Unbalanced Robertsonian translocations associated with Down's syndrome or Patau's syndrome: Chromosome subtype,proportion inherited,mutation rates,and sex ratio

Summary Summary data are presented on 168 D/21 and 131 G/21 translocation trisomies reported to the New York State Chromosome Registry. By combining these data with others from the literature it is estimated that about 59% of D/21 cases are the result of mutation in the parental generation; the rest are translocations inherited from parental carriers (39% maternal, 3% paternal). The proportion of mutants is about 10% greater for 14/21 cases and significant lower for 13/21 cases. Of G/21 cases 93% are mutant, about 6% of maternal origin, and 1% of paternal origin. All the mutant cases involve 21/21 rearrangements. Estimated mutation rates per 10⁵ gametes for translocation trisomies in affected livebirths are 0.1 for 21/13, 0.5 to 0.9 for 21/14, and 1.1 to 1.4 for 21/21. The rates for 21/15 and 21/22 translocation trisomies are probably all conservatively less than 0.1 per 10⁵ gametes. Of interchange trisomy Patau's syndrome, about 60% of cases are mutant; the rest are translocations inherited from a parental carrier (about 25% for 13/13 cases and about 45% for 13/14 cases. The estimated mutation rates for 13/13 and 13/14 interchange trisomies are each about 0.5 per 10⁵ gametes; the rate for 13/15 interchange trisomies is less than 0.1 per 10⁵ gametes. A male excess is observed for D/21 (sex ratio=1.70), and G/21 (sex ratio=1.38) interchange Down's syndrome, and a female excess for D/13 interchange Patau's syndrome (sex ratio =0.77), trends similar to those seen in the respective 47, trisomies associated with these phenotypes.     

The enantiomeric ratio: origin, determination and prediction

The enantiomeric ratio E =(k_cat^R/K_m^R) (k_cat^S/K_m^S) offers a concise representation of the enantioselective properties of an enzyme in reactions that involve chiral compounds. Both as a measure of the intrinsic selectivity of the catalyst, and as a parameter to model the performance of enzymatic processes for the production of enantiopure fine-chemicals, its merits have been well-recognized.

Several methods for the determination of E exist. The scope and limitations of these methods are evaluated in terms of accuracy and feasibility. There appears to be no single method that is both reliable and readily applicable in all cases. Complementary methods, however, are available.

The outstanding characteristics of the enantiomeric ratio as a quantitative measure of the effects of physical and chemical conditions on the intrinsic enantioselectivity of enzymes are presented in terms of the difference in Gibbs energies of the diastereomeric enzyme-substrate transition states. The prospects of molecular modeling strategies for the prediction of E are discussed.     

DNA replication fidelity: kinetics and thermodynamics

Mechanisms that control the fidelity of DNA replication are discussed. Data are reviewed for 3 steps in a fidelity pathway: nucleotide insertion, exonucleolytic proofreading, and extension from matched and mismatched 3′-primer termini. Fidelity mechanisms that involve predominately K_m discrimination, V_max discrimination, or a combination of the two are analyzed in the context of a simple model for fidelity. Each fidelity step is divided into 2 components, thermodynamics and kinetic. The thermodynamic component, which relates to free-energy differences between right and wrong base pair, is associated with a K_m discrimination mechanism for polymerase. The kinetic component, which represents the enzyme's ability to select bases for insertion and excision to achieve fidelity greater than that availablek from base pairing free-energy differences, is associated with a V_max discrimination mechanism for polymerase. Currently available fidelity data for nucleotide insertion and primer extension in the absence of proofreading appears to have relatively large K_m and small V_max components. An important complication can arise when analyzing data from polymerases containing an associated 3′-exonuclease activity. In the presence of proofreading, a V_max discrimination mechanisms is likely to occur, but this may be the result of two K_m discrimination mechanisms acting serially, one for nucleotide insertion and other for excision. Possible relationships between base pairing free energy differences measured in aqueous solution and those defined within the polymerase active cleft are considered in the context of the enzyme's ability to exclude water, at least partially, from the vicinity of its active site.     

