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PubMed2004

An observation of non-superimposable stereogeometrical features in a non-chiral one-component beta-Ala model peptide.

This paper describes the chemical synthesis and crystal molecular conformation of a non-chiral beta-Ala containing model peptide Boc-beta-Ala-Acc5-OCH3. The analysis revealed the existence of two crystallographically independent molecules A and B, in the asymmetric unit. Unexpectedly, while the magnitudes of the backbone torsion angles in both molecules are remarkably similar, the signs of the corresponding torsion angles are reverse therefore, inclining us to suggest the existence of non-superimposable stereogeometrical features in a non-chiral one-component beta-Ala model system. The critical mu torsion angle around CbetaH2-CalphaH2 bond of the beta-Ala residue represents a typical gauche orientation i.e., mu = 67.7 degrees in A and mu = -61.2 degrees in B, providing the molecule an overall crescent shaped topology. The observed conformation contrasts markedly to those determined for the correlated non-chiral model peptides: Boc-beta-Ala-Acc6-OCH3 and Boc-beta-Ala-Aib-OCH3 signifying the role of stereocontrolling elements since the stereochemically constrained Calpha, alpha-disubstituted glycyl residues (e.g., Acc5, Acc6, and the prototype Aib) are known to strongly restrict the peptide backbone conformations in the 3(10)/alpha-helical-regions ( phi approximately +/-60+/-20 degrees, psi approximately +/-30+/-20 degrees) of the Ramachandran map. Unpredictably, the preferred, phi, psi torsion angles of the Acc5 residue fall outside the helical regions of the Ramachandran map and exhibit opposite-handed twists for A and B. The implications of the semi-extended conformation of the Acc5 residue in the construction of backbone-modified novel scaffolds and peptides of biological relevance are highlighted. Taken together, the results indicate that in short linear beta-Ala containing peptides specific structural changes can be induced by selective substitution of non-coded linear- or cyclic symmetrically Calpha,alpha-disubstituted glycines, reinstating the hypothesis that in addition to conformational restrictions, the chemical nature of the neighboring side-chain substituents and local environments collectively influences the stabilization of folding-unfolding behavior of the two methylene units of a beta-Ala residue.

Amino Acid Sequence↗

PubMed2000

Dynamics of a mobile loop at the active site of Escherichia coli asparaginase.

Asparaginase II from Escherichia coli is well-known member of the bacterial class II amidohydrolases. Enzymes of this family utilize a peculiar catalytic mechanism in which a pair of threonine residues play pivotal roles. Another common feature is a mobile surface loop that closes over the active site when the substrates is bound. We have studied the motion of the loop by stopped-flow experiments using the fluorescence of tryptophan residues as the spectroscopic probe. With wild-type enzyme the fluorescence of the only tryptophan, W66, was monitored. Here asparagine induced a rapid closure of the loop. The rate constants of the process (100-150 s(-1) at 4 degrees C) were considerably higher than those of the rate-limiting catalytic step. A more selective spectroscopic probe was generated by replacing W66 with tyrosine and Y25, a component of the loop, with tryptophan. In the resulting enzyme variant, k(cat) and the rate of loop movement were reduced by factors of 10(2) and >10(3), respectively, while substrate binding was unaffected. This indicates that the presence of tyrosine in position 25 is essential for both loop closure and catalysis. Numerical simulations of the observed transients are consistent with a model where loop closure is an absolute prerequisite for substrate turnover.

Asparaginase↗

PubMed2002

Lead tetraacetate mediated one-pot multistage transformations: theoretical studies on the diverging behavior in the Hajos-Parrish and Wieland-Miescher series.

