Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Electrostatic contribution of surface charge residues to the stability of a thermophilic protein: benchmarking experimental and predicted pKa values

Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01). The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81) with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.     

Asp79 makes a large, unfavorable contribution to the stability of RNase Sa

The two most buried carboxyl groups in ribonuclease Sa (RNase Sa) are Asp33 (99% buried; pK 2.4) and Asp79 (85% buried; pK 7.4). Above these pK values, the stability of the D33A variant is 6kcal/mol less than wild-type RNase Sa, and the stability of the D79A variant is 3.3kcal/mol greater than wild-type RNase Sa. The key structural difference between the carboxyl groups is that Asp33 forms three intramolecular hydrogen bonds, and Asp79 forms no intramolecular hydrogen bond. Here, we focus on Asp79 and describe studies of 11 Asp79 variants. Most of the variants were at least 2kcal/mol more stable than wild-type RNase Sa, and the most interesting was D79F. At pH 3, below the pK of Asp79, RNase Sa is 0.3kcal/mol more stable than the D79F variant. At pH 8.5, above the pK of Asp79, RNase Sa is 3.7kcal/mol less stable than the D79F variant. The unfavorable contribution of Asp79 to the stability appears to result from the Born self-energy of burying the charge and, more importantly, from unfavorable charge-charge interactions. To counteract the effect of the negative charge on Asp79, we prepared the Q94K variant and the crystal structure showed that the amino group of the Lys formed a hydrogen-bonded ion pair (distance, 2.71A; angle, 100 degrees ) with the carboxyl group of Asp79. The stability of the Q94K variant was about the same as the wild-type at pH 3, where Asp79 is uncharged, but 1kcal/mol greater than that of wild-type RNase Sa at pH 8.5, where Asp79 is charged. Differences in hydrophobicity, steric strain, Born self-energy, and electrostatic interactions all appear to contribute to the range of stabilities observed in the variants. When it is possible, replacing buried, non-hydrogen bonded, ionizable side-chains with non-polar side-chains is an excellent means of increasing protein stability.     

Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome

DNAs from Mycobacterium tuberculosis, M. intracellulare, M. phlei and M. smegmatis were digested by restriction enzymes and hybridized with three probes consisting of the 5' (16S rRNA), the middle (16S and 23S rRNA), and the 3' (23S and 5S rRNA) portions of the Escherichia coli rrnB operon. The resulting hybridization patterns indicate that slow-growing Mycobacteria species (i.e., M. tuberculosis and M. intracellulare), with genome size 3.13 - 4.29 X 10(9) daltons, appear to possess only one rRNA operon, whereas fast-growing species (i.e., M. phlei and M. smegmatis), with genome size 4.30 - 5.20 X 10(9) daltons, appear to possess two rRNA operons.     

Standard measurements, elasticity values and tensile strength behavior of the human mandible, a contribution to the biomechanics of the mandible--I

This paper is intended to help to characterize the physiological behavior of compact bone structure, so that realistic account can be taken of it when performing finite element (FE) analysis with the aim of solving biomechanical problems. In addition, further knowledge about the complex mechanical behavior of bone structure is provided. In accordance with anthropometric considerations, 13 mandibles have been measured, and a standard mandible defined for use in representative model calculations. In 4 mandibles (fully dentulous, partly dentulous, edentulous), orthotropic mechanical behavior of the fresh bone was determined experimentally. A comparison was also made with published data, as well as our own unpublished data obtained in other bone structures. Orthotropic behavior of the bone structure was confirmed. The material data of the individual bone structure vary considerably, but remain within a certain range. The proportion of the direction-dependent data is, however, almost constant. Our data correspond well with published data, although a direct comparison with the data of the mandible was not possible. A comparison of the data with those from the lower and upper extremities shows significant differences in rigidity, Young's modulus, and extension. A gradation can be found--starting with a maximum--that reveals the following order: mandible, upper extremity, lower extremity. In the case of extension, this order is reversed.     

Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex

From a panel of monoclonal antibodies raised against fractions of rat liver nuclear envelopes (NEs), we have identified an antibody, RL30, which reacts with novel nuclear pore complex (NPC) antigens that are not O-glycosylated. By immunofluorescence staining of cultured cells, RL30 reacts exclusively with the NE in a punctate pattern that largely coincides with that of identified NPC proteins. RL30 labels only the cytoplasmic surface of the NPC in immunogold electron microscopy, predominantly in peripheral regions nearby the cytoplasmic ring. In immunoblots of isolated rat liver NEs and cultured rat cells, RL30 recognizes a 265-kD band, as well as a series of 175-265-kD bands in rat liver NEs that are likely to be proteolytic products of p265. Sequencing of peptides from the 175- and 265-kD RL30 antigens of rat liver revealed that they are both closely related to human Tpr, a protein whose amino-terminal 150-250 amino acids appear in oncogenic fusions with the kinase domains of the met, trk, and raf protooncogenes. We found that in vitro translation of human Tpr mRNA yields a major 265-kD band. Considered together, these data indicate that the 265-kD RL30 antigen in the NPC is the rat homologue of Tpr. Interestingly, Tpr contains an exceptionally long predicted coiled coil domain (approximately 1600 amino acids). The localization and predicted structure of Tpr suggest that it is a component of the cytoplasmic fibrils of the NPC implicated in nuclear protein import. Immunofluorescence microscopy shows that during NPC reassembly at the end of mitosis, Tpr becomes concentrated at the NE significantly later than O-linked glycoproteins, including p62. This indicates that reassembly of the NPC after mitosis is a stepwise process, and that the Tpr-containing peripheral structures are assembled later than p62.     

