Distribution of the Golgi apparatus in the mitosis of cultured tobacco cells as revealed by DiOC6 fluorescence microscopy

Summary We developed a new method for distinguishing the Golgi apparatus from the other membranous organelles which contain DNA, such as mitochondria and chloroplasts, under a fluorescence microscope. Thin sections of cells embedded in Technovit 8100 resin were stained with both 3,3-dihexyloxacarbocyanine iodide (DiOC₆) and 4,6-diamidino-3-phenylindole (DAPI), and those three membranous organelles were observed under an epifluorescence microscope. The Golgi apparatus, which do not contain DNA, were easily recognized when the two images stained with DiOC₆ and DAPI were superimposed using an image processor. Using this method, we investigated the dynamics of cellular membranes and organelles during the mitotic cycle of synchronized cultured tobacco cells BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2). The Golgi apparatus did not accumulate in the rim of the formating early cell plate at anaphase, while it accumulated near the maturing cell plate at telophase, and this accumulation seemed to be related to the maturation of cell plates. To confirm this hypothesis, synchronized BY-2 cells were treated with caffeine, which is known to inhibit the cell plate formation. Most of the cells treated with caffeine remained in a phase in which Golgi vesicles were accumulated at the equatorial plate, but the cell plate was only partially maturing. The Golgi apparatus accumulated only near the partially maturing cell plate, but not by the equatorial plate where the Golgi vesicles had accumulated.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DAPI 4,6-diamidino-3-phenylindole - LSD a modified Linsmaier and Skoog's medium containing 2,4-D     

An automated apparatus for the real-time monitoring of bioluminescence in plants

We developed an automated, high-throughput, bioluminescence-monitoring apparatus that can monitor 1920 individual plant seedlings under uniform light conditions. The apparatus is composed of five units: (i) a plate platform that can hold 20 96-well microplates under uniform light conditions, (ii) a scintillation counter, (iii) a robot that conveys plates between the plate platform and a scintillation counter, (iv) a sequence controller, and (v) an external computer that collects and analyzes bioluminescence data automatically. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms; neither was affected by the well position in a plate or the plate position on the platform. The apparatus is a powerful tool for both large-scale detailed analysis of gene expression and large-scale screening of mutants.     

Elektronenmikroskopische Untersuchungen von Differenzierungsvorgängen bei Moosen

Summary In meristematic cells of the gemma of Riella helicophylla and in young bud cells from the protonema of Funaria hygrometrica the cell plate is formed by fusion of small vesicles originating from the Golgi apparatus. These spherical vesicles of about 0.1 m diameter have an electron dense centre, probably consisting of pectic substances or their precursors. The endoplasmic reticulum producing multivesicular bodies participate in cell plate formation too. Another cytoplasmic component forming the cell plate are coated vesicles, the origin of which is the Golgi apparatus and perhaps also the endoplasmic reticulum. In view of these observations the question of whether the endoplasmic reticulum or the Golgi apparatus forms the cell plate must be answered in this way: both endoplasmic reticulum and Golgi apparatus supply material for growth of the cell plate. Multivesicular bodies, coated vesicles and other small vesicles of unknown nature participate in the formation of the primary wall.

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Zum Teil finanziert mit Sondermitteln des Landes Niedersachsen an Prof. Dr. M. Bopp.     

The preparation of silica gel thin layers with a new apparatus.

An apparatus for the preparation of uniform thin layer chromatography gel layers is described. The apparatus employs rubber cushion runners to compensate for differences in plate thickness and a gel applicator which functions independently of plate edge variability. Consistently uniform layers are prepared by careful establishment of the plate-to-applicator distance. Silica gel layers averaged 87% of the applied thickness with a variability of +/-2% within a single run and +/-8% between independent runs.     

The Preparation of Silica Gel Thin Layers with a New Apparatus

An apparatus for the preparation of uniform thin layer chromatography gel layers is described. The apparatus employs rubber cushion runners to compensate for differences in plate thickness and a gel applicator which functions independently of plate edge variability. Consistently uniform layers are prepared by careful establishment of the plate-to-applicator distance. Silica gel layers averaged 87% of the applied thickness with a variability of ±2% within a single run and ±8% between independent runs.     

