Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.


Escherichia coli K12 arabinose-binding protein mutants with altered transport properties.

The arabinose-binding protein (ABP) of Escherichia coli binds L-arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose-binding cleft were subjected to site-directed mutagenesis. Thirty-eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed with wild-type AraG and AraH from a plasmid, in a strain lacking the chromosomal araFGH operon, and the whole cell uptake parameters, Ven (maximum initial velocity of arabinose entry) and K(en) (concentration of arabinose yielding half-maximal entry) were determined. Twenty-four mutants had normal Ven values, 3 mutants had Ven and K(en) values twice wild type, and 11 mutants had Ven and K(en) values 20-50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutant binding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild-type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose-binding cleft, all of which are charged in the wild-type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex.


A kinetic model for binding protein-mediated arabinose transport.

A kinetic model is presented based on the simplest plausible mechanism for bacterial binding protein-dependent transport. The transport phenotypes of the 18 variant arabinose-binding proteins analyzed by Kehres and Hogg (1992, Protein Sci. 1, 1652-1660) (wild type and 17 mutants) are interpreted to mean that in wild-type arabinose uptake the forward transport rate (k(for)) greatly exceeds the dissociation rate (kund) of a binding protein docked with the AraG:AraH membrane complex, and that k(for) dominance is preserved in all of the binding protein surface mutants. The assumptions and predictions of the model are consistent with existing data from other periplasmic transport systems.


Comprehensive sequence analysis of the 182 predicted open reading frames of yeast chromosome III.

With the completion of the first phase of the European yeast genome sequencing project, the complete DNA sequence of chromosome III of Saccharomyces cerevisiae has become available (Oliver, S. G., et al., 1992, Nature 357, 38-46). We have tested the predictive power of computer sequence analysis of the 176 probable protein products of this chromosome, after exclusion of six problem cases. When the results of database similarity searches are pooled with prior knowledge, a likely function can be assigned to 42% of the proteins, and a predicted three-dimensional structure to a third of these (14% of the total). The function of the remaining 58% remains to be determined. Of these, about one-third have one or more probable transmembrane segments. Among the most interesting proteins with predicted functions are a new member of the type X polymerase family, a transcription factor with an N-terminal DNA-binding domain related to GAL4, a "fork head" DNA-binding domain previously known only in Drosophila and in mammals, and a putative methyltransferase. Our analysis increased the number of known significant sequence similarities on chromosome III by 13, to now 67. Although the near 40% success rate of identifying unknown protein function by sequence analysis is surprisingly high, the information gap between known protein sequences and unknown function is expected to widen and become a major bottleneck of genome projects in the near future. Based on the experience gained in this test study, we suggest that the development of an automated computer workbench for protein sequence analysis must be an important item in genome projects.


A database of protein structure families with common folding motifs.

The availability of fast and robust algorithms for protein structure comparison provides an opportunity to produce a database of three-dimensional comparisons, called families of structurally similar proteins (FSSP). The database currently contains an extended structural family for each of 154 representative (below 30% sequence identity) protein chains. Each data set contains: the search structure; all its relatives with 70-30% sequence identity, aligned structurally; and all other proteins from the representative set that contain substructures significantly similar to the search structure. Very close relatives (above 70% sequence identity) rarely have significant structural differences and are excluded. The alignments of remote relatives are the result of pairwise all-against-all structural comparisons in the set of 154 representative protein chains. The comparisons were carried out with each of three novel automatic algorithms that cover different aspects of protein structure similarity. The user of the database has the choice between strict rigid-body comparisons and comparisons that take into account interdomain motion or geometrical distortions; and, between comparisons that require strictly sequential ordering of segments and comparisons, which allow altered topology of loop connections or chain reversals. The data sets report the structurally equivalent residues in the form of a multiple alignment and as a list of matching fragments to facilitate inspection by three-dimensional graphics. If substructures are ignored, the result is a database of structure alignments of full-length proteins, including those in the twilight zone of sequence similarity.(ABSTRACT TRUNCATED AT 250 WORDS)


Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction.

Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size-exclusion high-performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 micrograms/mL, whereas the apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of < 20 micrograms/mL. In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect. Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of Ka = 2 x 10(7) M-1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 microM FAD. This association step obeys second-order kinetics with an association rate constant k = 7.4 x 10(3) M-1 s-1 at 20 degrees C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.


Characterization of the stabilizing effect of point mutations of pyruvate oxidase from Lactobacillus plantarum: protection of the native state by modulating coenzyme binding and subunit interaction.

Point mutations in the gene of pyruvate oxidase from Lactobacillus plantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions, or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased. The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 degrees C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.


Modeling the effects of mutations on the denatured states of proteins.

We develop a model for the reversible denaturation of proteins and for the effects of single-site mutations on the denatured states. The model is based on short chains of sequences of H (hydrophobic) and P (other) monomers configured as self-avoiding walks on the two-dimensional square lattice. The N (native) state is defined as the unique conformation of lowest contact energy, whereas the D (denatured) state is defined as the collection of all other conformations. With this model we are able to determine the exact partition function, and thus the exact native-denatured equilibrium for various solvent conditions, using the computer to exhaustively enumerate every possible configuration. Previous studies confirm that this model shows many aspects of protein-like behavior. The present study attempts to model how the denatured state (1) depends on the amino acid sequence, and (2) is changed by single-site mutations. The model accounts for two puzzling experimental results: (1) the replacement of a polar residue by a hydrophobic amino acid on the surface of a protein can destabilize a native protein, and (2) the "denaturant slope," m = partial delta G/partial c (where c is the concentration of denaturant--urea, guanidine hydrochloride), can sometimes change by as much as 30% due to a single mutation. The principal conclusion of the present study is that, under strong folding conditions, the denatured conformations that are in equilibrium with the native state are not open random configurations. Instead, they are an ensemble of highly compact conformations with a distribution that depends on the residue sequence and that can be substantially altered by single mutations. Most importantly, we conclude that mutations can exert their dominant effects on protein stability by changing the entropy of folding.


Environment-specific amino acid substitution tables: tertiary templates and prediction of protein folds.