Isolation and characterization of nuclear genes coding for subunits of the yeast ubiquinol-cytochrome c reductase complex

Nuclear genes coding for the M_r 17000, 14000 and 11000 subunits of the ubiquinol-cytochrome c reductase complex (complex III) in yeast have been isolated from a clone bank of yeast nuclear DNA by use of a mRNA hybridization-competition assay. This is based on our observations that levels of mRNAs for these subunits are much reduced during glucose repression and in cytoplasmic petite mutants and the procedure should be of general application for the isolation of other inducible or repressible genes coding for mRNAs present at low levels in the cell. A first characterization of the clones is presented. The genes are not closely linked in the genome and those coding for M_r 14000 and 11000 subunits are present in unique genomic environments, which suggests that there are only single copies of each in the nuclear genome.     

GENETIC CORRELATIONS IN A WILD RODENT: GRASS-EATERS AND FAST-GROWERS EVOLVE HIGH BASAL METABOLIC RATES

Basal metabolic rate (BMR), commonly used as a measure of the cost of living, is highly variable among species, and sources of the variation are subject to an enduring debate among comparative biologists. One of the hypotheses links the variation in BMR with diversity of food habits and life-history traits. We test this hypothesis by asking how BMR of a particular species, the bank vole Myodes (= Clethrionomys ) glareolus , would change under selection for high growth rate (measured as a postweaning body mass change; MD_PW ) and the ability to cope with a low-quality herbivorous diet (measured as body mass change during a four-day test; MD_LQD ). We show that both of the traits are heritable in the narrow sense ( MD_PW : h ²= 0.30; MD_LQD : h ²= 0.19), and are genetically correlated with mass-independent BMR (additive genetic correlation, r_A = 0.28 for MD_PW and 0.37 for MD_LQD ). Thus, both of the traits could change in response to a selection, and the selection would also result in a correlated evolution of the level of metabolism. The results are consistent with the hypothesis that a part of the interspecific variation in BMR evolved in response to selection for life-history and ecological traits such as food habits.     

A flow injection analysis system for fermentation monitoring and control

An automated analysis system for on-line fermentation monitoring is presented. The modular system consists of an in-line sterilizeable crossflow microfilter, a selection valve that allows injection of sample or standards, a degassing unit, a dilution module, and a FIA manifold with a spectrophotometric UV/VIS detector. In the dilution module samples are conditioned and diluted depending upon concentration of analyte and the working range of the analyzer. Methods for the monitoring of glucose, ethanol, ammonia and phosphate are described. Results from the monitoring of glucose and their use in fermentation control are presented. The maximal analysis frequency is 30 samples per hour including the dilution of 1 : 200. Detection limits are 5 mg/L for ethanol and glucose, 1 mg/L for phosphate and 50 mg/L for ammonia.     

Determination of loratadine and pheniramine from human serum by gas chromatography-mass spectrometry

In this work, a method for the determination of the antihistaminic drugs loratadine and pheniramine from human serum is presented. Serum samples are extracted under basic conditions with hexane-n-amyl alcohol (95:5, v/v), the analytes are reextracted into diluted hydrochloric acid and, after basification, are once again extracted into the organic phase. The samples are measured by GC-MS. The limits of detection of the assay are 0.5 ng/ml for loratadine and 2 ng/ml for pheniramine. The R.S.D.s in the day-to-day precision test for loratadine are 7.0% at 20 ng/ml and 12.4% at 2 ng/ml. For pheniramine, the R.S.D. are 6.4% at 300 ng/ml and 10.2% at 20 ng/ml.     

