Nucleus-associated microtubules help determine the division plane of plant epidermal cells: avoidance of four-way junctions and the role of cell geometry.

To investigate the spatial relationship between the nucleus and the cortical division site, epidermal cells were selected in which the separation between these two areas is large. Avoiding enzyme treatment and air drying, Datura stramonium cells were labeled with antitubulin antibodies and the three-dimensional aspect of the cytoskeletons was reconstructed using computer-aided optical sectioning. In vacuolated cells preparing for division, the nucleus migrates into the center of the cell, suspended by transvacuolar strands. These strands are now shown to contain continuous bundles of microtubules which bridge the nucleus to the cortex. These nucleus-radiating microtubules adopt different configurations in cells of different shape. In elongated cells with more or less parallel side walls, oblique strands radiating from the nucleus to the long side walls are presumably unstable, for they are progressively realigned into a transverse disc (the phragmosome) as broad, cortical, preprophase bands (PPBs) become tighter. The phragmosome and the PPB are both known predictors of the division plane and our observations indicate that they align simultaneously in elongated epidermal cells. These observations suggest another hypothesis: that the PPB may contain microtubules polymerized from the nuclear surface. In elongated cells, the majority of the radiating microtubules, therefore, come to anchor the nucleus in the transverse plane, consistent with the observed tendency of such cells to divide perpendicular to the long axis. In nonrectangular isodiametric epidermal cells, which approximate regular hexagons in section, the radial microtubular strands emanating from the nucleus tend to remain associated with the middle of each subtending cell wall. The strands are not reorganized into a single dominant transverse bar, but remain as a starlike array until mitosis. PPBs in these cells are not as tight; they may only be a sparse accumulation of microtubules, even forming along non-diametrical radii. This arrangement is consistent with the irregular division patterns observed in epidermal mosaics of isodiametric D. stramonium cells. The various conformations of the radial strands can be modeled by springs held in two-dimensional hexagonal frames, and by soap bubbles in three-dimensional hexagonal frames, suggesting that the division plane may, by analogy, be selected by minimal path criteria. Such behavior offers a cytoplasmic explanation of long-standing empirically derived "rules" which state that the new cell wall tends to meet the maternal wall at right angles. The radial premitotic strands and their analogues avoid taking the longer path to the vertex of an angle where a cross wall is already present between neighboring cells.(ABSTRACT TRUNCATED AT 400 WORDS)

An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus.

We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carota L.) using rhodaminyl lysine phallotoxin (RLP). In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation). These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells. The three-dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images. F-actin is present in interphase cells in four interconnected configurations: a meshwork surrounding the nucleus; thick cables in transvacuolar strands and deep in the cytoplasm; a finer network of bundles within the cortical cytoplasm; even finer filaments that run in ordered transverse array around the cell periphery. The actin network is organized differently during division but it does not disappear as do the cortical microtubules. RLP stains a central filamentous cortical band as the chromatin begins to condense (preprophase); it stains the mitotic spindle (as recently shown by Seagull et al. [Seagull, R. W., M. Falconer, and C. A. Weerdenburg, 1987, J. Cell Biol., 104:995-1004] for aldehyde fixed suspension cells) and the cytokinetic apparatus (as shown by Clayton, L., and C. W. Lloyd, 1985, Exp. Cell Res., 156:231-238). However, it is now shown that an additional network of F-actin persists in the cytoplasm throughout division associating in turn with the preprophase band, the mitotic spindle, and the cytokinetic phragmoplast.

Time-domain reflectometry: simultaneous measurement of soil water content and electrical conductivity with a single probe.

Two parallel metallic rods were used as a wave guide to measure the dielectric constant and electrical conductivity of soils having different electrical conductivities but the same water content. Measurements showed that the two parameters were sufficiently independent to permit simultaneous determinations of water content and bulk electrical conductivity.

Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.

Dissolution behaviour of sulphonamides into sodium dodecyl sulfate micelles: a thermodynamic approach.