Access to mates in a territorial ungulate is determined by the size of a male's territory, but not by its habitat quality

1. Territoriality is commonly associated with resource defence polygyny, where males are expected to gain access to females by anticipating how resources will influence female distribution and competing for resource-rich sites to establish their zone of dominance. 2. We tested this hypothesis in European roe deer (Capreolus capreolus) by simultaneously assessing the influence of resources on female distribution and the influence of female distribution on male distribution and breeding success using paternity analyses. 3. Females did not fully distribute themselves among male territories in relation to resources. As a result, relative female abundance in a male's territory depended on territory size, but not on its habitat quality. In turn, relative female abundance in a male's territory determined, at least partially, his breeding success. 4. Interestingly, male territory size, and hence access to females, was partly determined by male body mass (all males) and by residual antler size (subadults only). The latter result suggests that large antlers may be important to young males for establishing their first territory, which is then usually retained for all subsequent reproductive seasons. 5. To conclude, although territoriality of male roe deer has certainly evolved as a tactic for ensuring access to mates, our results suggest that it does not really conform to a conventional resource defence polygyny strategy, as males seem to gain no obvious benefit from defending a territory in an area of high habitat quality in terms of enhanced access to mates. 6. This may explain the stability of male territories between years, suggesting that male territoriality conforms to an 'always stay' and 'low risk-low gain' mating strategy in roe deer.     

Zooid size and growth rate of the bryozoan Cryptosula pallasiana Moll in relation to temperature, in culture and in its natural environment

The size of cheilostome bryozoan zooids has been widely discussed for its potential in inferring palaeotemperatures, based on correlations between zooid size and temperature. Studies in both the natural environment and under experimental laboratory conditions have shown that an increase in temperature significantly decreases zooid size in a range of bryozoan taxa. In order to test the effect of temperature on zooid size, the cheilostome bryozoan Cryptosula pallasiana was for the first time successfully cultured under laboratory conditions. C. pallasiana was grown at 14 °C and 18 °C using Rhodomonas sp. as a food organism. Zooid size, tentacle number and growth rate were measured over a period of 26 days. For comparison, zooids from colonies of C. pallasiana collected from the natural environment were measured in winter and summer months. Results showed that colonies grown in laboratory culture had significantly longer and wider zooids at 14 °C than at 18 °C. The specific growth rate of C. pallasiana doubled from 14 °C to 18 °C. Comparison of tentacle number in culture showed a significantly higher value at lower temperatures. This may be related to differing food availability, longer polypide life spans, or a shift of energy use at colder temperatures. In nature the zooids were significantly longer in colonies sampled in July than in January, a clear difference from laboratory results. The utility of cheilostome Bryozoa as indicators of environmental change and their potential for studies of paleotemperature are highlighted.     

Variation across amphibian species in the size of the nuclear genome supports a pluralistic, hierarchical approach to the C-value enigma

Amphibians have featured prominently in discussions of the C-value enigma, the still-unresolved puzzle regarding the evolution of genome size. Their wide range in nuclear DNA contents and diverse ecological and developmental lifestyles make them excellent subjects for addressing the key elements of the C-value enigma. However, in some cases the importance of work on amphibians appears to be overstated. This is especially true of claims that patterns of variation in salamanders support a particular theory of genome size evolution to the exclusion of others. This study provides a critical re-examination of some of these claims, as well as an investigation of the relationships between genome size, cell and nuclear size, and metabolism in amphibians. The results of these analyses, combined with an overview of previous amphibian genome size literature, strongly indicate the need for a pluralistic approach to the C-value enigma. In particular, it must be recognized that evolutionary forces operating and interacting at several levels of biological organization (of which the genome itself is one) are responsible for the observed patterns in amphibian genome size distributions.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79 , 329–339.     

