The loculus content and tapetum during pollen development in Lilium

 The ratio of loculus volume to the volume of the entire anther began to increase from the microspore mother cell stage and reached 32.3% at anthesis. The content of the loculus was examined in Lilium during pollen development and two waves could be distinguished. From the premeiotic stage until the vacuolated microspore stage, the loculus consisted of neutral polysaccharides, pectins and proteins. These substances originated from tapetal activity from the premeiotic stage until the young microspore stage. Dictyosomes and rough endoplasmic reticulum seemed to be involved in tapetal secretion, although, in some mitochondria, vesicles progressively developed as early as premeiosis and increased until the young microspore stage, which could reveal their involvement in the secretion process. At this stage, numerous cytoplasmic vesticles containing material similar to the locular material fused with the plasma membrane of the tapetum so that vesicle content was in contact with the loculus. It seems that tapetal and callose wall degradation at the late tetrad stage may also have contributed to the production of material in the loculus. From pollen mitosis to anthesis, the anther loculus contained mainly the pollenkitt which was synthesized in the tapetum between the young microspore stage and the vacuolated microspore stage. At the young microspore stage, proplastids divided and developed into elaioplasts and smooth endoplasmic reticulum (SER) increased dramatically. Pollenkitt had a double origin: some droplets were extruded directly from the plastid stroma through the plastid envelopes; the others were unsaturated lipid globules, which presumably derived from the interaction between SER saccules and plastids. Received: 2 September 1997 / Revision accepted: 12 March 1998     

A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha -adrenoceptor stimulation

The purpose of this study was to investigate the potential roleof mitogen-activated protein (MAP) kinase in smooth muscle contractionby monitoring MAP kinase activation, caldesmon phosphorylation, andcontractile force during agonist stimulation. Isometric tension inresponse to KCl and phenylephrine (PE) was measured from strips offerret aorta. MAP kinase activation was monitored by Western blot usinga phosphospecific p44/p42 MAP kinase antibody. Caldesmon phosphorylation was assessed using specific phosphocaldesmonantibodies. We report here that treatment of smooth muscle strips withPD-098059, a specific inhibitor of MAP kinase kinase, did notdetectably modify the KCl-evoked contraction but significantlyinhibited the contraction to PE in the absence of extracellularCa²⁺. In this experimentalcondition, where the contraction occurs in the absence of increases in20-kDa myosin light chain phosphorylation, PD-098059 also inhibitedsignificantly MAP kinase and caldesmon phosphorylation. Collectively,these results demonstrate a direct cause-and-effect relationshipbetween MAP kinase activation and Ca²⁺-independent smooth musclecontraction and support the concept of caldesmon phosphorylation as themissing link between both events.

     

Optimal Sequencing Rules for Some Large-Scale Flexible Manufacturing Problems Under the Manhattan and Chebychev Metrics

The purpose of this paper is to develop optimal tool partitioning policies and strip sequencing strategies for a class of flexible manufacturing problems. The problems under consideration involve a large number of operations to be performed by a series of tools on a two-dimensional object. For example, these operations could consist of drilling holes in a metallic sheet. Tools are arranged in a carousel or along a toolbar according to a predetermined sequence. Operations are performed by repeatedly moving the sheet to bring the hole locations under the tool. During each pass, as all operations involving a series of consecutive tools are executed, two main problems are to be solved: (1) how to move the sheet during each pass, (2) how to partition the tools into blocks of consecutive tools. A strip strategy is used to move the sheet. Given this policy, optimal strip widths and tool partitioning policies are determined jointly. Analytical solutions are derived under two metrics corresponding to different operating modes. A numerical example is provided.     

Determination of peptide contact points in the human angiotensin II type I receptor (AT1) with photosensitive analogs of angiotensin II

To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-L-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166-199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of [Sar1, Bpa8]AngII interacts with the second extracellular loop of the AT1 receptor.     

