Structure-function correlation in airway smooth muscle adapted to different lengths

Airway smooth muscle is able to adapt and maintain a nearly constant maximal force generation over a large length range. This implies that a fixed filament lattice such as that found in striated muscle may not exist in this tissue and that plastic remodeling of its contractile and cytoskeletal filaments may be involved in the process of length adaptation that optimizes contractile filament overlap. Here, we show that isometric force produced by airway smooth muscle is independent of muscle length over a twofold length change; cell cross-sectional area was inversely proportional to cell length, implying that the cell volume was conserved at different lengths; shortening velocity and myosin filament density varied similarly to length change: increased by 69.4% ± 5.7 (SE) and 76.0% ± 9.8, respectively, for a 100% increase in cell length. Muscle power output, ATPase rate, and myosin filament density also have the same dependence on muscle cell length: increased by 35.4% ± 6.7, 34.6% ± 3.4, and 35.6% ± 10.6, respectively, for a 50% increase in cell length. The data can be explained by a model in which additional contractile units containing myosin filaments are formed and placed in series with existing contractile units when the muscle is adapted at a longer length. muscle contraction; myosin filaments; ATPase activity; electron microscopy     

MHC class II beta sequence diversity in the deer mouse (Peromyscus maniculatus): implications for models of balancing selection

We studied population polymorphism at a major histocompatibility complex (MHC) class II beta gene in the deer mouse (Peromyscus maniculatus). We found that: (i) a single population of P. maniculatus has significantly higher levels of DNA and protein sequence diversity than worldwide samples from homologous genes in other taxa, including humans and mice; and (ii) the genealogy of allelic sequences in P. maniculatus deviates significantly from theoretical expectation under a model of symmetric balancing selection, in that alleles are relatively more divergent than expected. We suggest that the observation of high levels of pairwise allelic sequence divergence and deviation of the genealogy from theoretical expectation in P. maniculatus together provide support for a divergent allele advantage model for the maintenance of MHC polymorphism.     

Alpha1 Na,K-ATPase and Na,K,2Cl-cotransporte/D3mit3 loci interact to increase susceptibility to salt-sensitive hypertension in Dahl S(HSD) rats

BACKGROUND: Essential (multigenic) hypertension is a complex multifactorial disease whose genetic etiology has not been unraveled on a major locus-effect investigative paradigm. As with other complex genetic diseases, applying an interacting loci paradigm could be critical in the elucidation of genetic determinants. Having defined the alpha1 Na,K-ATPase (alpha1NK) as a hypertension susceptibility gene in Dahl salt-sensitive (Dahl S) rats, we determined whether alphaINK interacts with another renal epithelial Na transporter to increase susceptibility to salt-sensitive hypertension. We focused on alpha1NK and Na,K,2Cl-cotransporter (NKC) as an a priori candidate interacting gene pair because they comprise a functionally linked Na transport system in renal thick ascending limb of Henle (TALH) epithelial cells and exhibit altered function in prehypertensive Dahl S rats in contrast to Dahl salt-resistant normotensive (Dahl R) rats. MATERIAL AND METHOD: Cosegregation analysis of alphaNK and NKC loci was done in a (Dahl S x Dahl R) F2 cohort characterized for blood pressure by radiotelemetry using the D2mghII microsatellite marker in the alpha1NK gene and the D3mit3 microsatellite marker close to the NKC gene (NKC/D3mit3 locus). Single locus and digenic analyses were performed to establish the individual and interactive genetic contribution to salt-sensitive hypertension. Molecular analysis was then done to support the NKC gene as the likely candidate gene interacting with alpha1NK in Dahl salt-sensitive hypertension pathogenesis. RESULTS: Compared with respective single locus analysis, digenic analysis of 96 F2 (Dahl S x Dahl R) hybrid male rats revealed cosegregation of alpha1NK and NKC/D3mit3 loci as interacting pair with salt-sensitive hypertension with markedly increased significance for systolic (one-way ANOVA p = 10(-6)), diastolic (p = 10(-5)), and mean arterial (p = 10(-6)) blood pressures. Concordantly, two-way ANOVA detected interaction between alpha1NK and NKC loci in determining the levels of systolic (p = 0.004), diastolic (p = 0.008), and mean arterial (p = 0.006) pressures. To unravel potential NKC molecular dysfunction(s) involved in hypertension pathogenesis, we investigated putative differences between Dahl S and Dahl R rats in nucleotide sequence and isoform gene expression of the renal-specific Na,K,2Cl-cotransporter. Molecular analysis revealed an inversion of alternatively spliced NKC-isoform ratios (4B:4A:4F) between Dahl S and Dahl R prehypertensive kidneys supported by four mutations in intron-3 immediately upstream to alternatively spliced exons 4B, 4A, and 4F. No nucleotide changes were detected within the aminoacid encoding exons of NKC. CONCLUSIONS: Altogether, these current data and previous characterization of the role of the Q276L alpha1NK molecular variant in Dahl S hypertension provide cumulative compelling evidence that alpha1NK and NKC/D3mit3 loci interact to increase susceptibility to hypertension in Dahl S rats and that NKC is the likely candidate gene that interacts with alpha 1NK. More importantly, the data substantiate gene interaction as an operative mechanism in multigenic hypertension.     

