Fabrication of Chinese Traditional Medicines incorporated collagen biomaterials for human bone marrow mesenchymal stem cells.

Chinese Traditional Medicines (CTMs) are very popular for therapeutic applications to cure several chronic diseases. Many researchers are trying to discover the potential application and actual mechanism of CTMs in order to scientifically prove their effects for commercial use. One of the main functions of CTMs is to aid stem cell regeneration. Since, this study was focused to fabricate CTMs incorporated fish collagen film, which has good biocompatibility in mammalian cell growth and thus investigated the effect on human Mesenchymal stem cells (hMSCs) proliferation and differentiation. In this study, three types of CTMs such as Genistein, Icariin, and Naringin were used for film fabrication. Mechanical properties of collagen films were improved by the addition of CTMs, especially in Collagen-Naringin films. Solubility and In-vitro biodegradation of collagen films were enhanced by the hydrophobicity and chemical interaction of CTMs with collagen. The proliferation rate was accelerated in hMSCs cultured on CTMs incorporated collagen films in a dose- and time-dependent manner. Proliferation biomarkers such as Ki-67 and BrdU levels were higher in hMSCs cultured on CTMs incorporated collagen films. The proliferative and differentiation effect of CTMs was further confirmed by higher gene expression of Collagen I, Runx2, c-Fos, SMAD3 and TGF-ß1 in hMSCs. Overall, this study provides a new insight on novel biomaterial fabrication using CTMs and fish collagen for making a compatible platform for in-vitro stem cell culture.

A Triassic stem-salamander from Kyrgyzstan and the origin of salamanders.

The origin of extant amphibians remains largely obscure, with only a few early Mesozoic stem taxa known, as opposed to a much better fossil record from the mid-Jurassic on. In recent time, anurans have been traced back to Early Triassic forms and caecilians have been traced back to the Late Jurassic Eocaecilia, both of which exemplify the stepwise acquisition of apomorphies. Yet the most ancient stem-salamanders, known from mid-Jurassic rocks, shed little light on the origin of the clade. The gap between salamanders and other lissamphibians, as well as Paleozoic tetrapods, remains considerable. Here we report a new specimen of Triassurus sixtelae, a hitherto enigmatic tetrapod from the Middle/Late Triassic of Kyrgyzstan, which we identify as the geologically oldest stem-group salamander. This sheds light not only on the early evolution of the salamander body plan, but also on the origin of the group as a whole. The new, second specimen is derived from the same beds as the holotype, the Madygen Formation of southwestern Kyrgyzstan. It reveals a range of salamander characters in this taxon, pushing back the rock record of urodeles by at least 60 to 74 Ma (Carnian-Bathonian). In addition, this stem-salamander shares plesiomorphic characters with temnospondyls, especially branchiosaurids and amphibamiforms.

The last Palaeoproteus (Urodela: Batrachosauroididae) of Europe.

The Batrachosauroididae are an enigmatic group of salamanders known from the Cretaceous and Tertiary of North America and Europe. In Europe, the family is known only by two species of the genus Palaeoproteus. The genus has limited distribution in Western and Central Europe. In the present paper, we describe a new species, Palaeoproteus miocenicus, from the early late Miocene (11-9 Ma) of Austria and Ukraine, representing the youngest record of the family Batrachosauroididae from the Neogene of Europe. The new species differs from the Paleogene representatives of the genus by 12 characters, including large body size, the long anterior extension of the Meckelian groove and the size and shape of the odontoid process on the dentary. The µCT scanning of bones of the new species revealed novel features (e.g. anterior extension of Meckelian groove, interconnected network of canals and small cavities in atlas) observable only in this species. P. miocenicus inhabited aquatic environments, which existed under wet climatic conditions with mean annual precipitation higher than 900 mm. The new species expands the temporal range of the genus by at least 30 million years and enlarges the palaeogeographic distribution of the genus into Eastern Europe.

