Dynamics of breathing in the hypoxic awake lamb

Newborn mammals respond to hypoxia with an immediate hyperventilation that is rapidly dampened. Changes in mechanical properties of the respiratory system during hypoxia have been considered an important reason for this fall in minute ventilation (VE). We have studied the dynamic mechanical behavior of the respiratory system in eight unanesthetized intact newborn lambs (mean age 2 days) during normoxia and hypoxia (FIO2 = 0.08). Mouth pressure (P), airflow (V), and volume (V) were recorded while lambs were breathing through a leak-proof face mask and a pneumotachograph. Active compliance (C') and resistance (R') of the respiratory system were computed from P developed during an inspiratory effort against airway closure at end expiration and V and V of the preceding breaths. Tidal expiratory V-V curves were analyzed to estimate the elevation in functional residual capacity (FRC) over resting volume (Vr). After hypoxia, there was an immediate increase in VE in the first 2 min, from 0.49 to 1.13 l.kg-1.min-1, followed by a rapid decrease to 0.80. After 8 min of hypoxia, C' was unchanged. The inspiratory R' decreased during hypoxia, probably reflecting a drop in inspiratory laryngeal resistance. The expiratory V-V curves during hypoxia showed considerable braking, often with a double peak in expiratory V. This pattern was only occasionally seen during normoxia. In animals with a linear segment of the expiratory V-V curves the FRC-Vr difference could be calculated and averaged 1.93 ml/kg during normoxia and 3.47 during hypoxia. The recoil P of the respiratory system at end expiration was 0.75 cmH2O during normoxia vs. 1.63 cmH2O during hypoxia (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Constrained Unfolding of a Helical Peptide: Implicit versus Explicit Solvents

Steered Molecular Dynamics (SMD) has been seen to provide the potential of mean force (PMF) along a peptide unfolding pathway effectively but at significant computational cost, particularly in all-atom solvents. Adaptive steered molecular dynamics (ASMD) has been seen to provide a significant computational advantage by limiting the spread of the trajectories in a staged approach. The contraction of the trajectories at the end of each stage can be performed by taking a structure whose nonequilibrium work is closest to the Jarzynski average (in naive ASMD) or by relaxing the trajectories under a no-work condition (in full-relaxation ASMD—namely, FR-ASMD). Both approaches have been used to determine the energetics and hydrogen-bonding structure along the pathway for unfolding of a benchmark peptide initially constrained as an α-helix in a water environment. The energetics are quite different to those in vacuum, but are found to be similar between implicit and explicit solvents. Surprisingly, the hydrogen-bonding pathways are also similar in the implicit and explicit solvents despite the fact that the solvent contact plays an important role in opening the helix.     

Homology between a 173-kb Region from Mouse Chromosome 10, Telomeric to the Ifng Locus, and Human Chromosome 12q15

We sequenced a 173-kb region of mouse chromosome 10, telomeric to the Ifng locus, and compared it with the human homologous sequence located on chromosome 12q15 using various sequence analysis programs. This region has a low density of genes: one gene was detected in the mouse and the human sequences and a second gene was detected only in the human sequence. The mouse gene and its human orthologue, which are expressed in the immune system at a low level, produce a noncoding mRNA. Nonexpressed sequences show a higher degree of conservation than exons in this genomic region. At least three of these conserved sequences are also conserved in a third mammalian species (sheep or cow).     

KLF6 transcription factor protects hepatocellular carcinoma-derived cells from apoptosis

Hepatocellular carcinoma (HCC) is a major public health concern because of the absence of early diagnosis and effective treatments. Efficient diagnosis modalities and therapies to treat HCC are needed. Kruppel-like factor (KLF) family members, such as KLF6, are involved in cell proliferation and differentiation. KLF6 is inactivated in solid tumors, which may contribute to pathogenesis. However, KLF6 status in HCC is controversial. Thus, we undertook the characterization of KLF6 expression and function in HCC and HCC-derived cell lines. We found that HCC, HepG2 and HuH7 cells expressed KLF6 messenger ribonucleic acid and protein. Next, using RNA interference, we demonstrated that inhibiting KLF6 expression in vitro strongly impaired cell proliferation-induced G1-phase arrest, inhibited cyclin-dependent kinase 4 and cyclin D1 expression, and subsequent retinoblastoma phosphorylation. Finally, KLF6 silencing caused p53 upregulation and inhibited Bcl-xL expression, to induce cell death by apoptosis. Taken together, these data demonstrated that KLF6 is essential for HCC-derived cells to evade apoptosis.     

Impact of changing drug treatment and malaria endemicity on the heritability of malaria phenotypes in a longitudinal family-based cohort study

Despite considerable success of genome wide association (GWA) studies in identifying causal variants for many human diseases, their success in unraveling the genetic basis to complex diseases has been more mitigated. Pathogen population structure may impact upon the infectious phenotype, especially with the intense short-term selective pressure that drug treatment exerts on pathogens. Rigorous analysis that accounts for repeated measures and disentangles the influence of genetic and environmental factors must be performed. Attempts should be made to consider whether pathogen diversity will impact upon host genetic responses to infection.We analyzed the heritability of two Plasmodium falciparum phenotypes, the number of clinical malaria episodes (PFA) and the proportion of these episodes positive for gametocytes (Pfgam), in a family-based cohort followed for 19 years, during which time there were four successive drug treatment regimes, with documented appearance of drug resistance. Repeated measures and variance components analyses were performed with fixed environmental, additive genetic, intra-individual and maternal effects for each drug period. Whilst there was a significant additive genetic effect underlying PFA during the first drug period of study, this was lost in subsequent periods. There was no additive genetic effect for Pfgam. The intra-individual effect increased significantly in the chloroquine period.The loss of an additive genetic effect following novel drug treatment may result in significant loss of power to detect genes in a GWA study. Prior genetic analysis must be a pre-requisite for more detailed GWA studies. The temporal changes in the individual genetic and the intra-individual estimates are consistent with those expected if there were specific host-parasite interactions. The complex basis to the human response to malaria parasite infection likely includes dominance/epistatic genetic effects encompassed within the intra-individual variance component. Evaluating their role in influencing the outcome of infection through host genotype by parasite genotype interactions warrants research effort.     