A computational approach to the proposed mechanistic rationale of the lead tetraacetate mediated oxidative cleavage of selected unsaturated diols in the hydrindenediol (derived from the Hajos-Parrish ketone, abbreviated as HP) and octalindiol (derived from the Wieland-Miescher ketone, abbreviated as WM) series is presented. These two unexpected cascade-type transformations have been found to yield structurally different products despite the similarities in the starting compounds 1-HP and 1-WM, which differ only by the number of carbon atoms in the cycloalkane rings. The final product in the HP series is the complex ring-expanded molecule iii-6, while the final product in the WM series is the ring-expanded bisacetoxy diacetal 7. Calculations carried out at the B3LYP/6-31G level have revealed the fact that the transformations of i-6 to ii-6 and of i-7 to ii-7 are endothermic by 19.93 and 4.20 kcal/mol, respectively. The isomerization of compounds i to ii is more endothermic in the case of the HP series. Furthermore, calculations have shown the transformation of i to ii to be kinetically less favored in the HP series than in its analogue WM series. The activation barrier for the transformation of i-6 to ii-6 is calculated to be 22.09 kcal/mol, whereas the corresponding barrier in the WM series is 13.77 kcal/mol. The thermodynamic features of the reactions support the experimental findings as well: 7 is calculated to be more stable than iii-7, and iii-6 is more stable than 6, as expected. The diverging behavior in the mechanism of the interconversion of i to ii has been attributed to the stabilization of the positive charge by a carbonyl oxygen in ii-7 due to the flexibility of the seven-membered ring in contrast to the rigidity of the six-membered ring.

Journal Article↗

PubMed1999

A single domain thermophilic xylanase can bind insoluble xylan: evidence for surface aromatic clusters.

A clone expressing xylanase activity in Escherichia coli has been selected from a genomic plasmid library of the thermophilic Bacillus strain D3. Subcloning from the 9-kb insert located the xylanase activity to a 2.7-kb HindII/BamHI fragment. The DNA sequence of this clone revealed an ORF of 367 codons encoding a single domain type-F or family 10 enzyme, which was designated as XynA. Purification of the enzyme following over-expression in E. coli produced an enzyme of 42 kDa with a temperature optimum of 75 degrees C which can efficiently bind and hydrolyse insoluble xylan. The pH optimum of the enzyme is 6.5, but it is active over a broad pH range. A homology model of the xylanase has been constructed which reveals a series of surface aromatic residues which form hydrophobic clusters. This unusual structural feature is strikingly similar to the situation observed in the structure determined for the type-G xylanase from the Bacillus D3 strain and may constitute a common evolutionary mechanism imposed on different structural frameworks by which these xylanases may bind potential substrates and exhibit thermostability.

Amino Acid Sequence↗

PubMed1976

Conformational properties of adenylyl-3' leads to 5'-adenosine in aqueous solution.

A detailed 220-MHz NMR study has been made of the conformational properties for the homodinucleotide adenylyl-3' leads to 5'-adenosine, ApA, in D2O. Unambiguous signal assignments of all proton signals were made with the aid of selectively deuterated nucleotidyl units, ApA, ApA, and D-8ApA, and complete, accurate sets of NMR parameters were derived by simulation-iteration methods. Sets of limiting chemical shifts and coupling values were also obtained for ApA and constituent monomers 3'-AMP and 5'-AMP at infinite dilution and at identical ionization states for assessment of dimerization effects. Conformational properties were evaluated quantitatively for most of the conformational bonds of ApA and these are consistent with two compact folded dynamically averaged structures, a base-stacked right helical structure, I, characterized as anti, C3'-endo, g-, w,w' (320,330 degrees), g'g', gg, C3'-endo, anti, and a more loosely base-stacked loop structure, II, with anti, C3'-endo, g-, w,w' (80 degrees, 50 degrees), g'g', gg, C3'-endo, anti orientations. Dimerization produces a number of nucleotidyl conformational changes including a shift in ribose equilibrium C2'-endo (S) in equilibrium C3'-endo (N) in favor of C3'-endo in both Ap- and -pA (60:40 vs. 35:65 in monomers), a change in glycosidic torsion angle chiCN toward 0 degrees, and a greater locking-in of rotamers along bonds involved in the phosphodiester backbone. Moreover, there is clear evidence that the transitions from S leads to N forms and chiCN leads to 0 degrees are directly related to base stacking in ApA. Finally, ApA exists in solution as an equilibrium between I, II and an unstacked form(s) with as yet undetermined conformational features. Since C4'-C5', C5'-O5', and C3'-O3' bonds possess exceptional conformational stabilities, it is proposed that destacking occurs primarily by rotation about P-O5' and/or O3'-P. Predominant factors influencing the overall ApA conformation are thus base-base interaction and flexibility about P-O5' and O3'-P, with change of ribose conformation occurring in consequence of an alteration of chiCN, the latter in turn being governed by the need for maximum eta overlap of stacked adenine rings.

