The major veins of mesomorphic leaves revisited: tests for conductive overload in Acer saccharum (Aceraceae) and Quercus rubra (Fagaceae)

Many leaves survive the severing of their major veins in apparently excellent health. According to the classical explanation, the leaf minor veins provide "conductive overload," an excess of parallel conductive paths, rendering the major veins hydraulically dispensable. Whether such an excess of conductive paths exists has important implications for vascular design and for leaf response to vascular damage. We subjected leaves of Acer saccharum and Quercus rubra to cutting treatments that disrupted the major vein system and determined leaf survival, stomatal conductance (g), quantum yield of photosystem II (Φ(PSII)), and leaf hydraulic conductance (K(leaf)). For A. saccharum, the cuts led to the death of distal lamina. For Q. rubra, however, the treated leaves typically remained apparently healthy. Despite their appearance, the treated Q. rubra leaves had a strongly reduced K(leaf), relative to control leaves, and g and Φ(PSII) were reduced distal to the cuts, respectively, by 75-97% and 48-76%. Gas exchange proximal to the cuts was unaffected, indicating the independence of lamina regions and their local stomata. Analogous results were obtained with excised Q. rubra leaves. These studies demonstrate an indispensable, vital role of the major veins in conducting water throughout the lamina.     

Nicastrin is a resident lysosomal membrane protein

Nicastrin has been recently identified as part of the gamma-secretase complex that includes presenilin and other proteins. It is involved in the degradation of amyloid precursor protein to produce beta-amyloid peptides which are believed to be central to the pathophysiology of Alzheimer's disease. Previous reports have localized presenilin and nicastrin to the endoplasmic reticulum. However, during a proteomics-based characterization of lysosomal membrane proteins, a major spot observed on silver-stained IEF/SDS-PAGE gels was identified by mass spectrometric sequencing as nicastrin. Its M(r) corresponded to the reported mature M(r) for nicastrin, indicating that it is stable in the lysosomal environment. Furthermore, protease protection assays confirmed that nicastrin is contained in the outer lysosomal membrane, rather than in an internalized vesicle awaiting degradation, and that it is properly orientated with its amino-terminus facing the lysosomal lumen with its carboxyl-terminus facing the cytosol. We conclude that nicastrin is a resident lysosomal membrane protein.     

Iron-sulfur cluster biosynthesis. A comparative kinetic analysis of native and Cys-substituted ISA-mediated [2Fe-2S]2+ cluster transfer to an apoferredoxin target

ISA type proteins mediate cluster transfer to apoprotein targets. Rate constants have been determined for cluster transfer from Schizosaccharomyces pombe ISA to apo Fd. Substitution of the cysteine residues of ISA produced derivative proteins (C72A, C136A, and C138A) that were found to be at least as active in cluster transfer reactions as the native form at 25 degrees C (k(2) approximately 170 M(-1) min(-1) for native, k(2) approximately 169 M(-1) min(-1) for C72A, k(2) approximately 206 M(-1) min(-1) for C136A, and k(2) approximately 242 M(-1) min(-1) for C138A), although the yield of cluster transfer was found to be lower as a consequence of the enhanced lability of clusters in the derivative proteins. Minor variations in rate constant for the ISA Cys derivatives do not reflect any change in the affinity of binding to the apo Fd since k(2) was found to be independent of the concentration of apo Fd over the range of 1-25 microM. The pH dependence of cluster transfer rates was found to be similar for native and C136A ISA, with an observed pK(a) of 7.8 determined from the pH profiles for cluster transfer activity of each protein. The temperature dependence of the rate constant defining the cluster transfer reaction for the wild type versus this C136A ISA derivative is distinct (DeltaH* approximately 6.3 kcal mol(-1) and DeltaS* approximately -27.3 cal K(-1) mol(-1) for native and DeltaH* approximately 2.7 kcal mol(-1) and DeltaS* approximately -38.9 cal K(-1) mol(-1) for C136A ISA). Instability of the protein-bound cluster precluded a comparison with data from pH and temperature dependencies for the two other Cys derivatives. Experiments to determine the dependence of reaction rate constants on viscosity indicate cluster transfer is rate-limiting. A comparison of cross-species rate constants for cluster transfer to apo Fd targets from Homo sapiens and S. pombe demonstrated that the identity of the Fd is less critical for promoting cluster transfer from Sp ISA (at 25 degrees C, k(2) approximately 170 M(-1) min(-1) for Sp Fd and k(2) approximately 169 M(-1) min(-1) for Hs Fd). This contrasts with an earlier observation for ISU-mediated cluster assembly [Wu, S., et al. (2002) Biochemistry 41, 8876-8885], where the rates differed for Hs and Sp target Fd's, suggesting distinct binding sites for binding of holo ISA and ISU to apo Fd.     

Age-related changes in the biomolecular mechanisms of calvarial osteoblast biology affect fibroblast growth factor-2 signaling and osteogenesis

The ability of immature animals to orchestrate successful calvarial ossification has been well described. This capacity is markedly attenuated in mature animals and humans greater than 2 years of age. Few studies have investigated biological differences between juvenile and adult osteoblasts that mediate successful osteogenesis. To identify possible mechanisms for this clinical observation, we investigated cellular and molecular differences between primary osteoblasts derived from juvenile (2-day-old) and adult (60-day-old) rat calvaria. Data demonstrated that juvenile osteoblasts contain a subpopulation of less differentiated cells as observed by spindle-like morphology and decreased osteocalcin production. Juvenile, compared with adult, osteoblasts showed increased proliferation and adhesion. Furthermore, following rhFGF-2 stimulation juvenile osteoblasts increased expression of collagen I alpha 1 (5-fold), osteopontin (13-fold), and osteocalcin (16-fold), compared with relatively unchanged adult osteoblasts. Additionally, juvenile osteoblasts organized and produced more matrix proteins and formed 41-fold more bone nodules. Alternatively, adult osteoblasts produced more FGF-2 and preferentially translated the high molecular weight (22 kDa) form. Although adult osteoblasts transcribed more FGF-R1 and juvenile osteoblasts transcribed more FGF-R2 at baseline levels, juvenile osteoblasts translated more FGF-R1 and -R2 and showed increased phosphorylation. Collectively, these findings begin to explain why juvenile, but not adult, osteoblasts successfully heal calvarial defects.     

