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Biomedical subjects

H Savage

Publications and source records attributed to H Savage.

At least 19 recordsLinked to original sources
PubMed2000

Structural origins of the interfacial activation in Thermomyces (Humicola) lanuginosa lipase.

The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TlL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha(0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TlL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.

Crystallography, X-Ray↗
PubMed2000

Laser irradiative tissue probed in situ by collagen 380-nm fluorescence imaging.

BACKGROUND AND OBJECTIVE: There is no ideal method to detect molecular changes in situ of laser-irradiated tissue without removing a section of tissue for histologic examination. A new method is reported to evaluate laser-induced thermal tissue damage in situ by using 380-nm native fluorescence imaging under 340-nm excitation. STUDY DESIGN/MATERIALS AND METHODS: Native fluorescence imaging was performed on laser irradiated bovine tendon tissue and compared with light illuminated photography and histology with picrosirius red F3BA stain. RESULTS AND CONCLUSION: The results indicate that a decrease in collagen fluorescence at 380 nm was observed in laser-induced thermally damaged tissues. The injured region of tissue defined by its fluorescence image coincided with an area defined by photography and histology.

Animals↗
PubMed2000

Appetitive learning using visual conditioned stimuli in the pond snail, lymnaea.

Feeding can be conditioned in the pond snail Lymnaea stagnalis to two different visual stimuli (a black panel or a 5-mm black and white check surround) by pairing the potential conditioned stimulus (CS) with sucrose. Exclusion of chemical cues (associated with differences between the water in home tank and that in training apparatus) that could serve as CS is important for successful visual conditioning. A featureless gray surround, used as an alternative to the check (to which it was matched in luminance) in counterbalanced training designs, was discriminated from the check, showing that resolution (for which the eyes would be necessary) was occurring. The gray surround was largely ineffective as a CS. Single-trial learning was possible with the black panel, but not with the check; it is argued that this may be due to lack of prior experience of stimuli like the check. Conditioning of feeding has now been obtained in Lymnaea to chemical, tactile, and visual cues, opening the way to comparative studies of the neural circuitry underlying appetitive conditioning in different senses, so far explored in Lymnaea only for tactile CS. Such comparative studies are as yet largely lacking in invertebrates.

Animals↗
PubMed2000

A comparison of Cunyite and Fosterite NIR tunable laser tissue welding using native collagen fluorescence imaging.

OBJECTIVE: To evaluate the technique of native collagen fluorescence imaging for assessing the extent of welded areas for tissues exposed to different near-infrared (NIR) laser wavelengths. BACKGROUND: Native fluorescence imaging may be used to identify the distribution of collagen and elastin in tissues. Our past work demonstrated that different welding strengths were obtained under the same laser power conditions using different NIR wavelengths. The role of collagen in tissue welding experiments is not well understood. METHODS: Two new NIR tunable lasers were used to weld canine skin. The welded areas on the surface and in cross sections were analyzed by measuring the spatial distribution of native collagen fluorescence at 380 nm excited by 340 nm radiation. RESULTS: The results show that native collagen fluorescence imaging is a useful technique for analyzing the extent of tissue welds produced under a range of laser exposures. Fluorescence imaging reveals the depth of laser interaction with the tissue as well as evaluating collateral damage to the tissue surface. The welded volume obtained in skin using Cunyite laser exposure at 1,430 nm is deeper than that produced with Forsterite laser exposure at 1,250 nm. The post welded tensile strength for the same power density is greater for the Cunyite lasers. Ablated tissue on the surface is more prevalent with Forsterite laser welding at 1,250 nm than with Cunyite at 1,430 nm. CONCLUSION: Native collagen fluorescence can distinguish between tissue welds that have been produced by different NIR wavelengths. Tissue welding using 1,430 nm radiation is more effective than that using 1,250 nm.

Animals↗
PubMed2000

Fluorescence spectroscopic imaging to detect changes in collagen and elastin following laser tissue welding.

OBJECTIVE: A study was performed to evaluate the use of native fluorescence imaging to detect in situ molecular changes. SUMMARY BACKGROUND DATA: There is no ideal noninvasive method to monitor molecular changes in a local region at a laser weld joint without removing a section of tissue for histological examination. METHODS: Two sections of animal skin were welded together border to border using a Ti:sapphire laser beam (800 nm). Fluorescence imaging was performed on the cross section of the welded site at specific emission wavelengths (lambda c) for collagen at 380 nm and for elastin at 450 nm using excitation wavelengths (lambda e) of 340 nm, and 380 nm, respectively. RESULTS: A reduction of the collagen and elastin emission was observed in the fluorescence images of the welded region. These results were confirmed with histology using picrosirius red F3BA under polarized light and orcein stains. CONCLUSION: Optical spectroscopic imaging offers a new noninvasive detecting method for microscopic evaluation of laser tissue welding.