Adenine Nucleotides↗

PubMed1994

Binding of quinomycin antibiotic UK-65,662 to DNA: 1H-n.m.r. studies of drug-induced changes in DNA conformation in complexes with d(ACGT)2 and d(GACGTC)2.

Quinomycin antibiotic UK-65,662 binds selectively to the 5'-CpG-binding sites of the DNA duplexes d(ACGT)2 and d(GACGTC)2; the complexes have been studied in detail by 1H-n.m.r. spectroscopy and molecular-modelling techniques employing nuclear Overhauser effect-restrained energy minimization and molecular dynamics. Whereas the terminal A.T base pairs of the tetamer duplex d(ACGT)2 adopt a stable Hoogsteen alignment (characterized by a syn glycosidic conformation of the purine base), when internalized within the hexamer duplex d(GACGTC)2, the A.T base pairs revert to anti glycosidic torsion angles characteristic of the Watson-Crick hydrogen-bonding scheme. The energetics of base-pair stacking at the terminal 5'-GpA steps of the hexamer complex, with base pairs in the Watson-Crick alignment, are concluded to be important determinants of the adopted conformation, whereas an energetic preference for stacking interactions between terminal Hoogsteen A.T base pairs and the drug quinoline chromophores is evident in the tetramer complex. The internal G.C base pairs in both complexes are highly stabilized, as indicated by the very slow exchange rates of the guanine imino protons; in contrast, the flanking A.T base pairs are no more stable than in the ligand-free DNA duplexes. A large number of intermolecular nuclear Overhauser effects are indicative of many van der Waals contacts and hydrogen-bonding between the antibiotic and the minor groove of the central G.C base pairs in both complexes, indicating that interactions with the G.C base pairs in each duplex are very similar providing the essential features for recognition and tight binding. Despite the difference in the conformation of the A.T base pairs, stacking with the quinoline rings occurs primarily with the adenine bases in both complexes. Relative intensities of intranucleotide versus internucleotide nuclear Overhauser effects indicate that both duplexes are substantially unwound by drug binding (particularly at the CpG step) and this is confirmed by the structure calculations. Both duplexes have ladder-like structures that must lead to significant local distortions of the DNA conformation in vivo.

Anti-Bacterial Agents↗

PubMed2026

Causal circuit tracing reveals distinct computational architectures in single-cell foundation models: inhibitory dominance, biological coherence, and cross-model convergence.