The local environment of an amino acid in a folded protein determines the acceptability of mutations at that position. In order to characterize and quantify these structural constraints, we have made a comparative analysis of families of homologous proteins. Residues in each structure are classified according to amino acid type, secondary structure, accessibility of the side chain, and existence of hydrogen bonds from the side chains. Analysis of the pattern of observed substitutions as a function of local environment shows that there are distinct patterns, especially for buried polar residues. The substitution data tables are available on diskette with Protein Science. Given the fold of a protein, one is able to predict sequences compatible with the fold (profiles or templates) and potentially to discriminate between a correctly folded and misfolded protein. Conversely, analysis of residue variation across a family of aligned sequences in terms of substitution profiles can allow prediction of secondary structure or tertiary environment.


Atomic solvation parameters applied to molecular dynamics of proteins in solution.

A solvation energy function for use in the molecular simulation of proteins is proposed. It is based on the accessible surface areas of atoms in the protein and on atomic solvation parameters derived from empirical vapor-to-water free energies of transfer of amino acid side-chain analogs. The energy function and its derivatives were added to the CHARMM molecular simulation program (Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., & Karplus, M., 1983, J. Comput. Chem. 4(2), 187-217). The effect of the added energy term was evaluated by 110 ps of molecular dynamics on the 26-residue protein melittin. The melittin monomer and tetramer were studied both with and without the added term. With the added energy term the monomer partially unfolded, while the secondary structure of the tetramer was preserved, in agreement with reported experiments (Brown, L.R., Lauterwein, J., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 231-244; Lauterwein, J., Brown, L.R., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 219-230).


Equilibrium denaturation studies of mouse beta-nerve growth factor.

Equilibrium denaturation of dimeric mouse beta-nerve growth factor (beta-NGF) has been studied by monitoring changes in the protein's spectroscopic characteristics. Denaturation of beta-NGF in guanidine hydrochloride and urea resulted in an altered intrinsic fluorescence emission spectrum, fluorescence depolarization, and diminished negative circular dichroism. Native-like spectroscopic properties and specific biological activity are restored when denaturant is diluted from unfolded samples, demonstrating that this process is fully reversible. However, refolding of denatured beta-NGF is dependent on the three disulfide bonds present in the native protein and does not readily occur when the disulfide bonds are reduced. Graphical analysis and nonlinear least-squares fitting of beta-NGF denaturation data demonstrate that denaturation is dependent on the concentration of beta-NGF and is consistent with a two-state model involving native dimer and denatured monomer (N2 = 2D). The conformational stability of mouse beta-NGF calculated according to this model is 19.3 +/- 1.1 kcal/mol in 100 mM sodium phosphate at pH 7. Increasing the hydrogen ion concentration resulted in a 25% decrease in beta-NGF stability at pH 4 relative to pH 7.


pH dependence of the reverse reaction catalyzed by phosphofructokinase I from Escherichia coli: implications for the role of Asp 127.

The kinetics of the reverse reaction catalyzed by Escherichia coli phosphofructokinase, i.e., the synthesis of ATP and fructose-6-phosphate from ADP and fructose-1,6-bisphosphate, have been studied at different pH values, from pH 6 to pH 9.2. Hyperbolic saturations of the enzyme are observed for both substrates. The affinity for fructose-1,6-bisphosphate decreases with pH following the ionization of a group with a pK of 6.6, whereas the catalytic rate constant and perhaps the affinity for ADP are controlled by the ionization of a group with a pK of 6. Several arguments show that the pK of 6.6 is probably that of the carboxyl group of Asp 127, whereas the pK of 6 is tentatively attributed to the carboxyl group of Asp 103. The pK of 6.6 is assigned to the carboxyl group of Asp 127 in the free enzyme, and a simple model suggests that the same group would have an abnormally high pK, above 9.6, in the complex between phosphofructokinase and fructose-1,6-bisphosphate. It is proposed that the large pK shift of more than 3 pH units upon binding of fructose-1,6-bisphosphate is due to an electrostatic repulsion that could exist between the 1-phosphate group and the carboxyl group of Asp 127, which are close to each other in the crystal structure of phosphofructokinase (Shirakihara, Y. & Evans, P.R., 1988, J. Mol. Biol. 204, 973-994). The same interpretation would also explain the much higher affinity of the enzyme for fructose-1,6-bisphosphate when Asp 127 is protonated.(ABSTRACT TRUNCATED AT 250 WORDS)


Yeast ferrochelatase: expression in a baculovirus system and purification of the expression protein.

The terminal step of the heme biosynthetic pathway is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). In eukaryotes this enzyme is bound to the inner mitochondrial membrane with its active site facing the matrix side of the membrane. Previously this laboratory has characterized this enzyme via kinetic and protein chemical modification techniques, and with the recent cloning of the enzyme from yeast, mouse, and human sources it now becomes possible to approach structure-function questions by using site-directed mutagenesis. Of primary significance to this is the development of an efficient expression vector. This is of particular significance for ferrochelatase, as it is a low-abundance protein whose DNA coding sequence has a very low codon bias. In the current work we describe the production of yeast ferrochelatase in a baculovirus system. This system is shown to be an excellent one in which to produce large quantities of active ferrochelatase. The expressed enzyme is membrane associated and is not released into the growth medium either during or after virus development and cell lysis. The expressed protein can be purified in a procedure that requires only 1 day and makes use of a Pharmacia Hi Trap blue affinity column. The measured Km's for the substrates mesoporphyrin and iron are the same as those reported previously for the yeast enzyme. To our knowledge this is the first example of a mitochondrial membrane protein that has been expressed in a baculovirus system.


Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.


Characterization, primary structure, and evolution of lamprey plasma albumin.