Animals↗
PubMed1998

Crystallization and preliminary X-ray diffraction studies of phospho-adenylylsulfate (PAPS) reductase from E. coli.

PAPS reductase from E. coli is involved in sulfur metabolism and catalyses the reduction of phospho-adenylyl-sulfate (PAPS) to sulfite. The protein has been cloned, overexpressed and purified from E. coli. Crystallization experiments resulted in crystals suitable for X-ray diffraction. The crystals belong to the orthorhombic space group C2221 with cell dimensions a = 81.9, b = 97.4, c = 109.5 A, and contain one molecule per asymmetric unit. At cryogenic (100 K) temperatures the crystals diffract to a resolution limit of 2.7 A using a rotating anode and to 2.0 A at a synchrotron source.

Crystallization↗
PubMed1998

Efficient gene transfer to human squamous cell carcinomas by the herpes simplex virus type 1 amplicon vector.

BACKGROUND: This study evaluates the efficiency of herpes simplex virus (HSV) mediated gene transfer in human squamous cell carcinoma (SCC) cell lines in vitro and in vivo when delivered by selective intra-arterial perfusion. METHODS: Human head and neck SCC were exposed to HSV-LacZ and HSV-interleukin-2 (IL-2) and gene transfer and expression assessed by X-gal staining and enzyme-linked immunosorbent assay, respectively. Hamster cheek pouch tumors were perfused with HSV-LacZ or HSV-IL-2, by microcannulating the external carotid artery, and gene transfer determined. RESULTS: A ratio of 5 viral particles per tumor cell achieved gene transfer rates exceeding 50%. Interleukin-2 levels of 287 +/- 17 to 424 +/- 8.4 ng per million cells were achieved at a ratio of 2 viral particles per tumor cell. Selective intra-arterial perfusion of the HSV-IL-2 vector yielded IL-2 levels of 45.8 +/- 17.0 pg per g tumor. CONCLUSIONS: HSV amplicon vectors are efficient vehicles for gene transfer in vitro in human head and neck SCC cell lines and in vivo when introduced by selective intra-arterial perfusion.

Animals↗
PubMed1997

Behavioural lateralisation of the tetrapod type in the zebrafish (Brachydanio rerio).

Visual lateralisation resembling that found in a bird (domestic chick) is here demonstrated in a teleost (zebrafish, Brachydanio rerio). Zebrafish predominantly view objects with the body axis close to facing the object (0-20 degrees on either side of facing). Strange objects were viewed at first exposure chiefly with the right frontal field; so was a complex and unfamiliar scene made up of familiar components. In a second trial, using the same stimulus or scene, left frontal viewing tended to be used instead. A familiar partner (a fish of another species) was viewed left frontally. The domestic chick also uses the left eye to view familiar stimuli, shifting to the right when it has to decide what response is appropriate to the object at which it is looking. An empty scene in which nothing could be concealed (and so no response was called for) was viewed by zebrafish with the left eye from the start. In zebrafish and the chick, the right eye is used when it is necessary to inhibit premature response, in order to sustain viewing until a decision is reached, and the left is used when it is necessary to keep an eye on a familiar or clearly empty scene. The findings suggest homology of cerebral lateralisation in teleost fish and tetrapods.

Aggression↗
PubMed1997

Crystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide alpha hydrolases.