MOTIVATION: Sparse autoencoders (SAEs) decompose foundation-model activations into interpretable features, but the model-internal causal interactions between those features (i.e. what ablating one feature does to the others, as distinct from the biological causal structure of the underlying cells)-and how those model-internal relationships relate to biological structure-are uncharacterized in single-cell foundation models. RESULTS: We introduce model-internal causal circuit tracing-zeroing one SAE feature at a source layer and measuring the resulting change in all downstream SAE features, for each of 120 source features-and apply it to Geneformer V2-316M and scGPT whole-human across four conditions (96 892 ablation-derived edges, 80 191 forward passes). On annotation-selected source features, edges share GO/KEGG/Reactome/STRING/TRRUST ontology terms at 50.9%-68.5%, a 2.9-6.2× enrichment over a configuration-preserving permutation null (P<.002); on 20 randomly sampled source features this attenuates to 21.5%-26.3%-still 2.5-3.1× above null-quantifying the annotation-selection contribution. Inhibitory dominance (fraction of ablation edges with d<0, i.e. source activation supports downstream target) is 65.5%-89.4%. scGPT produces larger raw per-edge effects (mean |d|=1.40 versus 1.05); after feature-share normalization, Geneformer is stronger (paired gene-pair ratio 0.64 on 33 301 shared pairs). Cross-model consensus yields 1142 architecture-invariant domain pairs (ordered pairs of GO biological-process categories "A→B" each connected by at least one ablation edge in both models; 10.6× enrichment over permutation null; P<.001). Circuit edge magnitude explains <1% of the variance in marginal driver-gene coexpression on the same cells (R2=0.010, n=31 176): the graph encodes structure beyond bivariate correlation. Against a matched-cell-type ENCODE ChIP-seq prior, circuit-predicted transcription factor (TF)→target pairs are enriched 2.06× (Fisher OR 5.84), markedly higher than 1.12× against TRRUST; direct ChIP-seq-supported target pairs show 10-30× larger CRISPRi sign-bias-corrected excess than indirect pairs. Gene-level CRISPRi validation on Replogle K562 and the noncancer RPE1 arm (and a true primary-T-cell control from Shifrut E, Carnevale J, Tobin V et al. Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function. Cell 2018; 175: 1958-71.e15) after sign-bias correction shows excess over baseline of +0.03 and +0.35 percentage points on K562 and RPE1, respectively (baseline already 52%-56% from sign marginals); effect-magnitude Spearman correlations ρ≈0. Bootstrap and per-cell-type stability (N∈{50,100,200}; B cell, CD4 + T, macrophage) give Pearson r≥0.97 on shared edges with 100% sign agreement; edge Jaccard grows monotonically with sample size. The circuit graph is therefore highly reproducible as an effect-size map, cell type specific in edge identity, consistent with coexpression encoding, and weakly but detectably enriched for ChIP-seq-supported direct regulatory edges. AVAILABILITY AND IMPLEMENTATION: https://github.com/Biodyn-AI/bio-sae-circuits (Python). Archival DOI: 10.5281/zenodo.19,633,166 (Zenodo).

Humans↗

PubMed2001

Intrauterine contraception--what now and what next?

Rising each year by around 85 million, we have started this millennium with over 6,000 million, mostly utterly destitute, human beings on a finite planet. We will never meet human needs without stabilizing human numbers. In the sexual arena, all human beings (but especially the males of the species) are very bad at using their brains and their genitalia at the same time, i.e. putting the knowledge in their head about risks of conception or of sexually transmitted infections into appropriate preventive action when sexually aroused! Therefore, a very useful criterion for choice of methods all through the various stages of reproductive life is 'forgettability'. This is a great 'plus' point for the intrauterine route, especially the levonorgestrel intrauterine system (LNG-IUS). Its main contraceptive effects are local, by endometrial suppression and changes to the cervical mucus and uterotubal fluid, which impair sperm migration. The blood levels of levonorgestrel are about one-quarter of the peak levels in users of the progestin-only pill, and so gestagenic symptoms are uncommon and ovarian function is little altered. Most women continue to ovulate and, in the remainder, sufficient estrogen for health is produced from the ovary even if they become amenorrheic, as many do; this is primarily a local end-organ effect and should be seen as a benefit. Postmenopausal estradiol levels have not been detected, even in those who have no uterine bleeding. Although usable by selected nulliparae, the LNG-IUS is a particularly good choice for the parous woman, for whom I maintain it represents the future already here, which is not recognized by many providers. It has unsurpassed efficacy; 99.5% of women have not conceived after 5 years of use. Return of fertility is rapid and appears to be complete. Combining the best features of hormonal and intrauterine contraception without most ofthe problems of either, it fundamentally 'rewrites the textbooks' about intrauterine devices. Its gynecological benefits are impressive: the LNG-IUS user can expect a dramatic reduction in amount and, after the first few months, in duration of blood loss. Dysmenorrhea is also greatly benefited, in most cases. In summary, the LNG-IUS fulfils many of the criteria for an 'ideal' contraceptive. Adverse side-effects are few and, in general, they are not in the category 'hazardous'. Regarding the inconvenience of the first weeks of light post-insertion bleeding and the early-phase low incidence ofsteroidal side-effects, forewarned is forearmed!

