Receptive females mitigate costs of sexual conflict.

Males typically gain fitness from multiple mating, whereas females often lose fitness from numerous mating, potentially leading to sexual conflict over mating. This conflict is expected to favour the evolution of female resistance to mating. However, females may incur male harassment if they refuse to copulate; thus, greater female resistance may increase costs imposed by males. Here, I show that the evolution of resistance to mating raises fitness disadvantages of interacting with males when mating is harmful in female adzuki bean beetles, Callosobruchus chinensis. Females that were artificially selected for higher and lower remating propensity evolved to accept and resist remating, respectively. Compared with females that evolved to accept remating, females that evolved to resist it suffered higher fitness costs from continuous exposure to males. The costs of a single mating measured by the effect on longevity did not differ among selection line females. This study indicates that receptive rather than resistant females mitigate the fitness loss resulting from sexual conflict, suggesting that even though mating is harmful, females can evolve to accept additional mating.

Genetic basis of incidence and period length of circadian rhythm for locomotor activity in populations of a seed beetle.

Circadian rhythms are ubiquitous in a wide variety of organisms, although their genetic variation has been analyzed in only a few species. We found genetic differences in the circadian rhythm of adult locomotor activity among strains of the adzuki bean beetle, Callosobruchus chinensis, which differed in origin and have been maintained in isolation. All beetles in some strains clearly had free-running rhythms in constant darkness whereas most beetles in other strains were arrhythmic. The period of free-running rhythm varied from approximately 19 to 23 h between the strains. F(1) males from reciprocal crosses among strains with different periods of circadian rhythms had circadian periods that were intermediate between their parental strains. Segregation of the circadian rhythm appeared in the F(2) generation. These findings are consistent with the hypothesis that variation in the period length of circadian rhythm is explained by a major autosomal gene with additive effects and no dominance. This hypothesis was supported by the joint scaling test for the free-running period in the F(1) and F(2) generations. We discuss possible causes for genetic variation in circadian rhythm in the C. chinensis strains in terms of random factors and selection.

No genetic correlation between the sexes in mating frequency in the bean beetle, Callosobruchus chinensis.

Female multiple mating, which is common in animals, may have evolved not in response to fitness advantages to females but as a genetic corollary to selection on males to mate frequently. This nonadaptive hypothesis assumes a genetic correlation between females and males in mating frequency, which has received a few empirical investigations. We tested this hypothesis by observing the correlated response in male mating frequency in the adzuki bean beetle, Callosobruchus chinensis to artificial selection on female propensity to remate. Compared to control females, females from lines selected for increased or decreased female propensity to remate had, respectively, higher or lower mating frequency measured by the number of mating within a given period. This indicates that female receptivity to remating is genetically correlated with female mating frequency, and thus the artificial selection for female propensity to remate influenced female mating frequency. In contrast, males from the selected lines that diverged in female mating frequency did not vary significantly in their mating frequency. These results indicate that there is no genetic correlation between the sexes in mating frequency in C. chinensis. This study shows that the reason why females in C. chinensis remate despite suffering fitness costs cannot be explained by indirect selection resulting from selection on males to mate multiple times.

Persistent increase in chromosome instability in lung cancer: possible indirect involvement of p53 inactivation.

Karyotype and fluorescence in situ hybridization analyses have demonstrated the frequent presence of an altered static state of the number of chromosomes (ie, aneuploidy) in lung cancer, but it has not been directly established whether aneuploidy is in fact associated with a persistent increase in the rate of chromosomal losses and gains (ie, chromosome instability, or CIN). The study presented here used a panel of 10 lung cancer cell lines to provide for the first time direct evidence that CIN is a common feature in lung cancer cell lines in association with the presence of significant aneuploidy. In addition, we found that the CIN phenotype correlates well with the presence of p53 mutations. However, human papilloma virus 16-E6-directed inactivation of p53 in a representative non-CIN lung cancer cell line did not result in the induction of CIN, at least up to the 25th generation, suggesting that inactivation of p53 itself is unlikely to directly induce CIN in lung cancer cells. Interestingly, however, significant CIN could be induced in conjunction with the generation of aneuploid populations when the mitotic spindle formation was transiently abrogated in p53-inactivated cells. These results suggest that inactivation of p53 may allow lung cancer cells to go through an inappropriate second division cycle under certain forms of mitotic stresses, which would result in the induction of the CIN phenotype in conjunction with the generation of aneuploidy.

Hsp70 regulates the interaction between the peroxisome targeting signal type 1 (PTS1)-receptor Pex5p and PTS1.