The most abundant protein found in blood plasma from the sea lamprey (Petromyzon marinus) has the hallmarks of a plasma albumin: namely, high abundance, solubility in distilled water, a small number of tryptophans, and a high content of cysteines and charged residues. As in other vertebrate albumins, not all the cysteines are disulfide bonded. An unusual feature of this protein is its molecular weight of 175,000, roughly 2.5 times the size of other vertebrate albumins. Its amino acid sequence, deduced from a series of overlapping cDNA clones, can be aligned with other members of the gene family including plasma albumin, alpha-fetoprotein, and vitamin-D binding protein, confirming that it is indeed an oversized albumin. An unusual feature of the sequence is a 28-amino acid stretch consisting of a serine-threonine repeat with the general motif (STTT). Lamprey albumin contains a 23-amino acid putative signal peptide and a 6-residue putative propeptide, which, when cleaved, yield a mature protein of 1,394 amino acids with a calculated molecular weight of 157,000. The sequence also includes nine potential N-linked glycosylation sites (Asn-X-Ser/Thr), consistent with observation that lamprey albumin is a glycoprotein. If all the potential glycosylation sites were occupied by clusters of 2,000 molecular weight each, the total molecular weight would be 175,000. Like other members of the gene family, lamprey albumin is composed of a series of 190-amino acid repeats, there being seven such domains all together. Quantitative amino acid sequence comparisons of lamprey albumin with the other members of the gene family indicate that it diverged from an ancestral albumin prior to the gene duplications leading to this diverse group. This notion is confirmed by the pattern of amino acid insertions and deletions observed in a consideration of all domains that compose this family. Furthermore, it suggests that the invention of albumin antedates the vertebrate radiation.


A protease-sensitive site in the proposed Ca(2+)-binding region of human serum amyloid P component and other pentraxins.

Serum amyloid P component (SAP) is a decamer of 10 identical 25.5-kDa subunits. Limited proteolysis of SAP with alpha-chymotrypsin cleaves the subunit into two fragments of 18 and 7.5 kDa, although the fragments stay together in the decamer under nondenaturing conditions. Proteolysis does not occur in the presence of Ca2+ (10 mM). Cleavage with alpha-chymotrypsin prevents the Ca(2+)-dependent binding of SAP to zymosan extract, nucleosomes, and DNA. The alpha-chymotrypsin cleavage site identified is in a region of SAP that is highly conserved in members of the human C-reactive protein (CRP) family of proteins (pentraxins) to which SAP belongs and is similar to the Ca(2+)-binding site in calmodulin and related Ca(2+)-binding proteins (Nguyen, N.Y., Suzuki, A., Boykins, R.A., & Liu, T.-Y., 1986, J. Biol. Chem. 261, 10456-10465). Treatment of SAP with other proteases (trypsin, Pronase, and Nagarse protease) yields fragmentation patterns upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that are similar to those obtained with alpha-chymotrypsin. Two other members of the pentraxin family of proteins, hamster female protein and rabbit CRP, also exhibit similar fragmentation patterns on SDS-PAGE when treated with the various proteases. Recently, it has been shown that the homologous protein, human CRP, is cleaved in the same homologous position as cleavage of SAP by alpha-chymotrypsin, resulting in the loss of Ca(2+)-binding (as shown by equilibrium dialysis) and Ca(2+)-dependent binding reactivities (Kinoshita, C.M., Ying, S.-C., Hugli, T.E., Siegel, J.N., Potempa, L.A., Jiang, H.J., Houghten, R.A., & Gewurz, H., 1989, Biochemistry 28, 9840-9848).(ABSTRACT TRUNCATED AT 250 WORDS)


Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.

We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.


A proposed link between nitrogen and carbon metabolism involving protein phosphorylation in bacteria.

We demonstrate that certain phosphoryl transfer proteins of the bacterial phosphotransferase system (PTS), the fructose- and mannitol-specific IIA proteins or domains, are homologous to a class of proteins, one of which is known to affect transcription of some of the nitrogen-regulatory sigma 54-dependent operons in Klebsiella pneumoniae. The phosphorylatable histidyl residue in the homologous PTS proteins and the consensus sequence in the vicinity of the active-site histidine are fully conserved in all members that comprise this family of proteins. A phylogenetic tree of the eight protein members of this family was constructed, and a "signature" sequence that can serve for the identification of new protein members of this family is proposed. These observations suggest that PTS-catalyzed protein phosphorylation may provide a regulatory link between carbon and nitrogen assimilation in bacteria.


The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X-ray structures.

A high-precision solution structure of the elastase inhibitor eglin c was determined by NMR and distance geometry calculations. A large set of 947 nuclear Overhauser (NOE) distance constraints was identified, 417 of which were quantified from two-dimensional NOE spectra at short mixing times. In addition, a large number of homonuclear 1H-1H and heteronuclear 1H-15N vicinal coupling constants were used, and constraints on 42 chi 1 and 38 phi angles were obtained. Structure calculations were carried out using the distance geometry program DG-II. These calculations had a high convergence rate, in that 66 out of 75 calculations converged with maximum residual NOE violations ranging from 0.17 A to 0.47 A. The spread of the structures was characterized with average root mean square deviations (<rmsd>) between the structures and a mean structure. To calculate the <rmsd> unbiased toward any single structure, a new procedure was used for structure alignment. A canonical structure was calculated from the mean distances, and all structures were aligned relative to that. Furthermore, an angular order parameter S was defined and used to characterize the spread of structures in torsion angle space. To obtain an accurate estimate of the precision of the structure, the number of calculations was increased until the <rmsd> and the angular order parameters stabilized. This was achieved after approximately 40 calculations. The structure consists of a well-defined core whose backbone deviates from the canonical structure ca. 0.4 A, a disordered N-terminal heptapeptide whose backbone deviates by 0.8-12 A, and a proteinase-binding loop whose backbone deviates up to 3.0 A. Analysis of the angular order parameters and inspection of the structures indicates that a hinge-bending motion of the binding loop may occur in solution. Secondary structures were analyzed by comparison of dihedral angle patterns. The high precision of the structure allows one to identify subtle differences with four crystal structures of eglin c determined in complexes with proteinases.


Characterization of the structure and properties of the His 62-->Ala and Arg 38-->Ala mutants of yeast phosphoglycerate kinase: an investigation of the catalytic and activatory sites by site-directed mutagenesis and NMR.

