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PubMed1990

Elastin in alcoholic liver disease. An immunohistochemical and immunoelectron microscopic study.

Increased elastic stained material has been described in fibrotic and cirrhotic liver processes. The aim of this work was to follow the development and distribution of elastic fibers from 48 chronic alcoholic patients. Patients were scored for fibrosis as 0, without fibrosis or minimal (n = 5); 1, incipient or early fibrosis (n = 9); 2, fibrosis or incomplete cirrhosis (n = 12); and 3, cirrhosis (n = 22). Elastica staining was performed by orcein, resorcin-fuchsin and iron hematoxylin and confirmed by immunofluorescence staining with an anti-human elastin antibody (Institut Pasteur). Electron microscopy of representative cases of each group and electron microscopy of immunolabelled elastin (n = 5) were also performed. In early alcoholic fibrosis, oxytalan fibers were pointed out in terminal hepatic veins and in Disse space. In fibrous portal extensions and cirrhotic internodular septa, oxytalan and elaunin fibers represented the major elastin components in association with the alcoholic liver fibroplasia. Immunostaining with anti-elastin Ab exhibits the same distribution as with histochemical methods in portal and septal zones. Electron microscopy confirmed abundant microfibrillar bundles between collagen fibers that mesh and are in continuity with elaunin fibers. Immunoelectron microscopy confirmed elastin deposits in the amorphous material and in association with the microfibrillar material in the portal and septal zones and disclosed elastin even in the thin strands of fibrotic tissue. In conclusion, elastogenesis, mainly represented by oxytalan and elaunin fibers, develops in alcoholic disease and takes part, with collagen deposits, in the fibrotic process.

Collagen↗

PubMed1979

Retinal pigment epithelium. Interrelations of endoplasmic reticulum and melanolysosomes in the black mouse and its beige mutant.

By cytochemistry (acid phosphatase and tyrosinase activities) GERL, a specialized hydrolase-rich region of endoplasmic reticulum (ER), can be visualized in the cells of the mouse retinal pigment epithelium. Previously catalase cytochemistry permitted us to identify microperoxisomes, with numerous continuities to the ER. The present report reveals the extensive continuities of the ER to pigment granules in various stages of maturation. When the pigment granules, which we consider to be "melanolysosomes," first appear they consist of electron-opaque grains within dilated areas of the ER. As the dilations enlarge, fine fibrils appear in the ER cisternae. Thicker fibers develop from the fibrils; these fibers are generally obscured when melanin deposition occurs. At all stages, the melanolysosomes appear to be connected to the ER.

Acid Phosphatase↗

PubMed1990

[Chemo- and radiosensitivity testing of tumor tissue cultured in vitro. Development of a fiber culture system for cell harvesting].

Chemo- and radiosensitivity-testing of tumour cells should be performed in vitro under in vivo-like conditions. One problem is to grow cells to tissue-like cell densities in vitro. A hollow fiber reactor was prepared for cell growth to high densities. In order to obtain cells during and after sensitivity testing for quantitating cell damage, a harvesting fiber system was developed which can be inserted in the nutritional hollow fiber system. These hollow fibers have a diameter of 300 microns and a wall thickness of 10 microns. Small holes between 10 and 100 microns can be cut into the fibers for cell harvesting by electric sparks. Up to 60 holes per mm fiber length can be produced.

Cytological Techniques↗

PubMed2006

White and gray matter development in human fetal, newborn and pediatric brains.

Brain anatomy is characterized by dramatic growth from the end of the second trimester through the neonatal stage. The characterization of normal axonal growth of the white matter tracts has not been well-documented to date and could provide important clues to understanding the extensive inhomogeneity of white matter injuries in cerebral palsy (CP) patients. However, anatomical studies of human brain development during this period are surprisingly scarce and histology-based atlases have become available only recently. Diffusion tensor magnetic resonance imaging (DTMRI) can reveal detailed anatomy of white matter. We acquired diffusion tensor images (DTI) of postmortem fetal brain samples and in vivo neonates and children. Neural structures were annotated in two-dimensional (2D) slices, segmented, measured, and reconstructed three-dimensionally (3D). The growth status of various white matter tracts was evaluated on cross-sections at 19-20 gestational weeks, and compared with 0-month-old neonates and 5- to 6-year-old children. Limbic, commissural, association, and projection white matter tracts and gray matter structures were illustrated in 3D and quantitatively characterized to assess their dynamic changes. The overall pattern of the time courses for the development of different white matter is that limbic fibers develop first and association fibers last and commissural and projection fibers are forming from anterior to posterior part of the brain. The resultant DTMRI-based 3D human brain data will be a valuable resource for human brain developmental study and will provide reference standards for diagnostic radiology of premature newborns.

