Characterization of a thermo-osmotic gas transport mechanism in Alnus glutinosa (L.) Gaertn.

Summary A gas transport system based upon the physico-chemical effect of thermo-osmosis of gases in described for the black alder, Alnus glutinosa (L.) Gaertn. Air is transported through the alder's stem to the roots, thus improving O₂ supply to respiring tissues of the root system. The gas transport system is investigated by means of a tracer gas technique (11% ethane in air, v/v). Gas transport depends on any source of radiant heat generating a temperature difference between the tree's stems and the atmosphere. The amount of gas transported in leafless trees is four times higher than the amount of gas reaching the roots by gas diffusion. Two-thirds of the gas is transported in the wood, only one-third in the bark. Intercellular spaces inside the porous lenticels of the bark are responsible for this kind of gas transport. Their diameters are estimated by the effusion rates of different tracer gases to be in the range of 1 m.     

Structural characterization of tatiopterin, a novel pterin isolated from Methanogenium tationis

Cofactor extracts of Methanogenium tationis were screened for the presence of pterin-derivatives. Methanopterin, sarcinapterin and 7-methylpterin were absent, while 2-amino-4-hydroxy-pteridine and another blue fluorescent compound with a pterin spectrum were detected. The latter pterin was purified by ion exchange and reversed-phase column chromatography. The structure of this compound was elucidated by combining spectrophotometry, amino acid analysis and 1H-NMR spectroscopy. The pterin, which we named tatiopterin, was identified as an aspartyl derivative of sarcinapterin with a 7-proton instead of a 7-methyl group in the pterin moiety. The IUPAC name is: N-[-1'-(2'-amino-4'-hydroxy-7'-proton-6'-pteridinyl)ethyl]-4- [2',3',4',5'-tetrahydroxypent-1'-yl(5'----1')O-alpha- ribofuranosyl-5'-phosphoric acid]aniline, in which the phosphate group is esterified with alpha-hydroxyglutarylglutamylaspartic acid.     

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity

The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.     

Developmental potential of biopsied mouse blastocysts

The health of a preimplantation embryo can be diagnosed in one or more cells biopsied from the conceptus. Here, we tried to evaluate the impact of biopsy of some trophectoderm cells from hatching mouse blastocysts on their further in vitro implantation and early egg cylinder formation. Of 374 blastocysts evaluated 112 hours after hCG, 34% initiated hatching with a small number of mural, polar, or intermediate trophectoderm cells. Half of 59 embryos that underwent induction of hatching by zona puncturing herniated some cells through this opening. After removal of cells with a glass microneedle from spontaneously hatching blastocysts, viability assessed by vital FDA staining was impaired, as well as the in vitro zona pellucida shedding and implantation. When polar trophectoderm cells were biopsied, a significantly lower number of embryos reached the egg cylinder stage.     

Developmental change in follicular cell-enhanced amino acid uptake into mouse oocytes that depends on intact gap junctions and transport system Gly

Uptake of L-alanine, L-lysine, and choline into both preantral and antral mouse oocytes was enhanced by follicular cells. Follicular cells also enhanced glycine uptake into oocytes at the preantral stage of development, but no effect of these cells was observed at the antral stage. Glycine uptake was predominantly Na+ dependent and inhibited almost completely by 10 mM sarcosine, moderately by proline and its analog pipecolate, and poorly or not at all by other amino acids. By these criteria, glycine transport was mainly via system Gly in follicular cells and the oolemma at both the preantral and antral stages. Moreover, an increase in glycine transport via the oolemma between the preantral and antral stages was more than threefold larger than was the increase in transport of alanine or lysine. This relatively large increase in glycine-specific transport in the oolemma appears to obscure the ability of follicular cells to enhance glycine uptake into antral oocytes. In contrast to other amino acids, leucine uptake into oocytes was not enhanced by follicular cells unless 14 other amino acids were also present at their concentrations in mouse serum. An inhibitor of gap junctional communication, 18-alpha-glycyrrhetinic acid, abolished follicular cell-enhanced uptake of glycine and choline into preantral oocytes. Therefore, the extent to which follicular cells enhance uptake of a particular amino acid into oocytes depends on at least three physiologically important variables. Namely, enhancement may depend on the stage of follicular development, the presence of other amino acids in the environment, and gap junctional communication.     

5S rRNA sequences of representatives of the genera Chlorobium, Prosthecochloris, Thermomicrobium, Cytophaga, Flavobacterium, Flexibacter and Saprospira and a discussion of the evolution of eubacteria in general.

5S rRNA sequences were determined for the green sulphur bacteria Chlorobium limicola, Chlorobium phaeobacteroides and Prosthecochloris aestuarii, for Thermomicrobium roseum, which is a relative of the green non-sulphur bacteria, and for Cytophaga aquatilis, Cytophaga heparina, Cytophaga johnsonae, Flavobacterium breve, Flexibacter sp. and Saprospira grandis, organisms allotted to the phylum 'Bacteroides-Cytophaga-Flavobacterium' and relatives as determined by 16S rRNA analyses. By using a clustering algorithm a dendrogram was constructed from these sequences and from all other known eubacterial 5S RNA sequences. The dendrogram showed differences, as well as similarities, with respect to results obtained by 16S RNA analyses. The 5S RNA sequences of green sulphur bacteria were closely related to one another, and to a cluster containing 5S RNA sequences from Bacteroides and its relatives, including Cytophaga aquatilis. 5S RNA sequences of all other representatives of the 'Bacteroides-Cytophaga-Flavobacterium' phylum as distinguished by 16S RNA analysis failed to group with Bacteroides and related clusters. On the basis of 5S RNA sequences, Thermomicrobium roseum clustered with Chloroflexus aurantiacus, as was expected from 16S RNA analysis.     