Selenium Bioaccumulation Across Trophic Levels and Along a Longitudinal Gradient in Headwater Streams.

Toxic effects of selenium (Se) contamination in freshwaters have been well documented. However, study of Se contamination has focused on lentic and larger order lotic systems, whereas headwater streams have received little scrutiny. In central Appalachia, surface coal mining is a common Se source to headwater streams, thus providing a useful system to investigate Se bioaccumulation in headwater food chains and possible longitudinal patterns in Se concentrations. Toward that end, we assessed Se bioaccumulation in 2 reference and 4 mining-influenced headwater streams. At each stream, we sampled ecosystem media, including streamwater, particulate matter (sediment, biofilm, leaf detritus), benthic macroinvertebrates, salamanders, and fish, every 400 m along 1.2- and 1.6-km reaches. We compared media Se concentrations within and among streams and evaluated longitudinal trends in media Se concentrations. Selenium concentrations in sampled media were higher in mining-influenced streams compared with reference streams. We found the highest Se concentrations in benthic macroinvertebrates; however, salamanders and fish bioaccumulated Se to potentially harmful levels in mining-influenced streams. Only one stream demonstrated dilution of streamwater Se with distance downstream, and few longitudinal patterns in Se bioaccumulation occurred along our study reaches. Collectively, our results provide a field-based assessment of Se bioaccumulation in headwater food chains, from streamwater to fish, and highlight the need for future assessments of Se effects in headwater streams and receiving downstream waters. Environ Toxicol Chem 2020;39:692-704. © 2020 SETAC.

Structure and mode of action of a novel antibacterial peptide from the blood of Andrias davidianus.

Andrias davidianus is widely recognized in traditional medicine as a cure-all to treat a plethora of ailments. In a previous study, a novel antibacterial peptide named andricin B was isolated from A. davidianus blood. In this study, we investigated andricin B structure and its mode of action. Circular dichroism spectra suggested that andricin B adopts a random coil state in aqueous solution and a more rigid conformation in the presence of bacteria. Moreover propidium iodide/fluorescein diacetate double staining indicated that bacteria treated with andricin B were not immediately eliminated. Rather, there is a gradual bacterial death, followed by a sublethal stage. Scanning electronic microscope imaging indicates that andricin B might form pores on cell membranes, leading to the release of cytoplasmic contents. These results were consistent with flow cytometry analysis. Furthermore, Fourier transform infrared spectroscopy suggests that andricin B induces changes in the chemical properties in the areas surrounding these "pores" on the cell membranes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study suggested the new perspectives about the mode of action of antimicrobial peptide (AMP) active against sensitive bacteria. The AMP was able to be in a random coiled state in aqueous solution but to change to a more rigid one in the presence of sensitive bacteria. Exposure to AMP might not lead to immediate death of treated bacteria, rather bacteria concentration decreased gradually flattening at a sublethal stage. These findings will help people to understand better how the AMPs activate against sensitive bacteria.

Purification, characterization and application of a novel antimicrobial peptide from Andrias davidianus blood.

The Andrias davidianus has been known as a traditional Chinese medicine for a long time. Its blood is considered as a waste or by-product of the meat production industry. Although there are reports on isolation of the antimicrobial peptides from different resources, there are no reports of their isolation from A. davidianus blood. In this work, an antimicrobial peptide, andricin B, was isolated from the blood of A. davidianus by an innovative method in which the magnetic liposome adsorption was combined with reversed-phase high-performance liquid chromatography. The structure, antimicrobial activity and safety of andricin B were further investigated. Amino acid sequence was determined by N-terminal sequencing and found to be Gly-Leu-Thr-Arg-Leu-Phe-Ser-Val-Ile-Lys. Circular dichroism (CD) spectra and prediction of three-dimensional structure by bioinformatics software suggested the presence of a well-defined random coil conformation. Andricin B was found to be active against all bacteria tested in this study as well as some fungi. The minimum inhibitory concentrations (MICs) were in the range 8-64 µg ml-1 . Moreover, the haemolytic testing also suggested that andricin B could be considered safe at the MICs. Finally, andricin B was shown to inhibit the growth of Staphylococcus aureus in the cooked meat of A. davidianus. This study shows that andricin B is a promising novel antimicrobial peptide that may provide further insights towards the development of new drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the pioneer study on screening and isolation of antimicrobial peptide from the blood of Andrias davidianus. Here, we have developed a novel method by combining magnetic liposomes adsorption with reversed-phase high-performance liquid chromatography to purify and screen the antimicrobial peptides. From this screen, we identified a novel antimicrobial peptide which we name as andricin B. Andricin B is unique as it checks the growth of both Gram-positive and Gram-negative bacteria as well as few fungal species.