Adult↗

PubMed1983

Quantitative assessment of neural development in human premolars.

The number of nerve fibers entering a tooth gives an indication of the tooth's capacity to perform a sensory function. Nerve fiber development was quantitated from cross sections of the apical portions of 49 erupted human premolars at various stages of root development and in subjects up to 71 years of age. Neural development was incomplete in immature teeth, greatly variable in young mature teeth, and complete in older teeth. Myelinated axons changed in number but not in size during tooth development. There were significantly fewer myelinated axons in teeth with open and parallel apical foramina than in older teeth. Unmyelinated axons did not change significantly in number with development but fewer large axons were found in older teeth. The number of unmyelinated axons enclosed in a single boundary lamina tended to be lower in older teeth. As a physiologic correlate, threshold responses to electrical stimulation were also determined prior to premolar removal. Threshold stimulation decreased significantly with apical foramen maturation. A significant negative correlation was found between the threshold stimulus and the number of myelinated axons in fully developed teeth, but not in immature teeth.

Adolescent↗

PubMed1990

Development of muscle fiber types in the prenatal rat hindlimb.

Immunohistochemistry was used to examine the expression of embryonic, slow, and neonatal isoforms of myosin heavy chain in muscle fibers of the embryonic rat hindlimb. While the embryonic isoform is present in every fiber throughout prenatal development, by the time of birth the expression of the slow and neonatal isoforms occurs, for the most part, in separate, complementary populations of fibers. The pattern of slow and neonatal expression is highly stereotyped in individual muscles and mirrors the distribution of slow and fast fibers found in the adult. This pattern is not present at the early stages of myogenesis but unfolds gradually as different generations of fibers are added. As has been noted by previous investigators (e.g., Narusawa et al., 1987, J. Cell Biol. 104, 447-459), all of the earliest generation (primary) muscle fibers initially express the slow isoform but some of these primary fibers later lose this expression. In this study we show that loss of slow myosin in these fibers is accompanied by the expression of neonatal myosin. This switch in isoform expression occurs in all primary fibers located in specific regions of particular muscles. However, in other muscles primary fibers which retain their slow expression are extensively intermixed with those that switch to neonatal expression. Later generated (secondary) muscle fibers, which are interspersed among the primary fibers, express neonatal myosin, although a few of them in stereotyped locations later switch from neonatal to slow myosin expression. Many of the observed changes in myosin expression occur coincidentally with the arrival of axons in the limb or the invasion of axons into individual muscles. Thus, although both fiber birth date and intramuscular position are grossly predictive of fiber fate, neither factor is sufficient to account for the final pattern of fiber types seen in the rat hindlimb. The possibility that fiber diversification is dependent upon innervation is tested in the accompanying paper (K. Condon, L. Silberstein, H.M. Blau, and W.J. Thompson, 1990, Dev. Biol. 138, 275-295).

Acetylcholinesterase↗

PubMed2006

Multiple Eph receptors and B-class ephrins regulate midline crossing of corpus callosum fibers in the developing mouse forebrain.

Agenesis of the corpus callosum (CC) is a rare birth defect that occurs in isolated conditions and in combination with other developmental cerebral abnormalities. Recent identification of families of growth and guidance molecules has generated interest in the mechanisms that regulate callosal growth. One family, ephrins and Eph receptors, has been implicated in mediating midline pathfinding decisions; however, the complexity of these interactions has yet to be unraveled. Our studies shed light on which B-class ephrins and Eph receptors function to regulate CC midline growth and how these molecules interact with important guideposts during development. We show that multiple Eph receptors (B1, B2, B3, and A4) and B-class ephrins (B1, B2, and B3) are present and function in developing forebrain callosal fibers based on both spatial and temporal expression patterns and analysis of gene-targeted knock-out mice. Defects are most pronounced in the combination double knock-out mice, suggesting that compensatory mechanisms exist for several of these family members. Furthermore, these CC defects range from mild hypoplasia to complete agenesis and Probst's bundle formation. Further analysis revealed that Probst's bundle formation may reflect aberrant glial formations and/or altered sensitivity of CC axons to other guidance cues. Our results support a significant role for ephrins and Eph receptors in CC development and may provide insight to possible mechanisms involved in axon midline crossing and human disorder.

