Sucrose metabolism and IAA and ethylene production in muskmelon ovaries

Changes induced by the pollination of ovaries may be mediated by phytohormones and involve sudar-mediated by phytohormones and involve sugar-metabolizing enzymes. In order to further explore these relationships, soluble sugars, sucrose-phosphate synthase (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), acid and neutral invertases (EC 3.2.1.26), indole-3-acetic acid (IAA), and ethylene were investigated in muskmelon (Cucumis melo L.) ovaries sampled before, during, and after anthesis. The fresh weight of ovaries increased 100% within 48 h after pollination, but did not change significantly in the absence of pollination. While sugar content per ovary increased after pollination, sugar content per mg protein was unaffected. Sucrose was not detected in nonpollinated ovaries 48 h after anthesis. Free IAA content was highest in ovaries sampled 48h before anthesis. Pollination had no immediate effect on IAA content per mg protein in postanthesis ovaries. Although detected in all ovaries sampled, ethylene production increased significantly only in nonpollinated ovaries. Activity of sucrose-phosphate synthase was the same at all stages. The specific activities of sucrose synthase and the invertases were highest in nonpollinated ovaries. The increase in rate of sugar import into ovaries following pollination was not accompanied by an increase in the specific activity of any enzyme assayed, but was coincident with an increase in the total activity per ovary of surcose synthase and acid invertase. There appears to be no direct relationship between sucrose-metabolizing enzymes, IAA or ethylene in developing pollinated ovaries but the increase in sucrose cleavage activity in nonpollinated ovaries may be related to the increase in ethylene production.Mention of a specfic product does not imply an endorsement by the United States Depertment of Agriculture or Texas Aricultural Experiment Station over other suitable products.     

Activation of skeletal muscle nicotinic acetylcholine receptors

Summary and Conclusions Work over the past ten years has greatly increased our understanding of both the structure and function of the muscle nicotinic acetylcholine receptor. There is a strongly supported general picture of how the receptor functions: agonist binds rapidly to sites of low affinity and channel opening occurs at a rate comparable to the agonist dissociation rate. Channel closing is slow, so the channel has a high probability of being open if both agonist-binding sites are occupied by ACh. Results of expression studies have shown that each subunit can influence AChR activation and have given a structural basis for the major physiological change known for muscle AChR, the developmental change in AChR activation. These general statements notwithstanding, there are still major areas of uncertainty which limit our understanding. We have emphasized these areas of uncertainty in this review, to indicate what needs to be done.First, the quantitative estimates of rate constants are not as strongly supported as they should be. The major reasons are twofold—uncertainties about the interpretation of components in the kinetic data and difficulties of resolving brief events. As a result, any inferences about the functional consequences of structural alterations must remain tenuous.Second, the functional behavior of individual AChRs is not as well understood as it should be. The kinetic behavior of an individual receptor clearly can be complex (section II). In addition, there is evidence that superimposed on this complexity there may be stable and kinetically distinguishable populations of receptors (section III). Until the basis for the kinetically defined populations is clarified, kinetic parameters for receptors of defined structure cannot be unambiguously obtained.Finally, it is not surprising that the studies of AChR of altered structure have not given definitive results. Two reasons should be apparent from the preceding points: there is not a fully supported approach for kinetic analysis, and the normal population may not be clearly defined. An additional complication is also emerging, in that the available data support the idea that specific residues distributed over all subunits may influence AChR activation. This possibility renders the task of analysis that much more difficult.The muscle nicotinic AChR has served as a prototype for the family of transmitter-gated membrane channels, which includes the muscle and neuronal nicotinic receptors, the GABA_A, the glycine and possibly the non-NMDA excitatory amino acid receptor (Stroud et al., 1990). It is interesting to note that the functional properties of the GABA_A receptor, probably the best-studied of the other members of the family are rather similar. In particular, opentime and burst durations show multiple components interpreted as reflecting openings of singly and doubly liganded receptors (Mathers & Wang, 1988; Macdonald et al., 1989), the distribution of gaps indicates a relatively complex gating scheme (Twyman et al., 1990; Weiss & Magleby, 1989), and multiple kinetic modes are likely to exist (Newland et al., 1991). The situation with regards to the effects of GABA_A receptor subunit stoichiometry is more complex than for muscle AChR (e.g., Luddens & Wisden, 1991), perhaps similar to that found for neuronal nicotinic AChR (Papke et al., 1989; Luetje et al., 1990; Luetje & Patrick, 1991). Overall, it appears that the unresolved questions about the muscle nicotinic AChR are not indications that this is an exceptionally complicated transmitter-gated channel. Rather, it appears to be a relatively straightforward member of the family, and the lessons we learn from studying it are likely to be directly applicable to other receptors.We thank many friends for discussion, including Tony Auerbach, Paul Brehm, Jim Dilger, Meyer Jackson, and Chuck Stevens who told us about data before publication. Research in the authors' laboratories is supported by grants from the NIH (CL and JHS) and the AHA (CL).     

Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication

The spacer between the 16S and 23S rRNA genes of the chloroplast DNA has been implicated as an origin of replication in several species of plants. In the evening primrose, Oenothera, this site was found to vary greatly in size, with plastid genomes (plastomes) being readily distinguished. To determine whether plastome "strength" in transmission could be correlated with variation at oriB, the 16S rRNA-trnI spacer was sequenced from five plastomes. The size variation was found to be due to differential amplification (and deletion) of combinations of sequences belonging to seven families of direct repeats. From these comparisons, one short series of direct repeats and one region capable of forming a hairpin structure were identified as candidates for the factor that could be responsible for the differences between strong and weak plastome types. Ample sequence variation allowed phylogenetic inferences to be made about the relationships among the plastomes. Phylogenetic trees also could be constructed for most of the families of direct repeats. The amplifications and deletions of repeats that account for the size variation at oriB are proposed to have occurred through extensive replication slippage at this site.      

Loss of HSulf-1 expression enhances autocrine signaling mediated by amphiregulin in breast cancer

Heparan sulfate (HS) glycosaminoglycans are the oligosaccharide chains of heparan sulfate proteoglycans. The sulfation of HS glycosaminoglycan residues is required for its interaction with various heparin-binding growth factors to promote their biological activities to activate their high affinity receptor tyrosine kinases. We have identified HS glycosaminoglycan-6-O-endosulfatase HSulf-1 as a down-regulated gene in ovarian, breast, and several other cancer cell lines. Here we have shown that HSulf-1 inhibits autocrine activation of the EGFR-ERK (epidermal growth factor receptor-extracellular signal-regulated kinase) pathway induced by serum withdrawal in MDA-MB-468 breast cancer cells. Short hairpin RNA-mediated down-regulation of HSulf-1 in HSulf-1 clonal lines of MDA-MB-468 led to a significant increase in autocrine activation of ERK compared with vector only control. The autocrine signaling was also inhibited with neutralization antibodies against amphiregulin and HB-EGF, the heparin-binding growth factor family of the EGF superfamily. Furthermore, HSulf-1-mediated inhibition of autocrine signaling was associated with reduced cyclin D1 levels, leading to decreased S phase fraction and increased G(2)-M fraction, as well as increased cell death. Finally, evaluation of HSulf-1 expression levels in primary invasive breast tumors by RNA in situ hybridization indicated that HSulf-1 is down-regulated in the majority (60%) of tumors, with a predominant association with lobular histology. These data suggest a potential role of HSulf-1 down-regulation in mammary carcinogenesis.     

Genbank accession #: AJ131961

Plant Molecular Biology -     

pH-regulated Slo3 K+ channels: properties of unitary currents

Here we have examined the voltage and pH dependence of unitary Slo3 channels and used analysis of current variance to define Slo3 unitary current properties over a broader range of voltages. Despite complexity in Slo3 channel openings that precludes simple definition of the unitary conductance, average current through single Slo3 channels varies linearly with voltage at positive activation potentials. Furthermore, the average Slo3 unitary current at a given activation potential does not change with pH. Consistent with macroscopic conductance estimates, the apparent open probability of Slo3 channel exhibits a pH-dependent maximum, with limiting values around 0.3 at the most elevated pH and voltage. Estimates of Slo3 conductance at negative potentials support a weaker intrinsic voltage dependence of gating than is observed for Slo1. For the pH-regulated Slo3 K(+) channel, the dependence of macroscopic conductance on pH suggests that the pH-sensitive mechanism regulates gating in an allosteric manner qualitatively similar to regulation of Slo1 by Ca(2+). Together, the results support the view that the regulation of macroscopic Slo3 currents by pH reflects regulation of gating equilibria, and not a direct effect of pH on ion permeation. Specifically, both voltage and pH regulate a closed-open conformational change in a largely independent fashion.     

Echocardiographic quantification of myocardial function using tissue deformation imaging, a guide to image acquisition and analysis using tissue Doppler and speckle tracking

Recent developments in the field of echocardiography have allowed the cardiologist to objectively quantify regional and global myocardial function. Regional deformation (strain) and deformation rate (strain-rate) can be calculated non-invasively in both the left and right ventricle, providing information on regional (dys-)function in a variety of clinical settings. Although this promising novel technique is increasingly applied in clinical and preclinical research, knowledge about the principles, limitations and technical issues of this technique is mandatory for reliable results and for implementation both in the clinical as well as the scientific field. In this article, we aim to explain the fundamental concepts and potential clinical applicability of strain and strain-rate for both tissue Doppler imaging (TDI) derived and speckle tracking (2D-strain) derived deformation imaging. In addition, a step-by-step approach to image acquisition and post processing is proposed. Finally, clinical examples of deformation imaging in hypertrophic cardiomyopathy (HCM), cardiac resynchronization therapy (CRT) and arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) are presented.