Antimicrobial peptide from mucus of Andrias davidianus: screening and purification by magnetic cell membrane separation technique.

Andrias davidianus, the Chinese giant salamander, has been used in traditional Chinese medicine for many decades. However, no antimicrobial peptides (AMPs) have been described from A. davidianus until now. Here we describe a novel AMP (andricin 01) isolated from the mucus of A. davidianus. The peptide was recovered using an innovative magnetic cell membrane separation technique and was characterised using mass spectrometry and circular dichroism (CD) spectroscopy. Andricin 01 is comprised of ten amino acid residues with a total molecular mass of 955.1 Da. CD spectrum analysis gave results similar to the archetypal random coil spectrum, consistent with the three-dimensional rendering calculated by current bioinformatics tools. Andricin 01 was found to be inhibitory both to Gram-negative and Gram-positive bacteria. Furthermore, the peptide at the minimal bacterial concentration did not show cell cytotoxicity against human hepatocytes or renal cells and did not show haemolytic activity against red blood cells, indicating that is potentially safe and effective for human use. Andricin 01 shows promise as a novel antibacterial that may provide an insight into the development of new drugs.

Lesions of the dorsal striatum impair orienting behaviour of salamanders without affecting visual processing in the tectum.

In amphibians, visual information in the midbrain tectum is relayed via the thalamus to telencephalic centres. Lesions of the dorsal thalamus of the salamander Plethodon shermani result in impairment of orienting behaviour and in modulation of spike pattern of tectal neurons. These effects may be induced by an interruption of a tectum-thalamus-telencephalon-tectum feedback loop enabling spatial attention and selection of visual objects. The striatum is a potential candidate for involvement in this pathway; accordingly, we investigated the effects of lesioning the dorsal striatum. Compared to controls and sham lesioned salamanders, striatum-lesioned animals exhibited a significantly lower number of orienting responses toward one of two competing prey stimuli. Orienting towards stimuli was impaired, while the spike pattern of tectal cells was unaffected, because both in controls and striatum-lesioned salamanders the spike number significantly decreased at presentation of one prey stimulus inside the excitatory receptive field and another one in the surround compared to that at single presentation inside the excitatory receptive field. We conclude that the dorsal striatum contributes to orienting behaviour, but not to an inhibitory feedback signal onto tectal neurons. The brain area engaged in the feedback loop during visual object discrimination and selection has yet to be identified. Information processing in the amphibian striatum includes multisensory integration; the striatum generates behavioural patterns that influence (pre)motor processing in the brainstem. This situation resembles the situation found in rats, in which the dorsolateral striatum is involved in stimulus-response learning regardless of the sensory modality, as well as in habit formation.

Three ferritin subunit analogs in Chinese giant salamander (Andrias davidianus) and their response to microbial stimulation.

