Functional modulation of cardiac form through regionally confined cell shape changes

Developing organs acquire a specific three-dimensional form that ensures their normal function. Cardiac function, for example, depends upon properly shaped chambers that emerge from a primitive heart tube. The cellular mechanisms that control chamber shape are not yet understood. Here, we demonstrate that chamber morphology develops via changes in cell morphology, and we determine key regulatory influences on this process. Focusing on the development of the ventricular chamber in zebrafish, we show that cardiomyocyte cell shape changes underlie the formation of characteristic chamber curvatures. In particular, cardiomyocyte elongation occurs within a confined area that forms the ventricular outer curvature. Because cardiac contractility and blood flow begin before chambers emerge, cardiac function has the potential to influence chamber curvature formation. Employing zebrafish mutants with functional deficiencies, we find that blood flow and contractility independently regulate cell shape changes in the emerging ventricle. Reduction of circulation limits the extent of cardiomyocyte elongation; in contrast, disruption of sarcomere formation releases limitations on cardiomyocyte dimensions. Thus, the acquisition of normal cardiomyocyte morphology requires a balance between extrinsic and intrinsic physical forces. Together, these data establish regionally confined cell shape change as a cellular mechanism for chamber emergence and as a link in the relationship between form and function during organ morphogenesis.     

Visualization of Dynamic Sub-microsecond Changes in Membrane Potential

Direct observation of rapid membrane potential changes is critical to understand how complex neurological systems function. This knowledge is especially important when stimulation is achieved through an external stimulus meant to mimic a naturally occurring process. To enable exploration of this dynamic space, we developed an all-optical method for observing rapid changes in membrane potential at temporal resolutions of ～25 ns. By applying a single 600-ns electric pulse, we observed sub-microsecond, continuous membrane charging and discharging dynamics. Close agreement between the acquired results and an analytical membrane-charging model validates the utility of this technique. This tool will deepen our understanding of the role of membrane potential dynamics in the regulation of many biological and chemical processes within living systems.     

Adhesive thermosensitive gels for local delivery of viral vectors

Local delivery of viral vectors can enhance the efficacy of therapies by selectively affecting necessary tissues and reducing the required vector dose. Pluronic F127 is a thermosensitive polymer that undergoes a solution–gelation (sol–gel) transition as temperature increases and can deliver vectors without damaging them. While pluronics can be spread over large areas, such as the surface of an organ, before gelation, they lack sufficient adhesivity to remain attached to some tissues, such as the surface of the heart or mucosal surfaces. Here, we utilized blends of pluronic F127 and polycarbophil (PCB), a mucoadhesive agent, to provide the necessary adhesivity for local delivery of viral vectors to the cardiac muscle. The effects of PCB concentration on adhesive properties, sol–gel temperature transition and cytocompatibility were evaluated. Rheological studies showed that PCB decreased the sol–gel transition temperature at concentrations >1% and increased the adhesive properties of the gel. Furthermore, these gels were able to deliver viral vectors and transduce cells in vitro and in vivo in a neonatal mouse apical resection model. These gels could be a useful platform for delivering viral vectors over the surface of organs where increased adhesivity is required.     

Inhibition of photosystems I and II and enhanced back flow of photosystem I electrons in cucumber leaf discs chilled in the light

Pre-illumination of cucumber leaf discs at a chilling temperature in low-irradiance white light resulted in accelerated re-reduction of P700(+) [the special Chl pair in the photosystem I (PSI) reaction centre] when the far-red measuring light was turned off. Measurements (in +/- methyl viologen or +/- DCMU conditions) of the re-reduction half time suggest that accelerated re-reduction of P700(+) appeared to be predominantly due to charge recombination and only partly due to reductants sustained by previous cyclic electron flow around PSI. Apparently, charge recombination in PSI was greatly enhanced by inhibition of forward, linear electron flow. Inhibition of PSII electron transport was observed to occur to a lesser extent than that of PSI, but only if the measurement of PSII functionality was free from complications due to downstream accumulation of electrons in pools. We suggest that promotion of controlled charge recombination and cyclic electron flow round PSI during chilling of leaves in the light may partly prevent further damage to both photosystems.     

