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PubMed1992

Cell culture of Taxus as a source of the antineoplastic drug taxol and related taxanes.

Callus cultures of Taxus cuspidata and Taxus canadensis were induced using different tissue explants including green and red arils, seed contents, young stems and needles. Callus derived from stem segments displayed the best growth in defined media. The culture medium was supplemented with reducing agents and phenolic-binding compounds to inhibit callus darkening and subsequent growth reduction. T. cuspidata explant growth was affected by different concentrations and ratios of 2,4-D and kinetin. Callus tissues of T. cuspidata were extracted for taxol after 2 months in culture and analysed by HPLC. The presence of taxol (0.020 +/- 0.005% of the extracted dry weight) was indicated based on retention time, U.V. spectra, peak purity as assessed by photo-diode array spectroscopy and compared with an authentic taxol standard, as well as by 1H-NMR analysis. Suspension cultures of T. cuspidata were established from the callus cultures, subsequently immobilized onto glass fiber mats, and maintained as immobilized cultures for 6 months. The immobilized cell cultures also produced taxol at levels up to 0.012 +/- 0.007% of the extracted dry weight.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1992

Cellularity and fibrin mesh properties as a basis for ultrasonic tissue characterization of blood clots and thrombi.

This in vitro study was designed to evaluate the ability of ultrasonic tissue characterization (UTC) based on power spectrum analysis of backscattered radio-frequency echo signals to distinguish two prominent variables of thrombi: cellularity (primarily red cell content) and fibrin-mesh density. Six types of clots simulating thrombus components were prepared by varying red-cell and platelet concentrations and shear forces during clotting. Data were acquired with a linear-array transducer, digitized, and analyzed in terms of slope and intercept parameters obtained from normalized power spectra of radio-frequency echo signals. Increased cellularity and fibrin-mesh density both produced lower slope and higher intercept values, which permitted statistically significant discrimination of cellularity and mesh density in the six types of clots analyzed. Shearing forces and (to a lesser degree) platelet concentrations increased fibrin-mesh density. This study suggests that UTC based upon the power spectrum of echo signals may be used to detect and follow compositional differences that have clinical relevance in the diagnosis and follow-up of thrombi.

Blood Coagulation↗
PubMed2026

Identification and characterization of G protein-coupled receptors in the nocturnal halictid bee Megalopta genalis.

G protein-coupled receptors (GPCRs) are one of the largest families of membrane proteins in insects, regulating vision, neural signal transduction, and various physiological behaviors. Megalopta genalis exhibits a unique facultatively eusocial lifestyle and possesses adaptations for nocturnal activity; however, its GPCR family has not yet been systematically characterized. In this study, we performed genome-wide identification, phylogenetic analysis, and expression profiling of GPCRs in M. genalis by integrating genomic annotation and transcriptomic analysis. The results showed that a total of 99 GPCRs were identified in the genome of M. genalis, which were classified into four major families. Here, we show that M. genalis has undergone lineage-specific GPCR repertoire remodeling, marked by the expansion of novel orphan receptors and the systematic loss of multiple receptor subtypes, such as the neuropeptide receptors MIP-R and NPFR. Moreover, opsins have formed a diverse array of combinations and non-GPCR odorant receptors have undergone significant expansion via tandem duplication. Together, these features may represent part of the molecular repertoire associated with the adaptation of M. genalis to a nocturnal lifestyle. Furthermore, transcriptomic analysis revealed distinct spatiotemporal expression divergence within each of the Mth/Mthl and Fz GPCR families, suggesting functional specialization across development and adult tissues. This study provides the first systematic identification and initial functional characterization of GPCRs in M. genalis, revealing an evolutionary pattern characterized by the coexistence of contraction and expansion within the GPCR family. These findings lay a foundation for further studies aimed at elucidating the roles of these GPCRs in regulating M. genalis physiology and behavior.