The surface energies of four sulfonamides have been assessed from contact angle data, using the Lewis acid-base approach. From these data the free energy of adhesion between the drugs and sodium dodecyl sulfate (SDS) head groups and tails has been calculated. The most favored interaction was for adhesion to the SDS tails, rather than the head groups. The initial rotating disk dissolution rate (hereafter termed dissolution rate) of drug compacts has been measured in water and water with SDS micelles at a range of temperatures. The thermodynamic parameters of activation have been calculated from the rate data. Linear relationships exist between the enthalpy of transfer between water and SDS micelles and the free energy of adhesion between the drugs and both SDS head groups and SDS tails. The most nonpolar drugs had the most favored free energy of adhesion and the most favored enthalpy of transfer. The most polar drug had a disfavoured free energy of adhesion to the SDS head and a disfavoured enthalpy of transfer. This response demonstrates that the most important barrier to the passage from the aqueous fluid to the hydrophobic core of the micelle is the monopolar repulsion between the polar forces of the drug and head group surface energies. This provides a new insight into a possible mechanism of solubilization and offers the prospect of understanding even more complex partitioning behavior.

Determination of baseline human nasal pH and the effect of intranasally administered buffers.

The nose is becoming a common route of drug administration, however, little is known about the pH of the human nasal cavity. Local pH may have a direct effect on the rate and extent of absorption of ionizable compounds and hence this study was performed to investigate normal pH values and whether pH could be manipulated by various buffers. Twelve healthy volunteers participated in a study to measure pH in the anterior and posterior sites of the nasal cavity. Miniature pH electrodes were placed 3 cm apart in the nasal cavity and a baseline was recorded for 30 min once the pH had stabilized. One hundred microlitres of isotonic solution was sprayed into the nostril and the pH was measured for 4 h post-dose. The following five formulations were tested: formulation A--sodium chloride (0.9%) at pH 7.2; formulation B--sodium chloride (0.9%) at pH 5.8; formulation C--Sorensens phosphate buffer (0.06 M) at pH 5. 8; formulation D--Sorensens phosphate buffer (0.13 M) at pH 5.8 and formulation E--formulation as (c) but adjusted to pH 5.0. Each formulation also contained saccharin sodium (0.5%) as a taste marker for nasal clearance. The time at which each subject detected the taste of saccharin was noted. The 30-minute baseline recording prior to administration of the nasal spray formulation demonstrates that there was both considerable intersubject and intrasubject variation in nasal pH. The average pH in the anterior of the nose was 6.40 (+0. 11, -0.15 S.D.) when calculated from H(+) values. The pH in the posterior of the nasal cavity was 6.27 (+0.13, -0.18 S.D.). The overall range in pH was 5.17-8.13 for anterior pH and 5.20-8.00 for posterior pH. Formulation A caused the pH in the anterior part of the nasal cavity to reach a maximum of 7.06 in 11.25 min from the baseline of pH 6.14 (P<0.05). The mean baseline pH was 6.5 for the posterior part of the nose which did not change over the recording period. Formulation B caused the anterior pH to increase from pH 6. 60 to 7.25 within the first minute. This fell back to a mean pH of 7.07 over the first hour which was still significantly above the baseline. It remained at this value for the remainder of the recording period. The initial average posterior pH was 6.32 and again this did not significantly change over the recording period. Formulation C produced a sustained increase in anterior nasal pH from a baseline pH of 6.57-7.12. A small transient decrease was observed in the pH in the posterior of the nose but baseline pH of 6. 6 was re-established within 15 min post dose. Formulation D significantly reduced anterior nasal pH from 6.30 to 5.87 by 30 min reaching a pH of 5.95 by 90 min where it remained for the remainder of the recording period. The posterior baseline pH was 6.3 and introduction of the pH 5.8 buffer caused a slow increase over 90 min to pH 6.6. Formulation E increased anterior pH from 6.1 to 6.7 for the remainder of the recording period. It had an insignificant effect on posterior nasal pH. The mean (+/-S.D.) time to taste saccharin for formulations A to E was 13.42+/-10.21, 14.67+/-8.37, 11.67+/-8.08, 10.08+/-7.6, 9.80+/-6.73 min, respectively. There was no significant difference between the clearance times for the different formulations. In conclusion, average baseline human nasal pH is approximately 6.3. Nasal anterior pH can be decreased when buffers of 0.13 M and above are used. Mildly acidic solutions produce an increase in pH presumably due to reflux bicarbonate secretion. Posterior nasal pH was not altered by administration of any buffer except the 0.13 M buffer at pH 5.8. This produced a rise in posterior pH.