A C-terminal dimerization motif is required for focal adhesion targeting of Talin1 and the interaction of the Talin1 I/LWEQ module with F-actin

Focal adhesion complexes are plasma membrane-associated multicomponent complexes that are essential for integrin-linked signal transduction as well as cell adhesion and cell motility. The cytoskeletal protein Talin1 links integrin adhesion receptors with the actin cytoskeleton. Talin1 and the other animal and amoebozoan talins are members of the I/LWEQ module superfamily, which also includes fungal Sla2 and animal Hip1/Hip1R. The I/LWEQ module is a conserved C-terminal structural element that is critical for I/LWEQ module protein function. The I/LWEQ module of Talin1 binds to F-actin and targets the protein to focal adhesions in vivo. The I/LWEQ modules of Sla2 and Hip1 are required for the participation of these proteins in endocytosis. In addition to these roles in I/LWEQ module protein function, we have recently shown that the I/LWEQ module also contains a determinant for protein dimerization. Taken together, these results suggest that actin binding, subcellular targeting, and dimerization are associated in I/LWEQ module proteins. In this report we have used alanine-scanning mutagenesis of a putative coiled coil at the C-terminus of the Talin1 I/LWEQ module to show that the amino acids responsible for dimerization are necessary for F-actin binding, the stabilization of actin filaments, the cross-linking of actin filaments, and focal adhesion targeting. Our results suggest that this conserved dimerization motif in the I/LWEQ module plays an essential role in the function of Talin1 as a component of focal adhesions and, by extension, the other I/LWEQ module proteins in other multicomponent assemblies involved in cell adhesion and vesicle trafficking.     

Glycomic strategy for efficient linkage analysis of di-, oligo- and polysialic acids

Sialic acid polymers of glycoproteins and glycolipids are characterized by a high diversity in nature and are involved in distinct biological processes depending inter alia on the glycosidic linkages between the present sialic acid residues. Though suitable protocols are available for chain length and sialic acid determination, sensitive methods for linkage analysis of di-, oligo-, and polysialic acids (di/oligo/polySia) are still pending. In this study, we have established a highly sensitive glycomic strategy for this purpose which is based on permethylation of di/oligo/polySia after tagging their reducing ends with the fluorescent dye 1,2-diamino-4,5-methylenedioxybenzene (DMB). Using DMB-labeled sialic acid di/oligo/polymers glycosidic linkages could be efficiently determined and, optionally, the established working procedure can be combined with HPLC for in depth characterization of distinct di/oligo/polySia chains. Moreover, the outlined approach can be directly applied to mammalian tissue samples and linkage analysis of sialic acid polymers present in biopsy samples of neuroblastoma tissue demonstrating the usefulness of the outlined work flow to screen, for example, cancer tissue for the presence of distinct variants of di/oligo/polySia as potentially novel biomarkers. Hence, the described strategy offers a highly sensitive and efficient strategy for identification of glycosidic linkages in sialic acid di/oligo/polymers of glycoproteins and glycolipids.     

A New Methylene Blue Technic for Permanent Preparations

1. Tissues stained intra vitam with methylene blue are fixed in a 10% ammonium molybdate solution in physiological saline (or sea water if the tissue is from a marine animal). Fixation time is kept to a minimum. Washing also is reduced to a minimum.

2. Excess fluids are removed from tissues by blotting with a paper or cloth towel before they are put into the succeeding solution. Tissues are taken from the wash water, blotted and placed in a mixture of equal parts of absolute ethyl alcohol and n-butyl alcohol for 30 minutes. They are then blotted and transferred to n-butyl alcohol for 30 minutes. After blotting they are placed in a mixture of one part methyl salicylate and four parts xylene until cleared. Tissues may be mounted whole or prepared for sectioning by embedding in paraffin in the usual way.

3. Tissues fixed, washed, dehydrated and cleared as described retain nearly all of the stain; the time required is greatly reduced; there is no need to chill the dehydrating solutions; cell distortion is much reduced.     