Ferritin, an evolutionarily conserved iron-binding protein, plays important roles in iron storage and detoxification and in host immune response to invading stimulus as well. In the present study, we identified three ferritin subunit analog cDNAs from Chinese giant salamander (Andrias davidianus). All the three ferritin subunit cDNAs had a putative iron responsive element in the 5'-untranslated region. Two deduced ferritin subunits (designated as cgsFerH and cgsFerM) had the highest identity of 90% to H type subunit of vertebrate ferritins, while another deduced ferritin subunit (designated as cgsFerL) had the highest identity of 84% to L type subunit of vertebrate ferritins. The Chinese giant salamander ferritin (cgsFer) was widely expressed in various tissues, with highest expression for cgsFerH and cgsFerL in liver and highest expression for cgsFerM in spleen. Infection of Chinese giant salamander with A. davidianus ranavirus showed significant induction of cgsFer expression. Both lipopolysaccharide and iron challenge drastically augmented cgsFer expression in the splenocytes and hepatocytes from Chinese giant salamander. In addition, recombinant cgsFers bound to ferrous iron in a dose-dependent manner, with significant ferroxidase activity. Furthermore, the recombinant cgsFer inhibited the growth of the pathogen Vibrio anguillarum. These results indicated that cgsFer was potential candidate of immune molecules involved in acute phase response to invading microbial pathogens in Chinese giant salamander possibly through its regulatory roles in iron homeostasis.

Successful treatment of Batrachochytrium salamandrivorans infections in salamanders requires synergy between voriconazole, polymyxin E and temperature.

Chytridiomycosis caused by the chytrid fungus Batrachochytrium salamandrivorans (Bsal) poses a serious threat to urodelan diversity worldwide. Antimycotic treatment of this disease using protocols developed for the related fungus Batrachochytrium dendrobatidis (Bd), results in therapeutic failure. Here, we reveal that this therapeutic failure is partly due to different minimum inhibitory concentrations (MICs) of antimycotics against Bsal and Bd. In vitro growth inhibition of Bsal occurs after exposure to voriconazole, polymyxin E, itraconazole and terbinafine but not to florfenicol. Synergistic effects between polymyxin E and voriconazole or itraconazole significantly decreased the combined MICs necessary to inhibit Bsal growth. Topical treatment of infected fire salamanders (Salamandra salamandra), with voriconazole or itraconazole alone (12.5 µg/ml and 0.6 µg/ml respectively) or in combination with polymyxin E (2000 IU/ml) at an ambient temperature of 15 °C during 10 days decreased fungal loads but did not clear Bsal infections. However, topical treatment of Bsal infected animals with a combination of polymyxin E (2000 IU/ml) and voriconazole (12.5 µg/ml) at an ambient temperature of 20 °C resulted in clearance of Bsal infections. This treatment protocol was validated in 12 fire salamanders infected with Bsal during a field outbreak and resulted in clearance of infection in all animals.

Asbestos between science and myth. A 6,000-year story.

Asbestos was used in making pottery in Eastern Finland from around 4000 B.C. In the ancient era and in the Middle Ages, magic properties were frequently attributed to this mineral. In the first century A.D., the Latin encyclopaedist Pliny the Elder reported in his Historia Naturalis that asbestos protects against all poisonings, particularly that of magicians. Moreover, asbestos was often found in places of worship, in Rome as well as in Athens and in Jerusalem. In the Middle Ages asbestos was identified with some animals, such as the salamander and certain white rodents. With such appearance, the mineral  had a huge success in Western as well as in Eastern literature and the fine arts. Marco Polo (1254-1324) in the Milione tried to deny that asbestos was a salamander. Despite its noxious effects, asbestos continues to be used in much of the world. In the 21st century it seems to be maintaining its quality as a magic stone.

Salamander growth rates increase along an experimental stream phosphorus gradient.