Administration, Intravaginal↗

PubMed1999

Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311.

C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy.

Acid Phosphatase↗

PubMed2002

Transglutaminases: nature's biological glues.

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.

Animals↗

PubMed1991

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.

Catalysis↗

PubMed1993

Predominant and stable T cell responses to regions of myelin basic protein can be detected in individual patients with multiple sclerosis.

T lymphocytes from patients with multiple sclerosis (MS) recognize multiple myelin basic protein (MBP) epitopes. This situation complicates the design of specific immunotherapies. We investigated to which extent the T cell response to MBP is heterogeneous in single subjects in terms of preferentially recognized regions of the molecule, major histocompatibility complex (MHC) restriction, and stability over time. From each of nine patients with MS, a minimum of six MBP-specific T lymphocyte lines (TLL) were assayed for the proliferative response to a panel of overlapping peptides, encompassing the whole MBP. Predominant T cell recognitions of distinct MBP regions were present in three patients, all HLA-DR2+, independently of the clinical features of their disease. T cell reactivity was preferentially directed to residues 16-38 in one patient. In this case the response was also stable over time, during different phases of the disease. Predominant reactivity to residues 86-99 was detected in the two other DR2+ patients. In each of the patients with other HLA-DR haplotypes (DR2-), as well as in three DR2+ non-MS donors, the T cell response to MBP appeared to be considerably more heterogeneous. The HLA restriction element varied among TLL recognizing the same MBP region, even when raised from the same individual. The genomic HLA typing, performed on the DRB1 and DRB5 genes in the DR2+ subjects, showed no obvious correspondence between preferential responses to regions of MBP and HLA-DR2 subtypes. In this context, a simple, new method for the genomic typing of the HLA-DRB1 gene in individuals with the HLA-DR2 serological specificity is also described. We conclude that predominant and stable T cell responses to a single MBP region can be detected in some patients with MS. In these individuals, the MHC restriction of the T cell recognition of predominant regions appears to be variable. Polymorphisms of the HLA-DR2 gene products alone do not account for the selection of the dominant MBP T cell epitope.

Adolescent↗

PubMed2004

Precision grasps of children and young and old adults: individual differences in digit contact strategy, purchase pattern, and digit posture.

The evolutionary origins and variations of the precision grip, in which an object is held between the thumb and other digits, are poorly understood. This is surprising because the neural basis of this grasp pattern, including the motor cortex and pyramidal tract have received extensive study. Most previous work has shown that features of an object to be grasped (external factors) determine grasp patterns. The objective of the present study was to investigate individual differences (central factors) in use of the pincer and other precision grips. The grasping patterns of male and female young adults, older adults and children were examined as they reached (with both left and right hand) for five small beads (3-16 mm diameter). Frame-by-frame analysis of grasping indicated a high degree of variability in digit contact strategies, purchase patterns and digit posture both within and between subjects. (1) The contact strategies consisted of five variations, depending on whether the thumb or the index finger dragged or stabilized the bead for grasping. (2) Purchase patterns consisted of seven different types of precision grips, involving the thumb and various combinations of other digits. (3) There were four variations stemming from the posture of the non-grasping digits. Grip patterns of the left and right hands were correlated in individual subjects, as were strategies used for different bead sizes. Females displayed slightly more variability in grasp patterns than did males, and digit width (obtained from photocopies of the subjects' hands) was weakly correlated with the grasp patterns used. Although it was expected that the pincer would be used for all objects, it was preferentially used for only the smallest object except for older adults who used the pincer grasp on most objects. The variability in digit contact strategies, purchase patterns, and posture of the non-grasping digits indicates that central factors (innate or learning-induced architecture of the left parietal cortex) make important contributions to the selection of a grasping pattern. These individual differences are discussed in relation to the neural control of grasping and its potential contribution to understanding the evolution, development, and pathology of the precision grip.

Adolescent↗

PubMed2002

Comparative morphometry of L4 vertebrae: comparison of large animal models for the human lumbar spine.