Cumulative Energy and Global Warming Impact from the Production of Biomass for Biobased Products

The cumulative energy and global warming impacts associated with producing corn, soybeans, alfalfa, and switchgrass and transporting these crops to a central crop processing facility (called a "biorefinery") are estimated. The agricultural inputs for each crop are collected from seven states in the United States: Illinois, Indiana, Iowa, Michigan, Minnesota, Ohio, and Wisconsin. The cumulative energy requirement for producing and transporting these crops is 1.99 to 2.66 megajoules/kilo-gram (MJ/kg) for corn, 1.98 to 2.04 MJ/kg for soybeans, 1.24 MJ/kg for alfalfa, and 0.97 to 1.34 MJ/kg for switchgrass. The global warming impact associated with producing biomass is 246 to 286 grams (g) CO₂ equivalent/kg for corn, 159 to 163gCO₂ equivalent/kg for soybeans, 89 g CO₂ equivalent/ kg for alfalfa, and 124 to 147 g CO₂ equivalent/kg for switch-grass. The detailed agricultural data are used to assess previous controversies over the energy balance of bioethanol and, in light of the ongoing debates on this topic, provide a needed foundation for future life-cycle assessments.     

Examining water temperature proxies in<Emphasis Type="Italic"> Porites</Emphasis> corals from the Great Barrier Reef: a cross-shelf comparison

Cores from colonies of the coral species Porites sp. were collected from inshore, mid-shelf, and outer reef localities (central Great Barrier Reef) to test the robustness of the major elemental sea surface temperature (SST) proxies (B/Ca, Mg/Ca, Sr/Ca, U/Ca) to the influence of inshore processes. Time series analyses of Sr/Ca, U/Ca, B/Ca, and Mg/Ca are compared to sea surface temperature (SST) in order to provide calibrations for these elements. This study shows that there are significant variations between the corals with respect to some of the proxies. In some cases, variations of ~6 °C are observed for a single U/Ca value. This magnitude of variation is also seen in the Mg/Ca proxy and, to a smaller extent, in the B/Ca–SST relationship. In two of the corals, both Mg/Ca and U/Ca do not follow a seasonal signal. The Mg/Ca and U/Ca ratios for two inshore corals are significantly different than the offshore corals (lower and higher, respectively). The other two proxies (B/Ca and Sr/Ca) do not display any inshore vs. offshore variations except for one inshore site that did not have a clear seasonal signal for either of these proxies. The Sr/Ca–SST relationship is the most robust, with a temperature variation of ~2 °C for a single Sr/Ca value, which is within error for this technique.     

Dose responses of gibberellins

To determine the response type, published data for the most widely used bioassays for gibberellins have been analyzed by means of a computer program for estimating sensitivity parameters, or by interpolation. The dose response data are almost uniformly subsensitive, i. e. more than an 81-fold increase in external gibberellin concentration is required for a change from 10 to 90% of maximal response (S₉₀/S₁₀). Data for the interaction of gibberellins with artificial membranes are, in contrast, markedly ultrasensitive (S₉₀/S₁₀± 10). This difference further strengthens the view that lipid structures do not function as receptors for gibberellins. Most of the subsensitive dose responses for gibberellins can be quite precisely represented by cooperative isotherms. However, available data are insufficiently detailed for an unequivocal choice among cooperative, multiphasic or more complex kinetics.     

The Processing of Mammalian Haemopoietic Cells in Thin Layer Agar Cultures for Electron Microscopy

Human and mouse haemopoietic cells cultured by the thin layer agar technique have been studied with the electron microscope. To process colonies of haemopoietic cells or individual cells which appeared in these colonies, a special technique had to be developed. The technique presented covers methods of selection, isolation, and sectioning that were devised for this purpose.

Haemopoietic cells are cultured in small plastic Petri dishes containing a culture system with 0.25% agar. Cell colonies and individual cells intended for light as well as for electron microscopic study are examined and selected microscopically with the aid of a numbered grid which is placed under the closed Petri dish.