Animals↗
PubMed1989

Expression and tissue-specific assembly of human butyrylcholine esterase in microinjected Xenopus laevis oocytes.

Cholinesterases represent a ubiquitous, polymorphic family of acetylcholine hydrolyzing enzymes. The multileveled tissue-specific heterogeneity which characterizes these enzymes makes the cholinesterases an appropriate model for studying the mechanisms involved in regulating divergent pathways in protein biogenesis. For this purpose, a cDNA coding for human butyrylcholine esterase (BuChE) was subcloned into the SP 6 transcription vector. Synthetic mRNA transcribed from this construct was microninjected into Xenopus laevis oocytes alone, and in conjunction with poly(A)+ RNAs extracted from human brain or muscle. Injected BuChE-mRNA induced the biosynthesis of a protein exhibiting the catalytic activity, substrate specificity, and sensitivity to selective inhibitors characteristic of native human serum BuChE, and clearly distinct from the related enzyme acetylcholinesterase (AChE). The nascent BuChE was reproducibly distributed into low salt-soluble and detergent-extractable pools. Sucrose gradient analysis demonstrated that the nascent human enzyme was capable of limited subunit assembly, appearing as functional dimeric molecules in both of these fractions. Co-injection with brain or muscle-derived mRNAs facilitated higher order oligomeric assembly. Co-injected brain mRNA induced the appearance of tetramers while co-injected muscle mRNA induced the appearance of an array of heavy molecular forms, including a heavy 16 S form. These results indicate that the molecular determinants which distinguish BuChE from AChE are inherent to its primary amino acid sequence and that additional, tissue-specific protein(s) are involved in the modulation of subunit assembly within particular biological milieues.

Animals↗
PubMed1990

A tandem duplication causes the Kn1-O allele of Knotted, a dominant morphological mutant of maize.

Molecular and genetic techniques are used to define Kn1-O, a mutation which interferes with the normal differentiation of vascular tissue in leaves. Sequences associated with a previously cloned allele, Kn1-2F11, were used as hybridization probes in a Southern analysis of Kn1-O. By this analysis, Kn1-O lacks the Ds2 transposable element that causes Kn1-2F11 but instead is associated with a sequence duplication. Sequence and restriction analysis of genomic clones show that the duplication consists of a tandem array of two 17-kb repeats. Analysis of Kn1-O derivatives indicates that the duplication itself conditions the mutant phenotype; a severely knotted line, Kn1-Ox, has gained a repeat unit to form a triplication, whereas normal derivatives have either lost a repeat unit or sustained insertions that disrupt the tandem duplication. These insertions map near the central junction of the tandem duplication, suggesting that the mutant phenotype results from the novel juxtaposition of sequences. We discuss models that relate the tandem duplication of sequences to altered gene expression.

Alleles↗
PubMed1982

Effects of matrix macromolecules on chondrocyte gene expression: synthesis of a low molecular weight collagen species by cells cultured within collagen gels.

Chick-embryo sternal chondrocytes have been cultured within three-dimensional collagen gels as part of a study concerned with the effects of extracellular matrix macromolecules on chondrocyte gene expression. Data are presented indicating that chondrocytes cultured within such a collagenous environment synthesize significantly more of an hitherto unidentified, low molecular weight collagen species than do cells grown on plastic tissue-culture dishes in the conventional manner. This low molecular weight collagen species contains noncollagenous domains (as indicated by its decreased molecular size after mild pepsin digestion), is distinct from the known collagen types (as judged by CNBr peptide analysis), and forms part of the insoluble collagenous matrix produced by the chondrocytes. Cells growing within the gel tend to form colonies consisting of a linear array of cells reminiscent of the cellular organization in growth cartilage.

Animals↗
PubMed1991

Function of intraventricular human mesencephalic xenografts in immunosuppressed rats: an electrophysiological and neurochemical analysis.