The peroxisome targeting signal type 1 (PTS1) receptor, Pex5p, of the tetratricopeptide repeat (TPR) motif family is located mostly in the cytosol and mediates the translocation of PTS1 proteins to peroxisomes. As a step towards understanding the mechanisms of protein import into peroxisomes, we investigated the molecular mechanisms involved in PTS1 recognition by Pex5p with regard to requirement of energy and cytosolic factors, using cell-free synthesized acyl-CoA oxidase (AOx) as a PTS1 cargo protein, together with Pex5p and heat-shock protein (Hsp)70 from rat liver. Pex5p was partly associated with peroxisomes of rat liver, was resistant to washing with a high concentration of salt and to alkaline extraction and was inaccessible to protease added externally. Pex5p bound to AOx in an ATP-dependent manner. AOx synthesized in a cell-free translating system from rabbit reticulocyte lysate was imported into peroxisomes without being supplemented with Pex5p and Hsp70, implying that peroxisome-associated Pex5p was released from the membranes and functional in this in vitro import assay. Antibodies against Pex5p and Hsp70 inhibited AOx import. In contrast, AOx synthesized in a wheat-germ lysate required the external addition of Pex5p for import, in which Hsp70 augmented the AOx import. The TPR domain of Pex5p was revealed to bind to the N-terminal part in an Hsp70-independent manner, whereas mutual interaction of the TPR region was noted in the presence of Hsp70. Hsp70 interacted with the TPR domain of Pex5p. Moreover, Hsp70 and ATP synergistically enhanced the binding of Pex5p to the C-terminal PTS1-containing part of AOx, implying that Pex5p recognizes its cargo PTS1 protein by chaperone-assisted as well as energy-dependent mechanisms in vivo.

Molecular analysis of the mitotic checkpoint genes BUB1, BUBR1 and BUB3 in human lung cancers.

Our previous studies showed that mitotic checkpoint impairment is present in about 40% of human lung cancer cell lines but that mutations in the MAD mitotic checkpoint genes are infrequent. In the present study, we examined 44 lung cancer cases for the potential involvement of the other gene family involved in the mitotic checkpoint, i.e. BUB. We found that the BUB gene family members including BUB1, BUBR1 and BUB3 are not frequent targets for mitotic checkpoint defects in lung cancers, if present at all. Further studies are thus warranted to elucidate the molecular basis for the acquisition of mitotic checkpoint defects in order to better understand the molecular pathogenesis of lung cancers.

Abnormal haemoglobins, Hb Takamatsu and Hb G-Szuhu, detected during the analysis of glycated haemoglobin (HbA1C) by high performance liquid chromatography.

BACKGROUND: During medical checkups of two unrelated female outpatients during their annual health examination and one male inpatient suffering from cardiac failure the glycated haemoglobin (HbA1C) concentrations measured by high performance liquid chromatography (HPLC) were low, in spite of normal fasting plasma glucose concentrations. However, HbA1C concentrations measured by latex immunoagglutination and fructosamine concentrations were within the normal range. METHOD: Investigations were performed to elucidate the reasons for these discrepancies. RESULTS: Abnormal haemoglobins, Hb Takamatsu and Hb G-Szuhu, were found. The HPLC chromatogram showed an additional peak near HbA1a + b, which resulted in falsely low HbA1C concentrations. Isoelectric focusing analysis of the patients' haemoglobin disclosed abnormal haemoglobins, which migrated faster than normal HbA1 in the two female patients and slower in the male patient. The cDNA sequence and amino acid analyses of the haemoglobin alpha-chains and beta-chains indicated the presence of the haemoglobin variant beta 120 Lys-->Gln in the two female patients and beta 80 Asn-->Lys in the male patient; that is, Hb Takamatsu and Hb G-Szuhu. CONCLUSIONS: These cases show how these silent haemoglobin variants can result in falsely low HbA1C concentration readings when using HPLC.

Histological type-selective, tumor-predominant expression of a novel CHK1 isoform and infrequent in vivo somatic CHK2 mutation in small cell lung cancer.

Inactivation of p53, which represents the most prevalent genetic alteration in lung cancer, has been shown to play a crucial role in the acquisition of genomic instability. We examined 44 lung cancer specimens to search for mutations in the CHK1 and CHK2 genes, which have been suggested to play roles in regulating p53 after DNA damage. We found that the CHK2 gene was somatically mutated in lung cancer in vivo, although at a low frequency, and that a previously undescribed shorter isoform of CHK1 was expressed preferentially in small cell lung cancer in a tumor-predominant manner. Additional studies are warranted to investigate the functional significance of these changes as well as the potential involvement of other components in this important pathway to maintain genomic stability.

Hb Tsukumi [beta117(G19)His-->Tyr]: a new hemoglobin variant found in a Japanese male.