Animals↗

PubMed1996

Life on a string: development of the tetracycline fiber delivery system.

Development of new drug therapy is difficult for large companies and almost unheard of by individuals. This autobiographical account of the development of the tetracycline fiber delivery system for periodontal therapy (Actisite) relates experiences of a scientist between 1976 and 1994 which lead to FDA approval. In this account, the author acknowledges some of the key people, companies and institutions who were important in this development, makes observations concerning the process itself and offers suggestions for those who might consider commercial development of a scientific concept.

Drug Approval↗

PubMed2003

An ATP-binding cassette transporter GhWBC1 from elongating cotton fibers.

We have isolated a cDNA (GhWBC1) from cotton (Gossypium hirsutum) that encodes an ATP-binding cassette transporter of the WBC (white/brown complex) subfamily. Members of this subfamily are half-sized transporters and are reported to mediate lipid and drug excretion in human (Homo sapiens). GhWBC1 is highly expressed in developing fiber cells, but transcripts were also detectable in other tissues except roots. The transcript level peaked in rapidly expanding fibers from 5 to 9 DPA and then decreased. The GhWBC1 expression was weak in fiber cells of an li (ligon-lintless) mutant, which is defective in fiber cell elongation. These data indicate that GhWBC1 gene expression correlates with cotton fiber elongation. Transient expression of enhanced green fluorescence protein-GhWBC1 fusion protein in onion (Allium cepa) epidermal cells revealed plasma membrane localization. The GhWBC1 cDNA driven by a constitutive 35S promoter was introduced into Arabidopsis. About 13% of the transformants produced short siliques (SSs), whereas others had normal siliques (long siliques [LSs]). In siliques of SS lines, most embryos were severely shriveled, and only several seeds per silique could be found at maturity. The transgene expression level was higher in SS lines than in LS lines. Expression of AtWBC11, the closest homolog of GhWBC1 in Arabidopsis, was not altered in either SS or LS transgenic plants examined. These data suggest that GhWBC1 interferes with substance translocation that is required for Arabidopsis seed and silique development. Characterization of Arabidopsis WBC members, particularly AtWBC11, will help to dissect the role of GhWBC1 in cotton fiber development and elongation.

ATP-Binding Cassette Transporters↗

PubMed1988

Cellular electrophysiologic effects of vertebrate digitalis-like substances.

Substances structurally and functionally similar to digitalis glycosides are produced by several vertebrate species. There also is evidence for a digitalis-like substance of human origin. Standard microelectrode techniques were used to study the direct effects on the cellular electrophysiology of canine Purkinje fibers of 1) bufalin, an unconjugated cardiotonic steroid molecule that is produced by the toad Bufo marinus, and 2) an extract of human bile that showed digitalis-like immunoreactivity on radioimmunoassay. The goal of this study was to determine whether these substances have arrhythmogenic effects comparable with those seen with toxic doses of digitalis glycosides. Bufalin, 2 x 10(-8) M, significantly (p less than 0.05) reduced maximal diastolic potential, action potential amplitude and duration and maximal rate of rise of phase 0 (Vmax) within 40 min of onset of exposure. All six fibers developed delayed afterdepolarizations and two developed triggered rhythms. Ouabain was less potent, in that a 2 x 10(-7) M concentration was required to comparably reduce maximal diastolic potential, action potential amplitude and duration and Vmax within 30 min. These Purkinje fibers also developed delayed afterdepolarizations and triggered rhythms. A sample of an extract of human bile that showed digitalis-like immunoreactivity with an antibufalin serum also reduced maximal diastolic potential, action potential amplitude and duration and Vmax, and produced delayed afterdepolarizations and triggered activity. In contrast, immunologically unreactive bile extracts had no appreciable effect on the action potential. In summary, the cardiac toxicity of digitalis substances produced by lower vertebrates is comparable with that induced by the glycosides. Moreover, it appears that humans may produce digitalis-like substances that may be cardiotoxic.

