the microsomal fraction with only 2.8% in the granule fraction, and this latter amount also appeared to be associated with membranes upon further fractionation. Beta-N-acetylglucosaminidase was present in both the granule fraction and in the microsomal fraction with nearly the same specific activity. However, that present in the granule fraction was clearly associated with granules that distributed over a wide range of densities on a sucrose gradient. The calcium distribution was followed to attempt to determine its subcellular location; 19% was found in the same subfraction as the serotonin-containing granules, but at least 50% of the particulate calcium was associated with granules distinctly separate from the storage granules.

Preventive effect of pravastatin sodium, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on coronary atherosclerosis and xanthoma in WHHL rabbits.

In this paper, we examined whether the development of atherosclerosis in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model of familial hypercholesterolemia in man, could be prevented by the reduction of serum cholesterol levels. Pravastatin sodium (the generic name of CS-514), a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was used as a cholesterol-lowering drug. The drug was administered orally to 12 WHHL rabbits (2-3 months old) at a dose of 50 mg/kg per day for 24 weeks, and 13 animals were given water as control. In the treated group, serum cholesterol, phospholipid and triacylglycerol levels were significantly reduced by 28%, 32% and 16%, respectively, as compared with those of the control group. Although the prevention of development of the aortic atherosclerosis was not significant, the progression of coronary atherosclerosis was significantly prevented. The incidence of atherosclerosis in four main coronary arteries was reduced from 42% (control group) to 19% (treated group, P less than 0.01), and the development of lesion of coronary arteries evaluated by area of lesion was reduced from 19.7% (control group) to 9.1% (treated group, P less than 0.05). Histopathological findings supported the above observations. In addition, development of xanthoma in digital joints was also reduced from 90.4% (control group) to 58.3% (treated group, P less than 0.005). These results suggest that the development of coronary atherosclerosis and xanthoma in WHHL rabbit was reduced by continuous reduction of serum cholesterol levels treated with pravastatin sodium.

On the mechanism of induction of the enzyme systems for peroxisomal beta-oxidation of fatty acids in rat liver by diets rich in partially hydrogenated fish oil.

In this paper, we describe the early biochemical changes in liver cells that occur in rats fed a semisynthetic diet containing 20% (w/w) partially hydrogenated fish oil. Within hours the level of ornithine decarboxylase (ODC) increased, peaked at about 24 h (11-fold increase) and returned to subnormal levels within 48 h. The diet evoked a similar rapid increase in the cellular level of mRNA for the bifunctional enzyme of peroxisomal beta-oxidation (enoyl-CoA hydratase: beta-hydroxyacyl-CoA dehydrogenase (HD)) (12-fold), followed by increases in the specific content of HD protein (3-fold) and the capacity for beta-oxidation in peroxisomes (5.3-fold). The cellular level of long-chain acyl-CoA increased 2.1-fold. By contrast, no significant changes were observed in the specific activities of ornithine decarboxylase, peroxisomal beta-oxidation activity and microsomal omega-hydroxylation as well as the level of long-chain acyl-CoA in livers of rats fed (1 week) diets containing 20% (w/w) soybean oil with added 3 or 6% (w/w) of either elaidic acid (18:1(11) (trans)), brassidic acid (22:1(13) (trans)) or erucic acid (22:1(13) (cis)). Expression of normal levels of mRNA for the bifunctional enzyme was also found. Morphometric analyses revealed no proliferation of peroxisomes in these fatty acid-supplemented diets, in contrast to that observed with the partially hydrogenated fish oil diet. These results are consistent with the proposal (Flatmark, T., Christiansen, E.N. and Kryvi, H. (1983) Biochim. Biohys. Acta 753, 460-466) that components in dietary oils, different from C22:1 cis and trans fatty acids, are responsible for the pleiotropic responses evoked in target cells. Thus, the pattern of response induced by partially hydrogenated fish oil mimics those induced by xenobiotic compounds collectively termed peroxisome proliferators.