A new cell line from larval fat bodies of the bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Summary A new cell line, designated IOZCAS-Ha-I, was initiated from the fat body of larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. Spherical cells were predominant among the various cell types. The cell line showed a typical lepidopteran chromosome pattern ranging from 58 to 239 chromosomes in the majority of the cells, it was confirmed to have originated from the H. armigera by the DNA amplification-fingerprinting polymerase chain reaction (DAF-PCR) technique. The new cell line was only slightly susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses (NPV) from H. armigera.     

Determination of the thermogenesis curves and studies of the thermodynamics and thermokinetics of seed germination

The thermogenesis curves of the germination of different rice and tree seeds were determined and studied by using a newly constructed microcalorimeter. The thermogenesis curves of the germination of the seeds demonstrate the existence of physiological triphasic patterns, which include imbibition, activation and growth stages in the germination process. The thermodynamics and thermokinetics of the main growth phase of the growth stage in the germination process have been studied. The growth heat effect (deltaH), the growth rate constant (k), the growth inhibitory factor (s) and deceleration rate constant (beta) have been determined and calculated, In addition, the experimental thermokinetic equations of the growth stage in the seed germination process have been established.     

Role of Cysteine Residues in Human Plasma Phospholipid Transfer Protein

Phospholipid transfer protein (PLTP) belongs to a family of human plasma lipid transfer proteins that bind to small amphophilic molecules. PLTP contains cysteines at residues 5, 129, 168, and 318. Bactericidal/permeability-increasing protein, which is a member of the same gene family, contains an essential disulfide bond between Cys₁₃₅ and Cys₁₇₅; these residues, which correspond to Cys₁₂₉ and Cys₁₆₈ in PLTP, are conserved among all known members of the gene family. To identify the importance of these and the remaining cysteine residues to PLTP secretion and activity, each was replaced by a glycine by site-directed mutagenesis. The mutant as well as wild-type PLTP cDNAs were cloned into the mammalian expression vector pSV·SPORT1, and the PLTP cDNAs were transfected to COS-6 cells for expression. PLTP Cys₁₂₉ Gly and PLTP Cys₁₆₈ Gly were secretion incompetent. Neither PLTP mass nor activity was detectable in cell lysates and culture medium. Relative to wild-type PLTP, PLTP Cys₅ Gly and PLTP Cys₃₁₈ Gly exhibited similar specific activities but partially impaired PLTP synthesis and secretion. Intracellular PLTP appeared as two bands of 75 and 51 kDa corresponding to reported molecular masses for the glycosylated and nonglycosylated forms. The specific activities of PLTP Cys₅ Gly and PLTP Cys₃₁₈ Gly were similar in the cell lysates and medium, suggesting that glycosylation does not affect transfer activity.     

pH-dependent conformational changes of ferricytochrome c induced by electrode surface microstructure

pH-dependent processes of bovine heart ferricytochrome c have been investigated by electronic absorption and circular dichroism (CD) spectra at functionalized single-wall carbon nanotubes (SWNTs) modified glass carbon electrode (SWNTs/GCE) using a long optical path thin layer cell. These methods enabled the pH-dependent conformational changes arising from the heme structure change to be monitored. The spectra obtained at functionalized SWNTs/GCE reflect electrode surface microstructure-dependent changes for pH-induced protein conformation, pK_a of alkaline transition and structural microenvironment of the ferricytochrome c heme. pH-dependent conformational distribution curves of ferricytochrome c obtained by analysis of in situ CD spectra using singular value decomposition least square (SVDLS) method show that the functionalized SWNTs can retain native conformational stability of ferricytochrome c during alkaline transition.     

Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice

Mdx mouse, the animal model of Duchenne muscular dystrophy, lacks dystrophin and develops an X-linked recessive inflammatory myopathy characterized by degeneration of skeletal muscle fibers and connective tissue replacement. The present work aimed to assess whether gender dimorphism in mdx mice would influence skeletal muscle pathology at ages corresponding to main histological changes in the microenvironment of muscular tissue: myonecrosis, regeneration, and fibrosis. At the height of myonecrosis (6 weeks postnatal), skeletal muscles of male mdx mice showed increased sarcolemmal permeability, numerous inflammatory foci, and marked deposition of the extracellular matrix components (ECM) type I collagen and laminin. In contrast, age-matched mdx females showed mild ECM deposition, discrete myonecrosis, but increased numbers of regenerating fibers expressing the satellite cell marker NCAM. In contrast ovariectomized mdx females showed decreased numbers of regenerating fibers. Older (24 and 48 weeks postnatal) mdx females showed extensive fibrosis with increased sarcolemmal permeability and marked deposition of ECM components than corresponding males. These results suggest a role for female hormones in the control of myonecrosis probably by promoting regeneration of muscular tissue and mitigating inflammation especially at ages under the critical influence of sex hormones.     