We accidentally observed an abnormal elution pattern on high performance liquid chromatogram when we examined the Hb A1c level in a 65-year-old male patient who suffered from pneumoconiosis and alcoholic liver injury. The value of the glycated fraction was within the normal range but the elution patterns on high performance liquid chromatography varied with the glycohemoglobin analyzers. Isoelectrofocusing and urea-cellulose column chromatography showed an anomalous fast-moving beta chain estimated at approximately 47%. The instability test of the hemolysate was slightly positive. Structural analysis demonstrated that the mutant was consisted by a substitution of His-Tyr at beta117. This new variant was named Hb Tsukumi for the place of residence of the patient. Additionally, the nucleotide sequence showed a change of C-->T [CAC (His)-->TAC (Tyr)] at the first base in the 117th codon of the beta gene.

Hemoglobin Pitié-Salpétrière [beta 34 (B16) Val-->Phe] showing erythrocytosis and mild hemolysis in a Japanese man.

We report the first case of Hemoglobin Pitié-Salpétrière (Hb P-S) identified among the Japanese population. The patient was a 33-year-old man referred to us because of severe erythrocytosis and mild hemolysis. DEAE high-performance liquid chromatography showed an abnormal broad peak around Hb A2 peak. Isoelectrofocusing detected abnormal Hb at the position of the Hb F band, and the content of abnormal Hb was estimated at about 25%. An instability test according to the isopropanol precipitation method was positive, and the beta/alpha ratio of biosynthesized globin was slightly reduced. Structural analyses demonstrated the substitution of phenylalanine for valine at beta 34, which was also confirmed by DNA sequencing; that is a single base substitution of GTC-->TTC at codon 34 of beta chain. From these findings, the abnormal Hb was identified as being a high-oxygen-affinity variant, Hb P-S (beta 34 [B16] Val-->Phe). Hb P-S was detected in the patient's mother but not in his father, suggesting that the inheritance pattern is autosomal dominant. It was suggested that the slightly unstable state of Hb P-S caused by the looseness of alpha 1 beta 1 contact could result in mild hemolysis.

The mammalian peroxin Pex5pL, the longer isoform of the mobile peroxisome targeting signal (PTS) type 1 transporter, translocates the Pex7p.PTS2 protein complex into peroxisomes via its initial docking site, Pex14p.

In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p.

Hb Ube-2 in a diabetic case with an abnormally low HbA1C value.

A 69-year-old male diabetic patient had an abnormally low HbA1C value of 2.8%, which was inconsistent with his elevated fasting plasma glucose of 8.2 mmol/l. Hb analysis disclosed that the abnormal Hb was Hb Ube-2 [alpha68 (E17) Asn --> Asp] and it accounted for 21.5% of the total Hb. Since the glycated abnormal Hb emerged at the same position as did HbF on high performance liquid chromatography, the HbA1C value was falsely low. The present case demonstrates that Hb Ube-2 is one of the abnormal Hbs in which caution should be exercised when monitoring diabetic control.

[Hereditary hemoglobin H disease in Japanese siblings diagnosed by human parvovirus B19 infection].

Hereditary hemoglobin H (HbH) disease was diagnosed in 2 Japanese sisters who presented with aplastic crisis following acute human parvovirus B19 (HPV B19) infection. The proband, an 8-year-old girl, developed persistent fever and pallor, and samples of her peripheral blood showed hypochromic microcytic anemia. Other laboratory data were consistent with hemolytic anemia. Fever and signs of hypochromic microcytic anemia also developed in her sister 9 days later. Cation exchange HPLC analysis of their hemoglobin revealed abnormal hemoglobin migrating faster than HbF, a finding consistent with HbH. Although they presented neither arthralgia nor skin rash, we concluded that their aplastic crisis was induced by HPV B19, because HPV B19 DNA was detected in samples of their peripheral blood by PCR analysis, and HPV B19 IgM and IgG antibody titers were elevated. A genetic analysis of the alpha-globin gene in both sisters and their parents disclosed that the father was heterozygous for alpha-Thal-2, the mother, heterozygous for alpha-Thal-1, and the proband and her sister, double heterozygous for alpha-Thal-1 and alpha-Thal-2. alpha-Thal-2 is a 3.7 kb-deletion allele.

Transmembrane topology of the peroxin, Pex2p, an essential component for the peroxisome assembly.

