Protargol Research Articles

Morphological Redescription and Morphogenesis of Urosoma macrostyla (Wrześniowski, 1866) Berger, 1999 (Ciliophora, Hypotrichida)

The morphology and morphogenesis of the hypotrich ciliate Urosoma macrostyla (Wrześniowski, 1866) Berger, 1999, collected from a puddle in Harbin, China, were investigated using live observation and protargol impregnation. Based on previous and present studies, an improved diagnosis of U. macrostyla is supplied. It differs from its congeners mainly by the body shape, no cortical granules and number of macronuclear nodules. The ontogenesis of U. macrostyla is typical for species with such a somatic ciliary pattern: the oral primordium develops hypoapokinetally and FVT-anlagen develop in 5-streaks and primary mode. However, a unique characteristic in morphogenetic process is reported: anlagen for both the left and right marginal cirri occur de novo to the right of the parental structure which has never been seen in other oxytrichids. This characteristic was considered an apomorphy (Berger 1999). This indicates that U. macrostyla possibly has a high phylogenetic position within the genus Urosoma , or perhaps it represents a distinct subgenus.

Morphological Studies on Five Trachelocercids from the Yellow Sea coast of China, with a Description of Tracheloraphis huangi spec. nov. (Ciliophora, Karyorelictea)

The morphology and infraciliature of five trachelocercid ciliates: Tracheloraphis huang i spec. nov., T. colubis (Kahl, 1933) comb. nov., T. phoenicopterus (Cohn, 1866) Dragesco, 1960, T. oligostriata (Raikov, 1962) Foissner and Dragesco, 1996 and Trachelocerca incaudata Kahl, 1933, isolated from the intertidal zone of a beach at Qingdao, China, were studied in live and protargol impregnated specimens. Tracheloraphis huangi spec. nov. was distinguished from its congeners mainly by its single nuclear group composed of 25–30 round macronuclei and 29–37 somatic kineties. The poorly known T. colubis is redescribed including for the first time information on its infraciliature. An improved diagnosis is also provided. In light of its infraciliature, T. colubis is transferred to Tracheloraphis from the genus Trachelocerca . Additional data on other three species is supplied based on the Qingdao populations.

Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity

To assess the suitability of an antiseptic agent, both the microbicidal activity and the cytotoxic effect must be taken into consideration to derive biocompatible antibacterial agents. We defined the biocompatibility index (BI) by measuring the antibacterial activity against the test organisms Escherichia coli and Staphylococcus aureus and, in parallel, the cytotoxicity on cultured murine fibroblasts. The antiseptic agents tested were benzalkonium chloride (BAC), cetylpyridinium chloride (CPC), chlorhexidine digluconate (CHX), mild silver protein (MSP), octenidine dihydrochloride (OCT), polyhexamethylene biguanide (PHMB), povidone iodine in solution [PVP-I(s)], povidone iodine in ointment [PVP-I(o)], silver nitrate (AgNO(3)), silver (I) sulfadiazine (SSD) and triclosan (TRI). Assays were carried out for 30 min of exposure at 37 degrees C in the presence of cell culture medium containing 10% fetal bovine serum. The resulting dimensionless BI was defined as the ratio of the concentration at which 50% of the murine fibroblasts are damaged and the microbicidal effect producing at least 3 log(10) (99.9%) reduction. The resulting rank ordering of BI for the ratio of fibroblast cytotoxicity to E. coli toxicity was OCT > PHMB > CHX > PVP-I(o) > PVP-I(s) > BAC > CPC > TRI > MSP and that to S. aureus was OCT > PHMB > CHX > CPC > PVP-I(o) > BAC > PVP(s) > TRI > MSP. OCT and PHMB were the most suitable agents with a BI greater than 1. The BI presented may be a useful tool to evaluate antiseptic agents for use in clinical practice.

An iatrogenic enchondroma? An unusual X-ray finding

In this unusual case, accumulation of silver nitrate used to treat over-granulation in a finger injury led to a near-misdiagnosis of a bony tumour on X-ray. This underlines the need to support X-ray results with a full clinical assessment.

