Ultrastructure of the shoot apex of Chenopodium album and certain other seed plants.

The ultrastructure of cells of the vegetative shoot apices is described for Chenopodium album, Kalanchoë blossfeldiana and K. laxiflora, Bryophyllum daigremontianum, Nicotiana rustica, and N. tabacum (Maryland Mammoth), and Ginkgo biloba. A less intensive study was made of the last three listed. The structures and organelles usually associated with meristematic cells were observed: dictyosomes, plastids (in various stages of development), mitochondria, endoplasmic reticulum (ER), vacuoles, lipid droplets, and plasmalemma. In addition, spherosome-like structures were observed in all zones of the shoot apices. Also, multivesicular bodies were observed in C. album and B. daigremontianum. Ribosome density is greater in cells of the flank meristem. Proplastids, plastids with prolamellar bodies, or grana have a differential distribution in the apex, characteristic for a particular species. Confirmation could not be given to the concept that vacuoles arise as a series of local dilations in long extensions of the so called "smooth ER." The tonoplast and ER are distinguishable at the time of inception of a vacuole, although the tonoplast may arise from the ER. Rapid growth of a vacuole and/or fusion with other vacuoles may result in irregularly shaped prevacuoles. No vacuoles were observed to originate from cisternae of dictyosomes in the species studied.

The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism.

The rRNA methyltransferase ErmC' transfers methyl groups from S -adenosyl-l-methionine to atom N6 of an adenine base within the peptidyltransferase loop of 23 S rRNA, thus conferring antibiotic resistance against a number of macrolide antibiotics. The crystal structures of ErmC' and of its complexes with the cofactor S -adenosyl-l-methionine, the reaction product S-adenosyl-l-homocysteine and the methyltransferase inhibitor Sinefungin, respectively, show that the enzyme undergoes small conformational changes upon ligand binding. Overall, the ligand molecules bind to the protein in a similar mode as observed for other methyltransferases. Small differences between the binding of the amino acid parts of the different ligands are correlated with differences in their chemical structure. A model for the transition-state based on the atomic details of the active site is consistent with a one-step methyl-transfer mechanism and might serve as a first step towards the design of potent Erm inhibitors.

A novel, picomolar inhibitor of human immunodeficiency virus type 1 protease.

The design, synthesis, and molecular modeling studies of a novel series of azacyclic ureas, which are inhibitors of human immunodeficiency virus type 1 (HIV-1) protease that incorporate different ligands for the S1', S2, and S2' substrate-binding sites of HIV-1 protease are described. The synthesis of this series is highly flexible in the sense that the P1', P2, and P2' residues of the inhibitors can be changed independently. Molecular modeling studies on the phenyl ring of the P2 and P2' ligand suggested incorporation of hydrogen-bonding donor/acceptor groups at the 3' and 4-positions of the phenyl ring should increase binding potency. This led to the discovery of compound 7f (A-98881), which possesses high potency in the HIV-1 protease inhibition assay and the in vitro MT-4 cell culture assay (Ki = approximately 5 pM and EC50 = 0.002 microM). This compares well with the symmetrical cyclic urea 1 pioneered at DuPont Merck.

Strain differences in kinetic and thermal stability of two mouse brain tryptophan hydroxylase activities.

Levels of forebrain serotonin (5-HT), tryptophan, 5-hydroxyindoleacetic acid (5-HIAA) and hydroxylase cofactor (BH4) were comparable in two experimental mouse strains (A/J and C57Bl/6J) despite 2-3-fold differences in vitro in the relative activities of forebrain and midbrain tryptophan-5-monooxygenase (TPOH; EC 1.14.16.4). The enzyme activities did not differ with respect to Km for cofactor at saturating levels, but manifested different degrees of cooperativity with respect to cofactor when examined with BH4 concentrations within a physiological range. They differed also in the frequency and amplitude of kinetic variation around comparable mean velocity slopes across cofactor and time; in resistance to pre-incubation inactivation and responsiveness to its facilitation by calcium; in molecular weight heterogeneity as reflected in the distribution of molecular weight forms by gel diffusion chromatography; and in the number of peaks in power spectral analysis of kinetic variation patterns. Although the potential roles of small-molecule ligand and/or regulator proteins have not been ruled out, we hypothesize that differences in conformational stability underlie the differences in regulatory properties and make one enzyme activity more vulnerable to occlusive influences in vivo.

