A STUDY OF THE ULTRASTRUCTURAL LOCALIZATION OF HAIR KERATIN SYNTHESIS UTILIZING ELECTRON MICROSCOPIC AUTORADIOGRAPHY IN A MAGNETIC FIELD

The sites of the incorporation of labeled cystine into keratinizing structures were studied in electron microscopic autoradiographs. The tracer used was cystine labeled with S³⁵ emitting long-range ionizing particles. During exposure for 1 to 2 months, according to our method of electron microscopic autoradiography, emulsion-coated specimens were exposed to a static magnetic field which appeared to result in a marked increase in the number of reacted silver grains. In young Swiss mice receiving intraperitoneal injections at 1, 3, and 6 hours before biopsy, conventional autoradiography demonstrated that S³⁵-cystine was intensely localized in the keratogenous zone of anagen hair follicles, and that the radioactivity there increased in intensity progressively with time while the radioactivity in the hair bulb always remained very low. Our observations with electron microscopic autoradiography in a magnetic field appeared to indicate that at 3 and 6 hours after injection the S³⁵-cystine was directly and specifically incorporated into tonofibrils in the hair cortex and into amorphous keratin granules of the hair cuticle layer, possibly without any particular concentration of this substance in the other cellular components. There seemed to be an appreciable concentration of cystine in tonofibrils of the cuticle of the inner root sheath. However, trichohyalin granules in the hair medulla and inner root sheath failed to show any evidence of cystine concentration. The improved sensitivity of the electron microscopic autoradiography with S³⁵-cystine appeared to be partly due to the application of a static magnetic field. However, the reason for this could not be explained theoretically.     

Kinetics of polyglucose breakdown in Chlorobium

Washed cells of Chlorobium limicola f. thiosulfatophilum photoassimilate CO₂ and acetate into polyglucose, which is laid down within the cells as rosette-like granules. When the cells are incubated in the dark, the polyglucose is broken down. Experiments using electron microscopy and labelling with ¹⁴C-acetate have revealed that degradation of polyglucose occurs in such a way that all the granules are subject to degradation simultaneously and the polyglucose which has been formed most recently in the light, becomes metabolized in the dark first.     

Morphology of Hair Pigmentation in Wild Red Foxes, Silver Foxes, and Their Hybrids

The effects of dominant allele A ^r of locus Agoution the morphology of hair pigmentation were described in foxes. The A ^r allele was shown to determine the type of melanin and its content in hair with no effect on the morphology of pigment granules and their distribution throughout a hair. Using the method of electron spin resonance (ESR), the types of melanin (eumelanin and pheomelanin) and their content in the hair of red (A ^r A ^r EE) and silver (aaEE) foxes and their hybrids (A ^r aEE) were determined. In silver foxes, only one type of melanin (eumelanin) was found. In red foxes and their hybrids (which are phenotypically similar but darker than red foxes), both types of melanin (eu- and pheomelanin) were found. The highest melanin content was detected in the coat of silver foxes. In the hybrids, the total melanin content was lower than in silver foxes, but significantly higher than in red foxes. In red foxes, the contribution of pheomelanin to the total hair melanin content was twice as large as in the hybrids.     

Sodium rhodizonate staining of the keratogenous zone of the hair follicle and lingual papilla

Summary Exposure of frozen sections of guinea pig skin to a saturated aqueous solution of sodium rhodizonate buffered to pH 6.2 selectively induces a band of black granules in the keratogenous zone of hair follicles. The major staining is in the cortex, but to a lesser extent granules develop in the medulla and inner root sheath in the same zonal pattern. A similar granular staining is seen in the lingual papillae and naisl. Other structures in the skin and other organs do not show this. At pH 9.0 the sodium rhodizonate selectively stains the hair follicles a reddish brown. Although the affinity of the keratogenous zone of the hair follicle for sodium rhodizonate remains unexplained, one might postulate the stain is dependent upon the presence of an unusually high sulfhydryl content. In favor of this is the fact that the highest level of protein-bound sulfhydryl activity is in the exact site of the rhodizonate staining, and the fact that rhodizonic acid can oxidize sulfhydryl compounds to dark end products.Supported by a grant from the John A. Hartford Foundation.We are grateful to Mrs. Verna Stein for her technical assistance and Mr. William Tomlinson for his advice.     