Action Potentials↗

PubMed1995

Sex-specific differentiation of a male-specific abdominal muscle, the Muscle of Lawrence, is abnormal in hydroxyurea-treated and in fruitless male flies.

A prominent sex-specific abdominal muscle in male Drosophila is the Muscle of Lawrence (MOL), which is induced by male-specific innervation. We have examined MOL development in wild-type males, in males fed hydroxyurea to ablate the muscle precursors and in fruitless mutants, in which the MOL muscle develops aberrantly. One striking feature of MOLs in wild-type males was the presence of additional muscle nuclei compared with neighboring muscles or MOL-homologues in females. We tested whether muscle length and the sex-specific expression of a reporter gene depended critically on the number of nuclei present within a MOL fiber. MOL fibers developing from a reduced myoblast pool in hydroxyurea-affected hemisegments were recognizable by their attachment points and still contained more nuclei than did neighboring medial fibers, suggesting that these MOL fibers were able to actively recruit myoblasts nearly as well as wild-type MOLs. However, many of the hydroxyurea-affected MOL fibers were incapable of the normal male-specific expression of a muscle-specific reporter gene. We suggest that early events in MOL development, such as finding the correct muscle attachment points, are relatively insensitive to the number of MOL nuclei compared with later events, such as the sex-specific expression of a reporter gene. In fruitless mutant males, MOL-position fibers are smaller and had substantially fewer nuclei compared to wild-type MOLs. Since the number and distribution of muscle precursors was the same in fruitless mutant and wild-type animals, we propose that one fru+ function is to direct the male-specific recruitment of myoblasts into MOL-myotubes. However, fruitless+ must have more than one role in MOL fiber development, since simple reduction in the number of muscle nuclei, as demonstrated by the hydroxyurea ablations, is insufficient to account for all of the MOL muscle phenotypes in fruitless mutant males.

Abdominal Muscles↗

PubMed2003

Cotton-fiber germin-like protein. II: Immunolocalization, purification, and functional analysis.

Cotton (Gossypium hirsutum L.) contains a germin-like protein (GLP), GhGLP1, that shows tissue-specific accumulation in fiber. The fiber GLP is an oligomeric, glycosylated protein with a subunit size of approximately 25.5 kDa. Accumulation of GhGLP1 occurs during the period of fiber elongation [4-14 days post-anthesis (DPA)]. During early phases of fiber development (2-4 DPA), GhGLP1 localizes to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7-10 DPA), GhGLP1 localizes to the apoplast. In other plants, germins and GLPs have been reported to have enzymatic activities including oxalate oxidase (OxO), superoxide dismutase, and ADP-glucose pyrophosphatase. Cotton fiber extracts did not contain OxO activity, nor did intact fibers stain for OxO activity. A four-step purification protocol involving ammonium sulfate precipitation of a 1.0 M NaCl extract, ion-exchange chromatography on DEAE-Trisacryl M, lectin-affinity chromatography, and gel filtration chromatography resulted in electrophoretically pure GhGLP1. While 1.0 M NaCl extracts from 10-14 DPA fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a GLP accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.

Amino Acid Sequence↗

PubMed2001

Fiberoptic infrared radiometer for real time in situ thermometry inside an MRI system.

A warm body emits radiation whose intensity I is dependent on the surface temperature T. For T near room temperature this radiation is mostly in the mid-IR. Radiometric measurement of I is often used for non contact thermometry. Temperature measurements in situ and in remote locations can be carried out using optical fibers, but one needs fibers that are highly transparent in the mid-IR. Our group has developed fibers made of silver halides. These are flexible, nontoxic, not hygroscopic and highly transparent in the mid IR. These fibers served for the development of a novel fiberoptic radiometer. Using this radiometer we have measured the temperatures of samples while being imaged in an MRI system. The presence of the fibers inside the MRI system did not interfere with the operation of the MRI nor did not the MRI system affect the radiometer. The temperature measurements were made with accuracy of 0.3 degrees C, response time of 1 s and spatial resolution of 1 mm. Fiberoptic radiometers would be highly suitable for temperature measurement of human tissues, for example during surgical procedures done inside an MRI system.