Induction and decline of hepatic cytochromes P4501A1 and 1A2 in rats exposed to hyperoxia are not paralleled by changes in glutathione S-transferase-alpha.

We investigated the effects of hyperoxia on the activities of hepatic ethoxyresorufin O-deethylase (EROD) (CYP1A1), methoxyresorufin O-demethylase (MROD) (CYP1A2), and glutathione transferase-alpha (GST-alpha), and the status of protein thiols (PSH) in male Sprague-Dawley rats. Twenty-four h of hyperoxia more than doubled EROD and MROD activities, which were increased 7.6- and 3.3-fold, respectively, after 48 h of hyperoxia. The increases in EROD and MROD activities were paralleled by enhanced CYP1A1/1A2 apoproteins contents, as detected by Western analysis. At 60 h of hyperoxia, by which time hyperoxic Sprague-Dawley rats display marked respiratory distress, pulmonary edema, and other markers of pulmonary dysfunction, the activities and levels of hepatic CYP1A1 and 1A2 had declined dramatically and returned to levels observed in air-breathing control animals. Hepatic activities of GST-alpha, as well as PSH status, were not altered significantly in the hyperoxic animals at any time point. The marked induction and subsequent decline of hepatic CYP1A1/1A2 activities in rats exposed to hyperoxia suggest that these enzymes may contribute to the mechanisms of injury and/or to adaptive responses to hyperoxic exposures in vivo.

Interpretation of congruent and incongruent affective communications in paranoid schizophrenia.

OBJECTIVES: It was hypothesized that people with paranoid schizophrenia would differ from depressed and normal participants in their interpretation of complex communications in which the affect conveyed verbally was either congruent or incongruent with the affect conveyed non-verbally. DESIGN: A 3 (group) x 3 (positive, negative, neutral facial expression) x 3 (positive, negative, neutral verbal content) experimental design was used. There were eight participants per group, and the paranoid and depressed groups comprised inpatients in an acute psychiatric facility for either their first or second psychiatric episode. METHODS: Participants, tested individually, were asked to interpret the affect conveyed by the various communications presented. RESULTS: All participants interpreted most of the communications in a similar way. Paranoid schizophrenia patients, however, differed in their interpretation of communications in which negative feelings were expressed verbally. In contrast to both the normal and depressed groups, the paranoid schizophrenia group interpreted these communications as virtually devoid of any affect whatsoever. CONCLUSIONS: Paranoid schizophrenia patients show an information-processing bias in response to communications involving both congruent and incongruent negative verbal content. It is not obvious why the bias observed would be specific to negative verbal messages and not extend to negative non-verbal messages. Replication and further study are required.

The effect of structural changes in a polyamine backbone on its DNA-binding properties.

A novel analog of spermine, compound 1, 2, 6 bis(N-3-aminopropylmethanamine)-1-methoxy-4-methylbenzene, has been prepared which shows DNA binding which is altered from spermine in its base pair selectivity. A fluorescence spectroscopic assay is used to compare the complexation properties of compound 1, spermine, spermidine, putrescine, and berenil binding to calf thymus DNA, poly d(AT), and poly d(GC). The results are interpreted in terms of a major groove binding motif and compared with literature values for DNA dissociation constants.

The case for a polyphyletic origin of mitochondria: morphological and molecular comparisons.

The comparative morphology and pigmentation of protists suggest that those with tubular mitochondrial cristae belong to a different lineage than those with lamellar cristae and that the evolutionary divergence might have been very early. We propose that the difference in cristal morphology is the result of separate origins of the mitochondria from endosymbionts related to the Rhodospirillaceae (purple nonsulfur bacteria) but differing in the morphology of their internal membranes. Comparisons of the cytochromes c of protists and the Rhodospirillaceae and of 16s rRNA T1 oligonucleotide catalogs in the Rhodospirillaceae do not contradict, and in fact provide support for, the idea. More extensive evidence may be lacking simply because cytochromes c have been studied in very few protists with tubular mitochondrial cristae.