Study and mathematical modeling of the production of propionic acid by Propionibacterium acidipropionici immobilized in a stirred tank fermentor

A mathematical model was developed that describes production of propionic acid by fermentation of sweet whey with Propionibacterium acidipropionici immobilized in calcium polygalacturonate beads in a fermentor-type stirred tank. This mathematical model is constituted by a partial differential equations system, which fits consumption, production, growth and internal diffusion rates in the support. Fermentation was experimentally studied with free cells and immobilized cells, effective diffusivities of lactose and propionic acid were estimated in the support, and typical parameters of the model were obtained by nonlinear regression of the experimental data. The variance analysis shows that the combination of micro(max) and K(d) parameters is the source of variation most significative, also they were found to be the most sensitive parameters of the model. Finally, an effectiveness factor was calculated in order to assess the effect of mass transfer on the overall reaction rate observed.     

Modulation of HeLa cells spreading by the non-receptor tyrosine kinase ACK-2

The CDC42 regulated non-receptor tyrosine kinase ACK-2 has been associated with integrin signaling. In this report, the effect of ACK-2 on the modulation of cell spreading and motility was examined. HeLa cells expressing epitope-tagged wild type ACK-2 showed a slower rate of spreading on fibronectin when compared with untransfected cells. An ACK-2 protein lacking its SH3 domain was still capable of modulating HeLa cell spreading suggesting that its tyrosine kinase activity is sufficient to induce the observed phenotype. The ACK-2 effect on the rate of cell spreading did not involve inhibition of integrin-mediated activation of PI-3K signaling, since it did not alter membrane translocation of a GFP-PH-AKT domain (AKT pleckstrin homology domain) used as a reporter for PI-3K products induced by cell adhesion. The ACK-2 effect appears to be upstream from the adapter protein CrkII, since co-expression of CrkII and ACK-2 results in a neutralization of ACK-2 mediated effects on HeLa cell spreading. Similarly, co-expression of p130Cas, which interacts with the adapter protein CrkII, with ACK-2, also results in a partial reversion of the ACK-2 effects on cell spreading. CrkII mediated reversal of the ACK-2 induced phenotype requires the activity of the small GTPase, Rap1. Co-expression of ACK-2 and CrkII with a dominant negative form of Rap1 reverses the neutralization by CrkII suggesting that CrkII mediated activation of Rap1 is required. However, an active form of Rap1 is not sufficient to reverse the ACK-2 phenotype by itself. A role for Rac1 in ACK-2 effects was also established. An activated Rac1 protein neutralized the ACK-2 mediated inhibition of cell spreading. A direct measurement of cell motility by either a modified Boyden chamber or wounding assay demonstrates that ACK-2 overexpression increases the motility of the cells. These results suggest that ACK-2 modulates HeLa cells spreading upstream of pathways regulated by CrkII and that ACK-2 may regulate cell motility by controlling the activation of small GTPases such as Rap1 and Rac1.     