Solid pieces of human fetal mesencephalic tissue were grafted to the lateral ventricle adjacent to dopamine-depleted striata of rats immunosuppressed with cyclosporin A. Apomorphine-induced rotations were performed before and at monthly intervals after grafting. Reductions in rotations were seen at 2 months post-grafting and these reductions progressively increased. Spontaneously active dopaminergic cells were found within the grafts using extracellular single-unit recording techniques. Recordings of striatal cells ipsilateral to the graft revealed "normal" firing rates compared to those of neurons in the control striatum. In response to the local application of the dopamine antagonist cis-flupenthixol, both the dopaminergic and striatal neurons showed dose-dependent excitations. Potassium-evoked releases of electroactive species ipsilateral to the fetal human graft, measured using high-speed in vivo electrochemistry, revealed response amplitudes that were similar to control striatum when an electrode was placed adjacent to the graft; distal to the graft the responses showed smaller amplitudes but prolonged time courses. Much greater levels of dopamine and serotonin were detected in the grafts, compared to in normal rat substantia nigra, as measured with HPLC coupled to a 16-channel electrochemical array detector. Immunocytochemical studies using antibodies against tyrosine hydroxylase (TH), revealed not only TH-positive cells within the graft, but also a few positive neurons that migrated into the host striatum. Numerous TH-immunoreactive fibers penetrated into striatum and reinnervated its total volume. Taken together, these data suggest that intraventricular graft placement may be a highly efficacious technique for studying fetal brain tissues in terms of maturation, reinnervation, and function.

Analysis of Variance↗
PubMed1976

Analogue solution for electrical capacity of membrane-covered cubes in cubic array at high concentration.

Earlier measurements have shown that the equations derived for the resistance of suspension of spheres apply up to a volume concentration of 100% for close-packing forms. They have also shown, by a resistance-capacitance analogue, that they apply for the capacity of a close-packing of membrane-covered square cylinders in square array, approximating muscle and nerve. The present work is an extension of the two-dimensional to a three-dimensional array of membrane-covered cubes in a cubic array. It is found by measurements of a three-dimensional electrolytic analogue that the capacitance is indeed expressed by an extension of the analytic solution at low concentrations up to 100% volume concentration of the membrane-covered form, such as epithelial tissues. There is thus at least one example each of two-and three-dimensional forms which conform to the low concentration analysis up to 100% volume and so give a basis for the extensions to other and more complicated forms to complete a survey to work begun by Fricke in 1923.

Cell Physiological Phenomena↗
PubMed1990

Aggressive jejunal lymphoma of large granular lymphocytes. Immunohistochemical, ultrastructural, molecular, and DNA content analysis.

An unusual large cell lymphoma of the proximal jejunum with large granular lymphocyte (LGL) morphologic characteristics and T-helper/inducer cell phenotype is described. Although the cells strongly expressed Leu-7 (HNK-1), studies with antibodies directed against the more specific natural killer (NK) antigens, CD16 (Leu-11) and Leu-19, were negative. Ultrastructural analysis of the neoplastic cells demonstrated substantial numbers of electron-dense granules and rare parallel tubular arrays. Clonal rearrangement of the T-cell receptor beta chain gene and germline configuration of the immunoglobulin heavy chain gene confirmed the T-cell origin of the neoplastic cells. This lymphoma pursued an aggressive clinical course, with rapid dissemination to the lungs and central nervous system. DNA content analysis indicated that a similar DNA aneuploid population was present in the jejunal primary and lung tissue at recurrence. There was no evidence of nodal, peripheral blood, splenic, or bone marrow involvement. Morphologic and functional similarities between the lymphoid tissues of the gastrointestinal tract and lung have previously prompted a classification of the immune system into distinct peripheral somatic and mucosal components. Based on the distribution and migratory properties of the tumor cells in this case, the authors propose that this lymphoma arose from a minor mucosa-associated LGL subset that may be unrelated to circulating LGLs. In addition, these observations emphasize that prominent granulated cytomorphologic features may be seen in neoplastic disorders with the T-helper/inducer phenotype, as well as in the more widely recognized lymphoproliferative disorders of NK and cytotoxic/suppressor cell types.