The role of two "basic patch" residues, Arg-38 and His-62, in the catalytic function and anion-dependent activation of yeast 3-phosphoglycerate kinase (PGK) was investigated by site-directed mutagenesis. Steady-state kinetics and NMR experiments were conducted to characterize the functional properties and structural integrity of the R38A and H62A mutants. The results of these studies, in combination with earlier mutagenesis experiments, suggest that Arg-38 is the only catalytically essential residue among the conserved histidines and arginines of the basic patch. It appears that, similar to the remaining basic patch residues, His-62 is important for anion-dependent activation but not for enzyme activity. Cumulative evidence from this study and from previous mutagenesis experiments suggests that the basic patch region is in effect an extended anion binding site that encompasses both the catalytic and the general anion-binding site. It is proposed that substitution of any one of the basic patch residues results in an increased localization of the catalytic site. Substrate and product may still bind to this site, but a simultaneous binding of activatory anions, required for activation, has been impaired. NMR experiments suggest that the conformational changes observed upon binding of 3-PG to wild-type PGK are necessary for anion- and substrate-dependent activation.


Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability.

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.


The disulfide bond pairing of the pheromones Er-1 and Er-2 of the ciliated protozoan Euplotes raikovi.

The disulfide pairings of the two Euplotes raikovi pheromones Er-1 and Er-2 have been determined by chemical and mass spectrometric analyses. Cystine-linked peptides from thermolytic digestions of the native molecules were purified by reverse-phase high performance liquid chromatography and identified in the known sequences to make the assignments. The same pairing, Cys(I)-Cys(IV), Cys(II)-Cys(VI), and Cys(III)-Cys(V), was found in both pheromones, suggesting that this pattern occurs commonly throughout this family of molecules. This arrangement of disulfides indicates that the three-dimensional structure is defined by three loops, which can vary in size and charge distribution from one pheromone to another.


A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains.

A liquid chromatographic stationary phase was prepared by covalently binding to the surface of microparticulate silica gel functionality (benzylsilane), which mimics the side chain of the amino acid phenylalanine. The chromatographic retentions of the N-acetyl C-(N'-methyl) amides of various hydrophobic and amphiphilic amino acids on this stationary phase were measured using an aqueous mobile phase. A retention order of Gly < Ala < Cys < Val < Met < Pro < Ile < Leu < Tyr < Phe < Trp is seen at room temperature. Chromatographic retentions were used to derive free energies of adsorption of the amino acid derivatives on the chromatographic support relative to that of the glycine derivative. The temperature dependencies of the retention of aromatic and aliphatic amino acid derivatives differ in curvature, indicating a qualitative difference in the absorption mechanism. An adsorption model for retention is proposed, and arguments are made as to the suitability of an adsorption model for describing the contacts between amino acid side chains during the initial steps of protein folding.


Reversible dissociation and unfolding of the dimeric protein thymidylate synthase.

Conditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding-refolding conditions, demonstrating a monomer to dimer association step.


Characteristics of murine protoporphyrinogen oxidase.

Protoporphyrinogen oxidase (EC 1.3.3.4) (PPO) is the penultimate enzyme of the heme biosynthetic pathway. Mouse PPO has been purified in low yield and kinetically characterized by this laboratory previously. A new more rapid purification procedure is described herein, and with this protein we detect a noncovalently bound flavin moiety. This flavin is present at approximately stoichiometric amounts in the purified enzyme and has been identified by its fluorescence spectrum and high performance liquid chromatography as flavin mononucleotide (FMN). Fluorescence quenching studies on the flavin yielded a Stern-Volmer quenching constant of 12.08 M-1 for iodide and 1.1 M-1 for acrylamide. Quenching of enzyme tryptophan fluorescence resulted in quenching constants of 6 M-1 and 10 M-1 for iodide and acrylamide, respectively. Plasma scans performed on purified enzyme preparations did not reveal the presence of stoichiometric amounts of protein-bound metal ions, and we were unable to detect any protein-associated pyrroloquinoline quinone (PQQ). Data from circular dichroism studies predict a secondary structure of the native protein consisting of 30.5% alpha helix, 40.5% beta sheet, 13.7% turn, and 15.3% random coil. Denaturation of PPO with urea resulted in a biphasic curve when ellipticity is plotted against urea concentration, typical of amphipathic proteins.


An energy-based approach to packing the 7-helix bundle of bacteriorhodopsin.

Based on the heavy-atom coordinates determined by the electron microscopy for the seven main helical regions of bacteriorhodopsin with the all-trans retinal isomer, energy optimizations were carried out for helix bundles containing the all-trans retinal and 13-cis retinal chromophores, respectively. A combination of simulated annealing and energy minimization was utilized during the process of energy optimization. It was found that the 7-helix bundle containing the all-trans isomer is about 10 kcal/mol lower in conformational energy than that containing the 13-cis isomer. An energetic analysis indicates that such a difference in energy is consistent with the observation that absorption of a 570-nm proton is required for the conversion of a bacteriorhodopsin from its all-trans to 13-cis form. It was also found that the above conversion process is accompanied by a significant conformational perturbation around the chromophore, as reflected by the fact that the beta-ionone ring of retinal moves about 5.6 A along the direction perpendicular to the membrane plane. This is consistent with the observation by Fodor et al. (Fodor, S.P.A., Ames, J.B., Gebhard, R., van der Berg, E.M.M., Stoeckenius, W., Lugtenburg, J., & Mathies, R.A., 1988, Biochemistry 27, 7097-7101). Furthermore, it is interesting to observe that although the retinal chromophore undergoes a significant change in its spatial position, the orientation of its transition dipole changes only slightly, in accord with experimental observations. In other words, even though orientation of the retinal transition dipole is very restricted, there is sufficient room, and degrees of freedom, for the retinal chromophore to readjust its position considerably. This finding provides new insight into the subtle change of the retinal microenvironment, which may be important for revealing the proton-pumping mechanism of bacteriorhodopsin. The importance of electrostatic and nonbonded interactions in stabilizing the 7-helix bundle structure has also been analyzed. Electrostatic interactions favor an antiparallel arrangement among adjacent helices. Nonbonded interactions, however, drive most of the closely packed helices into an arrangement in which the packing angles lie around -160 degrees, a value very near the -154 degrees value computed earlier as the most favorable packing arrangement of two poly(Ala) alpha-helices (Chou, K.-C., Némethy, G., & Scheraga, H.A., 1983, J. Phys. Chem. 87, 2869-2881). The structural features of the 7-helix bundle and their relationship to those found in typical 4-helix bundle proteins are also discussed.(ABSTRACT TRUNCATED AT 400 WORDS)


Virulence factors in Serratia marcescens: cell-bound hemolysin and aerobactin.