Calibration↗

PubMed1980

The effect of ouabain on the isolated sinus node preparation of the rabbit studied with microelectrodes.

In 30 isolated, spontaneously beating right atrial preparations of the rabbit, the arrhythmogenic actions of ouabain were studied with microelectrodes. Ouabain (10(-6) M) uniformly produced the following events: (1) a gradual increase in sinus rate with periods of alternation of beta-to-beat atrial intervals, (2) a rapid rate and regular rhythm, (3) severe irregularity of rhythm, (4) total atrial arrest. Cycle length decreased from 394 msec +/- 43 (mean +/- SD) during control to 229 msec +/- 26 during the period of rapid rate and regular rhythm (P less than 0.001). Pretreatment with atropine or propranolol did not abolish this positive chronotropic action of ouabain. Maps of the spread of activation in the area of the sinus node in eight experiments revealed that the speeding up of rate is accompanied by a shift of dominant pacemaker site toward the sinoatrial border. In explanation, it is shown that sinoatrial border fibers develop strong diastolic depolarization, whereas dominant pacemaker fibers do so to a lesser extent or not at all. The atrial arrhythmias--best to be described as "digitalis-induced sinus tachycardia"--may represent the experimental counterpart of the so-called "paroxysmal atrial tachycardia with block" described as a common manifestation of digitalis intoxication in man.

Action Potentials↗

PubMed1998

Screening tool for hollow-fiber bioreactor process development

Fundamental research of factors affecting cell growth in hollow-fiber bioreactors is hindered by the lack of an efficient screening tool. To address this issue, a hollow-fiber micro-bioreactor has been developed. Hollow fibers with 10 kDa molecular weight cutoffs are housed within a piece of silicone tubing. Cells are inoculated within the hollow fibers which provides a 0.2-mL culture volume. The space between the fibers and silicone tubing (5 or 16 mL) is used as a medium reservoir sufficient to feed the cells for at least 24 h. Oxygenation is provided directly through the silicone tubing so that a pump for medium recirculation is not required. As a result, many conditions can be tested simultaneously in a single incubator. Three days after inoculation at 5 x 10(6) cells/mL in the micro-bioreactor, the rho 1D4 murine hybridoma cell line reached 2.8 x 10(7) cells/mL with an antibody concentration of 0.17 mg/mL. When inoculated at 5 x 10(7) cells/mL, the cell concentration reached 1.8 x 10(8)/mL after 3 days with an antibody concentration of 1.0 mg/mL. Results from a series of experiments with the micro-bioreactor suggested that the initial growth phase of this cell line in a hollow-fiber system is dependent on the serum concentration in the medium reservoir. This prediction was tested by simultaneously inoculating two production-scale hollow-fiber bioreactor systems. The cell side of the membrane for each bioreactor contained 10% serum, but serum was added to the reservoir side of only one of the bioreactors. The cells with only basal medium in the reservoir died after a few days, while the cells with 10% serum in the medium reservoir grew rapidly. These results demonstrate that the micro-bioreactor developed here can support good cell growth and that it can be used as a research tool to predict the performance of large-scale hollow-fiber systems.

Journal Article↗

PubMed2001

Roles of glutamate receptor delta 2 subunit (GluRdelta 2) and metabotropic glutamate receptor subtype 1 (mGluR1) in climbing fiber synapse elimination during postnatal cerebellar development.