Cells in the agar gel are fixed with glutaraldehyde which is pipetted directly onto the cultures. In order to facilitate their removal from the medium, the consistency of the agar solution is increased by evaporating liquid with controlled mild warming.

Pieces of agar containing colonies or single cells are cut out with a fine trephine and postfixed in osmium tetroxide. Agar pieces are embedded cell side up in a thin layer of Epon. After polymerization, the Epon-embedded pieces of agar are appropriately oriented at the head of flat embedding molds filled with fresh Epon. After another polymerization procedure, the top of the Epon blocks containing the cells are trimmed to a smooth surface with a glass knife.

The exact distance between the smooth surface of the blocks and the cells is measured by use of the vertical micrometer of a standard light microscope. The Epon layer around the specimen is trimmed away to expose selected cells for subsequent semi-thick and ultrathin sectioning. Sections are stained and examined microscopically.

With minor modifications the technique described also enables the processing of extremely small quantities of biological materials derived from other experiments for both light and electron microscopic observation.     

Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25

Vertebrates express two distinct families of SUMO proteins (SUMO1 and SUMO2/3) that serve distinct functions as posttranslational modifiers. Many proteins are modified specifically with SUMO1 or SUMO2/3, but the mechanisms for paralog selectivity are poorly understood. In a screen for SUMO2/3 binding proteins, we identified Ubiquitin Specific Protease 25 (USP25). USP25 turned out to also be a target for sumoylation, being more efficient with SUMO2/3. Sumoylation takes place within USP25's two ubiquitin interaction motifs (UIMs) that are required for efficient hydrolysis of ubiquitin chains. USP25 sumoylation impairs binding to and hydrolysis of ubiquitin chains. Both SUMO2/3-specific binding and sumoylation depend on a SUMO interaction motif (SIM/SBM). Seven amino acids in the SIM of USP25 are sufficient for SUMO2/3-specific binding and conjugation, even when taken out of structural context. One mechanism for paralog-specific sumoylation may, thus, involve SIM-dependent recruitment of SUMO1 or SUMO2/3 thioester-charged Ubc9 to targets.     

The step-size distance in muscle contraction: properties and estimates

The step-size distance in muscle contraction is obtained using the step-size distance equation z = u/n, where z is the step-size distance, u is the actin filament velocity and n is the ATPase rate of splitting. In a previous study a step-size distance of about 17 Å at no load was determined for intact frog muscle. Some properties of the step-size distance equation are described. We have now made estimates of the step-size distance z for a variety of muscles using existing physiological and biochemical data in the literature. The estimates are listed in Tables 1 and 2. We find that the step-size distances are clustered in the range 13–17 Å for nearly all muscles.     

Molecular dynamics simulation of the graphene–water interface: comparing water models

In this work, different water models (i.e. SPC/E, TIP3P, TIP4P/2005, TIP5P, SPC/Fw, TIP4P/2005f and SWM4_DP) are implemented to simulate water on neutral, negatively charged and positively charged graphene. In all cases ambient conditions are considered. Structural and dynamical properties for water are calculated to quantify the differences among various water models. The results show that SPC/E, TIP4P/2005, SPC/Fw, TIP4P/2005f and SWM4_DP water models yield a similar structure for interfacial water on graphene, whether it is neutral, negatively charged or positively charged. TIP5P is the model whose predictions for the structure of the interface deviate the most from those of the other models. Although qualitatively the results are for the most part similar, a large quantitative variation is observed among the dynamical properties predicted when various water models are implemented. Although experimental data are not available to discriminate the most/least accurate of the model predictions, our results could be useful for comparing results for interfacial water obtained implementing different models. Such critical comparison will benefit practical applications such as the development of energy-storage and water-desalination devices (e.g. electric double-layer capacitors), among others.