1. A total of 60 nosocomial isolates of Serratia marcescens were screened for the presence of markers related to virulence, i.e., cell-bound hemolysin and production of siderophore aerobactin. 2. No aerobactin-producing strains were found, and the incidence of cell-bound hemolysin was 97%. 3. Hemolysin-positive (58 strains) and hemolysin-negative (2 strains) Serratia marcescens showed the same LD50 (3 x 10(7) bacteria) in a test of virulence for mice. 4. These results indicate that cell-bound hemolysin is not a main factor of virulence for mice in Serratia marcescens.


Feeding behavior of different species of Chagas' disease vectors stimulated with blood sources at different temperatures.

1. The feeding behavior of six triatomid species toward latex condoms filled with blood at 26 +/- 1 and 36 +/- 1 degrees C was observed for 4 h. 2. The species studied were Triatoma infestans, Triatoma brasiliensis, Triatoma vitticeps, Triatoma pseudomaculata, Rhodnius prolixus and Panstrongylus megistus, and 34 to 348 insects were studied in each group (average, 190). All the life stages of each species were used. 3. Although most insects (80%, average for 6 species) preferred the warmer blood, 54% of the individuals of all species fed on blood kept at 26 degrees C. For Triatoma pseudomaculata, a higher proportion of insects fed on blood at 26 degrees C (92%) than on blood at 36 degrees C (65%). 4. These results suggest that bait containing insecticides which are effective through the digestive tract may be useful in the control of these vectors even at room temperature.


Immunoenzymatic quantitation of antibodies to Bothrops asper myotoxins after polyvalent antivenom administration in mice.

1. Two quantitative enzyme-immunoassays (EIA) for Bothrops asper myotoxin and anti-myotoxin antibodies, respectively, were utilized to study their in vivo distribution in mice (Swiss, 18 to 20 g). 2. After polyvalent antivenom (0.4 ml) administration by the iv route, there was an immediate peak in plasma anti-myotoxin antibodies which declined rapidly during the first hour, and then decreased more gradually. Anti-myotoxin antibodies were detected in muscular tissue (gastrocnemius) following iv injection of antivenom. After im injection of antivenom (0.4 ml), a slow and steady increase in plasma anti-myotoxin levels was observed, with a peak at 24 h. 3. Mice that received antivenom (0.4 ml) by the iv or im route 15 min after im injection of B. asper venom (100 micrograms) had lower levels of plasma anti-myotoxin antibodies than controls injected with antivenom only, suggesting that at least a fraction of the antibodies combines with myotoxins in vivo. 4. Myotoxin was not detected in plasma at any time after venom injection by the im (100 micrograms) or ip (40 micrograms) route. Following iv injection of 50 micrograms of purified myotoxin II, all plasma samples were also negative, at a detection limit of 10 ng/ml. 5. It was demonstrated that myotoxin II binds to mouse erythrocytes in vitro, a fact that could partially explain its rapid in vivo disappearance from plasma. 6. The present results on the distribution of anti-myotoxin antibodies in vivo are in agreement with previous experimental studies reporting the poor neutralization of myotoxicity induced by B. asper venom when antivenom is injected im, in comparison to i.v. injection.


Voluntary intake of "Tiquira", an alcoholic beverage prepared from fermented manioc, decreases immunoglobulin production and increases self-reactivity in mice.

We studied the effects of chronic voluntary ingestion of "Tiquira" (50%)--an alcoholic beverage prepared from fermented manioc, widely consumed in Maranhão,--on the natural immunological activity of young adult (2-3 months old) C57B1/6J mice (16-17 g) by evaluating the number of plaque-forming cells (PFCs) in the spleen and by titrating serum antibodies by ELISA. Voluntary ingestion of "Tiquira" for 30 days decreased immunoglobulin secretion in serum ( 1600 +/- 30 vs 193 +/- 20), caused an impressive reduction in the total number of PFCs in the spleen ( 482 +/- 22 vs 58 +/- 3) and increased the proportion of self-reacting antibody molecules in serum ( 119 +/- 16 vs 800 +/- 20) and self-reactive PFCs to mouse red blood cells (MRBC) in the spleen ( 183 +/- 14 vs 272 +/- 16; N = 10 animals per group). These preliminary results suggest that the voluntary ingestion of "Tiquira" for 30 days may interfere with immunological relations by decreasing the total number of immunoglobulin secreting cells and increasing the anti-self antibody production. Experiments are in progress to determine if ethanol or other substances present in "Tiquira" are responsible for the effects documented here.


Human leukocyte A and B antigen, gene and haplotype frequencies in the population of the city of São Paulo in Brazil.

1. Antigen, gene and haplotype frequencies are important data for population analysis, paternity exclusion testing, genetic studies, and for organ transplantation selection. 2. We have studied the Class I histocompatibility antigens of 617 unrelated individuals from the city of São Paulo, Brazil, to determine antigen and gene frequencies of 16 HLA-A and 28 HLA-B antigens. Estimated haplotype frequencies were also determined, as well as the genetic distances of this population from European Caucasian and Negro populations. 3. A previously unknown linkage disequilibrium was detected for A23-B49 and a clear trend towards antigen frequencies intermediate between Caucasoid and Negro populations was observed.


Relationship between immune response and clinical manifestations in patients with tuberculosis.

Cutaneous reactivity to purified protein derivative (PPD) and antibody levels were investigated in 122 adults and 28 children with tuberculosis. IgG anti-PPD levels (measured by ELISA and reported as absorbance at 405 nm) were higher in adult patients with the disease for more than one year (0.533 +/- 0.391, N = 38 vs 0.224 +/- 0.256, N = 50 in patients with the disease for less than one year) as well as in children with disseminated tuberculosis (0.138 +/- 0.137, N = 11, vs 0.072 +/- 0.055, N = 15 in children with localized disease). The cut-off values (mean + 2SD) for healthy children and adults were 0.09 and 0.22 absorbance at 405 nm. In both adult and pediatric patients, cutaneous reactivity to PPD was inversely correlated with antibody levels. The present data provide additional evidence for the existence of an unstable spectrum of immune response in tuberculosis patients in whom changes in clinical situation are dynamic.