BACKGROUND: Assimilatory sulphate reduction supplies prototrophic organisms with reduced sulphur for the biosynthesis of all sulphur-containing metabolites. This process is driven by a sequence of enzymatic steps involving phosphoadenylyl sulphate (PAPS) reductase. Thioredoxin is used as the electron donor for the reduction of PAPS to phospho-adenosine-phosphate (PAP) and sulphite. Unlike most electron-transfer reactions, there are no cofactors or prosthetic groups involved in this reduction and PAPS reductase is one of the rare examples of an enzyme that is able to store two electrons. Determination of the structure of PAPS reductase is the first step towards elucidating the biochemical details of the reduction of PAPS to sulphite. RESULTS: We have determined the crystal structure of PAPS reductase at 2.0 A resolution in the open, reduced form, in which a flexible loop covers the active site. The protein is active as a dimer, each monomer consisting of a central six-stranded beta sheet with alpha helices packing against each side. A highly modified version of the P loop, the fingerprint peptide of mononucleotide-binding proteins, is present in the active site of the protein, which appears to be a positively charged cleft containing a number of conserved arginine and lysine residues. Although PAPS reductase has no ATPase activity, it shows a striking similarity to the structure of the ATP pyrophosphatase (ATP PPase) domain of GMP synthetase, indicating that both enzyme families have evolved from a common ancestral nucleotide-binding fold. CONCLUSIONS: The sequence conservation between ATP sulphurylases, a subfamily of ATP PPases, and PAPS reductase and the similarities in both their mechanisms and folds, suggest an evolutionary link between the ATP PPases and PAPS reductases. Together with the N type ATP PPases, PAPS reductases and ATP sulphurylases are proposed to form a new family of homologous enzymes with adenine nucleotide alpha-hydrolase activity. The open, reduced form of PAPS reductase is able to bind PAPS, whereas the closed oxidized form cannot. A movement between the two monomers of the dimer may allow this switch in conformation to occur.

Acid Anhydride Hydrolases↗
PubMed1997

Polarization filter for biomedical tissue optical imaging.

A technique based on the degree that light is depolarized when propagating inside tissues is demonstrated for optical imaging in biomedical systems. The difference in the degree of polarization of the emerging light allows for the discrimination of different types of tissues. The technique was investigated in the transillumination and back-scattering geometry and in both cases the potential of this method to image and separate out different types of tissues is demonstrated.

Animals↗
PubMed1996

Two-dimensional structure of light harvesting complex II (LHII) from the purple bacterium Rhodovulum sulfidophilum and comparison with LHII from Rhodopseudomonas acidophila.

BACKGROUND: Within the membranes of photosynthetic bacteria, up to three types of light harvesting complexes (LHI, LHII, LHIII) are found. These complexes absorb photons and transfer the excitation energy to the photosynthetic reaction centre. The LH complexes comprise units that contain alpha and beta polypeptides with associated pigment molecules. RESULTS: The structure of LHII complex from Rhodovulum sulfidophilum has been examined to a resolution of 7 A using electron microscopy. The complex is a nonamer containing nine alphabeta subunits. These are arranged in two radially symmetric concentric cylinders, with the nine alpha chains positioned in the inner cylinder and the nine beta chains forming the outer cylinder. The 18 transmembrane helices are readily observed in the projection maps, along with 18 additional peaks attributed to the pigment molecules. CONCLUSIONS: The determination of more structures of LH complexes will uncover the full extent of the variability of the oligomerization states in different bacteria and also in the native membrane. The analysis of two-dimensional crystals allows a rapid determination of key structural features and the oligomeric state of the complex. Comparison of our structure determined by electron microscopy with the recently solved X-ray structure indicates that the results of the two methods are complementary.

Amino Acid Sequence↗
PubMed1995

Ultraviolet excitation fluorescence spectroscopy: a noninvasive method for the measurement of redox changes in ischemic myocutaneous flaps.

In this report, we discuss application of the noninvasive technology of ultraviolet fluorescence spectroscopy to the metabolic analysis of normal and compromised myocutaneous flaps. Acute changes in tissue redox states during ischemia and reperfusion were determined analysis of changes in the fluorescence spectrum of reduced nicotinomide adenine dinucleotide (NADH). Analysis of the system for NADH fluorescence showed good correlation between excitation spectra recorded at 450 nm from pure beta-NADH and those recorded from porcine rectus abdominis myocutaneous flaps. Sequential measurements of surface fluorescence were obtained from six flaps subjected to 6 hours of warm arterial ischemia and 4 hours of reperfusion. Results were compared with spectra obtained from six contralateral nonischemic control flaps. A significant mean increase in NADH fluorescence (49 percent; p < 0.05) was demonstrated within 30 minutes of vascular occlusion. Fluorescence intensity continued to increase throughout the ischemic period, reaching 320.5 percent of baseline values at 6 hours. Reperfusion resulted in the prompt return of fluorescence intensity to baseline levels. These results show that fluorescence spectroscopy of endogenous NADH is a sensitive and reliable indicator of vascular occlusion in experimental myocutaneous flaps.

Animals↗
PubMed1994

Tissue autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis.