Nutrient-driven perturbations to the resource base of food webs are predicted to attenuate with trophic distance, so it is unclear whether higher-level consumers will generally respond to anthropogenic nutrient loading. Few studies have tested whether nutrient (specifically, nitrogen [N] and phosphorus [P]) enrichment of aquatic ecosystems propagates through multiple trophic levels to affect predators, or whether N vs. P is relatively more important in driving effects on food webs. We conducted two-year whole-stream N and P additions to five streams to generate gradients in N and P concentration and N:P ratio (target N:P = 2, 8, 16, 32, 128). Larval salamanders are vertebrate predators of primary and secondary macroinvertebrate consumers in many heterotrophic headwater streams in which the basal resources are detritus and associated microorganisms. We determined the effects of N and P on the growth rates of caged and free-roaming larval Desmognathus quadramaculatus and the average body size of larval Eurycea wilderae. Growth rates and average body size increased by up to 40% and 60%, respectively, with P concentration and were negatively related to N:P ratio. These findings were consistent across both species of salamanders using different methodologies (cage vs. free-roaming) and at different temporal scales (3 months vs. 2 yr). Nitrogen concentration was not significantly related to increased growth rate or body size of the salamander species tested. Our findings suggest that salamander growth responds to the relaxation of ecosystem-level P limitation and that moderate P enrichment can have relatively large effects on vertebrate predators in detritus-based food webs.

Stoichiometry of a semi-aquatic plethodontid salamander: intraspecific variation due to location, size and diet.

Ecological stoichiometry provides a framework to investigate an organism's relationship to nutrient cycles. An organism's stoichiometry is thought to constrain its contribution to nutrient cycles (recycling or storage), and to limit its growth and reproduction. Factors that influence the stoichiometry of a consumer are largely unstudied, but what is known is that consumer stoichiometry is influenced by the elemental requirements of the consumer (e.g. for growth, reproduction and cell maintenance) and the availability of elements. We examined whole-body stoichiometry of larval southern two-lined salamanders (Eurycea cirrigera) and described the influence of location, body size, stoichiometry of diet items, and environmental nutrient supply on whole-body stoichiometry. Mean composition of phosphorous was 2.6%, nitrogen was 11.3%, and carbon was 39.6%, which are similar for other aquatic vertebrate taxa. The most significant predictor of whole-body stoichiometry was the site where the samples were collected, which was significant for each nutrient and nutrient ratio. Body size and stoichiometry of diet items were also predictors of Eurycea cirrigera stoichiometry. Our study suggests that spatial differences in environmental nutrient supply have a stronger influence on consumer whole-body stoichiometry among similar-sized larvae compared to life history traits, such as body size or diet.

Modulation of ionotropic glutamate receptor function by vertebrate galectins.

AMPA and kainate receptors are glutamate-gated ion channels whose function is known to be altered by a variety of plant oligosaccharide-binding proteins, or lectins, but the physiological relevance of this activity has been uncertain because no lectins with analogous allosteric modulatory effects have been identified in animals. We report here that members of the prototype galectin family, which are ß-galactoside-binding lectins, exhibit subunit-specific allosteric modulation of desensitization of recombinant homomeric and heteromeric AMPA and kainate receptors. Galectin modulation of GluK2 kainate receptors was dependent upon complex oligosaccharide processing of N-glycosylation sites in the amino-terminal domain and downstream linker region. The sensitivity of GluA4 AMPA receptors to human galectin-1 could be enhanced by supplementation of culture media with uridine and N-acetylglucosamine (GlcNAc), precursors for the hexosamine pathway that supplies UDP-GlcNAc for synthesis of complex oligosaccharides. Neuronal kainate receptors in dorsal root ganglia were sensitive to galectin modulation, whereas AMPA receptors in cultured hippocampal neurons were insensitive, which could be a reflection of differential N-glycan processing or receptor subunit selectivity. Because glycan content of integral proteins can be modified dynamically, we postulate that physiological or pathological conditions in the CNS could arise in which galectins alter excitatory neurotransmission or neuronal excitability through their actions on AMPA or kainate receptors.

Intraspecific variation overrides origin effects in impacts of litter-derived secondary compounds on larval amphibians.