STUDY DESIGN: Anatomic analysis of L4 vertebral morphometry comparing specimens harvested from humans and five common large animal species. OBJECTIVE: To compare fundamental structural similarities and differences in the vertebral bodies of commonly used experimental animals relative to human vertebrae. SUMMARY OF BACKGROUND DATA: Animal models are commonly used for assessment of spine fusion, instrumentation techniques, and vertebral bone biology. Among the animals used, the lumbar vertebrae exhibit considerable anatomic variability. The goal of this study was to determine which of the animals commonly used for spine research is best suited as an anatomic model for the human lumbar spine. METHODS: Morphometric features of the L4 vertebrae of five common research animals were compared with those of the human L4 vertebrae. Mature canines, immature pigs, mature micropigs, mature dairy goats, and mature sheep were analyzed. These species were chosen because they are commonly selected research animals, and most research facilities do not need to be modified to use them. The samples included ten L4 vertebrae of each animal species and seven human L4 vertebrae. Each specimen was meticulously cleaned of all soft tissue. The measurements were grouped into vertebral body parameters, neural canal dimensions, and pedicle and facet morphometery. The mean of each anatomic measurement was compared using a single factor analysis of variance and a Scheffe's post hoc test, with 0.05 denoting significance. RESULTS: The human vertebral body was significantly wider and deeper in the anteroposterior plane than any of the animals studied. However, the mean vertebral body height of the sheep and goat significantly exceeded that of the human specimens. The mean pedicle angle of every animal species was significantly greater than that of the human. The mean pedicle width of the micropig and goat were significantly narrower than the human pedicles, and the dog specimens lacked a definable pedicle altogether. There was no significant difference in mean pedicle width between any of the remaining species and the human specimens. Facet tropism and radius of curvature of the sheep and goat specimens differed significantly from the remaining selections. CONCLUSIONS: When posterior pedicle instrumentation is part of a testing protocol, the increased pedicle angle and lack of vertebral body depth found in all animals studied must be kept in mind. In addition, when testing interbody cages designed to stabilize the spine and promote fusion, one must be aware of the decreased vertebral body depth and width in these animals, as compared with humans. Physeal defects in the immature pig may alter specific biomechanical results during failure or fatigue testing, or in basic studies of vertebral bone material properties. In all cases, instrumentation and hardware must be sized appropriately to the selected model to provide meaningful results.

Age Factors↗

PubMed2005

Intracranial stereotactic positioning systems: Report of the American Association of Physicists in Medicine Radiation Therapy Committee Task Group no. 68.

Intracranial stereotactic positioning systems (ISPSs) are used to position patients prior to precise radiation treatment of localized lesions of the brain. Often, the lesion is located in close proximity to critical anatomic features whose functions should be maintained. Many types of ISPSs have been described in the literature and are commercially available. These are briefly reviewed. ISPS systems provide two critical functions. The first is to establish a coordinate system upon which a guided therapy can be applied. The second is to provide a method to reapply the coordinate system to the patient such that the coordinates assigned to the patient's anatomy are identical from application to application. Without limiting this study to any particular approach to ISPSs, this report introduces nomenclature and suggests performance tests to quantify both the stability of the ISPS to map diagnostic data to a coordinate system, as well as the ISPS's ability to be realigned to the patient's anatomy. For users who desire to develop a new ISPS system, it may be necessary for the clinical team to establish the accuracy and precision of each of these functions. For commercially available systems that have demonstrated an acceptable level of accuracy and precision, the clinical team may need to demonstrate local ability to apply the system in a manner consistent with that employed during the published testing. The level of accuracy and precision required of an individual ISPS system is dependent upon the clinical protocol (e.g., fractionation, margin, pathology, etc.). Each clinical team should provide routine quality assurance procedures that are sufficient to support the assumptions of accuracy and precision used during the planning process. The testing of ISPS systems can be grouped into two broad categories, type testing, which occurs prior to general commercialization, and site testing, performed when a commercial system is installed at a clinic. Guidelines to help select the appropriate tests as well as recommendations to help establish the required frequency of testing are provided. Because of the broad scope of different systems, it is important that both the manufacturer and user rigorously critique the system and set QA tests appropriate to the particular device and its possible weaknesses. Major recommendations of the Task Group include: introduction of a new nomenclature for reporting repositioning accuracy; comprehensive analysis of patient characteristics that might adversely affect positioning accuracy; performance of testing immediately before each treatment to establish that there are no gross positioning errors; a general request to the Medical Physics community for improved QA tools; implementation of weekly portal imaging (perhaps cone beam CT in the future) as a method of tracking fractionated patients (as per TG 40); and periodic routine reviews of positioning accuracy.