Partial characterization of hemagglutinin activity of the marine sponge Anthosigmella varians.

The marine sponge Anthosigmella varians contains proteins that agglutinate human erythrocytes irrespective of their ABO group antigens. The hemagglutination reaction depends on divalent cations and is not inhibited by L-arabinose, D-xylose, L-rhamnose, D-galactose, D-glucose, L and D-fucose, N-acetyl-D-galac-tosamine, N-acetyl-D-glucosamine, methyl-alpha-D-mannopiranoside, D-cellobiose, lactose, maltose, melibiose nor raffinose (33 mM each). A partial purification of the hemagglutinins with 31-fold increase in SA and 80% recovery of activity was obtained after gel filtration and ion-exchange gradient elution chromatography. Hemadsorption experiments carried out with the semipurified fraction using glutaraldehyde-fixed human erythrocytes suggest that proteins with molecular weight of 90 and 34 kDa participate in this reaction.


Biocytin as a retrograde tracer in the mammalian visual system.

We have successfully used biocytin as a retrograde tracer in the mammalian visual system. Retinal ganglion cells, pyramidal and stellate cortical neurons were labelled. Both pressure injections and gel implants were used successfully for retrograde labelling. Biocytin was detected using avidin conjugates and horseradish peroxidase histochemistry. Retrograde filling with biocytin proved to be more reliable and to allow better morphological resolution than other commonly used neurotracers such as horseradish peroxidase. The fine details of cell morphology observable by this method are comparable in many cases to the results obtained with intracellular tracer injections. The morphological resolution obtained with this method allows the study of brain microcircuits using extracellular deposits of biocytin.


Comparison of the anti-inflammatory effects of topically applied aspirin and indomethacin following photocoagulation of the rabbit iris.

1. A comparative study of the inhibitory action of topically applied indomethacin and aspirin (1% (w/v) 2 h before and immediately after trauma, totaling 4 mg each) on myosis, increase in intraocular pressure and breakdown of the blood aqueous barrier was performed. 2. Argon laser photocoagulation on the anterior surface of the left iris of pigmented rabbits, totaling 2,250 mJ, was used as traumatic stimulus. The untraumatized eye was used as a reference for measurements. 3. Four groups of 10 animals each (control, photocoagulated, photocoagulated plus indomethacin and photocoagulated plus aspirin) were studied. 4. Both drugs similarly inhibited the increase in the total protein concentration in aqueous humor and in intraocular pressure at 10, 20 and 40 min. Only aspirin showed some inhibitory action on the pupillary response 10 min after photocoagulation.


Properties of channels from rat liver gap junction membrane fractions incorporated into planar lipid bilayers.

1. Rat liver membrane fractions highly enriched for gap junctions can be incorporated into planar lipid bilayers exhibiting channel currents with both voltage-dependent and independent components. Voltage dependence, however, is only one of the characteristics of liver gap junction channels. Other features include poor ionic selectivity and sensitivity to calcium, pH, octanol and to some intracellularly applied antibodies. 2. To further test the junctional nature of channels from membrane fractions highly enriched in gap junctions incorporated into lipid bilayers we studied the sensitivity of these channels to uncoupling agents and determined channel selectivity properties. 3. We found the incorporated channels to be insensitive to calcium and octanol, and in most cases to pH in the range of 5-7, suggesting that either these agents do not interact directly with the junctional channels or that the corresponding gating regions are inactivated during the isolation and reconstitution procedures. 4. Attempts to block channel activity using polyclonal and monoclonal connexin 32 antibodies were generally unsuccessful, although one antibody (a monoclonal directed against the carboxy terminus portion of connexin32) blocked channel activity. 5. Selectivity measurements indicated that the incorporated channels were slightly cation selective (PNa = PK > PCl) and were permeable to large ions. 6. These results further support the idea that functional connexin32 gap junction channels are present in channel activity recorded from rat liver junctional membranes incorporated into planar bilayers.


Effect of estrogens on the adhesion of Trichomonas vaginalis to epithelial cells in vitro.

1. To further characterize the basic aspects of parasite-cell interaction, untreated and hormone-treated Madin-Darby canine kidney (MDCK) cells were allowed to interact with T. vaginalis. 2. Among the six hormones assayed, alpha-estradiol caused MDCK cells to become highly adhesive to T. vaginalis. Increases in parasite adhesion of 83% and 68% were obtained by previous incubation of the epithelial monolayers for 18 h with 1 ng/ml alpha-estradiol and 10 ng/ml 17-beta-estradiol, respectively. 3. The estrogenic response of MDCK cells was inhibited by tamoxifen, cycloheximide, puromycin, or 2-deoxy-D-glucose. 4. We conclude that MDCK cells possess receptors for estrogens and that T. vaginalis cytoadherence can be influenced by the hormonal cycle of its host.


[Evaluation of nutritional in 149 severely burned patients].

By means of random sampling we selected 149 cases with over 30% TBSA burn (> 10% full-thickness) for evaluation of nutriment supplementation during treatment. We analysed daily caloric supply, nitrogen balance, amount of blood transfusion, Hb, A/G, transferrin, leukocyte, lymphocyte, IgG, Fn and body weight. The data support our impression that hyperalimentation can promote wound healing and raise the survival rate.


[Effects of fat intake on immunomodulation in burned patients].

Since fatty acids influence prostaglandins synthesis, and both fatty acids and prostaglandins modulate immune function, we studied the effects of dietary fats on immunomodulation in severely burned patients. We observed that the administration of fish oil can affect immune response after burns, but a high fat intake seemed to markedly reduce the effect. With lower fat intake, fish oil induced a higher mitogenic response of the lymphocyte to PHA and higher blood IgG level in burned patients. These findings suggest that regulation of dietary lipids may play an important role in nutritional support after burn injury.


[A 5-year interval report on study of burn metabolism and nutrition].

