Conservation of δ-crystallin gene structure between ducks and chickens

A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization. A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks. Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons. Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes. Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events. Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.     

The pathogenesis of trehalose dimycolate-induced interstitial pneumonitis: III. Evidence for a role for T lymphocytes

Trehalose dimycolate, a glycolipid component of the cell walls of mycobacteria, induces interstitial pneumonitis and alveolar hemorrhages in C57BL/6 and C57BL/10 mice. Homozygous nude (nu/nu) mice of these backgrounds are not susceptible to this form of pulmonary injury. However, after administration of T-lymphocyte-enriched spleen cell preparations from syngeneic donors, homozygous nude mice become susceptible to trehalose dimycolate. The observations suggest that production of pulmonary lesions by this mycobacterial component is dependent on T lymphocytes. While the mechanisms are still under study, we propose that trehalose dimycolate can function as an activator of T lymphocytes and that products of activated T cells are responsible for production of the pulmonary lesions.     

Assignment of the αB-crystallin gene to human chromosome 11

Using a human αB-crystallin genomic probe and human-mouse somatic cell hybrids, the human αB-gene was assigned to chromosome 11 and further corroborated by in situ hybridization to normal metaphase chromosomes. This assignment confirmed and regionally mapped the locus to q22.3–23.1.     

Glutathione S-transferase and S-crystallins of cephalopods: Evolution from active enzyme to lens-refractive proteins

Our previous studies have shown that the S-crystallins of cephalopod (Ommastrephes sloani pacificus) eye lenses comprise a family of at least ten members which are evolutionarily related to glutathione S-transferase (GST, EC 2.5.1.18). Here we show by cDNA cloning that there are at least 24 different S-crystallins that are 46–99% identical to each other by amino acid sequence in the squid Loligo opalescens. In each species, all but one S-crystallin (SL11 in O. pacificus and Lops4 in L. opalescens) examined has an inserted central peptide of variable length and sequence. cDNA expression studies conducted in Escherichia coli showed that squid GST (which is expressed little in the lens) has very high enzymatic activity using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate; by contrast, SL20-1 of O. pacificus and Lops 12 of L. opalescens (which are encoded by abundant lens mRNAs) have no GST activity. Interestingly, SL11 and Lops4 have some enzymatic activity with the CDNB substrate. Site-specific mutations at Y7 or W38, both residues essential for activity of vertebrate GSTs, or insertion of the central peptide present in the inactive SL20-1, reduced the specific activity of squid GST by 30- to 100-fold. These data indicate that the S-crystallins consist of a family of enzymatically inactive proteins (when using CDNB as a substrate) which is considerably larger than previously believed and that GST activity was lost by gradual drift in sequence as well as by insertion of an extra peptide by exon shuffling. The results are also consistent with the idea that SL11 and Lops4 are orthologous crystallins representing the first descendants of the ancestral GST gene in the pathway which gave rise to the extensive S-crystallin family of lens proteins. Correspondence to: S.I. Tomarev     

The control of δ-crystallin gene expression during lens cell development: Dissociation of cell elongation,cell division, δ-crystallin synthesis,and δ-crystallin mRNA accumulation

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of δ-crystallin, and accumulate δ-crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved 6-day-old and freshly explanted 19-day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of δ-crystallin gene expression (as measured by δ-crystallin synthesis and δ-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether δ-crystallin gene expression is increased in lens epithelial cells stimulated to divide by treatment with fetal calf serum, as it is in those stimulated to elongate by treatment with serum. Three new findings were made in this study. First, the stimulation of δ-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of δ-crystallin synthesis is observed in lens epithelial cells during normal development and during serum starvation; in neither case is this decrease associated with a reduction in the number of δ-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of δ-crystallin synthesis, but can promote the accumulation of δ-crystallin mRNA. Thus, the relative proportion of δ-crystallin synthesized during chick lens development is not solely a function of the number of δ-crystallin mRNA sequences in the lens cells.     