Secondary compounds leached from plant litter can negatively affect aquatic amphibian larvae. Non-native plants and their potentially distinct secondary compounds may constitute cryptic threats to native amphibians. We used the availability of both native and introduced Phragmites australis (common reed) populations in North America to assess the importance of origin, intraspecific variation, and two purified classes of compounds (tannins and saponins; gradients 0-25 mg L(-1)) on two common and widespread amphibians (Ambystoma maculatum, spotted salamander, and Lithobates palustris, pickerel frog). In experiments with purified compounds, high tannin concentrations reduced A. maculatum survival and developmental rate while high saponin concentrations reduced survival, developmental rate, and size of L. palustris and reduced A. maculatum developmental rate. In experiments using leaf litter extracts of 14 different P. australis populations, A. maculatum larval survival varied among populations but plant origin (native or introduced) did not explain this variation. In contrast to the lack of effects of purified saponins, increases in saponin concentrations in P. australis leachates significantly decreased A. maculatum survival. Our results suggest: (1) secondary compounds can impact larval amphibian survival and development in species-specific ways; (2) impacts of P. australis on A. maculatum vary among P. australis populations, reflecting intraspecific variation in secondary chemistry; and (3) origin (whether the plant is native or introduced) is a poor predictor of P. australis effects on A. maculatum. Scientists and managers may need to move beyond considering origin as a predictive variable when managing plant communities to benefit amphibians.

Potential influence of plant chemicals on infectivity of Batrachochytrium dendrobatidis.

We explored whether extracts of trees frequently found associated with amphibian habitats in Australia and Arizona, USA, may be inhibitory to the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has been associated with global amphibian declines. We used salamanders Ambystoma tigrinum as the model system. Salamanders acquired significantly lower loads of Bd when exposed on leaves and extracts from the river red gum Eucalyptus camaldulensis, and loads were also low in some animals exposed on extracts of 2 oak species, Quercus emoryi and Q. turbinella. Some previously infected salamanders had their pathogen loads reduced, and some were fully cured, by placing them in leaf extracts, although some animals also self cured when housed in water alone. A significant number of animals cured of Bd infections 6 mo earlier were found to be resistant to reinfection. These results suggest that plants associated with amphibian habitats should be taken into consideration when explaining the prevalence of Bd in these habitats and that some amphibians may acquire resistance to the fungus if previously cured.

The role of the dorsal thalamus in visual processing and object selection: a case of an attentional system in amphibians.

In amphibians, the midbrain tectum is regarded as the visual centre for object recognition but the functional role of forebrain centres in visual information processing is less clear. In order to address this question, the dorsal thalamus was lesioned in the salamander Plethodon shermani, and the effects on orienting behaviour or on visual processing in the tectum were investigated. In a two-alternative-choice task, the average number of orienting responses toward one of two competing prey or simple configural stimuli was significantly decreased in lesioned animals compared to that of controls and sham-lesioned animals. When stimuli were presented during recording from tectal neurons, the number of spikes on presentation of a stimulus in the excitatory receptive field and a second salient stimulus in the surround was significantly reduced in controls and sham-lesioned salamanders compared to single presentation of the stimulus in the excitatory receptive field, while this inhibitory effect on the number of spikes of tectal neurons was absent in thalamus-lesioned animals. In amphibians, the dorsal thalamus is part of the second visual pathway which extends from the tectum via the thalamus to the telencephalon. A feedback loop to the tectum is assumed to modulate visual processing in the tectum and to ensure orienting behaviour toward visual objects. It is concluded that the tectum-thalamus-telencephalon pathway contributes to the recognition and evaluation of objects and enables spatial attention in object selection. This attentional system in amphibians resembles that found in mammals and illustrates the essential role of attention for goal-directed visuomotor action.

Extracts of the invasive shrub Lonicera maackii increase mortality and alter behavior of amphibian larvae.