Aged↗
PubMed1990

Analysis of purine nucleotides in muscle tissue by HPLC.

Optimal conditions for simultaneous analysis of the purine nucleotides adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine, adenosine, hypoxanthine, xanthine and uric acid in muscle samples by high-performance liquid chromatography (HPLC) were evaluated. A neutralized perchloric acid extract of freeze-dried human or rat skeletal muscle tissue was injected on to a reversed-phase silica column and eluted by a gradient composed of ammonium dihydrogen phosphate buffer and methanol. Good resolution for all the nucleotides was achieved within a retention time of about 20 min. Linearity for each of the nucleotides within the concentration intervals obtained in the samples was demonstrated. Purity of each peak was verified by use of the photodiode array technique. Reproducibility for biological samples with variation coefficients below 3.6% for ATP, ADP, AMP, inosine and hypoxanthine and 6.7% for IMP was obtained. The stability of the compounds after extraction was specifically addressed. Storing of frozen extracts at -20 degrees C for 24 h gave acceptable values, while storage for 7 days cannot be recommended. Storage of unfrozen extracts at 4 degrees C was acceptable for (up to) 7 h. This technique provides a sensitive, convenient and reliable method for simultaneous analysis of a large number of purine nucleotides in small skeletal muscle biopsies, provided that certain precautions are taken with respect to the instability of these metabolites.

Animals↗
PubMed1979

Intracellular calcium deposition in brain following electrical stimulation.

The effects of electrical stimulation of the cat cerebral cortex have been evaluated by light and electron microscopy following a wide variety of stimulation parameters (QD/ph of 10 - 300 muC/cm2/ph). Platinum or rhodium disc electrode arrays were bilaterally implanted subdurally on the parietal cortex and subjected to 36-hour stimulations (9 hrs./day for 4 days). Prominent among the degenerative changes shown by electron microscopy were dense crystalline inclusions that were identified as calcium hydroxyapatite (CHA) crystals by electron diffraction and energy dispersive X-ray analysis. The appearance of intracellular calcification generally paralleled the onset of other degenerative changes in stimulated tissue, including gliosis, mitochondrial swelling, lipid inclusions, degenerating cells, neuronal loss, and phagocytic activity. A preferential deposition of calcium was noted in mitochondria of several cell types and in postsynaptic dendrites. The mechanism of the apparently electroresponsive calcium deposition is obscure; however, a plausible explanation is that increased cyclic AMP levels, known to occur with electrical stimulation of nervous tissue, result in enhanced calcium plasmalemmal permeability.

Animals↗
PubMedDate not supplied

A new look into the microscope: proliferation and regression of myocardial capillaries.

The key to understanding the mechanics of capillary proliferation probably lies in comprehension of the normal capillary growth during the early postnatal period when most of the terminal vessels in mammalian hearts are being formed. Similarly, stimulated cardiac growth is accompanied mainly, but not exclusively, by capillary proliferation if it takes place in the young, growing organism. The opposite situation, decreased capillarization of the heart, is commonly found in several experimental and clinical situations. The cause of lower capillary density is more often due to the growth of capillaries which is slower than the rate of growth of the remaining tissue, rather than to a disappearance of the existing capillaries. This brief review is followed by the discussion of how to evaluate the myocardial capillary supply based on the theoretical analysis of the effect of alterations in capillary surface density on myocardial PO2. The final part deals with confusing teminology in the field, with the following terms discussed in depth: diffusion distance, maximal diffusion distance and hexagonal array.

Animals↗
PubMed1987

Intercellular junctions between fibroblasts in connective tissues of the eye of macaque monkeys. A thin section and freeze fracture analysis.