Regulation of ERK1 and ERK2 by glucose and peptide hormones in pancreatic beta cells

We showed previously that ERK1/2 were activated by glucose and amino acids in pancreatic beta cells. Here we examine and compare signaling events that are necessary for ERK1/2 activation by glucose and other stimuli in beta cells. We find that agents that interrupt Ca2+ signaling by a variety of mechanisms interfere with glucose- and glucagon-like peptide (GLP-1)-stimulated ERK1/2 activity. In particular, calmodulin antagonists, FK506, and cyclosporin, immunosuppressants that inhibit the calcium-dependent phosphatase calcineurin, suppress ERK1/2 activation by both glucose and GLP-1. Ca2+ signaling from intracellular stores is also essential for ERK1/2 activation, because thapsigargin blocks ERK1/2 activation by glucose or GLP-1. The glucose-sensitive mechanism is distinct from that used by phorbol ester or insulin to stimulate ERK1/2 but shares common features with that used by GLP-1.     

Roles for an Epo receptor Tyr-343 Stat5 pathway in proliferative co-signaling with kit

Erythroid progenitor cell expansion depends upon co-signaling by Epo receptor (EpoR) and Kit, but underlying mechanisms are incompletely understood. To quantitatively analyze EpoR contributions to co-signaling, phosphotyrosine (Tyr(P)) mutants were expressed as human epidermal growth factor (hEGF) receptor-mEpoR EE chimeras at matched and physiological levels in FDCW2 hematopoietic progenitor cells and were assayed for proliferative activities in the absence or presence of endogenous Kit stimulation. Two Tyr(P)-null (but Jak2-coupled) EpoR forms each retained 相似文献   

Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss

Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in primary mouse fibroblasts in vitro and in 95% of regenerating hepatocytes in vivo. Without CENP-E, diminished levels of BubR1 are recruited to kinetochores and BubR1 kinase activity remains at basal levels. CENP-E binds to and directly stimulates the kinase activity of purified BubR1 in vitro. Thus, CENP-E is required for enhancing recruitment of its binding partner BubR1 to each unattached kinetochore and for stimulating BubR1 kinase activity, implicating it as an essential amplifier of a basal mitotic checkpoint signal.     

Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A

To test the hypothesis that fast anterograde molecular motor proteins power the slow axonal transport of neurofilaments (NFs), we used homologous recombination to generate mice lacking the neuronal-specific conventional kinesin heavy chain, KIF5A. Because null KIF5A mutants die immediately after birth, a synapsin-promoted Cre-recombinase transgene was used to direct inactivation of KIF5A in neurons postnatally. Three fourths of such mutant mice exhibited seizures and death at around 3 wk of age; the remaining animals survived to 3 mo or longer. In young mutant animals, fast axonal transport appeared to be intact, but NF-H, as well as NF-M and NF-L, accumulated in the cell bodies of peripheral sensory neurons accompanied by a reduction in sensory axon caliber. Older animals also developed age-dependent sensory neuron degeneration, an accumulation of NF subunits in cell bodies and a reduction in axons, loss of large caliber axons, and hind limb paralysis. These data support the hypothesis that a conventional kinesin plays a role in the microtubule-dependent slow axonal transport of at least one cargo, the NF proteins.     

Immunization with Th-CTL fusion peptide and cytosine-phosphate-guanine DNA in transgenic HLA-A2 mice induces recognition of HIV-infected T cells and clears vaccinia virus challenge

We evaluated immunogenicity of a novel Th-CTL fusion peptide composed of the pan DR Th epitope and a CTL epitope derived from HIV-pol in two transgenic HLA-A*0201/K(b) mouse models. The immunogenicity of peptides of this structure is highly dependent on coadministered cytosine-phosphate-guanine DNA. Initial evaluations of peptide-specific immunity are based on results of chromium release assay, intracellular cytokine, and tetramer staining. Significant cytotoxic T cell responses are found upon a single immunization with as low as 0.1 nmol both peptide and cytosine-phosphate-guanine DNA. Splenocytes from immunized mice recognize naturally processed HIV-pol expressed from vaccinia virus (pol-VV). Translation of immunologic criteria into more relevant assays was pursued using systemic challenge of immunized mice with pol-VV. Only mice receiving both peptide and DNA together successfully cleared upward of 6 logs of virus from ovaries, compared with controls. Challenge with pol-VV by intranasal route of intranasal immunized mice showed a significant reduction in the levels of VV in lung compared with naive mice. A convincing demonstration of the relevance of these vaccines is the robust lysis of HIV-infected Jurkat T cells (JA2/R7/Hyg) by immune splenocytes from peptide- and DNA-immunized mice. This surprisingly effective immunization merits consideration for clinical evaluation, because it succeeded in causing immune recognition and lysis of cells infected with its target virus and reduction in titer of highly pathogenic VV.