Water-soluble phytochemicals produced by invasive plants may represent novel elements of invaded ecosystems that can precipitate a variety of direct and indirect effects on native organisms. Phenolic compounds in particular are a common plant defense, and these compounds may have disproportionate impacts on amphibians compared to other taxa. We coupled an exploration of invasive plant extract effects on larvae of four amphibian species (the salamander Ambystoma maculatum, the toad Anaxyrus americanus, and the frogs Hyla sp. and Lithobates blairi) with behavioral observations designed to determine whether behavior can ameliorate the negative effects of exposure to invasive plant extracts. Larvae were reared in extracts of the widespread invasive Amur honeysuckle (Lonicera maackii), mixed native leaf litter, and a water control. Anaxyrus americanus tadpoles reared in L. maackii extracts were more likely to die than tadpoles reared in native extracts, but differences in mortality following rearing in native and exotic extracts were not significant for the other three species. Anaxyrus americanus and L. blairi tadpoles made more trips to the surface in L. maackii extracts than in native extracts, consistent with the hypothesis that exotic extracts may interfere with respiratory physiology and suggesting that L. blairi can behaviorally ameliorate the negative effects of L. maackii extracts. Our study highlights both a direct and indirect pathway by which invasive plant extracts may alter the ecological dynamics of native fauna.

Organic preservation of fossil musculature with ultracellular detail.

The very labile (decay-prone), non-biomineralized, tissues of organisms are rarely fossilized. Occurrences thereof are invaluable supplements to a body fossil record dominated by biomineralized tissues, which alone are extremely unrepresentative of diversity in modern and ancient ecosystems. Fossil examples of extremely labile tissues (e.g. muscle) that exhibit a high degree of morphological fidelity are almost invariably replicated by inorganic compounds such as calcium phosphate. There is no consensus as to whether such tissues can be preserved with similar morphological fidelity as organic remains, except when enclosed inside amber. Here, we report fossilized musculature from an approximately 18 Myr old salamander from lacustrine sediments of Ribesalbes, Spain. The muscle is preserved organically, in three dimensions, and with the highest fidelity of morphological preservation yet documented from the fossil record. Preserved ultrastructural details include myofilaments, endomysium, layering within the sarcolemma, and endomysial circulatory vessels infilled with blood. Slight differences between the fossil tissues and their counterparts in extant amphibians reflect limited degradation during fossilization. Our results provide unequivocal evidence that high-fidelity organic preservation of extremely labile tissues is not only feasible, but likely to be common. This is supported by the discovery of similarly preserved tissues in the Eocene Grube Messel biota.

Immunohistochemical localization of calbindin-D28k and calretinin in the brainstem of anuran and urodele amphibians.

Calbindin-D28k (CB) and calretinin (CR) are calcium binding proteins present in distinct sets of neurons; they act as buffers regulating the concentration of intracellular calcium. CB and CR immunohistochemistry was studied in the brainstem of anuran and urodele amphibians in combination with other markers (choline acetyltransferase, tyrosine hydroxylase, and nitric oxide synthase), which served to clarify the localization and signature of many cell groups. CR labeled the retinorecipient layers of the optic tectum, and CB and CR labeled distinct tectal cell populations. The two proteins were largely complementary in the torus semicircularis and marked auditory and lateral line sensory regions, depending on the species. CB and CR in the mesencephalic and isthmic tegmentum specified the boundaries of basal and medial longitudinal bands. In the cerebellum, CB labeled Purkinje cells in all species, whereas CR was mainly found in fibers and labeled Purkinje cells only in Rana. In the parabrachial region, CB and CR allowed the distinction of the laterodorsal tegmental nucleus, isthmic nucleus, locus coeruleus, and rostral octavolateral nuclei. The distribution of CB- and CR-immunoreactive cells in the reticular formation and central gray was consistent with the current models of brainstem segmentation in amphibians. CR was found in the auditory fibers and nuclei in Rana and in mechanosensory lateral line fibers in Xenopus and urodeles, whereas CB mainly labeled vestibular fibers and nuclei in all species. These results highlight the anatomical complexity of the amphibian brainstem and help in an understanding of its regional organization that is not cytoarchitectonically evident.