Direct visualization of clay microfabric signatures driving organic matter preservation in fine-grained sediment

We employed direct visualization of organic matter (OM) sequestered by microfabric signatures in organo-clay systems to study mechanisms of OM protection. We studied polysaccharides, an abundant class of OM in marine sediments, associated with the nano- and microfabric of clay sediment using a novel application of transmission electron microscopy, histochemical staining (periodic acid-thiosemicarbazide-silver proteinate), and enzymatic digestion techniques. We used two experimental organo-clay sediment environments. First, laboratory-consolidated sediment with 10% chitin (w/w) added was probed for chitin before and after digestion with chitinase. Second, fecal pellets from the polychaete Heteromastus filiformis were used as a natural environment rich in clay and polysaccharides. Sections of this material were probed with silver proteinate for polysaccharides before and after digestion with a mixture of enzymes (amylase, cellulase, chitinase, dextranase, and pectinase). In both environments, chitin or other polysaccharides were found within pores, bridging clay domains, and attached to clay surfaces in undigested samples. Digested samples showed chitin or polysaccharides more closely associated with clay surfaces and in small pores. Our results imply protective roles for both sorption to clay surfaces and encapsulation within clay microfabric signatures.

Critical Observations on the Neurotoxicity of Silver

Silver is a xenobiotic element with no recognized trace metal value in the human body. It is absorbed into the body through the lungs, gastrointestinal tract, mucus membranes of the urinogenital tract, and through the skin, mainly in the form of silver protein complexes. Although silver is metabolized throughout the soft tissues, available evidence from experimental animal studies and human clinical reports has failed to unequivocally establish that it enters tissues of the central nervous system or is a cause of neurotoxic damage. Argyria characterized by deposition of particles of silver sulfide or silver selenide is the principle contraindication for using silver in medical devices or occupationally. This presents discoloration of the skin but is not regarded as a health risk or manifestation of toxicity. No evidence is available to demonstrate the toxic risk of silver to the peripheral nervous system, although silver sulfide deposits have been identified in the region of cutaneous nerves. Transitory silver sulfide deposits seen in the tissues of the blood–brain and blood–CSF barriers are mostly lysosomally bound or deposited on basement membranes or collagen without toxic effect. Silver is mostly excreted from the body in the urine and feces. Further research is indicated to evaluate the role of metal binding proteins including metallothioneins as cytoprotectants for neurological tissue.

A Case of Generalized Argyria Caused by the Use of Silver Protein as a Disinfection Medicine

A Case of Generalized Argyria Caused by the Use of Silver Protein as a Disinfection Medicine

Nanotechnologic Nutraceuticals: Nurturing or Nefarious?

The use of nutraceuticals (herbal medicines, minerals, and vitamins) has increased dramatically, and it is estimated that approximately one third of Americans consume some form of dietary supplementation (1). Adverse clinical side effects of nutraceutical ingestion have been well documented (2), as have interferences from these agents in laboratory tests (3). Colloidal suspensions of metal particles (e.g., copper, gold, platinum, silver, molybdenum, palladium, titanium, and zinc) have been marketed as oral health supplements (2)(4). Metal colloids are reactive and can act as reducing agents, bind to proteins, and denature enzymes, and they are efficacious as bactericides in topical formulations (2); however, the oral administration of metallic colloids, in particular colloidal silver protein, has been reported to have toxic effects (2). Despite this, dozens of companies sell metal colloids as nutritional supplements. …

Ultrastructural analysis of the behavior of the dimorphic fungus Microbotryum violaceum in fungus-induced anthers of female Silene latifolia flowers

The development of male organs is induced in female flowers of the dioecious plant Silene latifolia by infection with the fungus Microbotryum violaceum. Stamens in a healthy female flower grow only to stage 6, whereas those in an infected female flower develop to the mature stage (stage 12), at which the stamens are filled with fungal teliospores instead of pollen grains. To investigate these host-parasite interactions, young floral buds and fungus-induced anthers of infected female flowers were examined by electron microscopy following fixation by a high-pressure freezing method. Using this approach, we found that parasitic hyphae of this fungus contain several extracellular vesicles and have a consistent appearance up to stage 8. At that stage, parasitic hyphae are observed adjacent to dying sporogenous cells in the infected female anther. At stage 9, an increased number of dead and dying sporogenous cells is observed, among which the sporogenous hyphae of the fungus develop and form initial teliospores. Several types of electron-dense material are present in proximity to some fungi at this stage. The initial teliospores contain two types of vacuoles, and the fungus cell wall contains abundant carbohydrate, as revealed by silver protein staining. The sporogenous cell is probably sensitive to infection by the fungus, resulting in disruption. In addition, the fungus accelerates cell death in the anther and utilizes constituents of the dead host cell to form the mature teliospore.