Nuclear and cytoplasmic chloroplast mutants induced by chemical mutagens in Mimulus cardinalis: Genetics and ultrastructure.

The modes of inheritance of chemically induced chlorophyll-deficient phenotypes in Mimulus cardinalis reveal that the chloroplast is controlled by the genome and the plastome. Three of the chlorophyll-deficient mutants in M. cardinalis are inherited through nuclear recessive genes and two are inherited through plastome genes. One chlorophyll-deficient mutant was sterile and could not be analyzed genetically. Ultrastructural analysis of the six mutant types reveals that each possesses a unique defective chloroplast type(s) in comparison to the genotypically and phenotypically normal chloroplasts. Based on plastid ultrastructure it seems reasonable to assume that the mutations, genome and plastome, are non-allelic or at least significantly different forms of the same allele. The isolation of these types of mutants provide suitable material needed to study the effects of specific biochemical blocks and the elucidation of developmental pathways leading to chloroplast biogenesis. The mutants also provide valuable information concerning the interrelationship between the nucleic acid of the genome and the plastome.

The comparative aspects of cell wall chemistry in the green algae (Chlorophyta).

The origin of a cell wall was an event of fundamental importance in the evolution of plants. In the green algae, cell walls apparently had independent origins in at least three lines of evolution. In this paper, the components of the cell wall were determined and compared in four filamentous green algae representing the charophycean, chlorophycean and ulvacean evoluationary lines. The walls of all four have hydroxyproline-containing proteins which separate into five or six bands upon SDS gel electrophoresis. Variation does exist, with the charophyte possessing fast moving electrophoretic bands and high hydroxyproline content, the chlorophytes having intermediate movement of bands and lower hydroxyproline content, and the ulvaecean representative possessing slow moving bands and a very low, if not questionable, hydroxyproline and saccharide content. Qualitative and quantitative estimates of wall proteins and sugars have been determined and compared. A hypothetical scheme of cell wall evolution based on these data, those of previous analyses, and recent phylogenetic schemes is presented. Although sound conclusions cannot be made until more information is available, the scheme might help to emphasize the areas most in need of additional research.

Light-harvesting pigment-protein complex deficiency in Hosta (Liliaceae).

A yellow-leaved plastome mutant of Hosta (Hosta sieboldii Ingram complex, Liliaceae) known as 'Wogan Gold' lacks normal granal stacks, but has numerous stroma lamellae extending throughout the chloroplast. The chlorophyll a/b ratio is 0.76 in the mutant and 2.9 in wild type. The mutant contains a qualitatively normal pattern of other photosynthetic co-pigments. SDS-polyacrylamide gel electrophoresis showed a deficiency in the photosystem (PS) II light-harvesting complex. Since PS II is localized mainly in the granal region, the absence of the light-harvesting complex may explain the loss of granal stacking in this mutant.

Development of the cell wall in Tetraselmis: role of the Golgi apparatus and extracellular wall assembly.

The green algal flagellate, Tetraselmis, is a key transition organism in the phylogeny of green algae. It has been proposed that the cell wall of Tetraselmis arose evolutionarily from the fusion of scales and that this event secondarily caused the alteration of some cytoplasmic processes such as mitotic and cytokinetic mechanisms. Ultrastructural and developmental studies of the cell wall were performed with several strains of Tetraselmis. Two major wall types are reported. The wall of type 1 cells consists of a thick inner region covered by a layer of regularly repeating subunits of 26 nm, comparable to the subunits found in the median W2-W6 layer of Chlamydomonas. The more elaborate type 2 cell wall consists of a thick median wall layer, homologous to the type 1 inner wall, with additional inner and outer strata of hairs, grains and scales. Development of the cell wall begins in the endomembrane system, particularly the Golgi apparatus, where fibrillar tufts and electron-dense droplets are synthesized, modified and transported to the outside. Here, the tufts and droplets are displaced around the protoplast and assemble in several steps to yield the intact wall. Edge-growth assembly of the wall occurs here synchronously with cytoplasmic developments to yield the characteristic anterior flagellar pit. Models explaining various aspects of this development are discussed. When released from the cell, the wall subunits are not completely comparable to stellate scales, but appear to correspond to developmental stages of scales in green flagellates possessing body scales.

