Aging varies greatly within a single genus: A demographic study of Rhododendron spp. in botanic gardens.

PREMISE: There is mounting evidence that age matters in plant demography, but also indications that relationships between age and demographic rates may vary significantly among species. Age-based plant demographic data, however, are time-consuming to collect and still lacking for most species, and little is known about general patterns across species or what may drive differences. METHODS: We used individual birth and death records for 12 Rhododendron species from botanic gardens and conducted Bayesian survival trajectory analyses to assess how mortality changed with age. We calculated the demographic measures of aging rate, life-span equality, and life expectancy for each species, and assessed their relationships with the climatic conditions at species' sites of ancestral origin and with taxonomic group (subgenus). RESULTS: We found substantial among-species variation in survival trajectories, with mortality increasing, decreasing, or remaining constant with advancing age. Moreover, we found no relationships between demographic measures and ancestral climatic conditions but there were statistically significant differences among taxonomic groups in the rate of change in mortality with age (aging rate). CONCLUSIONS: We conclude that demographic consequences of aging can differ qualitatively, even among species in the same genus. In addition, taxonomic trends in aging rates indicate they may be genetically determined, though evolutionary drivers are still unclear. Furthermore, we suggest there is untapped potential in using botanic garden records in future studies on plant life history.

Data gaps and opportunities for comparative and conservation biology.

Biodiversity loss is a major challenge. Over the past century, the average rate of vertebrate extinction has been about 100-fold higher than the estimated background rate and population declines continue to increase globally. Birth and death rates determine the pace of population increase or decline, thus driving the expansion or extinction of a species. Design of species conservation policies hence depends on demographic data (e.g., for extinction risk assessments or estimation of harvesting quotas). However, an overview of the accessible data, even for better known taxa, is lacking. Here, we present the Demographic Species Knowledge Index, which classifies the available information for 32,144 (97%) of extant described mammals, birds, reptiles, and amphibians. We show that only 1.3% of the tetrapod species have comprehensive information on birth and death rates. We found no demographic measures, not even crude ones such as maximum life span or typical litter/clutch size, for 65% of threatened tetrapods. More field studies are needed; however, some progress can be made by digitalizing existing knowledge, by imputing data from related species with similar life histories, and by using information from captive populations. We show that data from zoos and aquariums in the Species360 network can significantly improve knowledge for an almost eightfold gain. Assessing the landscape of limited demographic knowledge is essential to prioritize ways to fill data gaps. Such information is urgently needed to implement management strategies to conserve at-risk taxa and to discover new unifying concepts and evolutionary relationships across thousands of tetrapod species.

Analysis of public discourse on heart transplantation in Japan using social network service data.

The clarification of public concerns regarding heart transplantation is important for improving low organ donation rates in Japan. In the present study, we used the Twitter data of 4986 tweets (between August 2015 and January 2016) and 1429 tweets (between April 2016 and May 2016) to analyze public discourse on heart transplantation in Japan and identify the reasons for low organ donation rates. We manually categorized all tweets relevant to heart transplantation into nine categories and counted the number of tweets in each category per month. During the study period, the most popular category of tweets was related to the media, followed by money (tweets questioning or even criticizing the high price of fundraising goals to go overseas for heart transplantations), while some tweets were misconceptions. We also conducted a sentiment analysis, which revealed that the most popular negative tweets were related to money, while the most positive tweets were related to reports on the favorable outcomes of recipients. Our results suggest that listening to concerns, providing correct information (particularly for some misconceptions), and emphasizing the outcomes of recipients will facilitate an increase in the number of people contemplating heart transplantation and organ donation.

Filamentous protein of basal cell epithelioma: characteristics in vivo and in vitro.