Effect of ibuprofen on blood pressure control by propranolol and bendrofluazide.

The effect of 1600 mg/day ibuprofen in two groups of patients with hypertension controlled by either propranolol or bendrofluazide was studied in a double-blind, double-placebo, randomized crossover trial. No significant difference in blood pressure was found at the end of the crossover period in either group, suggesting that the routine co-administration of ibuprofen does not attenuate the anti-hypertensive effect of thiazide diuretics or propranolol. Significant weight gain, attributable to fluid retention, had occurred in the bendrofluazide-treatment group by the end of the drug-free washout period. No significant change in mean weight occurred in the crossover stages in either group, although substantial weight gain was noted during ibuprofen treatment in two patients given bendrofluazide and one given propranolol. Biochemical variables were unaffected by ibuprofen throughout the crossover period. This study suggests that ibuprofen may be administered routinely to patients receiving thiazides or propranolol without loss of control of the anti-hypertensive action of these drugs but it is recommended that individuals are monitored for possible weight gain or an increase in diastolic blood pressure.

LX4211, a dual SGLT1/SGLT2 inhibitor, improved glycemic control in patients with type 2 diabetes in a randomized, placebo-controlled trial.

Thirty-six patients with type 2 diabetes mellitus (T2DM) were randomized 1:1:1 to receive a once-daily oral dose of placebo or 150 or 300 mg of the dual SGLT1/SGLT2 inhibitor LX4211 for 28 days. Relative to placebo, LX4211 enhanced urinary glucose excretion by inhibiting SGLT2-mediated renal glucose reabsorption; markedly and significantly improved multiple measures of glycemic control, including fasting plasma glucose, oral glucose tolerance, and HbA(1c); and significantly lowered serum triglycerides. LX4211 also mediated trends for lower weight, lower blood pressure, and higher glucagon-like peptide-1 levels. In a follow-up single-dose study in 12 patients with T2DM, LX4211 (300 mg) significantly increased glucagon-like peptide-1 and peptide YY levels relative to pretreatment values, probably by delaying SGLT1-mediated intestinal glucose absorption. In both studies, LX4211 was well tolerated without evidence of increased gastrointestinal side effects. These data support further study of LX4211-mediated dual SGLT1/SGLT2 inhibition as a novel mechanism of action in the treatment of T2DM.

Study of the management of suspected cardiac infarction by British immediate care doctors.

A study of the management by immediate care general practitioners of 511 patients suspected of suffering from acute myocardial infarction showed that the median time of arrival after the onset of chest pains was 60.2 minutes. One hundred and eleven patients died of cardiac infarction within 48 hours of the onset of chest pain; 23 died in the presence of the general practitioner.

Ancient chronology. Astronomical orientation of the pyramids.

Spence speculates that Egypt's pyramid builders found true north by using a plumb line: when the stars Kochab and Mizar were seen on the same vertical, one was facing north. As evidence in support of this hypothesis, she points to the proposed interstar-line precession past the north celestial pole at a rate of 27' per century (cy). We argue that a mathematical error affects this result, which when corrected points more strongly to a different pair of stars. This suggests that the conventional ancient chronology, instead of being compressed, may actually have to be expanded slightly.

Three-dimensional organization of chromosomes of Crepis capillaris by optical tomography.

The three-dimensional (3D) organization of chromosomes of Crepis capillaris (2n = 6) has been investigated. Root tips were fixed, macerated with enzymes and gently separated without squashing. The cells were then stained with DAPI and optically sectioned under computer control. Sections were stored as video images and processed to remove noise and out-of-focus information. Computer modelling was then used to trace the paths of each chromosome and to display the paths as a 3D wire diagram. In all, 88 sets of anaphase chromosomes were modelled from 47 optically sectioned cells. The models and the coordinates of the chromosomes were then analysed to detect non-random arrangements or preferential associations of particular pairs of chromosomes. The methods used have significant advantages over electron microscope tomography for the analysis of 3D chromosome arrangement; in particular, the large number of samples allowed more thorough statistical tests to be performed on the data obtained. No evidence was found for either non-random arrangements or homologous association and, moreover, the distances between the two larger pairs of homologues were larger than for other pairs of chromosomes. These results conflict with previous results for this and other plant species where the material was squashed before measurements were taken. We found no evidence of haploid genome separation.