Studies on intestinal permeability of cirrhotic patients by analysis lactulose and mannitol in urine with HPLC/RID/MS

The method for separation and determination of lactulose (L) and mannitol (M) in urine was developed by HPLC with a refractive index detector (RID). The linearity ranged from 5 to 1000 microg/mL for L and M, respectively. Recoveries ranged from 93.1% to 97.1%. The intra- and inter-day relative standard deviations of peak area were between 0.8-1.4% (n=3) and 1.4-3.6% (n=3). The limits of detection were obtained with 1.40 microg/mL for L and 1.65 microg/mL for M. The ratios of L/M in the urine samples for the spontaneous ascitic fluid infection (SAI), sterile ascitic fluid (SA) patients, and healthy volunteers (HV) were determined. The results showed well the correlations among the L/M ratio, intestinal permeability (IP) and the illness status of patients, and also indicated lactulose could improve the IP of SAI patients. The peaks of L and M in chromatograms were identified by electrospray ionization/mass spectrometry (ESI/MS), which ensured the accurate measurement of the ratio L/M. This method presented a rapid, accurate, and practical technique for determining IP in clinical practice and investigating the pathology of hepatocirrhosis.     

A microcalorimetric study of the effect of La3+ on mitochondria isolated from Star-Cross 288 chicken liver tissue cells

The thermogenic curves of the metabolism of mitochondria isolated from the liver of chicken Star-Cross 288 and the effect of La³⁺ on it were studied by using an LKB-2277 BioActivity Monitor, ampoule method, at 37°C. After isolation from the chicken liver tissue, mitochondria still have metabolic activity and can live for a long time by using the stored nutrients. From the thermogenic curves, we obtained the thermokinetic equations under different condition. The kinetic study shows that La³⁺ has changed the metabolism completely.     

Citrate,Beneficial or Deleterious in the CNS?

Cerebellar granule neurons were incubated with or without glucose (3 mM) in the presence or absence of citrate (20 mM) using normoxic and/or hypoxic incubation conditions. During 4 h of hypoglycemia and also during hypoxia plus hypoglycemia, citrate increased lactate dehydrogenase (LDH) leakage from the cells and decreased mitochondrial activity, the latter was also the case in the presence of glucose. After 24 h of hypoglycemia, however, citrate decreased LDH leakage slightly, possibly due to its metabolism in the tricarboxylic acid cycle under these conditions. It should be noted that during mild hypoxia plus hypoglycemia a reduced LDH leakage was observed when compared to hypoglycemia alone. The 4 h low oxygen period did protect the neurons also during the 20 h re-oxygenation period. The present study might indicate that incubation of brain cell cultures in an atmosphere of air (30% oxygen) and 5% CO₂, which is used in most laboratories, can be toxic and that oxygen concentration should be lowered considerably to mimic conditions in the brain.     

Inheritance of alleles for glutelin alpha-2 subunit genes in rice and identification of their corresponding cDNA clone

Rice glutelins consist of acidic (alpha) and basic (beta) subunits which are further separated into three polypeptide components assigned as alpha-1, alpha-2, and alpha-3 subunit components and beta-1, beta-2 and beta-3 subunit components. Nine rice mutant lines with a decreased amount of the glutelin alpha-2 subunit component (alpha-2L) were obtained by screening about 6,800 potential mutant lines derived from the fertilized egg treatment with N-methyl-N-nitrosourea (MNU) using the SDS-PAGE method. The mutants were classified into three types of the increased alpha-1 subunit (alpha-1H/alpha-2L), the decreased beta-2 subunit (beta-2L/alpha-2L) and the increased alpha-3 subunit (alpha-3H/alpha-2L) represented by EM278, CM1707 and EM659, respectively. Iso-electric focus (IEF) analysis revealed that all of the mutants had an extremely low amount of a polypeptide with a 6.71 pI value, whereas a polypeptide with either a 6.50 pI value or with a 6.90 pI value increased significantly in alpha-1H/alpha-2L mutants or in alpha-3H/alpha-2L mutants, respectively. The beta-2L/alpha-2L mutants had a decreased amount of a basic polypeptide with a 8.74 pI value. Genetic analysis revealed that the three types of mutants were controlled by a single incomplete dominant gene respectively, and the three are alleles. The gene was temporarily named glu4, which was found to be located on chromosome 1 linked with the eg and spl6 genes. Two-dimensional electrophoresis analysis revealed that the glu4 encoded polypeptides of pI 6.71/alpha-2 and pI 8.74/beta-2. Amino acid sequence analysis suggested that the mutated acidic polypeptide was the product of a GluA subfamily gene. Northern and RT-PCR analyses revealed that glu4 corresponded to the GluA-1 gene.