The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) pattern of the citrate-soluble fibrous protein isolated from human basal cell epitheliomas showed polypeptides with molecular weights of 43,000, 45,000, and 59,000. These polypeptides, found in the protein of normal epidermis, were present in a different proportion in this tumor tissue. Basal cell epitheliomas were cultured by the use of killed murine 3T3 feeder layers but only when 10(-9) M cholera toxin was present. The SDS-PAGE pattern of fibrous protein from cultured tumor cells was identical to the pattern of cultured normal human epidermal cells, and cornified cell envelopes were found in both types of cultures. Electron microscopy showed stratified cells that contained desmosomes, tonofilaments, and keratohyalin granules.

Immunohistochemical localization of caspase-3 correlates with clinical outcome in B-cell diffuse large-cell lymphoma.

Although B-cell diffuse large-cell lymphoma (DLCL) can respond to chemotherapy and radiotherapy, a large number of patients are still resistant to treatment. Caspase-3 is an enzyme crucial to the apoptotic process and may be important in the clinical outcome of these patients. The pattern of caspase-3 expression was studied in 54 cases of DLCL using immunohistochemistry and quantitative reverse-transcription PCR. Tumor cells displayed both a diffuse cytosolic and a punctate cytosolic staining for caspase-3. Kaplan-Meier survival curves indicated that tumor cells with a diffuse cytosolic expression of caspase-3 correlated with a poor prognosis (P > 0.0004). In addition, a punctate cellular localization was associated with complete response to treatment (P = 0.011). Cases with a small percentage of lymphoma cells expressing caspase-3 also tended to show poor survival (P > 0.09). Levels of caspase-3 mRNA were not significant (P > 0.17), although a weak trend was observed similar to the immunohistochemical analysis. The pattern of expression of caspase-3 was also assessed with respect to terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) positivity in both reactive lymph nodes and B-cell DLCL cases. Our results suggest that TUNEL-positive cells are not caspase-3-positive and that there is no correlation between DLCL cases with a high degree of DNA fragmentation and caspase-3 immunostaining. Furthermore, a survival curve indicated that a high TUNEL positivity was associated with a poor survival probability (P < 0.02) and a poor response to treatment (P = 0.04). These results confirm the dynamic nature of caspase-3 expression in DLCL and suggests that the pattern of expression of the enzyme has prognostic significance.

Purification and properties of a brain enzyme which deiminates proteins.

The deimination of guanidyl groups of peptides, proteins and other arginine-containing compounds is catalyzed by enzymes found in mammalian brain and epidermis. In cow, the brain and epidermal enzymes differ kinetically and physically, but both may be quantitated by measuring the production of benzoyl citrulline ethyl ester from benzoyl-arginine ethyl ester. The brain enzyme has been purified to apparent homogeneity, as judged by the presence of only one 85,000 dalton band in purified preparations when examined by SDS-polyacrylamide gel electrophoresis. An antibody raised to this band precipitates pure and partially purified brain enzyme but not partially purified epidermal enzyme, using the Ouchterlony technique. The antibody bound to an insoluble matrix removes brain enzyme activity from solution but not epidermal enzyme activity. The Km of the brain enzyme for benzoyl-arginine ethyl ester is about 0.33 mM.

Cytokeratins of the bovine hoof: classification and studies on expression.

The bovine hoof has been examined as a model for the study of keratinized skin appendages. We characterized the keratin polypeptides of hoof bed and matrix and compared them to epidermis using two-dimensional electrophoresis and immunoblot techniques. Both hoof tissues express keratins 6 and 16 (as described by Franke et al. (1981) J. Mol. Biol. 153, 933-959) and b2 and a1-4 which are previously undescribed proteins unique to the bovine hoof. Keratins of hoof matrix and bed share one or more common antigenic components as defined by immunoblot analysis. Hoof matrix expresses keratins 7 and 14, which are absent in hoof bed, and also expresses a greater number of isoelectric variants of keratin 6. Biopsies of hoof bed and matrix transplanted onto athymic mice both made hard hoof and underwent active keratin synthesis as evidenced by incorporation of [3H]leucine. Indirect immunofluorescence studies of the grafts showed that they had the histology and immunoreactivity previously noted for hoof bed and matrix. The two-dimensional gel electrophoretic patterns of both grafts were similar and expressed keratins b2 and a1-4. We conclude that a unique group of keratins exists in hoof. Furthermore, while hoof matrix is the major contributor to hard hoof, hoof bed epidermis maintains the capacity to make hard hoof and may contribute to the synthesis of the hoof plate in vivo. The ability to graft hoofs onto athymic mice provides an opportunity for the study of a number of aspects of hoof formation.