Equipment Failure Analysis↗

PubMed1996

Selecting infusion devices for use in ambulatory care.

Intravenous infusion devices commonly used in home care and ambulatory care settings are reviewed and factors to consider in selecting a device are suggested. The type of therapy to be administered, the patient or caregiver's ability to understand and carry out instructions, staff time required for patient teaching and drug and device preparation, drug stability, frequency of doses, reservoir volume, control of flow rate, type of venous access, cost and availability of devices and supplies, and reimbursement should be considered. Cost-effectiveness of a device can be evaluated only by analyzing all of the costs associated with administering a medication. Decisions must be based on an individual agency's needs, but usually one type of single-dose infuser and one brand of electronic ambulatory-care infusion pump can meet the needs of most of an agency's patients. For patients self-administering up to four doses per day, appropriate methods may include slow intravenous injection (i.v. push), infusion from minibags and tubing, and the use of elastomeric infusers, electronic or mechanical syringe pumps, or a new device based on infusion across a bioelectric membrane. Some of these types of infusers can also be used for continuous infusion. Syringe pumps are reliable and affordable and control infusion rates well, but infusion volume is limited and these devices must be recovered, cleaned, and tested between patients. Although elastomeric pumps may have higher price tags than other devices, they are simple for patients to use and dispose of. While elastomeric devices have tubing permanently attached, the disposable tubing of some other devices is detachable and can be reused if institutional policy permits. Electronic ambulatory-care infusion pumps can meet a wide range of infusion requirements. They vary in size, weight, ability to detect occlusions, features, and reliability. All use proprietary infusion sets, but the costs of sets and disposable supplies vary. Some pumps can be used for a single infusion mode, such as patient-controlled analgesia (PCA); others offer two or more modes (for example, continuous, intermittent, and PCA). "Multichannel" pumps can be used for simultaneous infusion of up to four medications; the rate of each infusion is programmed separately, and some multichannel devices offer multiple infusion modes. Some can be programmed remotely by telephone. Before an agency decides on a pump, it can investigate current users' and rental firms' experiences with the device. Also, all relevant personnel should try programming the device. Determining which devices to use requires a comparison of features that are pertinent to the particular agency or institution and a cost analysis that considers acquisition, reimbursement, patient training time, and the cost of disposable supplies.

Ambulatory Care↗

PubMed2002

Cation charge and size selectivity of the C2 domain of cytosolic phospholipase A(2).