Fine structure of the mandibular gland in Japanese field vole (Microtus montebelli)

The mandibular glands of the Japanese field vole were examined by light microscopy, and transmission and scanning electron microscopies. The acinar cells contained light and coarse secretory granules, and reacted with PAS and stained slightly with AB; they were considered to be seromucous in nature. The acinar epithelium was composed of light and dark cells containing many secretory granules. The intercalated duct cells consisted of light cells possessing a few dense granules. A few cytoplasmic crystalloides of moderate density were observed in occasional light cells. The striated ducts were comprized of two distinct portions, a secretory portion and a typical striated portion without secretory granules. The epithelium secretory portion consisted of light and dark cells containing acidophilic granules and exhibited a sexual dimorphism in these granules: The male epithelia contained the granules of low to high densities, while the female epithelia had only dense granules being smaller than those in the males. The epithelium of typical striated portion was composed of light and dark cells containing fine vacuoles and vesicles.     

Stage-specific crosstalk between light, auxin, and ethylene during low-pH-induced root hair formation in lettuce (Lactuca sativa L.) seedlings

In the light, transfer of lettuce seedlings precultured on liquid medium at pH 6.0 to fresh medium at pH 4.0 induces root hair formation. However, no root hairs form in the dark. Here, we investigated how light induces root hair formation. Randomization of the transverse cortical microtubule (CMT) arrays which occurs in root epidermal cells in the light prior to root hair initiation was not observed in the dark. However, addition of indole-3-acetic acid (IAA) or 1-aminocyclopropane-1-carboxylic acid (ACC) induced CMT randomization and root hair formation. In these cases, CMT randomization occurred in almost the same time-dependent manner as under light. However, root hair initiation was delayed for several hours in the dark. These results suggest that light promotes CMT randomization and root hair initiation via auxin and ethylene signaling but light additionally influences root hair initiation independently of these signaling mechanisms. Furthermore, addition of a microtubule-depolymerizing drug in the dark disrupted the transverse CMT arrays and initiated root hair formation; however, root hair elongation was still suppressed. Root hairs elongated when IAA or ACC was applied with the drug. These results suggest that light also promotes root hair elongation via auxin and ethylene signaling.     

Chromatophoromas in a pine snake

Iridophoroma and melanophoroma were diagnosed in an adult male pine snake. Light microscopic examination of irregularly thickened white and black portions of abnormal scales demonstrated two distinctive populations of pigment-containing cells. Pigment cells within abnormal-appearing white scales had needle-shaped granules that were dark amber in color while black portions were composed of pigment cells typical of melanophores, with dark black, round granules. Both populations of cells showed junctional activity, and clusters of both neoplastic pigment cell types were found in adjoining areas of the epidermis. By electron microscopy, the pigment cell with amber-colored granules contained reflecting platelet profiles typical of iridophores while pigment cells with dark round granules contained melanosomes. At a junctional area between abnormal white and black scales, mosaic chromatophores containing reflecting platelet profiles and melanosomes were observed. At 1 1/2 years following initial diagnosis, the snake died and neoplastic iridophores were found at multiple visceral sites; there was no evidence of metastases of melanophores to any organ. The two pigment cell tumors are believed to have developed from either stem cells destined to become iridophores and melanophores or from prexisting iridophores and melanophores in the dermis.     

Characteristics of serial cross-sections of hairs of the Japanese monkey (Macaca fuscata fuscata)

The characteristics of serial cross-sections of hairs collected from an adult male Japanese monkey were investigated. Cross-sections were made of five to eight pieces per hair. The shapes of the cross-sections were elliptical or rounded on the whole. The fibre indices of the sections ranged from 83 to 100. In particular, those of proximal (basal) sections were close to 100. The hair diameter was 86.4 μ at maximum and 27.2 μ at minimum. A tendency was observed for the longer hairs to have thicker diameters. The changes in thickness along the fibre shaft were slightly different in relation to hair length. The thickest point was at around the middle of the fibre in the intermediate hair, somewhat towards the top of the central part in the long hair, and somewhat towards the base in the short hair. The hair of the Japanese monkey, however, was considered to be scanty in changes along the fibre shaft in comparison with many other animals. Medullae could scarcely be seen in the short hair and in the terminal and proximal sections of all hairs. Their shapes in cross-section were not uniform and rough at the margins. The fibre-medulla indices were generally less than 30 and smaller than those of many other mammals. Pigmentary granules were observed in all sections examined. The granules were black-grey in sections of the black-grey coloured part and yellow in the yellowish sections. They were dense in distal sections and scarce in sections close to the base. The cross-sectional appearance of the thickest part of the long hair was considered to be useful for hair identification, since it was good in pigmentation and medullation and relatively small fibre index.     