A mutation in the silver gene leads to defects in melanosome biogenesis and alterations in the visual system in the zebrafish mutant fading vision

Forward genetic screens have been instrumental in defining molecular components of visual function. The zebrafish mutant fading vision ( fdv) has been identified in such a screen due to defects in vision accompanied by hypopigmentation in the retinal pigment epithelium (RPE) and body melanocytes. The RPE forms the outer most layer of the retina, and its function is essential for vision. In fdv mutant larvae, the outer segments of photoreceptors are strongly reduced in length or absent due to defects in RPE cells. Ultrastructural analysis of RPE cells reveals dramatic cellular changes such as an absence of microvilli and vesicular inclusions. The retinoid profile is altered as judged by biochemical analysis, arguing for a partial block in visual pigment regeneration. Surprisingly, homozygous fdv vision mutants survive to adulthood and show, despite a persistence of the hypopigmentation, a partial recovery of retinal morphology. By positional cloning and subsequent morpholino knock-down, we identified a mutation in the silver gene as the molecular defect underlying the fdv phenotype. The Silver protein is required for intralumenal fibril formation in melanosomes by amylogenic cleavage. Our data reveal an unexpected link between melanosome biogenesis and the visual system, undetectable in cell culture.

Epitope Mapping of the Melanosomal Matrix Protein gp100 (PMEL17): RAPID PROCESSING IN THE ENDOPLASMIC RETICULUM AND GLYCOSYLATION IN THE EARLY GOLGI COMPARTMENT

Melanosomes, specific organelles produced only by melanocytes, undergo a unique maturation process that involves their transition form amorphous rounded vesicles to fibrillar ellipsoid organelles, during which they move from the perinuclear to the distal areas of the cells. This depends upon the trafficking and processing of gp100 (also known as Pmel17 and the silver protein), a protein of great interest, because it elicits immune responses in melanoma patients but in which specific function(s) remains elusive. In this study, we have used biochemical and immunochemical approaches to more critically assess the synthesis, processing, glycosylation, and trafficking of gp100. We now report that gp100 is processed and sorted in a manner distinct from other melanosomal proteins (such as tyrosinase, Tyrp1 and Dct) and is predominantly delivered directly to immature melanosomes following its rapid processing in the endoplasmic reticulum and cis-Golgi. Following its arrival, gp100 is cleaved at the amino and at the carboxyl termini in a series of specific steps that result in the reorganization of immature melanosomes to the fibrillar mature melanosomes. Once this structural reorganization occurs, melanogenic enzymes begin to be targeted to the melanosomes, which are then competent to synthesize melanin pigment.

Sonographic Appearance of Injection and Natural Edema in the Udder of Show Cows

Informal reports indicate that foreign materials are injected into sites of fore and rear udder attachment of show cows to augment the contour of the udder. These substances cause localized edema. This study evaluated the sonographic appearance and pattern of distribution of edema produced by subcutaneous injection with mild silver protein suspension compared to natural causes.

Ultrasonographic appearance of edema caused by injections in the mammary gland attachments of dairy cows.

To determine the ultrasonographic appearance and detectability of edema induced by SC injection of mild silver protein suspension in the mammary gland attachments of dairy cows. Prospective study. 6 lactating cows. In each cow, the number of quarters that received injections was randomly assigned. A mild silver protein susoension was injected SC into cranial and caudal mammary gland attachment sites. The number of injections and volume injected were determined on the basis of the appearance of the mammary gland and the desired subjective visual effect. Seventeen sites were chosen for injection and 7 sites did not receive injections. Ultrasonographic images were obtained 1 day prior and 6 days after injections were started. Cows received injections 1, 3, and 5 days after initial sonography. The sonographer was unaware of which sites received injections. Ultrasonography revealed alternating hypoechoic and hyperechoic bands at injection sites. Certain injections caused the intimal surface of the subcutaneous abdominal vein to develop a corrugated appearance. All injection sites were correctly identified ultrasonographically (100% sensitivity, 100% specificity) with a positive and negative predictive value of 1.0. Results suggest that mild silver protein suspension injected SC to enhance the appearance of the mammary glands of dairy cows can be readily detected by ultrasonography. Detection of injection sites should be made on the basis of the distribution and ultrasonographic appearance of edema.