This paper introduces the essential experiences concerning studies of burn metabolism and nutrition in our institute in the past five years. 1. Three new and practical animal models were developed for studying gastro-enteral nutrition in burns. 2. With indirect calorimetry, resting energy expenditure (REE) of 92 burn adult patients were measured and analyzed, and on the basis of which a new formula for calculating nutritional supplement in Chinese burn adults was proposed: kcal/day = 1,000 x M2 (body surface area) + 25 x % TBSA (total burn surface area). 3. Through experimental and clinical studies, it was found that antiouperoxide agents (such as SOD, CAT), tolbutamide, glutamine and Chinese herb decoction Sizunzituang all exhibited modulating effects on postburn metabolism and nutrition, e.g. decreasing catabolism, reducing negative nitrogen balance, stimulating secretion of insulin, enhancing tissue utilization of glucose, maintaining the mass of enteral mucosa and improving functions of viscerae. 4. A new less irritating, simple and easy-to-introduce nasal-enteral nutrition tube was devised, which could pass through the pylorus easily into the duodenum usually within 6hrs without using a stylet. It would be useful in the early postburn enteral nutrition supplementation.


[Problems in parenteral nutrition and metabolic support for burned patients].

Four problems in parenteral nutrition and metabolic support for burns were discussed in this paper. They were: 1. Estimation of the nutrition requirement, especially energy and protein requirements for burned patients. 2. Selection of the macronutrients for parenteral nutrition with burns. 3. Principles of the prescription of parenteral nutrition. 4. Experiences of parenteral nutrition for burned patients. Because of the safety, effectiveness and convenience, the importance and advantage of peripheral parenteral nutrition were emphasized. Some advances in this field were discussed.


[Gastrointestinal dysfunction and peroral nutrition after severe burns].

The patient with severe burns is characterized by a hypermetabolic response, and degree of which is proportional to the magnitude of burns. Major thermal injury is often associated with gastrointestinal dysfunction. Failure to provide adequate caloric intake by both enteral and parenteral routes will results in pronounced weight loss, delayed wound healing, depression of host resistance and cellular dysfunction. We analysed 676 burn patients with TBSA over 50%, and found regular changes in gastrointestinal function with endoscopy and also at autopsy. A new dietary programme is described in the article.


[Changes in plasma free amino acid concentration in burned patients with sepsis].

The sequential changes in plasma free amino acid concentration were analyzed and compared in burned patients with sepsis (n = 12) and without sepsis (n = 19). After burn injury, phenylalanine, methionine, lysine, and the Phe/Tyr ratio were significantly increased in two groups (P < 0.05-0.01). Threonine, serine, histidine, arginine, proline and BCAA/AAA ratio were significantly decreased in two groups (P < 0.05-0.001). The Phel Tyr ratio in patients with sepsis was much higher than that in patients without sepsis on postburn days 14 and 21 (P < 0.05), while the BCAA/AAA ratio in patients with sepsis was much lower than that in patients without sepsis on postburn day 14 (P < 0.01). The level of proline in patients with sepsis was much higher than that in patients without sepsis on postburn days 3 and 7 (P < 0.05). It is suggested that these results, in collaboration with other clinical and laboratory findings, may be helpful in foretelling the probable development of sepsis in patients with major burns.


[Reverse flow axial island flap in the mastoid region].

Anatomical and clinical studies on the reverse flow axial island flap in the mastoid region were reported. The studies in the dissection of 9 cadavers with a total of 17 sides and the observations in 6 operations proved that the blood in the superfacial temporal artery could flow reversely into the postauricular artery through the anastomotic branches to nourish the mastoid island flap. So the island flap wit a fascia pedicle containing the superficial temporal vessels could be transferred to various region of the face through subcutaneous tunnel to repair deformities of the orbit, nose, eyes and cheek in one stage operation. This reverse axial island flap has been utilized in 18 cases. 16 flaps survived. All cases were followed up for 2 to 7 years. The results were satisfactory.


[Island flap with anastomosis of small saphenous vein to great saphenous vein].

We have devised a small saphenous island flap and successfully used in 6 patients for repairing soft tissue defect of the hell and the sole. After its transference, the small saphenous vein was anastomosed to the large saphenous vein over the ankle in order to obtain the blood supply from the small saphenous vein and blood return through the large saphenous vein. The advantage of this flap is that the vessels are superficial with large caliber so that it is easy to do anastomosis without any influence to the donor area. We believe that this flap can be used to repair soft tissue defect of the leg and foot. In this article, the operative method and advantages and shortcoming are introduced.


[Double island flaps from middle and ring fingers for the repair of soft tissue defects of the thumb (clinical and anatomical studies)].

This article reports the clinical application of double island flaps from the middle and ring fingers for the repair of soft tissue defects of the thumb in 16 cases and the results of the study in applied anatomy with 23 palms from adult cadavers. The double island flaps based on the pedicle of the second digital palmar common artery and the associated digital palmar common nerves have proved to be suitable materials for the repair of the soft tissue defects of the thumb. The method is applicable to the defects of soft tissue distal to the MP joint of the thumb. The repaired thumb is considered to have satisfactory contour and function.


[Monitoring of renal function in burn patients with radioimmunological evaluation].

Urinary beta 2-microglobulin, albumin, IgG, Tamm-Horsfall mucoprotein, and glycoprotein were determined on PBD 0, 1, 3, 7, 14, 21, 27. It was found that the amount of albumin was double that of normal in patients with burn extent of 30%-50% TBSA, while IgG showed no change. In patients with over 50% TBSA burns, Alb was five-fold and IgG four-fold of normal. They probably indicated the degree of damage to the glomeruli. In 27 patients with infection, beta 2-m was increased, indicating damage to the proximal convoluted tubules. Glycoprotein showed a tendency to increase in moderate and severe burns, while it was markedly reduced in very large burns or renal failure, indicating damage to the ascending limb of thick medullary loop. The changes in the amount of urinary protein were more sensitive in reflecting early functional changes of the kidney.


[Repair of bedsores and ulcers with gluteus maximus musculocutaneous flap].