Pigment synthesis in the Himalayan mouse

The effect of temperature on pigment synthesis in adult and juvenile Himalayan mice was investigated. Since pigment synthesis only occurs in actively growing hair, adult mice were plucked to induce hair growth. The extent of darkening of the hair was recorded by photography against a reference scale. The presence of pigment granules in hair follicles was investigated histologically. Housing adult and juvenile mice at 15 degrees C results in the synthesis of pigment in growing hair follicles whereas housing at 30 degrees C results in the absence of pigment granules in the growing hair follicles.     

Is Blepharisma hyalinum Truly Unpigmented?1

ABSTRACT. To answer whether Blepharisma hyalinum is truly unpigmented, the organism must be established in culture as pointed out by Giese in 1973. Accordingly, the present study deals with B. hyalinum kept in culture since its isolation in 1975. The organism still remains colorless after growth in the dark; however, it contains cortical granules resembling pigment granules in colored species. A comparative study was therefore undertaken of B. hyalinum and B. steini; both species have a compact macronucleus, though of different shape. Crude pigment was extracted with acetone from organisms grown in the dark for three weeks and the maxima were measured by absorption. Purified pigment was obtained from TLC-plate preparations and the absorption maxima were measured after removal of lipids with chloroform. No maxima characteristic of blepharismin were found in extracts of B. hyalinum, but these were present in extracts of B. steini. Electron microscopy of the cortical region revealed membrane-bound granules in both species; these granules differed in content but not in their capacity to extrude. In B. hyalinum all granules had a homogenous electron-dense substructure; in B. steini the granules had a net-like granulated substructure of varying electron density. This difference corresponds to that published on “pigment” granules in albino and pigmented strains of B. undulans. Our conclusions are that B. hyalinum is unpigmented (and a valid separate species) and that the cortical granules may serve other functions than that of storing blepharismin.     

Individuals with dark eyes and hair exhibit higher pain sensitivity

Emerging evidence suggests that some phenotypic features, such as eye or hair colour, might predict pain. We investigated if light and dark eye and hair colour would influence pain in 60 healthy subjects divided in groups of 15 according to their eye–hair colour and gender. Pressure pain thresholds (PPTs), cold pressor test (CPT), and quality of the perceived pain were assessed. Findings indicated that dark pigmentation phenotype is more sensitive in response to CPT.     

Synthesis,storage and degradation of polyglucose in chlorobium thiosulfatophilum

Cultures of Chlorobium thiosulfatophilum form polyglucose during growth. The polyglucose is laid down within the cells as rosette-like granules, which are made up from smaller grains. The size of each granule appears to be limited to less than 30 nm, since an increase in polyglucose content leads to more granules being formed rather than an increase in granule size.The polyglucose in washed cells is fermented in the dark to acetate, propionate, caproate and succinate, of which acetate by far comprises the largest fraction (68%). During incubation of washed cells without hydrogen donor, the level of polyglucose decreases regardless of whether the cells are incubated in the dark or in the light. Since the products formed from polyglucose under the two different conditions are not the same, it is suggested that polyglucose in the dark serves as an energy source, whereas when in the light the role of polyglucose is mainly to provide the cell with reducing power.     

The use of hair as an indicator of occupational 14C contamination

This paper presents a study in which the specific activity of ¹⁴C in hair has been investigated as an easily determined bio-indicator of the integrated ¹⁴C exposure (over several months). The study includes 28 Swedish workers handling ¹⁴C-labelled compounds, or working in a ¹⁴C-enriched environment. Hair samples from personnel at a Swedish nuclear power plant showed very low levels of ¹⁴C contamination, if any. In contrast, personnel at the investigated research departments showed ¹⁴C levels in hair of up to 60% above the natural specific activity of ¹⁴C. Much higher levels, up to 80 times the natural specific activity of ¹⁴C, were found in hair from individuals working at a pharmaceutical research laboratory. This contamination was, however, not solely an internal contamination. There were indications that most of the ¹⁴C in the hair originated from airborne ¹⁴C-compounds, which were adsorbed onto the hair. The difficulties in removing this external ¹⁴C contamination prior to analysis are discussed, as are the possibilities of using accelerator mass spectrometry to analyse various types of samples for retrospective dose assessment.     

Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (< 550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in "blue light toxicity" as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.     