Direct and indirect evidence for polypeptide absorption by the teleost gastrointestinal tract

The ability of the gastrointestinal tract of chinook salmon, Oncorhynchus tshawytscha , to absorb polypeptides in vivo was investigated by reference to the appearance of orally administered adrenocorticotropic hormone (ACTH) within the blood of fish previously treated with dexamethasone (3μg g^−l body weight) in order to suppress endogenous ACTH secretion. Further, since cortisol presence within plasma is dependent upon the availability of ACTH, dexamethasone blockade of endogenous ACTH secretion, in conjunction with subsequent measurements of plasma cortisol levels, provides a means by which biological patency of absorbed exogenous ACTH may be demonstrated. Levels of ACTH and cortisol in plasma of dexamethasone-treated salmon were therefore measured for a period of 360 min immediately following oral intubation of ACTH (15μg g⁻¹ body weight). Peak plasma presence of ACTH-like immunoreactivity (676.6 ± 33.6 pg ml⁻¹ plasma) and cortisol (227.1 ± 29.0 ng ml⁻¹ plasma) were recorded 120 min after ACTH administration. Results from the experimental groups were compared to those of 15 control treatments. Since administration of ACTH to chinook salmon elicited a consistent and significant elevation in not only plasma ACTH but also cortisol presence, it is contended that the salmonid gut expresses an ability to absorb polypeptides of dietary origin. The significance of these findings with respect to the oral administration of biologically active peptides and proteins to fish of importance to aquaculture is discussed.     

Quantitative histochemical analysis of glucose-6-phosphatase activity in rat liver using an optimized cerium-diaminobenzidine method

We have optimized a cerium-diaminobenzidine-based method for histochemical analysis of glucose-6-phosphatase (G6Pase) activity and have determined quantitative data on the zonal distribution pattern in the liver acinus of fasted male rats. In the cerium-diaminobenzidine technique, cerium instead of lead ions is used as capturing reagent for the enzymatically liberated phosphate. For light microscopy, the primary reaction product, cerium phosphate, is then visualized by conversion into cerium perhydroxide using hydrogen peroxide and subsequent oxidative polymerization of diaminobenzidine to diaminobenzidine brown as the final reaction product. Variation of the substrate (glucose-6-phosphate) concentration in the incubation medium yielded in periportal zones a KM value of 2.3 +/- 0.7 mM and a Vmax value of 0.96 +/- 0.18 (expressed as mean integrated absorbance). In perivenous zones a KM value of 1.1 +/- 0.4 mM and a Vmax value of 0.51 +/- 0.08 were calculated. The cytophotometric analysis performed in this study demonstrated for the first time that a functional difference of G6Pase, the key enzyme for gluconeogenesis, exists in the periportal and perivenous zones of the liver acinus. Periportal zones contain twice as many enzyme molecules (high Vmax) as perivenous zones, but the affinity for the substrate is twice as low. This may have important implications for the concept of metabolic zonation of the liver and also for glucose homeostasis in the blood.     

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.     

IL-1 alpha or tumor necrosis factor-alpha stimulate release of three NAP-1/IL-8-related neutrophil chemotactic proteins in human dermal fibroblasts

Human dermal fibroblasts in culture secrete three protein-like neutrophil chemotactic factors, when stimulated either with human rIL-1 alpha or IL-1 beta; not, however, after incubation with LPS. These three fibroblast-derived neutrophil-activating proteins (FINAP) could be purified by subsequently performed reversed phase and size exclusion HPLC. By high resolution SDS-PAGE, all the proteins were shown to migrate with an Mr of 6,700 (alpha-FINAP), 3,600 (beta-FINAP), and 5,300 (gamma-FINAP). All purified cytokine preparations were found to be chemotactic for human neutrophils. In addition, all FINAP induced release of lysosomal enzymes in neutrophils. Deactivation of chemotaxin-elicitable enzyme release showed cross-desensitization of all FINAP with NAP-1/IL-8. Western blot analysis of alpha-FINAP by using mAb against neutrophil-activating protein (NAP)-1/IL-8 reveals immunologic cross-reactivity with NAP-1/IL-8. By amino-terminal amino acid sequence analysis alpha-FINAP could be identified as the 77-residue extended form of NAP-1/IL-8 containing the 79-residue form as a minor contaminant. Whereas beta-FINAP has been found to be a truncation product of alpha-FINAP, gamma-FINAP shows identity with authentic melanoma growth stimulatory activity with respect to retention time upon reversed phase HPLC, high resolution SDS-PAGE, and biologic properties, as well as amino-terminal amino acid sequence. These data show that human dermal fibroblasts may actively participate in inflammatory reactions by secretion of proinflammatory cytokines.