An ultrastructural study of cell plate modifications induced by 2,6-dichlorobenzonitrile in onion root meristems

Summary The effect of 2,6-dichlorobenzonitrile on cytokinesis of meristematic cells of onion root during both treatment and recovery has been studied by electron microscopic techniques. 2,6-dichlorobenzonitrile interferes with cell plate formation in such a way that Golgi apparatus vesicles of treated cells appear to be different than controls and seem to coalesce as anomalous partial cell plates. During recovery, an apparently normal progression of cytokinesis is observed and abnormal portions of the cell plate are retained. Nuclear constrictions are observed frequently during recovery as a result of temporal alterations in cytokinesis. Our results show that 2,6-dichlorobenzo-nitrile induces anomalous and/or incomplete cell plates, which might be caused by an altered function of Golgi apparatus.     

Validation of a colorimetric assay for the in vitro screening of inhibitors of angiotensin-converting enzyme (ACE) from plant extracts.

A new method for the in vitro screening of plant extracts with potential angiotensin-converting enzyme (ACE) inhibitory activity is proposed. The method is based on the cleavage of the substrate hippuryl-glycyl-glycine by ACE and subsequent reaction with trinitrobenzenesulfonic acid to form 2,4,6-trinitrophenyl-glycyl-glycine, whose absorbance is determined at 415 nm in a microtitre plate reader. Rabbit lung dehydrated by acetone was employed as an enzyme source. Validation of the method showed satisfactory intra-day (CV = 7.63%) and inter-day precision (CV = 13.61%), recovery (97-102.1%), sensitivity (IC50 = 14.1 nmol/l) and linearity in the range 7.5-120 mmol/l of glycyl-glycine (r2 = 0.9921). Besides, the method showed good correlation with a HPLC assay already established for the screening of ACE inhibitors (r = 0.9935 and 0.9034, respectively, for captopril solutions and for plant extracts). The method involves only inexpensive reagents and apparatus.     

A Modification of the Mycoplasma Serum-Drop Growth Inhibition Test

A modification of the serologic mycoplasma growth inhibition test is described. It is based on application of a drop of antiserum directly onto the centre of an inoculated agar plate provided with a slightly concave agar surface. The special apparatus devised for the preparation of these plates is described. This modified serum-drop method is more sensitive than other growth inhibition tests used today and is equally specific.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

挂榜山小鲵成体和亚成体舌器的形态特征

以透明骨骼双色法对挂榜山小鲵(Hynobius guabangshanensis)成体和亚成体舌器的形态特征进行观察和详细描述。结果表明,在成体中,下舌软骨中部交叉;角舌软骨末端被第一下鳃骨和第一角鳃骨的愈合体遮盖;具有基鳃软骨角状突;第二角鳃骨骨化;尾舌骨骨化,呈"一"字形。在亚成体中,下舌软骨中部未交叉;第一下鳃骨和第一角鳃骨单独存在;无基鳃软骨角状突;第二角鳃骨未骨化;具有第三、第四对角鳃骨,且均为软骨;尾舌骨未骨化,成"1"字形。这些形态特征的改变可能由舌器在成体和亚成体阶段的功能所引起。     

Comparative tests of evolutionary trade-offs in a palinurid lobster acoustic system

Communication structures vary greatly in size and can be structurally and behaviorally integrated with other systems. In structurally integrated systems, dramatic changes in size may impose trade-offs with the size of neighboring structures. In spiny lobsters (Palinuridae), there is a fivefold difference in size of the antennular plate, on which sound producing apparatus is located, such that the antennular plate reaches 38% carapace length in some sound producers (Stridentes) compared to only 4% carapace length in non-sound producing spiny lobsters (Silentes). We examined whether this major variation in antennular plate size imposes trade-offs with the adjoining antennae, specifically in the context that the signal producing structures and antennae are both used in predator defense. We recorded and analyzed lobster sounds in order to test whether size increases in the acoustic morphology were correlated with production of particular signal features. Antennal and antennular plate structures were measured across the family, including both Stridentes and Silentes. Phylogenetic comparative methods were used to test for correlated evolutionary change among the structures and signal features. We analyzed the phylogenetic relationships of the Palinuridae based on morphological characters and ribosomal DNA evidence (16S, 18S and 28S nuclear and mitochondrial ribosomal RNA gene regions). We found that the number of sound pulses was positively correlated with length of the sound producing apparatus. Opposite to the predicted trade-offs, we found that the size of the antennular plate was positively correlated with size of the surrounding antennae within Stridentes. Nevertheless, when Stridentes were compared to Silentes, the latter had relatively larger antennae for a given antennular plate size than did the sound producing taxa. These results suggest that body size does not limit size increases in acoustic structures within Stridentes, however the presence and associated constructional costs of a sound producing apparatus may impose a trade-off when taxa with and without the apparatus are compared. Alternatively, since both systems are used in predator defense, this pattern may indicate greater selection for antennal force production in Silentes, which lack the additional acoustic mode of predator defense.     