Twenty four hour variations in subcellular structures of rat pancreatic islet B-, A- and D-cells,and of portal plasma glucose and insulin levels

Summary Variations in stomach weights, plasma glucose and insulin levels in the portal vein, and in subcellular structures of rat pancreatic islet B-, A- and D-cells were examined over 24 h. The variation in stomach weights paralleled plasma glucose levels, indicating that the levels may be influenced by intestinal glucose absorption. Plasma insulin levels increased from the late dark to the early light periods, whereas they decreased from the late light to the early dark periods. The variation in plasma insulin levels was in the opposite sense to that in the relative numbers of B-cell granules. The decrease in the relative numbers of A-cell granules occurred between the late dark and early light periods. The relative numbers of D-cell granules decreased before and after the decreases in B- and A-cell granules. The variation in D-cell granules appears to correspond to the inhibitory effect of somatostatin. The relative amounts of rough endoplasmic reticulum (rER) in each cell varied in a reciprocal manner to those of B-cell granules. Moreover, the variation in plasma insulin levels coincided with variations in rER of hepatocytes and pancreatic acinar cells. The changes in rER of each cell may correlate with the trophic effect of insulin.     

Mechanisms leading to cortical reaction in the mammalian egg

Activation of the mammalian egg results in cortical reaction (CR), which is correlated with an increase in intracellular Ca²⁺ concentration and PKC activation. The CR is a gradual rather then an “all or none” response, and can be regulated by different concentrations of parthenogenetic activators. To evaluate the biological significance of parthenogenetic induced CR, rat eggs were fertilized or activated by different concentrations of ionomycin and TPA. Cortical granules (CG) were monitored by electron microscopy, while the CG exudate was visualized by Lens culinaris lectin and Texas Red, using light and confocal microscopy. The ability of the CR to trigger a full block to polyspermy was examined in an IVF system. Our study demonstrates the existence of light and dark CG, which differ by number, distribution in the egg cortex, and sensitivity to parthenogenetic activators. Sperm penetration or high concentration of activators, trigger depletion of both light and dark CG, leading to a full CR. Low concentration of activators altered the CG density, the ratio of dark/light CG, and induced partial CR that was sufficient to cause a block to polyspermy. The results imply that Ca²⁺ rise or PKC activation have different effects on light and dark CG. In recently fertilized or parthenogenetically activated eggs, CG exudate appeared as evenly distributed spots, whereas in more advanced stages of fertilization the exudate was scattered as patchy aggregates. This observation suggests a difference in the dispersion of CG exudate after fertilization as compared to parthenogenetic activation. Mol. Reprod. Dev. 51:295–303, 1998. © 1998 Wiley-Liss, Inc.     

Incorporation of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair of mice

The incorporation of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair of newborn mice was investigated in order to contribute to the validation of PhIP in hair as a suitable biomarker for human dietary exposure. Black mice (C57BL/6J; 7-9 days old) were given graded doses of [³H]-PhIP subcutaneously during the start of the hair growth period. The distribution of [³H]-PhIP and incorporation into hair were investigated by tape-section autoradiography. Almost all the radioactivity in hair represented PhIP as shown by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). A dose-response proportionality of incorporation into hair was found when incorporation was determined by liquid scintillation counting. Autoradiography showed that PhIP was rapidly cleared from the skin, but remained for at least 28 days in the part of the hair shafts which was formed during the exposure period. The present results obtained using the mouse as a model, further support the suggestion that PhIP in hair may be a suitable biomarker for human exposure to dietary PhIP.     

Validation of a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair using LC-MS/MS and its application to human and rat hair

Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC-MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair pigmentation did not have any significant effect on the degree of the deposition of drugs and their metabolites in hair.     

Chemical Characterization of Melanins in Sheep Wool and Human Hair

The color of hair and wool in mammals and feathers in birds is mostly determined by the quantity and quality of melanins that are synthesized in follicular melanocytes and transferred to keratinocytes. There are two chemically distinct types of melanin pigments: the black to brown eumelanins and the yellow to reddish pheomelanins. Melanins in sheep wool and human hair of various colors were characterized by HPLC methods to estimate 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units in eumelanins and benzothiazine units in pheomelanins. Melanins were also characterized by spectrophotometric methods after differential solubilization in alkalies. It was demonstrated that 1) black wool in Asiatic sheep contains eumelanin with the DHICA content similar to black mouse melanin, while black to brown melanins from human hair contain much lower ratios of DHICA-derived units, comparable to the slaty mutation in mice, 2) dark brown to brown hair in human contains eumelanin whose chemical properties are indistinguishable from those of black hair, 3) dark red wool and red human hair contain pheomelanic pigments whose chemical properties are rather different from those of yellow pheomelanins in mice, and 4) light brown, blonde, and red hairs in human can be differentiated from each other with this methodology.