The polypeptide composition of epidermal prekeratin.

An alpha-fibrous protein, prekeratin, has been isolated from cow snout epidermis with citrate buffer, pH 2.65. Using acrylamide electrophoresis with 0.1% sodium dodecyl sulfate, prekeratin can be shown to contain three polypeptide chains of different molecular weights. The two faster migrating components are very similar with a mol. wt of about 47,000 while the slower one has a mol. wt of about 58,000. Chromatography on a number of molecular sieve and exchange resins does not separate the components, but use of Sepharose 2B with 0.1 M Tris, pH 9.0, containing 10% propanol gives two peaks of protein. The first and major peak contains all three components while the second has only the two with the faster mobility. The two more rapidly migrating components and the slower one were isolated by acrylamide electrophoresis, and the latter has an amino acid composition more compatible with a non-helical protein. Enzymatic digestion with tosyl-L-phenylalanine chloromethylketone-treated (TPCK-)trypsin shows that the component of mol. wt 58,000 is more susceptible to hydrolysis than the other two. These data suggest that prekeratin is not homogenous in composition and consists of several interacting polypeptide chains. One of these components would appear to be non-helical in structure.

Purification of thymidine phosphorylase from human amniochorion.

Thymidine phosphorylase (thymidine : orthophosphate deoxyribosyltransferase, EC 2.4.2.4) has been purified 1500-fold from extracts of human amniochorion. The purified enzyme catalyzes the phosphorolysis of deoxythymidine and to a lesser extent deoxyuridine but not deoxycytidine nor uridine. Discontinuous gel electrophoresis of the freshly purified enzyme shows a band containing 95% of the stainable protein. Gradient gel electrophoresis resolves the preparations into an active fraction with an apparent molecular weight of about 120 000 and a heavier less active or inactive fraction of about 180 000. Storage of the enzyme results in a decrease of the 120 000 dalton component, a loss in activity, and an apparent increase in the high molecular weight component. Sodium dodecyl sulfate gel electrophoresis shows only a single subunit of about 58 000 daltons which does not change on storage. These data are consistent with an active enzyme dimeric in structure which is capable of being converted to a less active form larger in molecular weight and possibly trimeric or tetrameric in structure.

Partial purification and specificity of an arginine-converting enzyme from bovine epidermis.

An enzyme which catalyzes the conversion of intraprotein arginine residue to intraprotein citrulline residue is present in bovine snout epidermis. This arginine-converting enzyme has been partially purified by (NH4)2SO4 preciptation chromatography on DEAE-cellulose and chromatography on Sephadex G-200. The enzyme is active at neutral pH, requires Ca2+ and a reducing agent and has an apparent molecular weight of 69 000. Its substrates include histone, polyarginine, S-carboxymethyl cysteine-hair keratin, S-carboxymethyl cysteine epidermal keratin and prekeratin and S-carboxymethyl cysteine-trichohyalin. A large number of proteins, synthetic and naturally occurring peptides, and other guanidine-containing compounds were substrates of the arginine-converting enzyme.

Epidermal proteins of cultured human and bovine keratinocytes.

Bovine and human epidermal cells were cultured on mitomycin C treated fibroblasts. The cells were carried through four passages and found to synthesize fibrous proteins and insoluble cell envelopes. Acid buffer soluble fibrous protein, prekeratin, and urea soluble fibrous protein were both identified and the latter was the major component in older cultures. Some of the prekeratin polypeptides of intact tissue were not found in cultured cells, but the ones that were present corresponded to those of whole tissue. X-ray diffraction, amino acid analysis and immunological techniques were used to establish that the polypeptides were keratins. The insoluble cell envelopes had a higher proline and 1/2 cystine content than the fibrous protein, similar to what is found in whole epidermis. Histidase, a characteristic enzyme marker of whole epidermis, was not observed in cultured cells. These studies indicate that differentiation occurs in cultured cells but it may not be as complete as in intact tissue.

