Competitive habitat utilization in the damselfly,Mnais nawai (Zygoptera: Calopterygidae) coexisting with a related species,Mnais pruinosa

Reproductive behaviors related to habitat utilization were studied in males of the damsefly,Mnais nawai, which has two male forms, territorial orange-winged males (nawai) and non-territorial pale-orange-winged males (sahoi), at the upper part of a mountain stream where they partiallycoexist with a related species,Mnais pruinosa, which also has two male forms, territorial orange-winged males (esakii) and non-territorial hyaline-winged males (strigata). These two species showed parapatric distribution; the lower part of the stream was occupied byM. nawai, and the upper part byM. pruinosa. In the present study, cross-matings occurred between bothMnais species, although normal intraspecific matings occurred more frequently than cross-matings. Territorial males of both species copulated with conspecific females that entered their territory and guarded the ovipositing females, probably to avoid sperm displacement resulting from subsequent copulations. Severe competition for oviposition sites by territorial males even occurred between the two species. On the other hand, non-territorial males of both species have alternative mating strategies (including several tactics such as sneaking, takeover and interception). The possible benefits from conflict among territorial males of both species is discussed.     

Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E₂) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E₂ pellets were removed, but administration of progesterone (P), 5-dihydrotestosterone (DHT), or continued implantation of E₂ pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E₂, P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Establishing apoptosis resistant cell lines for improving protein productivity of cell culture

The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA- containing SV40 ori and immunoglobulin gene for transiently expressing protein. The bcl–2 expressing COS–1 cells produced more protein than the mock transfected COS–1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.     

O uptake rate measurements as a novel tool to study shear effects on suspended strawberry cells

Despite the same mean volumetric power input of 19 and 47 W/m 3 , the specific oxygen uptake rate (OUR S ) of strawberry ( Fragaria ananassa) cell suspensions was higher in bioreactors equipped with a Rushton Turbine (4.4 and 6.2 3 10 -5 mol O 2 /kg.s) than with an anchor stirrer (2.5 and 4.6 3 10 -5 mol O 2kg.s). The increase in OUR S was caused by stress-activated respiration and appeared to be correlated with the locally dissipated power input. OUR S was corrected for the increase in surface through aggregate break-up and reached a maximum of 6.0 3 10 -5 mol O 2kg.s when agitating with approximately 200 kW/m 3 locally dissipated power input.     

Host range phenotype induced by mutations in the internal ribosomal entry site of poliovirus RNA.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.     

The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

Differential effects of body weight, hyperinsulinemia and oral glucose load on serum C-peptide/insulin molar ratio.

Serum C-peptide immunoreactivity (CPR)/immunoreactive insulin (IRI) molar ratio was determined in 136 subjects without renal, hepatic and thyroid disorders, at fasting, and during the initial period of 75 g-oral glucose tolerance test. The subjects were divided into 4 groups based on their body weight and age; Group A, young (< 55 years) and normal body weight (body mass index [BMI, kg/m2] < or = 25) subjects; Group B, young and overweight (BMI > 25) subjects; Group C, aged (> or = 55 years) and normal body weight (BMI < or = 25) subjects; Group D, aged and overweight subjects. Fasting CPR/IRI ratio and absolute CPR level negatively correlated in Groups B and D but not in A and C. After oral glucose load with elevation of insulin, CPR/IRI ratio invariably declined in all groups and significant negative correlation between CPR/IRI and CPR was found in Groups A, B and D but not in C. Slope of the regression lines obtained for correlation between CPR/IRI ratio and CPR were significantly steeper at fasting compared to the post-stimulation phase. CPR/IRI ratio is affected by hyperinsulinemia and oral glucose load but not by obesity alone. Assuming that CPR/IRI ratio reflects hepatic extraction of insulin, the insulin clearance at fasting is progressively reduced with increasing insulin secretion in overweight subjects: failure to detect such phenomenon in normal body weight subjects may be due to a narrower CPR range in this population. Insulin metabolism at fasting and during glucose stimulation is likely to be regulated by distinct factors.     

Effects of chair restraint on plasma enzyme values in the rhesus monkey (Macaca mulatta)

The purpose of this paper was to examine the effect of chair restraint on plasma enzyme values in the rhesus monkey. Six monkeys were restrained to the monkey chair for eight hours. Creatine phosphokinase (CK) value increased significantly three hours after the onset of restraint and LDH value did eight hours after the onset of restraint. The increase in CK, GOT and GPT values continued for 1 or 2 days after the release from restraint. On the other hand, these plasma enzyme values in non-restraint monkeys showed almost no changes. These results indicate that it is necessary to establish a proper method for the adjustment to chair restraint in the rhesus monkey.