Climbing fiber (CF) synapse formation onto cerebellar Purkinje cells (PCs) is critically dependent on the synaptogenesis from parallel fibers (PFs), the other input to PCs. Previous studies revealed that deletion of the glutamate receptor delta2 subunit (GluRdelta2) gene results in persistent multiple CF innervation of PCs with impaired PF synaptogenesis, whereas mutation of the metabotropic glutamate receptor subtype 1 (mGluR1) gene causes multiple CF innervation with normal PF synaptogenesis. We demonstrate that atypical CF-mediated EPSCs (CF-EPSCs) with slow rise times and small amplitudes coexisted with typical CF-EPSCs with fast rise times and large amplitudes in PCs from GluRdelta2 mutant cerebellar slices. CF-EPSCs in mGluR1 mutant and wild-type PCs had fast rise times. Atypical slow CF responses of GluRdelta2 mutant PCs were associated with voltage-dependent Ca(2+) signals that were confined to PC distal dendrites. In the wild-type and mGluR1 mutant PCs, CF-induced Ca(2+) signals involved both proximal and distal dendrites. Morphologically, CFs of GluRdelta2 mutant mice extended to the superficial regions of the molecular layer, whereas those of wild-type and mGluR1 mutant mice did not innervate the superficial one-fifth of the molecular layer. It is therefore likely that surplus CFs of GluRdelta2 mutant mice form ectopic synapses onto distal dendrites, whereas those of wild-type and mGluR1 mutant mice innervate proximal dendrites. These findings suggest that GluRdelta2 is required for consolidating PF synapses and restricting CF synapses to the proximal dendrites, whereas the mGluR1-signaling pathway does not affect PF synaptogenesis but is involved in eliminating surplus CF synapses at the proximal dendrites.

Aging↗

PubMed1992

Mechanisms of nascent fiber formation during avian skeletal muscle hypertrophy.

This study examined two putative mechanisms of new fiber formation in postnatal skeletal muscle, namely longitudinal fragmentation of existing fibers and de novo formation. The relative contributions of these two mechanisms to fiber formation in hypertrophying anterior latissimus dorsi (ALD) muscle were assessed by quantitative analysis of their nuclear populations. Muscle hypertrophy was induced by wing-weighting for 1 week. All nuclei formed during the weighting period were labeled by continuous infusion of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analog, and embryonic-like fibers were identified using an antibody to ventricular-like embryonic (V-EMB) myosin. The number of BrdU-labeled and unlabeled nuclei in V-EMB-positive fibers were counted. Wing-weighting resulted in significant muscle enlargement and the appearance of many V-EMB+ fibers. The majority of V-EMB+ fibers were completely independent of mature fibers and had a nuclear density characteristics of developing fibers. Furthermore, nearly 100% of the nuclei in independent V-EMB+ fibers were labeled. These findings strongly suggest that most V-EMB+ fibers were nascent fibers formed de novo during the weighting period by satellite cell activation and fusion. Nascent fibers were found primarily in the space between fascicles where they formed a complex anastomosing network of fibers running at angles to one another. Although wing-weighting induced an increase in the number of branched fibers, there was no evidence that V-EMB+ fibers were formed by longitudinal fragmentation. The location of newly formed fibers in wing-weighted and regenerating ALD muscle was compared to determine whether satellite cells in the ALD muscle were unusual in that, if stimulated to divide, they would form fibers in the inter- and intrafascicular space. In contrast to wing-weighted muscle, nascent fibers were always found closely associated with necrotic fibers. These results suggest that wing-weighting is not simply another model of regeneration, but rather produces a unique environment which induces satellite cell migration and subsequent fiber formation in the interfascicular space. De novo fiber formation is apparently the principal mechanism for the hyperplasia reported to occur in the ALD muscle undergoing hypertrophy induced by wing-weighting.

Aging↗

PubMed1974

Boron Deficiency in Unfertilized Cotton (Gossypium hirsutum) Ovules Grown in Vitro.

Boron deficiency and phytohormone interactions have been studied in unfertilized cotton (Gossypium hirsutum) ovules grown in vitro. Such ovules required exogenous indoleacetic acid and/or gibberellic acid for fiber elongation. Boron also was required for maintenance of fiber elongation and normal morphogenesis throughout 14 days of culture. The amount of exogenous boron necessary for maximum fiber elongation varied among experiments, presumably in relation to endogenous boron levels at anthesis. Some ovular epidermal cells distant from the liquid medium could be induced to elongate into fiber even after 6 days in boron-deficient medium in response to the later addition of boron.Boron deficiency, in the presence of exogenous indoleacetic acid, was characterized by lack of fiber development on the inundated ovular surface and reduced fiber growth on the ovular surface exposed to air. In the presence of gibberellic acid, boron deficiency was characterized by complete absence of fiber and callusing of the entire ovular surface. When both indoleacetic acid and gibberellic acid were added, the lack of boron resulted in proliferation of callus laterally and upward from the inundated epidermis, accumulation of brown pigments (presumably phenolic compounds) in the callus, and restriction of fiber to a small area of the upper ovular surface.

Journal Article↗