Pmch-Deficiency in Rats Is Associated with Normal Adipocyte Differentiation and Lower Sympathetic Adipose Drive

The orexigenic neuropeptide melanin-concentrating hormone (MCH), a product of Pmch, is an important mediator of energy homeostasis. Pmch-deficient rodents are lean and smaller, characterized by lower food intake, body-, and fat mass. Pmch is expressed in hypothalamic neurons that ultimately are components in the sympathetic nervous system (SNS) drive to white and interscapular brown adipose tissue (WAT, iBAT, respectively). MCH binds to MCH receptor 1 (MCH1R), which is present on adipocytes. Currently it is unknown if Pmch-ablation changes adipocyte differentiation or sympathetic adipose drive. Using Pmch-deficient and wild-type rats on a standard low-fat diet, we analyzed dorsal subcutaneous and perirenal WAT mass and adipocyte morphology (size and number) throughout development, and indices of sympathetic activation in WAT and iBAT during adulthood. Moreover, using an in vitro approach we investigated the ability of MCH to modulate 3T3-L1 adipocyte differentiation. Pmch-deficiency decreased dorsal subcutaneous and perirenal WAT mass by reducing adipocyte size, but not number. In line with this, in vitro 3T3-L1 adipocyte differentiation was unaffected by MCH. Finally, adult Pmch-deficient rats had lower norepinephrine turnover (an index of sympathetic adipose drive) in WAT and iBAT than wild-type rats. Collectively, our data indicate that MCH/MCH1R-pathway does not modify adipocyte differentiation, whereas Pmch-deficiency in laboratory rats lowers adiposity throughout development and sympathetic adipose drive during adulthood.     

Overweight Is an Independent Risk Factor for Reduced Lung Volumes in Myotonic Dystrophy Type 1

Background

In this large observational study population of 105 myotonic dystrophy type 1 (DM1) patients, we investigate whether bodyweight is a contributor of total lung capacity (TLC) independent of the impaired inspiratory muscle strength.

Methods

Body composition was assessed using the combination of body mass index (BMI) and fat-free mass index. Pulmonary function tests and respiratory muscle strength measurements were performed on the same day. Patients were stratified into normal (BMI < 25 kg/m²) and overweight (BMI ≥ 25 kg/m²) groups. Multiple linear regression was used to find significant contributors for TLC.

Results

Overweight was present in 59% of patients, and body composition was abnormal in almost all patients. In overweight patients, TLC was significantly (p = 2.40×10⁻³) decreased, compared with normal-weight patients, while inspiratory muscle strength was similar in both groups. The decrease in TLC in overweight patients was mainly due to a decrease in expiratory reserve volume (ERV) further illustrated by a highly significant (p = 1.33×10⁻¹⁰) correlation between BMI and ERV. Multiple linear regression showed that TLC can be predicted using only BMI and the forced inspiratory volume in 1 second, as these were the only significant contributors.

Conclusions

This study shows that, in DM1 patients, overweight further reduces lung volumes, as does impaired inspiratory muscle strength. Additionally, body composition is abnormal in almost all DM1 patients.     

Modeling the proteome of a Marek's disease transformed cell line: a natural animal model for CD30 overexpressing lymphomas

Marek's disease (MD) in the chicken, caused by the highly infectious MD alpha-herpesvirus (MDV), is both commercially important and a unique, naturally occurring model for human T-cell lymphomas overexpressing the Hodgkin's disease antigen, CD30. Here, we used proteomics as a basis for modeling the molecular functions and biological processes involved in MDV-induced lymphomagenesis. Proteins were extracted from an MDV-transformed cell line and were then identified using 2-D LC-ESI-MS/MS. From the resulting 3870 cellular and 21 MDV proteins we confirm the existence of 3150 "predicted" and 12 "hypothetical" chicken proteins. The UA-01 proteome is proliferative, differentiated, angiogenic, pro-metastatic and pro-immune-escape but anti-programmed cell death, -anergy, -quiescence and -senescence and is consistent with a cancer phenotype. In particular, the pro-metastatic integrin signaling pathway and the ERK/MAPK signaling pathways were the two predominant signaling pathways represented. The cytokines, cytokine receptors, and their related proteins suggest that UA-01 has a regulatory T-cell phenotype.     

Heterostereocomplexes prepared from d-PLA and l-PLA and leuprolide. II. Release of leuprolide

Reversible stereoselective complexes were spontaneously obtained from mixing acetonitrile solutions of enatiomeric d-poly(lactic acid) (d-PLA), l-poly(lactic acid) (l-PLA), and leuprolide, a l-configured nonapeptide LHRH analogue. The complex spontaneously aggregated and precipitated in high yields (>90%) from acetonitrile solution, forming uniform, porous microparticles. The stereocomplex microparticles showed a continuous release of the interlocked peptide for a period of one to three months under physiological conditions. Various factors, including method of complex formation, molecular weight of PLA, leuprolide:polymer and d-PLA:l-PLA complex ratios, and additives, influenced the release pattern of leuprolide from the stereocomplexes. Continuous release of leuprolide for over 100 days was observed for certain stereocomplex compositions. In vivo evaluation of the leuprolide loaded stereocomplexes in rats by monitoring testosterone levels in the blood of rats after subcutaneous injection showed low testosterone levels for over 42 days.