Identification and characterization of ZIIBC, a complex formed by cellular factors and the ZII site of the Epstein-Barr virus BZLF1 promoter.

The transition from latency to lytic Epstein-Barr virus replication is dependent on the Epstein-Barr virus BZLF1 gene product. Genetic and biochemical attempts to link cellular second-messenger signaling pathways that trigger this transition with the subsequent viral gene cascade have identified functional elements within the BZLF1 promoter (Zp) that appear to bind undefined cellular transcription factors. One of these previously identified sites, ZII, has homology to consensus AP-1 and CREB binding sites, implying a role for these factors in the inductive process. We have identified and characterized ZIIBC, a ZII site binding complex that is distinct from the factors previously proposed to bind this site. Active ZIIBC was found to be present in both uninduced and chemically induced cell extracts at approximately equivalent concentrations. Analysis of the DNA sequence requirements for the binding of ZIIBC to the ZII site shows that sequences homologous to AP-1 and CREB consensus sites are necessary but not sufficient for complex formation. Although the components of ZIIBC that directly contact DNA were found to be of the same molecular masses (26 and 36 kDa) in both uninduced and chemically induced cell extracts, a slight mobility difference between DNA-protein complexes formed by these two types of extracts is observable and indicates that ZIIBC is directly affected by chemical induction. The effects of ZIIBC binding to the ZII site on expression from Zp were evaluated, and they suggest that ZIIBC plays a critical role in the regulation of Zp expression.

Localization of ribosomal and telomeric DNA sequences in intact plant nuclei by in-situ hybridization and three-dimensional optical microscopy.

We have combined the use of three-dimensional (3-D) fluorescence microscopy and computer image processing of images with in-situ hybridization to analyse the 3-D organization of interphase nuclei in plants. In sections of root tips of Pisum sativum, using cDNA probes, we have shown that telomeres are arranged around the nuclear periphery and that the ribosomal genes in this species appear to exist in discrete, 3-D domains.

Three-dimensional localisation of DNA in the nucleolus of Spirogyra by correlated optical tomography and serial ultra-thin sectioning.

Spirogyra nucleoli were shown by three-dimensional optical microscopy of DAPI fluorescence to contain DNA with a pattern and distribution matching those of the fibrillar centres. This was confirmed using different species with nucleoli showing different sizes of fibrillar centre. Much lower levels of fluorescence were seen corresponding to the dense fibrillar component. Nearly all the DAPI fluorescence arises from the fibrillar centres or from regions very close to their surface, indicating that this is the site of nucleolar transcription.

Template requirements for the initiation of adenovirus DNA replication.

The first step in the replication of the adenovirus genome is the covalent attachment of the 5'-terminal nucleotide, dCMP, to the virus-encoded terminal protein precursor (pTP). This reaction can be observed in vitro and has been previously shown to be dependent upon either viral DNA or linearized plasmid DNA containing viral terminal sequences. Plasmids containing deletions or point mutations within the viral terminal sequence were constructed by site-directed mutagenesis. In the case of linear double-stranded templates, pTP-dCMP formation required sequences located within the first 18 base pairs of the viral genome. This sequence contains a segment of 10 base pairs that is conserved in all human adenovirus serotypes. Point mutations within the conserved segment greatly reduced the efficiency of initiation, while a point mutation at a nonconserved position within the first 18 base pairs had little effect. Single-stranded DNAs can also support pTP-dCMP formation in vitro. In contrast to the results obtained with duplex templates, experiments with a variety of single-stranded templates, including phage M13-adenovirus recombinants, denatured plasmids, and synthetic oligodeoxynucleotides, failed to reveal any requirements for specific nucleotide sequences. With single-stranded templates containing no dG residues, the specific deoxynucleoside triphosphate requirements of the initiation reaction were altered.