Therapeutic strategies distinguish community based primary care physicians from rheumatologists in the management of osteoarthritis.

Using an osteoarthritis (OA) case study, we described the drug therapy that primary care physicians prescribe for uncomplicated OA of the hip, and for OA complicated by a history of gastropathy or renal insufficiency. To produce "gold standard" criteria against which to interpret previous results, the same instrument was administered to 126 rheumatologists selected at random from the membership of the American College of Rheumatology. Virtually all rheumatologists prescribed nonsteroidal antiinflammatory drugs (NSAID); 76% specified doses large enough to have significant antiinflammatory effects. In contrast, 65% of the primary care physicians recommended NSAID therapy in a suboptimal antiinflammatory dose (p = 0.055 for the rheumatologist-primary care physician difference). For OA complicated by a history of either gastropathy or renal insufficiency, rheumatologists were more likely than primary care physicians to adopt a therapeutic strategy that did not inhibit prostaglandin synthesis (p < 0.001 for both). Differences also were noted in the ancillary therapies employed by the 2 groups for managing uncomplicated OA. Educational interactions between rheumatologists and primary care physicians could benefit by recognition of the differing perspectives on NSAID dosing, the avoidance of NSAID induced side effects, and ancillary therapies that appear to differentiate subspecialists and generalists.

Discovery of a new cyclooxygenase-2 lead compound through 3-D database searching and combinatorial chemistry.

Using a combination of computational and combinatorial chemistry methodologies, a phenothiazine compound was discovered that is a selective inhibitor of cyclooxygenase-2 and serves as a lead compound for a potentially novel series of anti-inflammatory compounds.

Molecular basis for potentiation of bleomycin-mediated degradation of DNA by polyamines. Experimental and molecular mechanical studies.

The bleomycin-mediated degradation of DNA is stimulated (amplified) by certain DNA binding compounds, such as polyamines, that distort the double helix. Computer modelling studies suggest that putrescine (1), spermidine (2), and spermine (3) bind preferentially on the floor of the major groove of (dGdC)5.(dGdC)5. This interaction results in a bend of the oligomer helix toward the major groove and enlargement of the minor groove, both effects being in the order 1 less than 2 less than 3. These polyamine-induced distortions, as obtained from theoretical studies, parallel the experimental values of the amplification activities of 1-3 in the bleomycin-mediated degradation of poly(dGdC).poly(dGdC). The amplification mechanism of non-competitive binding of amplifier molecules in the major groove, and bleomycin in the minor groove, is proposed. It is suggested that the amplifier-induced conformational changes of the DNA helix increase affinity of the activated bleomycin complex toward the DNA minor groove and, consequently, result in an increased efficiency of the bleomycin-mediated degradation of the helix.

Comparison of Staphylococcus aureus and Escherichia coli infusion in conscious rats.

The purpose of this study was to compare the pathophysiology of bacteremia produced by intravenous infusion of either a Gram-positive (Staphylococcus aureus) or a Gram-negative (Escherichia coli) organism. Conscious, unrestrained, instrumented rats received S. aureus, E. coli, or sterile saline over 120 min, followed by a 240-min monitoring period. The infusates produced 90% (S. aureus,) 80% (E. coli), and 0% (saline) mortality at 24 hr. Neither bacterial group produced hypotension during the entire 360-min study period. E. coli produced early tachycardia and increased glucose, followed by decreased stroke volume and increased lactate and pO2. S. aureus caused early tachycardia followed by decreased pH, stroke volume, and cardiac output and increased lactate and systemic vascular resistance. Respiration rate and central venous pressure were not affected by either bacterial infusion. Compared to E. coli, S. aureus produced decreased pH, glucose, pO2, heart rate, and cardiac output and increased lactate, hematocrit, pCO2, and systemic vascular resistance. These data document quantitative differences in the acute response of the conscious rat to bacteremia caused by these isolates of E. coli and S. aureus.