Quantitative correlation between the residual activity of β-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease

Summary A previously suggested model for the correlation between residual activity of a lysosomal enzyme and the turnover rate of its substrate(s) has been extended to a discussion of substrate accumulation rates in individual cells and whole organs. With these considerations, much of the observed variability in age of onset and clinical phenotype, as well as the phenomenon of pseudodeficiency, can be understood as the consequences of small differences in the residual activity of the affected enzyme. In order to experimentally verify the basic assumptions on which this model rests, studies were performed in cell culture. The radiolabeled substrates ganglioside G_M2 and sulfatide were added to cultures of skin fibroblasts with different activities of -hexosaminidase A or arylsulfatase A, respectively, and their uptake and turnover measured. In both series of experiments, the correlation between residual enzyme activity and the turnover rate of the substrate was essentially as predicted: degradation increased steeply with residual activity, to reach the control level at a residual activity of approximately 10–15% of normal. All cells with an activity above this critical threshold had a normal turnover. Comparison of the results of these feeding studies with the clinical status of the donor of each cell line basically confirmed our notions but also revealed the limitations of the cell culture approach.     

A Re-Analysis of the Routes of Coronado and Oñate into the Plains in 1541 and 1601

Abstract

A re-study based upon translations of the original documents is made of the many published routes suggested for Coronado’s and Oñate’s routes into the Plains. The point is made that disagreements among historians stem from the differing initial point of reference, a bridge thrown across a large river. Others have identified this river as the Pecos but Schroeder feels the evidence shows this is the Canadian River. Coronado’s route is then traced across the Plains to the Cimarron River. From this point to Quivira Schroeder is in agreement with other historians that the route was along the Arkansas River. He also agrees with Wedel’s archaeological location of Quivira as being in the Rice-McPherson County area of Kansas. The separate routes of Coronado’s army and party are then traced back to the Pecos Pueblo along what Schroeder deduces to have been more or less a straight line across the Oklahoma-Texas Panhandles.

Oñte’s later trip into the Plains is compared with Coronado’s. Considered in some detail are the intervening location of the Apachean and Plains Caddoan groups in relation to each other as suggested by the statements of the various chroniclers. The conclusion is reached that Oñate’s Quivira was located farther south than that visited by Coronado. Schroeder feels that Wedel’s archaeological work supports this view and concludes that Oñate’s Quivira extended slightly farther south along the Chicaskia River near Ponca City, Oklahoma.     

A critical evaluation of the predicted and X-ray structures of α-Lactalbumin

The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine α-Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browneet al. (1969) and later by Warmeet al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blakeet al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon α-Lactalbumin derived from X-ray crystallography (Acharyaet al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on C^α of residues 1–95, 1.1 Å).     

A comparative calorimetric and spectroscopic study of the effects of cholesterol and of the plant sterols β-sitosterol and stigmasterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed comparative DSC and FTIR spectroscopic measurements of the effects of β-sitosterol (Sito) and stigmasterol (Stig) on the thermotropic phase behavior and organization of DPPC bilayers. Sito and Stig are the major sterols in the biological membranes of higher plants, whereas cholesterol (Chol) is the major sterol in mammalian membranes. Sito differs in structure from Chol in having an ethyl group at C24 of the alkyl side-chain, and Stig in having both the C24 ethyl group and trans-double bond at C22. Our DSC studies indicate that the progressive incorporation of Sito and Stig decrease the temperature and cooperativity of the pretransition of DPPC to a slightly lesser and greater extent than Chol, respectively, but the pretransition persists to 10 mol % sterol concentration in all cases. All three sterols produce essentially identical effects on the thermodynamic parameters of the sharp component of the DPPC main phase transition. However, the ability to increase the temperature and decrease the cooperativity and enthalpy of the broad component decreases in the order Chol > Sito > Stig. Nevertheless, at higher Sito/Stig concentrations, there is no evidence of sterol crystallites. Our FTIR spectroscopic studies demonstrate that Sito and especially Stig incorporation produces a smaller ordering of the hydrocarbon chains of fluid DPPC bilayers than does Chol. In general, the presence of a C24 ethyl group in the alkyl side-chain reduces the characteristic effects of Chol on the thermotropic phase behavior and organization of DPPC bilayer membranes, and a trans-double bond at C22 magnifies this effect.     

Comparison of the properties of concanavalin A and anti-α-d-glucose antibodies

Concanavalin A and anti-α-d-glucose antibodies form precipitin complexes with antigens havingα-d-glucose as terminal units. The sedimentation rates, molecular weights, gel electrophoretic mobilities, isoelectric points, and immunoglobulin type of Con A andα-Ab have been determined. The interactions of the compounds with antigens in the presence of potential inhibitors have been compared. The data show that the interaction of Con A with glucose units occurs with hydrogen bonding at hydroxyl groups at C1, 3,4, and 6 and van der Waals bonding at the pyranose ring oxygen. In theα-Ab complex with glucose units, in addition to the above bond types, a hydrogen bond at the hydroxyl at C2 occurs and this bond is essential for interaction.     

