��3 Integrin in Cardiac Fibroblast Is Critical for Extracellular Matrix Accumulation during Pressure Overload Hypertrophy in Mouse

The adhesion receptor β3 integrin regulates diverse cellular functions in various tissues. As β3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of β3 integrin in cardiac fibrosis by using wild type (WT) and β3 integrin null (β3−/−) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, β3−/− mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from β3−/− mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in β3−/− mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and β3−/− mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that β3−/− cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in β3−/− cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that β3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.     

Ldh activity in Euphausia mucronata and Calanus chilensis: implications for vertical migration behaviour

Specific lactic dehydrogenase (ldh) activity was measured inEuphausia mucronata and Calanus chilensis. Both key pelagicspecies inhabit areas of the Humboldt Current System that havepermanent subsurface oxygen minimum layer. The specific ldhactivity in E. mucronata was two orders of magnitude higherthan that of C. chilensis, consistent with E. mucronata’sability to conduct daily vertical migrations through the oxygenminimum layer. In contrast, C. chilensis is restricted to inhabitingoxygenated waters over the oxygen minimum layer without carryingout daily vertical migrations across it. A positive correlation(P < 0.0001) was found between the specific ldh activityand body size in E. mucronata. This study also reports the apparentkinetic properties (V_max and K_m) of ldh and the effect of temperatureand pH on the specific activity for both species.     

The absence of prostaglandin e1 returned confluent cultures of highly proliferative murine polycystic kidney principal cells to a normal proliferation level

Constitutively high proliferation, loss of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA)-regulated proliferation, and half-normal cAMP levels were observed previously in principal cells from the C57BL/6J- Cyc1\[cf12\]cpk\[cf1\] (cpk) model of autosomal recessive polycystic kidneys disease (PKD) cultured in defined medium supplemented with prostaglandin E1 (PGE1). Because PGE1 can up- or down-regulate renal cAMP production depending upon its receptor coupling; cAMP exerted both PKA-dependent and PKA-independent effects on cell proliferation; proliferation is considered to be a component of cystogenesis; and PGE1 resulted in loss of tubular structures and formation of cystic structures in gel culture of Madin Darby Canine Kidney cells; the effect of removing PGE1 on murine principal cell proliferation was examined. Proliferation was measured in filter-grown cultures of cystic (cpk) and noncystic (C57) principal cells from cpk and C57BL/6J mice, respectively. Lack of PGE1 had no effect on subconfluent C57 and cpk cultures or confluent C57 cultures but had a dramatic effect on confluent cpk cultures. Without PGE1, cpk proliferation was comparable with the low C57 level. In PGE1-deficient medium, differences were observed between confluence conditions and cell types for responses to a cAMP analog and a PKA activity inhibitor that suggested altered regulation of both PKA-dependent and PKA-independent cell proliferation. Cyclic adenosine monophosphate-dependent differences reported here, and previously, support the idea that the combination of mutant PKD gene product, altered PGE1 responsiveness, and altered PKA targeting contributes to activation of a cystogenic signaling pathway that regulates principal cell proliferation and is involved in pathogenesis.     

The effect of viral concentrate addition on the respiration rate of Chaetoceros gracilis cultures and microplankton from a shallow bay (Coliumo, Chile)

Experiments were conducted to test the possible effects of viralconcentrate additions on the respiration rates of both Chaetocerosgracilis and a natural microplankton community (<200 µm)from a shallow bay located in central-south Chile (Coliumo Bay,36°32'S, 72°57'W). Each experiment was started by adding2 ml of viral concentrate to a C. gracilis culture, microplanktoncommunity or bacterioplankton community (nominal size 0.2–1.0µm) contained in a 125 ml borosilicate bottle. The incubationslasted 24 h at in situ temperatures and included a minimum offive replicates and five controls. Respiration was measuredas oxygen consumption using a semi-automatic photometric Winklermethod. Samples were taken from the experimental bottles toassess chlorophyll a, ATP concentration and bacterioplanktonabundance throughout the incubation period. Our results showthat the addition of viral concentrates can affect the respirationrates of both natural microplankton communities and C. graciliscultures. When subjected to a viral concentrate addition, therespiration rates of the natural microplankton community decreasedby 12–50% or increased by 29%, depending on the experiment,whereas bacterioplankton respiration rates increased by 92%and decreased by 78%, and C. gracilis rates increased by ~4%and decreased by ~7%.     

Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV

The organizational relationship between the recently identified alpha 3 chain of basement membrane collagen (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B.G. (1987) J. Biol. Chem. 262, 7874-7877) and collagen IV was determined. This was accomplished by the identification of subunits in hexamers of the NC1 domain of collagen IV that were immunoprecipitated with antibodies prepared against subunits M1, corresponding to alpha 1(IV)NC1 and alpha 2(IV)NC1, and M2, corresponding to alpha 3NC1, and by amino acid sequence analysis. The presence of at least two distinct types of hexamers was revealed, one enriched in M1 and the other enriched in M2, but in both types, M1 and M2 coexist. Evidence was also obtained for the existence of heterodimers comprised of M1 and M2. These results indicate that M2 is an integral component of the NC1 hexamer of collagen IV. The amino acid sequence of the NH2-terminal region of M2 was found to be highly related to the collagenous-NC1 junctional region of the alpha 1 chain of collagen IV. Therefore, M2 is designated alpha 3(IV)NC1 and its parent chain alpha 3(IV). These findings lead to a new concept about the structure of collagen IV: namely, 1) collagen IV is comprised of a third chain (alpha 3) together with the two classical ones (alpha 1 and alpha 2); the alpha 3(IV) chain exists within the same triple-helical molecule together with the alpha 1(IV) and alpha 2(IV) chains and/or within a separate triple-helical molecule, exclusive of alpha 1(IV) and alpha 2(IV) chains, but connected through the NC1 domains to the classical triple-helical molecule comprised of alpha 1(IV) and alpha 2(IV) chains. Additionally, a portion of those triple-helical molecules exclusive of alpha 1(IV) and alpha 2(IV) chains may be connected to each other through their NC1 domains; and 3) the epitope to which the major reactivity of autoantibodies are targeted in glomerular basement membrane in patients with Goodpasture syndrome is localized to the NC1 domain of the alpha 3(IV) chain.     

Use of novel nest boxes by carmine bee‐eaters (Merops nubicus) in captivity

Carmine bee‐eaters make attractive additions to zoo aviaries but breeding programs have had challenges and limited success. The objectives of this study were to document nesting behavior of Carmine bee‐eaters in a captive setting and compare reproductive success between a novel nest box (plastic, 17 × 30 × 22 cm) and a PVC pipe model used previously (30 cm long, 8 cm in diameter). Three bee‐eater pairs were given access to seven nest chambers (six novel boxes, one PVC model). Behavioral observations occurred during a 15‐min period in the morning or afternoon before egg production and continued until chicks fledged for a total of 87 observation periods (21.75 hr). All occurrences by an individual bird entering or exiting a nest tunnel, food provision, and the time (min) spent inside a nest cavity were documented. Additionally, daily temperature within each nest chamber was recorded. Before eggs were produced the average daily temperature (23.02°C) within the nest chambers did not differ, suggesting that nest cavity choice was not influenced by temperature. No differences were detected among pairs in percent of observed time spent inside their nest cavities or number of times a nest tunnel was entered during the incubation or fledging periods. During incubation females spent a greater percent of observed time inside the nest cavity than males (P=0.02). During the fledging period food provision did not differ between the pairs, however males entered their nest tunnels more often per hour than females (P=0.03), and males tended to provide food more often than females (P=0.053). Two pairs nested in novel nest boxes and successfully fledged one chick each. The pair that nested in the PVC model did not fledge a chick. A nest box that aids in keeping eggs intact is essential for breeding bee‐eaters in captivity, and maintaining captive populations will provide opportunities for zoo visitors to enjoy these birds and will reduce the need to remove birds from the wild. Zoo Biol 0:1–13, 2007. © 2007 Wiley‐Liss, Inc.     

Extensive homology between the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen

We have determined the complete primary structure for the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen; which have 229 and 227 amino acids, respectively. The amino acid sequences are 63% identical and conservatively substituted in 28 positions. A striking feature of these peptides is that the first half of each sequence is homologous with the second half, 37% in alpha 1(IV) and 36% in alpha 2(IV). These results suggest that the carboxyl-terminal peptides of type IV collagen are closely related in their structure and evolution. Presumably, they were first derived by internal duplication of a common ancestral DNA sequence which later, by gene duplication, gave rise to the two different but homologous carboxyl-terminal peptides of type IV collagen.