C2 domains regulate numerous eukaryotic signaling proteins by docking to target membranes upon binding Ca(2+). Effective activation of the C2 domain by intracellular Ca(2+) signals requires high Ca(2+) selectivity to exclude the prevalent physiological metal ions K(+), Na(+), and Mg(2+). The cooperative binding of two Ca(2+) ions to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)-alpha) induces docking to phosphatidylcholine (PC) membranes. The ionic charge and size selectivities of this C2 domain were probed with representative mono-, di-, and trivalent spherical metal cations. Physiological concentrations of monovalent cations and Mg(2+) failed to bind to the domain and to induce docking to PC membranes. Superphysiological concentrations of Mg(2+) did bind but still failed to induce membrane docking. In contrast, Ca(2+), Sr(2+), and Ba(2+) bound to the domain in the low micromolar range, induced electrophoretic mobility shifts in native polyacrylamide gels, stabilized the domain against thermal denaturation, and induced docking to PC membranes. In the absence of membranes, the degree of apparent positive cooperativity in binding of Ca(2+), Sr(2+), and Ba(2+) decreased with increasing cation size, suggesting that the C2 domain binds two Ca(2+) or Sr(2+) ions, but only one Ba(2+) ion. These stoichiometries were correlated with the abilities of the ions to drive membrane docking, such that micromolar concentrations of Ca(2+) and Sr(2+) triggered docking while even millimolar concentrations of Ba(2+) yielded poor docking efficiency. The simplest explanation is that two bound divalent cations are required for stable membrane association. The physiological Ca(2+) ion triggered membrane docking at 20-fold lower concentrations than Sr(2+), due to both the higher Ca(2+) affinity of the free domain and the higher affinity of the Ca(2+)-loaded domain for membranes. Kinetic studies indicated that Ca(2+) ions bound to the free domain are retained at least 5-fold longer than Sr(2+) ions. Moreover, the Ca(2+)-loaded domain remained bound to membranes 2-fold longer than the Sr(2+)-loaded domain. For both Ca(2+) and Sr(2+), the two bound metal ions dissociate from the protein-membrane complex in two kinetically resolvable steps. Finally, representative trivalent lanthanide ions bound to the domain with high affinity and positive cooperativity, and induced docking to PC membranes. Overall, the results demonstrate that both cation charge and size constraints contribute to the high Ca(2+) selectivity of the C2 domain and suggest that formation of a cPLA(2)-alpha C2 domain-membrane complex requires two bound multivalent metal ions. These features are proposed to stem from the unique structural features of the metal ion-binding site in the C2 domain.

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PubMed2005

Sporadic amyotrophic lateral sclerosis: a hypothesis of persistent (non-lytic) enteroviral infection.

Because of recently reported reverse transcriptase polymerase chain reaction evidence of enterovirus in sporadic amyotrophic lateral sclerosis (SALS) and because of newly available anti-enteroviral drugs binding enteroviral capsids, it is reasonable to re-formulate an enteroviral hypothesis of SALS using recent advances in molecular virology. Viral persistence is non-lytic and non-cytopathic infection that evades host's immune surveillance. Enteroviruses are known to cause persistent as well as lytic infection both in vitro and in vivo. Both virion as well as host factors modulate between persistent and lytic infection. Apoptosis, or programmed cell death, is a process of active non-necrotic cell death. It has complex interplay with viruses and may be either promoted or opposed by them. Apoptosis is a major factor in motor neuron death in SALS. Viral tropism is the process by which viruses select and propagate to target cells. It is controlled by capsid conformation and surface receptors on host cells. Enteroviruses have a region on their capsids known as the canyon which docks on such receptors. Docking induces conformational changes of the capsid and genome release. Poliovirus, tropic for motor neurons, docks on the poliovirus receptor, about which much is known. The virus penetrates the motor system focally after crossing either the blood-muscle or the blood-brain barriers. It propagates bidirectionally along axons and synapses to contiguous motor neurons, upper as well as lower, which sequester infection and create avenues for spread over long distances. If chronic and persistent rather than acute and lytic, such viruses trafficking in a finite system of non-dividing cells and inducing apoptosis would cause cell death that summates linearly rather than exponentially. Taken together, these explain signature clinical features of SALS - focal onset weakness, contiguous or regional spread of weakness, confinement to upper and lower motor neurons, and linear rates of progression. The hypothesis predicts the following testable investigations: 1) viral detection may be possible by applying amplification technology to optimally acquired nervous tissue processed by laser microdissection; 2) genetic susceptibility factors such as cell surface receptor polymorphisms may combine with sporadic exposure and chance penetration of the motor system in SALS; 3) a transgenic animal model might be created by inserting such genetic factors into an animal host and inoculating intramuscularly rather than intracerebrally biochemical fractions of SALS motor neurons at vulnerable periods in the developmental life cycle of the transgenic host; and 4) continual long-term administration of anti-enteroviral agents called capsid-binding compounds which stabilize capsids and prevent genome release might be efficacious.

Amyotrophic Lateral Sclerosis↗