The capacity to identify subclinical neoplastic disease of the upper aerodigestive tract (UADT) using tissue auto-fluorescent spectroscopy would significantly contribute to cancer screening. Rats received N-nitrosomethyl benzylamine (NMBA), a carcinogen shown to cause esophageal malignancies. Following sacrifice at early weekly intervals, gross assessment of esophageal mucosa of NMBA-exposed rats was indistinguishable from saline-treated controls. Histopathologic evaluation, however, revealed NMBA-induced preneoplastic changes in the epithelium. Concurrent with these changes, the NMBA-exposed rats demonstrated specific alterations in autofluorescence. These results demonstrate that NMBA-induced esophageal premalignancy can be distinguished by autofluorescent properties. The capacity to detect alterations in autofluorescence may allow more sensitive screening of UADT mucosa at risk for cancer development.

Animals↗
PubMed1990

The in vivo biologic effect of interleukin 2 and interferon alfa on natural immunity in patients with head and neck cancer.

Given the association of deficient natural immunity with the risk of metastatic disease, the ability to activate natural killer cell function may have a therapeutic significance. The effect of continuous infusion of interleukin 2 plus intramuscular interferon alfa on natural immune status was, therefore, analyzed in eight patients with head and neck cancer. Also evaluated was the effect of interleukin 2-interferon alfa therapy on lymphokine-activated killer cell activity as well as total lymphocyte count, percent of lymphocyte subsets, and levels of both circulating immune complexes and antibody classes. Both the percent and absolute number of natural killer cells (ie, CD56+ CD3- lymphocytes) within peripheral blood as well as natural killer cell activity against K562 targets increased significantly with treatment. The remaining immune parameters were not significantly altered. The demonstrated capacity to modulate natural immune function supports the potential use of interleukin 2-containing regimens as a preventive measure against metastatic disease in patients with head and neck cancer.

Adult↗
PubMed1989

Structure of insulin: results of joint neutron and X-ray refinement.

Neutron diffraction data for porcine 2Zn insulin were collected to 2.2 A resolution from a single crystal deuterated by slow exchange of mother liquor. A joint neutron/X-ray restrained-least-squares refinement was undertaken using the neutron data, as well as the 1.5 A resolution X-ray data collected previously. The final R factors were 0.182 for the X-ray data and 0.191 for the neutron data. Resulting atomic coordinates were compared with the initial X-ray model, showing a total r.m.s. shift of 0.36 A for the protein and 0.6 A for the solvent. Protonation of a number of individual amino acids was investigated by analysis of the neutron maps. No D atoms were found between the carboxylates of Glu B13 which make an intermolecular contact, suggesting nonbonded interaction rather than the predicted hydrogen bond. Amide hydrogen exchange was investigated in a refinement of their atomic occupancies. Regions of unexchanged amide groups were found in the center of the B helices. The results of this study emphasize the limited amount of information available in neutron diffraction studies of proteins at resolution lower than 2 A.

Animals↗
PubMed1986

Water structure in vitamin B12 coenzyme crystals. I. Analysis of the neutron and x-ray solvent densities.

The disordered solvent distribution in crystals of vitamin B12 coenzyme was examined using the methods of high-resolution neutron and x-ray diffraction. One set of neutron (0.95 A) and two sets of x-ray (0.94 and 1.1 A) data were collected and the resulting models were extensively refined using least-squares and Fourier syntheses. The solvent regions were analyzed in two stages: first, main sites were assigned to the well defined regions of solvent density and refined using least squares; second, continuous sites were assigned representing the more disordered diffuse and elongated regions of solvent density. During the analysis an acetone molecule was also located. Water networks were formulated from the assigned sites in the above models and also from those assigned in the original structure determination (Lenhert, 1968), using criteria that included hydrogen bonding (derived from small crystal hydrates), van der Waals contact distances, side-chain disorder, water molecule orientations, and the presence or absence of foreign solvent. The well established networks extend throughout all the solvent regions of the crystal with interesting orientational arrangements of the individual waters around both polar and apolar groups of the coenzyme molecule. The networks were seen to be consistent among each of the four models in terms of occupying relatively similar positions. However, the occupancy values of the individual networks varied between the models; some networks were clearly visible in one but attenuated in another. The specific details of the water structure (bonding geometries, short-range nonbonded contacts, orientations of the waters, polar and apolar interactions, etc.) are described in the following paper.

Models, Molecular↗