Evaluation of virus decontamination techniques for porcine embryos produced in vitro

The objective of this study was to explore approaches to decontaminate embryos either contaminated naturally or under experimental conditions with different viruses. Embryos were obtained from in vitro maturation and fertilisation of porcine oocytes. After 7 days of development, morula and blastocyst stages were exposed for 1 h to the following viruses: encephalomyocarditis virus (EMCV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and bovine viral diarrhea virus (BVDV) at an infectivity of 100 TCID50/mL. Embryos samples were treated with different washing procedures, which all included the following standard washing solutions: PBS+0.4% BSA (five times for 10 s), Hank's+0.25% trypsin (two times for 60-90 or 120-150 s, or one time of 5 min), Hank's+0.1 mg/mL DNase 1+20 U/mL RNase One (one time of 30 min) and PBS+0.4% BSA again (five times for 10s). Two new approaches were used to improve trypsin treatment, 0.1% hyaluronidase (one time for 5 min) instead of trypsin and a pre-incubation with oviductal cells. Therefore, in the first experiment, oocytes received standard maturation treatments and in the second, they were also co-incubated with oviductal cells for the last 3 h of maturation. The effectiveness of the different washing techniques in removing viruses was evaluated by polymerase chain reaction (PCR) analysis. In the first experiment, trypsin treatment did not eliminate PRRSV, PPV, PCV, and EMCV from contaminated embryos. Surprisingly, treatment with hyaluronidase eliminated all tested viruses. In the second experiment, all viruses tested were removed from the oocytes following the different enzymatic treatments. In conclusion, in vitro embryo decontamination was more effective following exposure to oviductal secretions and hyaluronidase eliminated more virions than trypsin in washing techniques.     

Selective blockade of NF-kappa B activity in airway immune cells inhibits the effector phase of experimental asthma

Knockout mice studies have revealed that NF-kappaB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-kappaB also plays a role in the effector phase of airway allergy and whether inhibiting NF-kappaB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-kappaB activity in the lungs during OVA challenge. Intratracheal administration of NF-kappaB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-kappaB activity in the airways upon OVA provocation. NF-kappaB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-kappaB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-kappaB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.     

The Type III secretion system of Xanthomonas fuscans subsp. fuscans is involved in the phyllosphere colonization process and in transmission to seeds of susceptible beans

Understanding the survival, multiplication, and transmission to seeds of plant pathogenic bacteria is central to study their pathogenesis. We hypothesized that the type III secretion system (T3SS), encoded by hrp genes, could have a role in host colonization by plant pathogenic bacteria. The seed-borne pathogen Xanthomonas fuscans subsp. fuscans causes common bacterial blight of bean (Phaseolus vulgaris). Directed mutagenesis in strain CFBP4834-R of X. fuscans subsp. fuscans and bacterial population density monitoring on bean leaves showed that strains with mutations in the hrp regulatory genes, hrpG and hrpX, were impaired in their phyllospheric growth, as in the null interaction with Escherichia coli C600 and bean. In the compatible interaction, CFBP4834-R reached high phyllospheric population densities and was transmitted to seeds at high frequencies with high densities. Strains with mutations in structural hrp genes maintained the same constant epiphytic population densities (1 x 10(5) CFU g(-1) of fresh weight) as in the incompatible interaction with Xanthomonas campestris pv. campestris ATCC 33913 and the bean. Low frequencies of transmission to seeds and low bacterial concentrations were recorded for CFBP4834-R hrp mutants and for ATCC 33913, whereas E. coli C600 was not transmitted. Moreover, unlike the wild-type strain, strains with mutations in hrp genes were not transmitted to seeds by vascular pathway. Transmission to seeds by floral structures remained possible for both. This study revealed the involvement of the X. fuscans subsp. fuscans T3SS in phyllospheric multiplication and systemic colonization of bean, leading to transmission to seeds. Our findings suggest a major contribution of hrp regulatory genes in host colonization processes.     

Inter-MITE polymorphisms (IMP): a high throughput transposon-based genome mapping and fingerprinting approach

Miniature inverted-repeat transposable elements or MITEs represent a large superfamily of transposons that are moderately to highly repetitive and frequently associated with plant genes. These attributes were exploited in the development of a powerful marker technology called Inter-MITE polymorphism, or IMP, which involves the amplification between two adjacent MITEs. In this report, we describe the utility of the IMP approach in the mapping and fingerprinting of the barley genome. MITEs were systematically mined from barley genomic gene sequences by computer-assisted database searches and structural analysis. Barley MITEs include members of the Stowaway family and a new family we have named Barfly. Using these barley MITEs, a total of 88 IMP markers were mapped onto an existing barley RFLP map that was based on a doubled-haploid segregating population between Hordeum vulgare and Hordeum spontaneum. We demonstrate that the IMP approach can be effectively applied in the fingerprinting of barley cultivars and for genetic similarity analysis. We also provide evidence that barley MITE-based primers can be effectively used in the mapping and fingerprinting of other cereals, suggesting that the IMP approach has broad applicability. Received: 24 March 2000 / Accepted: 28 July 2000