Ultrastructure and function of Sertoli cell processes in the Korean native goat.

The ultrastructure of Sertoli cell in the testes of Korean native goats was investigated by scanning and transmission electron microscopy to elucidate the relationship between germ cell and Sertoli cell processes in the spermiogenetic cycle. Type-A Sertoli cells at stage V of the spermiogenetic cycle and type-B Sertoli cells at stage VII of the spermiogenetic cycle were used for analysis. Morphologically, the Sertoli cell processes were classified into sheet-like and slender cord-like processes. The sheet-like process originated solely from the Sertoli cell column while the slender cord-like process projected either from the Sertoli cell column or the sheet-like process. Periodic acid (PA)-thiocarbohydrazide (TCH)-silver protein (SP)-physical development (PD)-positive granules were found diffusively both in the sheet-like and slender cord-like processes near the round spermatid, whereas they had accumulated near the head of the elongated spermatid. The morphological variation and glucoconjugate histochemical reaction of the Sertoli cell processes reflect nourishment, movement and transformation of the spermatogenic cells in accordance with spermiogenesis.

Silver Proteinate Impregnation in Under Two Hours

A new silver impregnation technique for demonstrating nerve fibers in the brain is described. This silver impregnation technique, being argyrophilic, includes mordant and oxidizing steps, followed by decolorizing. The silver proteinate impregnation may be repeated if stronger staining is desired. Toning, developing and fixation steps complete the procedure. The silver impregnation, reduction and development are conducted in a water bath at 60°C after an initial boost with a microwave oven at 480 watts for 35 sec (equivalent to 4 kcal of energy). No silver ion “enhancers” (such as copper shots) are needed. The procedure can be completed in less than 2 hours with consistent results. (The J Histotechnol 24:259, 2001)Submitted: March 20, 2001; Accepted May 4, 2001

Silver Proteinate Impregnation in Under Two Hours

A new silver impregnation technique for demonstrating nerve fibers in the brain is described. This silver impregnation technique, being argyrophilic, includes mordant and oxidizing steps, followed by decolorizing. The silver proteinate impregnation may be repeated if stronger staining is desired. Toning, developing and fixation steps complete the procedure. The silver impregnation, reduction and development are conducted in a water bath at 60°C after an initial boost with a microwave oven at 480 watts for 35 sec (equivalent to 4 kcal of energy). No silver ion “enhancers” (such as copper shots) are needed. The procedure can be completed in less than 2 hours with consistent results. (The J Histotechnol 24:259, 2001)Submitted: March 20, 2001; Accepted May 4, 2001

Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia transcription factor.

H(2)O(2) and other reactive oxygen species are key regulators of many intracellular pathways. Within mammalian skin, H(2)O(2) is formed as a byproduct of melanin synthesis, and following u.v. irradiation. We therefore analyzed its effects on melanin synthesis. The activity of the rate-limiting melanogenic enzyme, tyrosinase, decreased in H(2)O(2)-treated mouse and human melanoma cells. This inhibition was concentration- and time-dependent in the B16 melanoma model. Maximal inhibition (50-75%) occurred 8-16 hours after a 20 minute exposure to 0.5 mM H(2)O(2). B16 cells withstand this treatment adequately, as shown by a small effect on glutathione levels and a rapid recovery of basal lipid peroxidation levels. Enzyme activities also recovered, beginning to increase 16-20 hours after the treatment. Inhibition of enzyme activities reflected decreased protein levels. mRNAs for tyrosinase, tyrosinase-related protein 1, dopachrome tautomerase, silver protein and melanocortin 1 receptor also decreased after H(2)O(2) treatment, and recovered at different rates. Downregulation of melanocyte differentiation markers mRNAs was preceded by a decrease in microphthalmia transcription factor (Mitf) gene expression, which was quantitatively similar to the decrease achieved using 12-O-tetradecanoylphorbol-13-acetate. Recovery of basal Mitf mRNA levels was also observed clearly before that of tyrosinase. Therefore, oxidative stress may lead to hypopigmentation by mechanisms that include a microphthalmia-dependent downregulation of the melanogenic enzymes.

Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin.

Melanocytes produce two chemically distinct types of melanin pigments, eumelanin and pheomelanin. These pigments can be quantitatively analyzed by acidic permanganate oxidation or reductive hydrolysis with hydriodic acid to form pyrrole-2,3,5-tricarboxylic acid or aminohydroxyphenylalanine, respectively. About 30 coat color genes in mice have been cloned, and functions of many of those genes have been elucidated. However, little is known about the interacting functions of these loci. In this study, we used congenic mice to eliminate genetic variability, and analyzed eumelanin and pheomelanin contents of hairs from mice mutant at one or more of the major pigment loci, i.e., the albino (C) locus that encodes tyrosinase, the slaty (Slt) locus that encodes tyrosinase-related protein 2 (TRP2 also known as dopachrome tautomerase, DCT), the brown (B) locus that encodes TRP1, the silver (Si) locus that encodes a melanosomal silver protein, the agouti (A) locus that encodes agouti signaling protein (ASP), the extension (E) locus that encodes melanocortin-1 receptor, and the mahogany (Mg) locus that encodes attractin. We also measured total melanin contents after solubilization of hairs in hot Soluene-350 plus water. Hairs were shaved from 2-3-month-old congenic C57BL/6J mice. The chinchilla (c(ch)) allele is known to encode tyrosinase, whose activity is about one third that of wild type (C). Phenotypes of chinchilla (c(ch)/c(ch)) mice that are wild type or mutant at the brown and/or slaty, loci indicate that functioning TRP2 and TRP1 are necessary, in addition to high levels of tyrosinase, for a full production of eumelanin. The chinchilla allele was found to reduce the amount of pheomelanin in lethal yellow and recessive yellow mice to less than one fifth of that in congenic yellow mice that were wild type at the albino locus. This indicates that reduction in tyrosinase activity affects pheomelanogenesis more profoundly compared with eumelanogenesis. Hairs homozygous for mutation at the slaty locus contain 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-poor melanin, and this chemical phenotype was retained in hairs that were mutant at both the brown locus and the slaty locus. Hair from mice mutant at the brown locus, but not at the slaty locus, do not contain DHICA-poor melanin. This indicates that the proportion of DHICA in eumelanin is determined by TRP2, but not by TRP1. Mutation at the slaty locus (Slt(lt)) was found to have no effect on pheomelanogenesis, supporting a role of TRP2 only in eumelanogenesis. The mutation at silver (si) locus showed an effect similar to brown, a partial suppression of eumelanogenesis. The mutation at mahogany (mg) locus partially suppressed the effect of lethal yellow (Ay) on pheomelanogenesis, supporting a role of mahogany in interfering with agouti signaling. These results show that combination of double mutation study of congenic mice with chemical analysis of melanins is useful in evaluating the interaction of pigment gene functions.

Development of bone canaliculi during bone repair

We recently found that silver impregnation staining with protargol (silver protein), that is, a modified Bodian method, is useful for histologically identifying the details of bone canaliculi structure, using thin sections of decalcified bone tissues. With this staining method, we conducted the present study to assess the development of bone canaliculi during the process of intramembranous ossification using a fracture-like stimulation model of the rat femur. After making a drill-hole in the cortex of the rat femur, decalcified thin sections were obtained after 3, 5, 7, and 14 days by the standard paraffin-embedding procedure. Silver staining for bone canaliculi was performed using our previously reported technique. The results showed that woven bone covered the fracture surface of the cortex after 5 days, then immature lamellar bone attached to the woven bone after 7 days, and finally the lamellar bone matured and became thick with appositional growth after 14 days. The osteocytes in the woven bone appeared at an early stage of bone repair and developed a few canaliculi that were short and irregularly distributed in the osteoid matrix, while the osteocytes in the lamellar bone at a late stage formed many bone canaliculi that were long and regularly distributed in mature bone matrix. Therefore, we concluded that woven bone osteocytes may be necessary for induction of the lamellar bone osteocytes followed by active appositional growth of the lamellar bone at the early stage of bone repair, and also that both bone tissues could be clearly distinguished from one another based on the pattern of development of bone canaliculi by the osteocytes, as seen with the use of our sensitive staining method.

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO Red staining

The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies. Copyright © 2000 John Wiley & Sons, Ltd.