The presence of citrulline in epidermal proteins.

Citrulline is present in the stratum corneum proteins of human, cow snout, pig snout and guinea pig epidermis but is absent from the stratum corneum proteins of frog, mouse, turtle, rat and hamster epidermis. The amino acid is released by acid hydrolysis and ranges from 1.7 to 5.5 residues per thousand residues of protein amino acid. Protein derived citrulline co-chromatographs with authentic L-citrulline on an amino acid analyzer, on Dowex-50, on Dowex-2 and on thin-layer chromatography. Dansylated material co-chromatographed with authentic dansyl-L-citrulline in two thin-layer chromatography systems. Labelling experiments have shown that the protein bound citrulline is derived from protein bound arginine and probably results from enzymatic conversion of the guanido group to the ureido group.

Isolation of the polypeptide chains of prekeratin.

Stratum corneum alpha protein, the principal structural protein of the epidermis, and its precursor prekeratin have been studied. Both proteins have similar patterns on sodium dodecyl sulfate electrophoresis and consist of several polypeptide chains (designated A, A', B, B'). These subunits have been isolated by chromatography on diethylaminoethyl cellulose in 8 M urea and characterized. Immunological studies have shown that the four subunits could be grouped into two distinct immunological groups referred to as the A and B groups and analysis of the cyanogen bromide fragments of purified components appeared to support such a hypothesis. X-ray diffraction studies have shown that at least one A component and one B component are necessary for the production of a typical alpha X-ray diffraction pattern. Circular dichroism studies have shown that the polypeptide chains of either class have low ellipticity when compared to the intact molecule, indicating that both chains are necessary for the formation of an alpha-helical structure.

A new class of soluble basic protein precursors of the cornified envelope of mammalian epidermis.

The cornified envelope has been shown to be formed beneath the plasma membrane as a result of the cross-linking of soluble and membrane-associated precursor proteins by transglutaminase. We have obtained a monoclonal antibody which reacts with the periphery of cells in the upper layers of human epidermis by indirect immunofluorescence (IIF) following immunization of mice with cornified envelopes of cultured human keratinocytes. The antibody also stained the cell peripheries of bovine, rat and mouse epidermis as well as stratified epithelium. Neutral buffer extracts of human cultured keratinocytes and epidermis examined under denaturing conditions contained polypeptides of molecular weight 14,900 and 16,800 which reacted with the antibody, and an additional component of molecular weight 24,800 was found in cultured cells. The polypeptides were shown to have a pI of about 9.0. Under non-denaturing conditions the two lower-molecular-weight polypeptides had an apparent molecular weight of 30,000, while the 24,800 protein had one of 60,000. Incubation of the polypeptides under conditions that activate transglutaminase resulted in a disappearance of the polypeptides or the formation of cross-linked products. Basic polypeptides with somewhat different pI values and molecular weights were identified in neutral buffer extracts of bovine and rat epidermis. The HCE-2 antibody appears to identify a new class of basic protein precursors of mammalian cornified envelope.

The fibrous protein of fish epidermis.

Fibrous proteins have been isolated from fish epidermis and shown to have physical and chemical properties similar to those of mammalian epidermal keratins. These results are most consistent with our hypothesis that hard keratins evolved late in evolution and there was not a common precursor of mammalian epidermal and hard keratin in primitive vertebrates.

A comparative study of the immunologic properties of hoof and nail fibrous proteins.