The stoichiometry and antenna size of the two photosystems in marine green algae, Bryopsis maxima and Ulva pertusa, in relation to the light environment of their natural habitat

The stoichiometry and antenna sizes of the two photosystems in two marine green algae, Bryopsis maxima and Ulva pertusa, were investigated to examine whether the photosynthetic apparatus of the algae can be related to the light environment of their natural habitat. Bryopsis maxima and Ulva pertusa had chlorophyll (Chl) a/b ratios of 1.5 and 1.8, respectively, indicating large levels of Chl b, which absorbs blue-green light, relative to Chl a. The level of photosystem (PS) II was equivalent to that of PS I in Bryopsis maxima but lower than that of PS I in Ulva pertusa. Analysis of Q(A) photoreduction and P-700 photo-oxidation with green light revealed that >50% of PS II centres are non-functional in electron transport. Thus, the ratio of the functional PS II to PS I is only 0.46 in Bryopsis maxima and 0.35 in Ulva pertusa. Light-response curves of electron transport also provided evidence that PS I had a larger light-harvesting capacity than did the functional PS II. Thus, there was a large imbalance in the light absorption between the two photosystems, with PS I showing a larger total light-harvesting capacity than PS II. Furthermore, as judged from the measurements of low temperature fluorescence spectra, the light energy absorbed by Chl b was efficiently transferred to PS I in both algae. Based on the above results, it is hypothesized that marine green algae require a higher ATP:NADPH ratio than do terrestrial plants to grow and survive under a coastal environment.     

Growth, development and temporal variation in the onset of six Chironex fleckeri medusae seasons: a contribution to understanding jellyfish ecology

Despite the worldwide distribution, toxicity and commercial, industrial and medical impacts jellyfish present, many aspects of their ecology remain poorly understood. Quantified here are important ecological parameters of Chironex fleckeri medusae, contributing not only to the understanding of an understudied taxon, the cubozoa, but also to the broader understanding of jellyfish ecology. C. fleckeri medusae were collected across seven seasons (1999, 2000, 2003, 2005-07 and 2010), with growth rates, temporal variation in the medusae season onset and differences in population structure between estuarine and coastal habitats quantified. With a mean of 2 September ± 2 d (mean ± 95% confidence limits), the earliest date of metamorphosis was temporally constrained between seasons, varying by only 7 d (30 August to 5 September). Juvenile medusae appeared to be added over an extended period, suggesting polyp metamorphosis was an ongoing process once it commenced. At a maximum of 3 ± 0.2 mm d(-1) IPD, medusae growth to an asymptotic size of ~190 mm IPD was rapid, yet, with the oldest medusae estimated to be ~78 d in age, medusae did not appear to accumulate along the coastline. Furthermore, a greater proportion of juveniles were observed along the coastline, with estuarine populations typified by larger medusae. With key aspects of C. fleckeri's ecology now quantified, medusae season management protocols can be further developed.     

Overwintering leaves of a forest-floor fern, Dryopteris crassirhizoma (Dryopteridaceae): a small contribution to the resource storage and photosynthetic carbon gain

BACKGROUND AND AIMS: Dryopteris crassirhizoma is a semi-evergreen fern growing on the floor of deciduous forests. The present study aimed to clarify the photosynthetic and storage functions of overwintering leaves in this species. METHODS: A 2-year experiment with defoliation and shading of overwintering leaves was conducted. Photosynthetic light response was measured in early spring (for overwintering leaves) and summer (for current-year leaves). KEY RESULTS: No nitrogen limitation of growth was detected in plants subjected to defoliation. The number of leaves, their size, reproductive activity (production of sori) and total leaf mass were not affected by the treatment. The defoliation of overwintering leaves significantly reduced the bulk density of rhizomes and the root weight. The carbohydrates consumed by the rhizomes were assumed to be translocated for leaf production. Photosynthetic products of overwintering leaves were estimated to be small. CONCLUSION: Overwintering leaves served very little as nutrient-storage and photosynthetic organs. They partly functioned as a carbon-storage organ but by contrast to previous studies, their physiological contribution to growth was found to be modest, probably because this species has a large rhizome system. The small contribution of overwintering leaves during the short-term period of this study may be explained by the significant storage ability of rhizomes in this long-living species. Other ecological functions of overwintering leaves, such as suppression of neighbouring plants in spring, are suggested.     

Seed size in relation to phylogeny,growth form and longevity in a subalpine meadow on the east of the Tibetan plateau

This study examined seed size distribution, and seed size in relation to phylogeny, growth form and longevity, where seed size is expected to be approximately of a log-normal frequency distribution and correlate dwith phylogeny, growth form and longevity. We made use of a data set of 229 species from alpine meadow on the east of the Tibetan plateau. Species spanned 10⁴ range of seed size, and the frequency of seed mass classes on a logarithmic scale produced an approximately normal distribution, but largely shifted towards smaller-sized seeds compared to those of the temperate zone and the tropics. It was evident that seed size was strongly related to phylogeny, where order, family, genus and species accounted for 2%, 32.9%, 48.9% and 16.2% of total variation in log seed mass. However, both growth form and longevity did not account for variation in log seed mass, which was opposite to the previously reported patterns of trait correlation. The implications of our results are discussed.     

Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen

Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.