Thin-section electron microscopy and freeze fracture were used in the analysis of the intercellular junctions between fibroblasts in connective tissues of the eye of Macaca mulatta, M. fascicularis, and M. arctoides. Fibroblasts located in the subconjunctival loose connective tissue, anterior sclera, scleral spur, iris stroma, ciliary body stroma, and posterior choroid were jointed by three kinds of junctions. Gap junctions were of different sizes and frequently composed of a small number of connexons organized in polygonal aggregates or linear arrays. Tight junctions were represented by isolated strands and never composed a continuous belt around the cells. Intermediate junctions were seen in thin sections but did not have any representation in the interior of the plasma membrane. It remains to be established whether, as is the case in other tissues, pathologic conditions of the eye are accompanied by some changes in the morphology and distribution of intercellular junctions between fibroblasts.

Animals↗
PubMed1989

Effects of tumor necrosis factor and epidermal growth factor on cell morphology, cell surface receptors, and the production of tissue inhibitor of metalloproteinases and IL-6 in human microvascular endothelial cells.

The effect of human TNF on cultured human microvascular endothelial (HME) cells was examined. Incubation with TNF alone transformed the morphology of HME cells from a cobblestone-like appearance into a disordered array of criss-crossed, elongated, spindle-shaped cells. Coadministration of epidermal growth factor (EGF) and TNF caused even more dramatic morphologic changes than TNF alone. Addition of basic fibroblast growth factor or insulin-like growth factor-I showed rather weak effects on cell morphology than EGF. Cell growth of HME cells was stimulated up to two-fold by TNF whereas addition of EGF additively enhanced the growth rate. Treatment of HME cells with 10 ng/ml EGF increased the binding of 125I-TNF, and Scatchard analysis showed increased TNF-R number by EGF treatment. Cellular response to TNF in the absence or presence of EGF was assessed by analyzing SDS-PAGE patterns of secreted proteins from HME cells. TNF enhanced the secretion of a protein of molecular weight 25,000 Da (25 kDa) which was found to be IL-6. In contrast, secretion of a polypeptide of 29 kDa was significantly increased when HME cells were treated with EGF, but not with TNF. Coadministration of TNF and EGF synergistically increased the secretion of the 29-kDa protein. This 29-kDa protein was found to be tissue inhibitor of metalloproteinases when assayed with antitissue inhibitor of metalloproteinases antibody. TNF and EGF also enhanced secretion of collagenase with Mr of approximately 55 kDa. Increased steady state levels of the inhibitor mRNA were observed when HME cells were treated with EGF, and coadministration of TNF further increased the levels. The morphologic transformation of HME cells by TNF and/or EGF is discussed in relation to their expression of the secreted proteins.

Cell Division↗
PubMed1989

Buffering of muscle tissue PO2 levels by the superposition of the oxygen field from many capillaries.

High resolution measurement in both exercised skeletal and cardiac tissue made radially outward from capillaries and longitudinally parallel to capillaries by Gayeski and Honig (1986, 1986a,b) and Honig and Gayeski (1987) indicate shallow variation of tissue PO2 and the absence of strong causal relation between the PO2 at a point and the proximity of that point to the nearest active capillary. Proposed as a model for the analysis of this tissue PO2 distribution, so contrary to the expectations of Krogh type models, are a class of multicellular tissue cylinder models. Each cylinder is penetrated by many parallel capillaries. In order to better represent the natural irregularities of the skeletal and cardiac tissue both with regard to radial placement and the stagger of the capillary inlets, the following types of models both with and without yoglobin are examined: regular square arrays where the capillary PO2 levels are random, uniform capillary PO2 levels but random capillary positions, and those with both the capillary PO2 levels and the positions are random. The results of the model calculations show that the superposition of the oxygen diffusion fields of all the capillaries produce a tissue PO2 distribution with the properties: (1) lower tissue PO2 levels than those predicted by Krogh theory, (2) significant non-local contributions to the PO2 at a point in the tissue which greatly reduces this correlation between PO2 at a point and its proximity to an active capillary, (3) shallow transcellular PO2 variation.