The peroxisome biogenesis factor, peroxin Pex2p, is an integral membrane protein of peroxisomes [Tsukamoto, T., Miura, S., and Fujiki, Y. (1991) Nature 350, 77-81]. As a step toward elucidating the structure and biological function of Pex2p, we determined the transmembrane topology of Pex2p by expressing epitope-tagged rat Pex2p in COS-7 cells. Pex2p tagged with myc at the C-terminus was detected as a punctate staining pattern, when the cells were permeabilized with 50 microg/ml of digitonin, under which conditions intra-peroxisomal proteins such as PTS1-proteins are inaccessible to exogenous antibodies. N-terminally flag-tagged Pex2p was likewise detected upon the same treatment. These results strongly suggest that both the N- and C-terminal parts of Pex2p are exposed to the cytosol. The transmembrane orientation of Pex2p was also assessed by using rat liver peroxisomes and Pex2p region-specific antibodies. The two types of antibodies used, raised to the N- (amino acid residues 1-131) and C-terminal part (residues 226 to the C-terminus), respectively, specifically recognized Pex2p and immunoprecipitated intact, whole peroxisomes. Pex2p was not recognized by the antibodies when the peroxisomes were treated with Proteinase K. Furthermore, in situ crosslinking studies involving bifunctional reagents revealed an apparently dimeric form of Pex2p. Therefore, Pex2p is anchored to the peroxisomal membrane by two membrane-spanning segments, with its N- and C-terminal regions exposed to the cytosol.

[Hemoglobinopathy in Japan: detection and analysis].

By means of systematic abnormal Hb surveys of people living in eight districts (Okayama, Shimane, Kagawa, Hyogo, Osaka, Nara, Aichi, and Yamanashi Prefectures) using isoelectric focusing, the number of abnormal Hbs discovered and identified among 301,190 subjects was 128. The frequency was estimated to be about one per 2,350. Recently, however, abnormal Hb carriers have been found among individuals with high or low levels of Hb A1c or with an abnormal HPLC pattern. These carriers were found when glyco-HPLC was used to determine the HbA1c level for detection and diagnosis of diabetes mellitus in physical check-ups of inhabitants in each prefecture. To date, 1,227 cases of abnormal Hbs have been detected by isoelectric focusing and glyco-HPLC. These Hbs were analyzed by protein chemistry and DNA analytical methods. The most typical Hbs found in Japan have been Hb J-Cape Town (an alpha-chain variant) and Hb Riyadh (a beta-chain variant). Homozygotes for Hb J-Cape Town, Hb Ube-2, Hb Hamadan, Hb G-Szuhu and Hb Takamatsu have been discovered among abnormal Hb carriers. The carriers of Hb M-Hyde Park and Hb Koln, which cause hemolytic anemia due to their molecular instability, have been found in some parts of Japan, but by clinical examination rather than by Hb survey. On the other hand, alpha- and beta-thals have often been detected in blood samples with a high or low HbA2 level or in abnormal Hb such as Hb H, during systematic surveys for abnormal Hb. Peripheral blood examinations of these patients revealed typical data, a low MCV, MCH, and sometimes low Hb levels. The diagnosis was made by DNA analyses of the nucleotide sequencing of the beta-globin gene for beta-thals and of the alpha-globin gene arrangement for alpha-thals. beta-Thals found in Japanese mainly involve a point mutation, such as-31CapA-->G(in ATA box) and beta 90 codon GAG-->TAG(creation of a stop codon). The alpha-thals had genotypes of -alpha 3.7/alpha alpha for alpha-thal-2, -SEA/alpha alpha for alpha-thal-1 and -SEA/-alpha 3.7 for Hb H disease.

The peroxin Pex14p. cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis.

Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14). The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40-42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

Hb Oita [alpha45(CE3)His-->Pro]: a new silent hemoglobin variant.

We describe a new alpha chain variant accidentally found in a 49-year-old female living in Usa City, Oita Prefecture, Japan. An abnormally low Hb A1c value of 2.5% (normal range: 4.8-6.3%) was found while she was treated with glucocorticoid for Fisher syndrome. The patient was also diagnosed as having an iron deficiency anemia, but otherwise showed a normal hemogram. An abnormal hemoglobin was not detectable by isoelectrofocusing and high performance liquid chromatographic methods, but appeared as a fast-moving alpha chain abnormality by urea-carboxymethyl cellulose column chromatography of the globin, from which the content of the abnormal hemoglobin was estimated at approximately 20%. The instability test of the hemolysate was normal. Structural studies demonstrated that the abnormal hemoglobin had an amino acid substitution of His-->Pro at alpha45. It is a new variant and was named Hb Oita or alpha45(CE3) His-->Pro. Additionally, sequence analysis showed a nucleotide change from A-->C at the second base in the 45th codon of the alpha2 gene, CAC(His)-->CCC (Pro). The beta/alpha ratio was 0.51 (normal range: 0.9-1.2). Her mother and a son did not have the abnormal hemoglobin variant; her father was deceased and no sample was available to verify the inheritance of the variant in this kindred.