A comparison of theβ A- andβ B-globin gene clusters of sheep

Domestic sheep have two common alleles at the adult beta-globin locus, beta A and beta B. Here we report the structure of the beta-globin locus of A-haplotype sheep. The locus consists of 12 genes, organized as a triplicated 4-gene set: 5' epsilon 1-epsilon II-psi beta I-beta C-epsilon III-epsilon IV-psi beta II-beta A-epsilon V-epsilon VI-psi beta III-beta F 3'. This arrangement is identical to that of the closely related goat locus. Sheep with the B haplotype have a locus arrangement consisting of a duplicated four-gene set, lacking the beta C gene as well as three other genes present in A sheep and goats. In order to understand the evolutionary history of the B sheep locus, we have sequenced the beta B gene from these sheep, and the beta C gene from A-haplotype sheep, and compared the sequences to those of the sheep beta A, goat beta C, and beta A, and cow adult beta genes. Our results indicate that the beta B gene has diverged recently from the beta A gene, and therefore the beta B locus structure may have resulted from a recent deletion from a triplicated locus.     

The Pharmacology of the Cannabinoid System—A Question of Efficacy and Selectivity

Our knowledge of the function of the cannabinoid system in the body has been aided by the availability of pharmacological agents that affect its function. This has been achieved by the design of agents that either directly interact with the receptor (agonists and antagonist/inverse agonists) and agents that indirectly modulate the receptor output by changing the levels of the endogenous cannabinoids (endocannabinoids). In this review, examples of the most commonly used receptor agonists, antagonists/inverse agonists, and indirectly acting agents (anandamide uptake inhibitors, fatty acid amide hydrolase inhibitors, monoacylglycerol lipase inhibitors) are given, with particular focus upon their selectivity and, in the case of the directly acting compounds, efficacy. Finally, the links between the endocannabinoid and cyclooxygenase pathways are explored, in particular, with respect to agents whose primary function is to inhibit cyclooxygenase activity, but which also interact with the endocannabinoid system.     

Heterogeneity in the molecular basis of three types of hereditary persistence of fetal hemoglobin and the relative synthesis of the Gγ and Aγ types of γ chain

Restriction endonuclease analyses of DNA from one Black ^GA-HPFH homozygote and four Black and one Indian ^GA-HPFH heterozygotes have identified three different HPFH types which are the result of large deletions including the and genes. Two of the types are comparable to those characterized previously, but the third, which is present in the Indian heterozygote, shows a distinct difference in the size of the deletion. The 5 end point of the deletion in this type III ^GA-HPFH extends 0.5–1.0 kb beyond the 5 end point of one of the Black types of HPFH (type I). Each of the three types is associated with a distinct ratio between the ^G and the ^A chains, an observation supported by family data. The highest ratio is found in the heterozygote with the Indian type III ^GA-HPFH, with 69.3% ^G chains, while the averages for the other types were 50.7% ^G (type I) and 32.3% ^G (type II).This research was supported in part by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution 0764 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912. P. K. Sukumaran was on leave from the Bai Jerbai Wadia Hospital for Children, Parel, Bombay, India.     

Volcanic impacts on the Holocene vegetation history of Britain and Ireland? A review and meta-analysis of the pollen evidence

Volcanic ash layers show that the products of Icelandic volcanism reached Britain and Ireland many times during the Holocene. Historical records suggest that at least one eruption, that of Laki in a.d. 1783, was associated with impacts on vegetation. These results raise the question: did Icelandic volcanism affect the Holocene vegetation history of Britain and Ireland? Several studies have used pollen data to address this issue but no clear consensus has been reached. We re-analyse the palynological data using constrained ordination with various representations of potential volcanic impacts. We find that the palynological evidence for volcanic impacts on vegetation is weak but suggest that this is a case of absence of evidence and is not necessarily evidence of absence of impact. To increase the chances of identifying volcanic impacts, future studies need to maximise temporal resolution, replicate results, and investigate a greater number of tephras in a broader range of locations, including more studies from lake sediments.     