Decline of neural tube defects cases after a folic acid campaign in Nuevo León,México

BACKGROUND: Nuevo León is a state in northeastern Mexico, near the border of Texas. Mean mortality rate from 1996-98 due to anencephaly cases was 0.6/1,000. In 1999 a surveillance program for the registry and prevention of neural tube defects (NTD) cases was initiated. METHODS: Cases were obtained from hospitals and OB-GYN clinics by immediate notification, death certificates, or fetal death registries. Only isolated cases of NTD were included. In August 1999 a folic acid campaign was initiated with the free distribution of the vitamin to low-income women with a recommendation to take a 5.0-mg pill once a week. Number of cases and rates from 1999 to 2001 were compared (chi(2) test). RESULTS: After 2 years there has been a significant reduction in the number of cases and rates. In 1999 there were 95 NTD cases and in the years 2000 and 2001 there were only 59 and 55 respectively (P < 0.001). NTD rate decreased from 1.04/1,000 in 1999 to 0.58/1,000 in 2001. Anencephaly and spina bifida rates decreased from 0.55/1,000 to 0.29/1,000 and from 0.47/1,000 to 0.22/1,000 respectively, from 1999-2001. Decrease of female cases was higher than male cases for both phenotypes. CONCLUSION: After 2 years there was a 50% decrease in the incidence of anencephaly and spina bifida cases with a significant reduction of infant mortality and disability. These results encourage us to propose the use of a single tablet of 5.0-mg of folic acid per week as an alternative to supplementation on a daily basis.     

The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone,and not mimicked by rh-TNF-alpha,IL-1beta and IFN-gamma

It is becoming widely accepted that the inflammatory response is involved in neurodegenerative disease. In this context, we have developed an animal model of dopaminergic system degeneration by the intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation. To address the importance of the inflammatory response in the LPS-induced degeneration of nigral dopaminergic neurones, we carried out two different kinds of studies: (i) the possible protective effect of an anti-inflammatory compound, and (ii) the effect of the intranigral injection of inflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) on dopaminergic neurones viability. Present results show that dexamethasone, a potent anti-inflammatory drug that interferes with many of the features characterizing pro-inflammatory glial activation, prevented the loss of catecholamine content, Tyrosine hydroxylase (TH) activity and TH immunostaining induced by LPS-injection and also the bulk activation of microglia/macrophages. Surprisingly, injection of the pro-inflammatory cytokines failed to reproduce the LPS effect. Taken together, our results suggest that inflammatory response is implicated in LPS-induced neurodegeneration. This damage may be due, at least in part, to a cascade of events independent of that described for TNF-alpha/IL-1 beta/IFN-gamma.     

Gene introgression into Coffea arabica by way of triploid hybrids (C. arabica x C. canephora)

Interspecific triploid hybrid plants between the tetraploid species Coffea arabica L. and the diploid species C. canephora P. were backcrossed to C. arabica. Although characterised by a low production and an important fruit dropping, all attempted crosses (ie, 6) generated BC(1) progenies. Flow cytometric analysis of the nuclear DNA content revealed that most of the BC1 individuals were nearly tetraploid. Among the male gametes produced by the interspecific triploid hybrids, those presenting a high number of chromosomes appeared strongly favoured. Only pollen mother cells having nearly 22 chromosomes were effective, the others leading to deficient endosperm and fruit dropping. Molecular markers (ie, microsatellite and AFLP) combined with evaluations of morphological characteristics and resistance to leaf rust were applied to verify the occurrence of gene transfer from C. canephora into C. arabica, and to estimate the amount of introgression present in BC(1) individuals. The results reveal a strong deficiency in the C. canephroa alleles indicating a severe counter-selection against the introgression of genetic material from C. canephora into C. arabica by way of triploid hybrids. However, introgressants displaying desirable traits such as a high resistance to leaf rust were obtained. The low level of introgression could be an advantage by facilitating the recovery of the recurrent parent and possibly reducing the number of required backcrosses. On the other hand, this could be a limitation when attempting the transfer of a complex trait or several simply inherited traits.