An automated filtration assay for protein kinase C ligands

[3H]Phorbol dibutyrate ([3H]PDBu) binding to soluble mouse brain protein kinase C (PKC) was established in a 96-well microtiter plate assay. [3H]PDBu-PKC receptor complexes were rapidly aspirated from wells, filtered, and washed onto glass fiber filter mats using an automated cell harvester. Results were compared to a modification of a previously described assay in which components were incubated in tubes, and manually delivered and washed onto filters with a manifold filtration apparatus. Both 96-well plate and tube assays gave qualitatively and quantitatively similar results since: (i) [3H]PDBu binding to PKC was phosphatidylserine (PS) dependent and calcium stimulatable; (ii) the amounts of [3H]PDBu bound by filters with each technique at receptors excess were similar, 3.2 +/- 0.3 and 3.1 +/- 0.4 pmol respectively; and (iii) the affinities of [3H]PDBu for PKC were comparable; Kd's were 1.95 +/- 0.3 and 2.2 +/- 0.55 nM, respectively. The 96-well plate assay was more accurate and rapid than the tube assay. The microtiter plate assay was adapted for use with [N,N-dimethyl-3H]N,N-dimethylstaurosporine ([3H]DMS). With [3H]PDBu and [3H]DMS as ligands, the 96-well plate method was used for the rapid discrimination of agents which bound selectively at the regulatory and/or catalytic domains of PKC.     

A COMPARISON STUDY BETWEEN OXYRASE ANAEROBIC AGAR PLATES AND CONVENTIONAL ANAEROBIC CHAMBER FOR THE ISOLATION AND IDENTIFICATION OF ANAEROBIC BACTERIA FROM CLINICAL INFECTIONS

Misplaced confidence in the broad-spectrum antibiotics, increased resistance among previously predictable anaerobic antibiograms, and the push to maximize productivity of available space and downsizing trends has created a need for a simplified cost-effective, and superior method for the isolation and identification of anaerobic bacteria. In this study, the Oxyrase anaerobic plate system which requires no extraneous apparatus to create an anaerobic environment was compared to an anaerobic chamber in the isolation of anaerobic bacteria from 212 consecutive wound specimens. Brucella blood agar and KVL agar plates were used in this comparison study. RapID ANA II, AP120A, special potency disks, and GLC were used for identification. Of the 212 specimens cultured, 87 yielded anaerobic bacteria comprising 182 strains. Thirty-nine strains failed to grow in the anaerobic chamber but grew on the OxyPlates^(TM). These strains were predominantly Peptostreptococcus species (28%), Eubacterium species (20%), and Propionibacterium species (20%). Fourteen strains failed to grow on the OxyPlates, but grew in the anaerobic chamber. No trend was noted and all organisms in this category grew on the OxyPlates from other specimens. In conclusion, the Oxyrases anaerobic plate system appears to be an excellent alternative to the conventional anaerobic chamber in the isolation and identification of clinically significant anaerobes found in human samples, obviating the need for separate anaerobic-aerobic workstations, expensive anaerobic apparatus, and additional incubator space.     

Gas-phase hydrolysis of proteins and peptides

A simplified gas-phase hydrolysis procedure for proteins and peptides is described. The apparatus consists of a glass vacuum desiccator, a ceramic plate, and a Teflon ring. The method was shown to give reproducible compositions for hydrolysis of human serum albumin and microanalysis of α-melanocyte stimulating hormone including the quantitation of as little as one residue of tryptophan. It minimizes sample handling and allows for the simultaneous hydrolysis of a large number of samples.     