PCR-based assays of mendelian polymorphisms from anonymous single-copy nuclear DNA: techniques and applications for population genetics

This paper outlines a PCR-based approach for population genetics that offers several advantages over conventional Southern blotting methods for revealing restriction-fragment-length polymorphisms (RFLPs) in nuclear DNA. Primers are constructed from clones isolated from a nuclear DNA library, and these primers subsequently are employed in in vitro syntheses of homologous regions. Amplified products are then screened directly for RFLPs by using gel-staining procedures. Population applications for this PCR-based approach, including potential strengths and weaknesses, are exemplified by two RFLP data sets generated to estimate (a) male-mediated gene flow in the green turtle (Chelonia mydas) and (b) geographic population genetic structure in the American oyster (Crassostrea virginica). Restriction assays of amplified products from 14 or 15 independent primer pairs in each species revealed polymorphisms at several loci that proved highly informative in the population genetic analyses. In general, the Mendelian polymorphisms produced by this PCR-based approach will provide useful genetic markers for population studies, particularly in situations where simpler and less expensive allozyme methods have failed, for whatever reason, to provide adequate information.      

The isocitrate dehydrogenase phosphorylation cycle: Regulation and enzymology

Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by phosphorylation and dephosphorylation. This phosphorylation cycle controls the flow of isocitrate through the glyoxylate bypass, a pathway which bypasses the CO₂ evolving steps of the Krebs' cycle. IDH is phosphorylated at a single serine which resides in its active site. Phosphorylation blocks isocitrate binding, thereby inactivating IDH. The IDH phosphorylation cycle is catalyzed by a bifunctional protein kinase/phosphatase. The kinase and phosphatase reactions appear to be catalyzed at the same site and may share some catalytic steps. A variety of approaches have been used to examine the IDH phosphorylation cycle in the intact organism. The picture which has emerged is one of an exquisitely sensitive and flexible system which is capable of adapting efficiently to the environment both inside and outside the cell. © 1993 Wiley-Liss, Inc.     

Electrical and adaptive properties of rod photoreceptors in bufo marinus. II. Effects of cyclic nucleotides and protaglandins

Substances known to alter cyclic nucleotide levels in cells were applied to the isolated toad retina and effects on rod electrical and adaptive behavior were studied. The retina was continually superfused in control ringer’s or ringer’s containing one or a combination of drugs, and rod activity was recorded intracellularly. Superfusion with cGMP, Bu(2)GMP, isobutylmethylxanthine (IBMX; a phosphodiesterase inhibitor), or PGF(2α) (a prostaglandin) caused effects in rods that closely match those observed when extracellular Ca(2+) levels were lowered. For example, short exposures (up to 6 min) of the retina to these substances caused depolarization of the membrane potential, increase in response amplitudes, and some changes in waveform; but under dark-adapted or partially light-adapted conditions receptor sensitivity was virtually unaffected. That is, the position of the V-log I curve on the intensity axis was determined by the prevailing light level, not by drug level. These drugs, like lowered extracellular Ca(2+), also decreased the period of receptor saturation after a bright-adapting flash, resulting in an acceleration of the onset of membrane and sensitivity recovery during dark adaptation.

Long-term (6-15 min) exposure of a dark-adapted retina to 5 mM IBMX or a combination of IBMX and cGMP caused a loss of response amplitude and a desensitization of the rods that was similar to that observed in rods after a long-term low Ca(2+) (10(-9)M) treatment. Application of high (3.2 mM) Ca(2+) to the retina blocked the effects of applied Bu(2)cGMP. PGE(1) superfusion mimicked the effects of increasing extracellular Ca(2+). The results show that increased cGMP and lowered Ca(2+) produce similar alterations in the electrical activity of rods. These findings suggest that Ca(2+) and cGMP are interrelated messengers. We speculate that low Ca(2+) may lead to increased intracellular cGMP, and/or that applied cGMP, and/or that applied cGMP may lower cytosol Ca(2+), perhaps by stimulating Ca(2+)- ATPase pumps in the outer segment.