We have studied the localization of epidermal (soft) and nail and hoof (hard) fibrous keratins in the various anatomic regions of bovine hoof and human nail. Indirect immunofluorescence was performed on frozen sections of various parts of hoof and nail with antibodies demonstrated to be specific for hard and soft fibrous keratins by the Ouchterlony technique. The antibody to hard fibrous keratin reacted with the upper region of hoof bed and matrix tissue but not perihoof epidermis. The antibody to soft fibrous keratin reacted with hoof bed, matrix tissue, and perihoof epidermis. Electrophoretic analysis of the fibrous proteins of hoof bed indicated they contained both soft and hard fibrous keratins while matrix tissue contained only hard keratins. Immunoblot analysis of matrix fibrous proteins indicated that most of the polypeptides reacted with both antibodies. These results indicate that the antibody to soft fibrous keratin cross-reacts with hard fibrous keratin but the antibody to hard fibrous keratin appears to be specific. Immunologic studies, therefore, must be correlated with electrophoretic studies in order to define the localization of the various types of fibrous keratins. Similar results were obtained with human nail.

The occurrence and immunology of peptidylarginine deiminase and its preparation from bovine epidermis by an improved method.

Peptidylarginine deiminase activity has been found in some tissues from at least one representative of each vertebrate class, suggesting that the occurrence of the enzyme throughout the vertebrates is widespread. Using a three-step procedure including affinity chromatography on arginine agarose, a greatly improved purification of bovine epidermal peptidylarginine deiminase is presented. The purified enzyme preparation contains a 70-75 kD band and several minor components when examined by sodium dodecyl sulfate electrophoresis. A polyclonal antibody raised to the enzyme of bovine brain cross-reacts with human and newborn rat epidermis by indirect immunofluorescence. This cross-reactivity is markedly diminished by absorption of the antibody to the purified brain enzyme.

Fibrous protein of human epidermis.

The fibrous proteins of the malpighian layer of human epidermis (prekeratin) have been isolated with citrate buffer, pH 2.65, and shown to consist of 7 polypeptide chains varying in molecular weight from 45,000 daltons to 67,000. Some variation in the number and amount of the components was observed in prekeratin prepared from the epidermis of different individuals. The fibrous proteins of the stratum corneum were isolated with Tris buffer, pH 9.0, containing 6 M urea and 0.1 M mercapto-ethanol and were found to have a pattern similar to prekeratin but not identical to it. However, fibrous protein isolated from the superficial layers of the stratum showed a considerably different pattern indicating that there was post-translational modification of the protein in the late stages of keratinization. These data show that human keratin has the same heterogeneity which was observed previously in cow epidermis. This was further confirmed by studying the polypeptide chain content of prekeratin from a large number of lesions showing benign epidermal hyperplasia, where considerable variation in composition was observed.

The fibrous protein of fish epidermis.

Fibrous proteins have been isolated from fish epidermis and shown to have physical and chemical properties similar to those of mammalian epidermal keratins. These results are most consistent with our hypothesis that hard keratins evolved late in evolution and there was not a common precursor of mammalian epidermal and hard keratin in primitive vertebrates.

The growth and differentiation of cultured newborn rat keratinocytes.

Keratinocytes were cultured from adult and newborn rat epidermis using the 3T3 feeder cell technique. By modifying culture conditions a long-lived line of newborn rat keratinocytes was developed which showed a plating efficiency of 40% and a doubling time of 16 h. The cells produced stratified colonies with tonofilaments, desmosomes, cell envelopes, and keratohyaline granules. When the cells were grown on a collagen gel they formed a thick stratum corneum and many keratohyaline granules. The fibrous proteins synthesized by the newborn rat cultured keratinocytes were different than those of newborn rat epidermis but similar to those of adult rat cultured keratinocytes. A histidine-rich basic protein was identified by immunologic techniques but it appeared to be more heterogeneous than that of newborn rat epidermis. A cell envelope precursor protein was identified by dansyl cadaverine incorporation studies and was identical to a major envelope precursor of newborn rat epidermis. The growth characteristics, colony morphology, and biochemical markers did not change for up to 40 passages and there was no evidence of malignant transformation. Because of their case of growth and long-term survival these cells are useful for studying a variety of problems related to keratinization.