Animals↗
PubMed1991

A parsley 4CL-1 promoter fragment specifies complex expression patterns in transgenic tobacco.

The 4CL-1 gene is one of two highly homologous parsley genes encoding 4-coumarate:coenzyme A ligase, a key enzyme of general phenylpropanoid metabolism. Expression of these genes is essential for the biosynthesis of both defense-related and developmentally required phenylpropanoid derivatives. We examined the developmental regulation of the 4CL-1 promoter by analyzing the expression of 4CL-1-beta-glucuronidase fusions in transgenic tobacco plants. A 597-base pair 4CL-1 promoter fragment specified histochemically detectable expression in a complex array of vegetative and floral tissues and cell types. The activity of a series of 5' deleted promoter fragments was analyzed in parsley protoplasts and transgenic tobacco plants. Deletions past -210 base pairs led to a drastic decline in beta-glucuronidase activity in protoplasts and loss of tissue-specific expression in transgenic tobacco. These results were put into the context of potential protein-DNA interactions by in vivo footprint analysis of the 4CL-1 promoter in parsley cells. Loss of promoter activity in parsley protoplasts and transgenic tobacco was correlated with the deletion or disruption of the distal portion of a large (100-base pair) footprinted region within the first 200 base pairs of the 4CL-1 promoter.

Base Composition↗
PubMed1992

Apparent fusion of basement membranes in colorectal carcinoma.

Previous studies on colorectal carcinomas indicate that consistent differences in epithelial basement membrane (EBM) integrity are present between the tumour centre and periphery. We report that within the tumour centre, EBM staining between back-to-back (BTB) neoplastic glands (i.e., adjacent glands in direct contact with no intervening connective tissue) generally follows a pattern different from that of EBM staining at the tumour:stromal interface (TSI). Such distinctions are important, since the factors responsible for EBM deficiencies may vary with intra-tumoural location, as may the prognostic significance of these deficiencies. Analysis of paraffin sections from 130 colorectal carcinoma cases showed that EBM staining between BTB glands is generally weaker and more discontinuous than at the TSI, sometimes appearing as a linear array of immunostained granules on high-resolution light microscopy. By double-labelling immunofluorescence analysis of cryostat sections from 30 cases, a decrease in type-IV collagen:laminin staining intensity ratio was found between BTB glands. Hence, the composition of EBM between BTB glands appears to be abnormal. As much recent evidence indicates that epithelial:mesenchymal interactions play an essential role in EBM formation, the demonstration of immunostained EBM fragments between BTB glands requires an explanation: We suggest that the synthesis of EBM between BTB glands involved previously intervening stromal (mesenchymal) cells, and that EBM fusion and dissolution occur between BTB glands following the displacement of these cells.

Basement Membrane↗
PubMed1986

Isolation of JC virus capsomer-like structures from progressive multifocal leukoencephalopathy brain.

Brain tissue from a patient with progressive multifocal leukoencephalopathy (PML) was analyzed by molecular biological and electron-microscopic techniques. Viral DNA was isolated directly from brain tissue, cloned into a plasmid vector, and subjected to restriction endonuclease analysis. The pattern of restriction fragments identified by gel electrophoresis was almost indistinguishable from that of prototype JC virus. By this procedure the etiologic agent of PML in this patient was identified without the isolation of infectious virus. After centrifugal clarification of brain homogenates, high speed centrifugal pellets were studied by electron microscopy. Large numbers of 9-nm polygonal particles, sometimes in paracrystalline arrays, were observed. It was thought likely that these particles were capsomer subunits of 41-43 nm JC virus virions. That the particles were capsomers was supported by negative stain electron microscopy, including reconstruction studies with simian virus 40.

Aged↗