Evolutionary implications of intron–exon distribution and the properties and sequences of the RPL10A gene in eukaryotes

The RPL10A gene encodes the RPL10 protein, required for joining 40S and 60S subunits into a functional 80S ribosome. This highly conserved gene, ubiquitous across all eukaryotic super-groups, is characterized by a variable number of spliceosomal introns, present in most organisms. These properties facilitate the recognition of orthologs among distant taxa and thus comparative studies of sequences as well as the distribution and properties of introns in taxonomically distant groups of eukaryotes. The present study examined the multiple ways in which RPL10A conservation vs. sequence changes in the gene over the course of evolution, including in exons, introns, and the encoded proteins, can be exploited for evolutionary analysis at different taxonomic levels. At least 25 different positions harboring introns within the RPL10A gene were determined in different taxa, including animals, plants, fungi, and alveolates. Generally, intron positions were found to be well conserved even across different kingdoms. However, certain introns seemed to be restricted to specific groups of organisms. Analyses of several properties of introns, including insertion site, phase, and length, along with exon and intron GC content and exon–intron boundaries, suggested biases within different groups of organisms. The use of a standard primer pair to analyze a portion of the intron-containing RPL10A gene in 12 genera of green algae within Chlorophyta is presented as a case study for evolutionary analyses of introns at intermediate and low taxonomic levels. Our study shows that phylogenetic reconstructions at different depths can be achieved using RPL10A nucleotide sequences from both exons and introns as well as the amino acid sequences of the encoded protein.     

A Delicate Adjustment: Wallace and Bates on the Amazon and “The Problem of the Origin of Species”

For over a century it has been believed that Alfred Russel Wallace and Henry Walter Bates set out for the Amazon in 1848 with the aim of “solving the problem of the origin of species”. Yet this enticing story is based on only one sentence. Bates claimed in the preface to his 1863 book that Wallace stated this was the aim of their expedition in an 1847 letter. Bates gave a quotation from the letter. But Wallace himself never endorsed or repeated this story. Many writers have acknowledged that this letter still survives. Yet the wording is different from that quoted by Bates and the letter says nothing of an expedition. It is argued that the sentence given by Bates is not a genuine quotation from this or any other Wallace letter but was modified by Bates to promote his own reputation. More significantly, this leads to the conclusion that there was a very sudden and dramatic shift in the way species were thought of and discussed after Darwin’s Origin of species appeared. Something called “the problem of the origin of species” (and similar variants) never occurred before Darwin’s book but exploded in frequency immediately after it. A profound change in how species origins were discussed happened which no one seemed to notice.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

A method for the separation of peptides and α-amino acids

1. Peptides and alpha-amino acids, occurring in mixtures from various sources, can be separated into one fraction containing the amino acids and several peptide fractions. This is achieved by chelation of the mixture with Cu(2+) ions and subsequent chromatography of these chelates over the acetate form of diethylaminoethylcellulose or triethylaminoethylcellulose. 2. The amino acid fraction is obtained by elution with 0.01m-collidine-acetate buffer, pH8.0. 3. Peptide fractions are eluted with 0.01m-collidine-acetate buffer, pH4.5, 0.17n-acetic acid and 0.1n-hydrochloric acid respectively. 4. With the exception of aspartic acid and glutamic acid, which are partly found in the acidic peptide fraction, the amino acids are completely separated from the peptides. 5. Contamination of the acidic peptide fraction with glutamic acid and aspartic acid can be largely avoided by previous addition of an excess of arginine. 6. Copper is removed from the eluates by extraction with 8-hydroxyquinoline in chloroform.     

Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A–type lamins

Background

The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression.

Methods

In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson–Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses.

Results

Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression.

Conclusions

These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics.

General significance

Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.     

Isolation and characterization of the four major cysteine-proteinase components of the latex of carica papaya L. reactivity characteristics towards 2,2′-dipyridyl disulfide of the thiol groups of papain,chymopapains A and B,and papaya peptidase A

High-quality spray-dried latex of Carica papaya L was fractionated by using SP-Sephadex-C50. The four major cysteine-proteinase components—papain(E.C.3.4.22.2), chymopapains A and B(jointly designated currently as E.C.3.4.22.6), and papaya peptidase A—were isolated and characterized by protein chemical methods and by study of their thiol groups using2,2′-dipyridyl disulfide as a two-protonic-state titrant and reactivity probe. Papain and papaya peptidase A each contain one thiol group/molecule, which in each case is part of the catalytic site, as evidenced by high reactivity toward2,2′-dipyridyl disulfide in acidic media. Chymopapains A and B each contain two thiol groups/molecule, only one of which is essential for catalytic activity. The reactivities of the thiol groups of these enzymes toward2,2′-dipyridyl disulfide at pH4 and10 and activity loss analysis by Tsou Chen-Lu plots each provides a ready means of distinguishing among the four cysteine proteinases. The nonessential thiol groups of chymopapains A and B readily undergo irreversible oxidation. The reactivity characteristics of the essential thiol groups of the four enzymes suggest the presence of somewhat similar interactive cysteine-histidine catalytic center systems in papain, papaya peptidase A, and chymopapain B but a different type of catalytic center environment in chymopapain A.