Site-specific characterization of the N-linked glycans of murine prion protein by high-performance liquid chromatography/electrospray mass spectrometry and exoglycosidase digestions

The murine prion protein PrP gene encodes a protein of 254 amino acids with two consensus sites for Asn-linked glycosylation at codons 180 and 196. A partial site-specific study of the N-linked glycans from hamster PrP has previously been carried out by mass spectrometry [Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., and Prusiner, S. B. (1993) Biochemistry 32, 1991-2002] and revealed that the glycosylation at Asn-181 (equivalent to mouse 180) is heterogeneous, comprising over 30 glycoforms. The identification of the glycosylated peptide spanning Asn-197 was not reported. Recent technical advances in electrospray mass spectrometry now provide the sensitivity to detect low femtomole quantities of glycopeptides with >5000 mass resolution and 30 ppm mass measurement [Medzihradszky, K. F., Besman, M. J., and Burlingame, A. L. (1998) Rapid Commun. Mass Spectrom. 12, 472-478]. This performance coupled with stepwise exoglycosidase digestion has been employed to establish the differential nature of the structural complexity (glycoforms) of the glycans at Asn-180 and Asn-196 from a single strain infected with the ME7 strain. Some sixty structures have been found characterized by neutral and sialylated bi-, tri-, and tetraantennary complex-type bearing outer-arm alpha(1-3)-fucosylation (the Lewisx and sialyl-Lewisx epitopes), core alpha(1,6) fucosylation, and the presence of terminal HexNAc residues. The Lewisx trisaccharide is the major nonreducing structure at Asn-180, and significant amounts of both Lewisx and sialyl Lewisx epitopes are observed at Asn-196. The abundance of the Lewisx and sialyl Lewisx epitopes on murine PrPSc may indicate a role for these structures in the normal function of PrPC or the pathophysiology of PrPSc.     

Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either ³H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (¹⁴C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1–10 g ml^–1) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1–6 h. The results were expressed as pmol min^–1 (mg cell protein)^–1, n= 6–8 for each value. Basal ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means ± S.E.M.) 32.14 ± 1.34 and 13.48 ± 1.86 pmol min^–1 (mg cell protein)^–1, respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes. Typically, ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes in the presence of insulin were 58.57 ± 4.49 and 29.52 ± 3.41 pmol min^–1 (mg cell protein^–1), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 g ml^–1) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in ³H-deoxy-D-glucose and ¹⁴C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 g ml^–1. Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of ³H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin. (Mol Cell Biochem 261: 99–104, 2004)     

Zinc-deficient rats have more limited bone recovery during repletion than diet-restricted rats

The objective of this study was to investigate the effects of dietary zinc deficiency and diet restriction on bone development in growing rats, and to determine whether any adverse effects could be reversed by dietary repletion. Weanling rats were fed either a zinc-deficient diet ad libitum (ZD; <1 mg zinc/kg) or nutritionally complete diet (30 mg zinc/kg) either ad libitum (CTL) or pair-fed to the intake of the ZD group (DR; diet-restricted) for 3 weeks (deficiency phase) and then all groups were fed the zinc-adequate diet ad libitum for 3, 7, or 23 days (repletion phase). Excised femurs were analyzed for bone mineral density (BMD) using dual-energy x-ray absorptiometry, and plasma was analyzed for markers of bone formation (osteocalcin) and resorption (Ratlaps). After the deficiency phase, ZD had lower body weight and reduced femur BMD, zinc, and phosphorus concentrations compared with DR; and these parameters were lower in DR compared with CTL. Femur calcium concentrations were unchanged among the groups. Reduced plasma osteocalcin in ZD and elevated plasma Ratlaps in DR suggested that zinc deficiency limits bone formation while diet restriction accelerates bone resorption activity. After 23 days of repletion, femur size, BMD, and zinc concentrations remained lower in ZD compared with DR and CTL. Body weight and femur phosphorus concentrations remained lower in both ZD and DR compared with CTL after repletion. There were no differences in plasma osteocalcin concentrations after the repletion phase, but the plasma Ratlaps concentrations remained elevated in DR compared with CTL. In summary, both ZD and DR lead to osteopenia during rapid growth, but the mechanisms appear to be due to reduced modeling in ZD and higher turnover in DR. Zinc deficiency was associated with a greater impairment in bone development than diet restriction, and both deficiencies limited bone recovery during repletion in growing rats.