A Method of Eliminating the Electrification of Paraffin Ribbons

An apparatus for effectively eliminating the electrification of paraffin ribbons can be constructed for about $2. The essential parts are: a Ford T-TT induction coil to which is connected two, 4-inch brass or copper strips. To the end of one strip is soldered a copper disc 1 1/2 inches in diameter, which is then covered with heavy tinfoil. To the second strip is soldered one-half of a similar disc and its surface is also covered with several layers of tinfoil. The tinfoil should extend 1/2 inch beyond the straight edge of the half circle copper plate. The foil extending is cut to present 15 or 20 pointed projections. The coil is supplied with current from a toy train transformer yielding 5 to 12 volts or 4 to 6 amps. When the coil is in operation the two discs should be moved apart to a distance just beyond which the spark fails to jump. The apparatus is so set that the two brass strips are parallel to and 1 or 2 inches above the microtome knife. A bell push-button may be inserted into the circuit so that the operation of the apparatus may be controlled by the foot, leaving the hands free to handle the ribbon.     

Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.     

Multichannel plating unit for high-throughput plating of cell cultures

High-throughput genomic approaches to gene function or target identification have led to the development and implementation of the 96-well format for many standard molecular biology manipulations. The apparatus described here, a Multichannel Plating Unit, is designed to plate out individual cultures efficientlyfrom standard 96-well culture blocks. Following transformation, aliquots of culture are loaded onto sterile beads that are rolled along individual channels of agar media. After the beads traverse the channel, they drop into the exit alley for disposal via an exit pore. The apparatus presented has 12 individual lanes, and the spacing is compatible with a standard 12-channel pipettor Thus, the unit allows for the rapid plating of 12 individual cultures at a time. For one 96-well block of transformants, this method reduces the labeling and plating effort from 96 culture dishes that are spread individually to eight multichannel plates. The savings in time, materials, and storage space is significant     

SCAMPs highlight the developing cell plate during cytokinesis in tobacco BY-2 cells

We previously demonstrated that rice (Oryza sativa) SECRETORY CARRIER MEMBRANE PROTEIN1 (OsSCAMP1)-yellow fluorescent protein in transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 cells locates to the plasma membrane and to motile punctate structures, which represent the trans-Golgi network/early endosome and are tubular-vesicular in nature. Here, we now show that SCAMPs are diverted to the cell plate during cytokinesis dividing Bright Yellow-2 cells. As cells progress from metaphase to cytokinesis, punctate OsSCAMP1-labeled structures begin to collect in the future division plane. Together with the internalized endosomal marker FM4-64, they then become incorporated into the cell plate as it forms and expands. This was confirmed by immunogold electron microscopy. We also monitored for the Golgi apparatus and the prevacuolar compartment (PVC)/multivesicular body. Golgi stacks tend to accumulate in the vicinity of the division plane, but the signals are clearly separate to the cell plate. The situation with the PVC (labeled by green fluorescent protein-BP-80) is not so clear. Punctate BP-80 signals are seen at the advancing periphery of the cell plate, which was confirmed by immunogold electron microscopy. Specific but weak labeling was observed in the cell plate, but no evidence for a fusion of the PVC/multivesicular body with the cell plate could be obtained. Our data, therefore, support the notion that cell plate formation is mainly a secretory process involving mass incorporation of domains of the trans-Golgi network/early endosome membrane. We regard the involvement of multivesicular late endosomes in this process to be equivocal.     

A method for horizontal polyacrylamide slab gel electrophoresis

We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.     

Apparatus for the Simultaneous Handling of Numerous Histological Specimens

An apparatus has been devised consisting of a series of bakelite plates 2 3/4 inches in diameter which can be clamped together one on top of the other. Each contains 37 depressions 1/4 inch in diameter, with a 1/16 inch hole at the bottom of each passing completely thru the plate. Each depression can contain its own specimen, its identity established by the number of the plate and the position of the depression. Several plates can be clamped together and run simultaneously thru the necessary reagents, thus handling large numbers of specimens without losing the identity of any of them.