From 1984 to 1991, 5 cases of bedsores and 1 case of ulcer resulted from irradiation in gluteal region were repaired with gluteus maximus musculocutaneous flaps. All 5 cases of bedsores were the result of paraplegia. After a myocutaneous flap was transferred, the donor area was directly sutured without skin grafting. The excision wound in one patient reached 18 cm x 12 cm in size, however it was still repaired with total gluteus maximus musculocutaneous flap, and the donor area was also immediately closed with sutures. All of the patients were healed by first intention. For non-paraplegic patients it was deemed contra-indicated to use a total maximus gluteus musculocutaneous flap, and instead one half of the muscle was used, in order to avoid impairment of function of the hip joint.


[Early fluid therapy for dogs with severe burn-blast combined injury].

Fifty male mongrel dogs were inflicted with 25% III degree burn combined with moderate blast injury. They were divided into five groups; four treatment groups (with various amounts of infused fluid and sodium) and one control group. Each group consisted of 10 animals. The results indicated that every kind of treatment was effective. However, the regime of crystalloid plus whole blood (infused 8 h after injury) was the best. Generally speaking, fluid therapy should be given carefully. The optimal amount of sodium given was 0.3 mmol.kg-1.1%.BSA-1. The amount of water-2-2.5ml.kg-1% BSA-1. After treatment with the Parkland formula, the pulmonary water content was high, so it is not worth recommending. Continuous measurement of the viscosity of plasma, microhematocrit and the amount of sodium in urine and blood were simple and useful.


[Changes in the concentration of plasma cardionatrin and morphological changes of atrial cardiocytes after severe burn].

The purpose of this experiment is to investigate the changes in plasma concentration cardionatrin of and morphological changes in atrial cardiocytes in rats after severe burn by means of radioimmunoassay, immunocytochemistry, electron microscopy and morphometry. The results showed that: (1) At 1, 2 h postburn the plasma cardionatrin concentrations were significantly increased, at 6-48 h significantly decreased and at 72, 168 h returned to normal. (2) The changes in plasma cardionatrin concentration were related to the alterations of secretory function in atrial cardiocytes. (3) The pathological changes in atrial cardiocytes were characterized by appearing early. The pathological changes and alterations of secretory function of atrial cardiocytes ran a dynamic course, which could be roughly divided into three phases, the phase of stress, the phase of secretory inhibition and injury, and the phase of recovery. (4) It might be one of the forms of atrial cardiocytes secreting cardionatrin that the atrial specific granules with intact limiting membrane were excreted from the defect of plasmalemma.


[Experimental study on monitor of microcirculation of random skin flaps and early division of their pedicles].

In a series of 30 random skin flaps formed in pigs, the skin capillary blood flow was measured with laser doppler flowmetry (LDF) pre-, and postoperatively and within 48 hours after pedicle division. The results showed that the change in the microcirculation can be divided into three phases: (1) rapid rising, (2) plateau, and (3) slow rising. The plateau stage may be considered as the critical period. Division of the flap before this "critical" period causes flap necrosis, while division after the period resulted in flap survival (100 percent). Comparison of preoperative and postoperative LDF values can be regarded as a reliable objective criterion for division of the pedicle.


The corrosion behavior of Nd2Fe14B and SmCo5 magnets.

Rare earth magnets have corrosive problems associated with their use in prostheses in various fields including orthodontics. The purpose of this study is to investigate the corrosion behavior of an Nd2Fe14B magnet and a SmCo5 magnet in an oral environment. The relations among the attractive force changes, the released elements, the weight changes and the anodic polarization measurements of the magnets were examined under immersions in 1% NaCl, 1% lactic acid, 0.05% HCl, 0.1% Na2S and Greenwood's artificial saliva at 37 degrees C for forty-two days. The results showed that the rare earth magnets underwent high corrosive assaults and large attractive force reductions by the immersions in 1% lactic acid and 0.05% HCl. The problem of corrosion of the magnets could be overcome by sealing them within laser-welded stainless steel capsules.


Phase transformation of octacalcium phosphate in vivo and in vitro.

Octacalcium phosphate (OCP) was implanted in both thigh muscle pouches of ddY mice for 3 weeks, and was soaked in a simulated body fluid (SBF) and 1% NaCl solution maintained at 37 degrees C for 1 day, 1 week and 3 weeks. After implantation or soaking, the specimens were characterized by scanning electron microscopic analyses, X-ray diffractometry, and infrared absorption spectroscopy. X-ray diffraction study showed that OCP had been completely transformed to apatite with low crystallinity such as the mouse's femur after a 3-week implantation in the mouse body, and an apatic crystal was formed after soaking in the NaCl solution. The IR absorption spectra revealed that CO3 was incorporated in the apatite formed from OCP in both the mouse body and the NaCl solution, while OCP changed little after soaking in the SBF. It seems that the magnesium ion retards the transformation of OCP to apatite.


A new procedure of the tensile fatigue test for dental materials.

A new tensile fatigue test using a stainless steel screw was introduced. A transparent PMMA rod was selected as the test material. A hole was drilled and tapped into the rod; a screw was put into the rod with dental cement or without cementation. Three types of cyclic loads were applied to the test piece; the number of times the load was applied when the initial crack was observed and when the final fracture occurred were recorded. Surface observation showed that actual fatigue fractures occurred in the test piece. The preferred applied load waveform was the cyclic haver-triangles load because of the small coefficient of variance. Fatigue strengths of test pieces with and without cementation were different. Cementation improved the fatigue properties, and the type of cement had an effect on the fatigue properties. Although the number of final fractures minus the number of initial cracks was the constant regardless of the type of cement used.


A new method for assessment of marginal sealability of dental restorations.

A new testing system was developed for assessment of marginal sealability of dental restorations. It was constructed with a thermocycle loading line and a leakage detecting line partitioned from each other by the restoration under evaluation. The hot and cold solutions of rhodamine B as a tracer were alternately circulated in the thermocycle loading line by the switching of six solenoid valves. Marginal leakage of the tracer into distilled water circulating in the detecting line was periodically monitored by a spectrometer while thermocycling the restoration. The leakage data could be analyzed on the basis of an empirical formula. In application of this method, the durability of a complete seal against thermal changes was estimated on three teeth each of which was restored with an inlay, an amalgam or a glass ionomer.


Influence of composition and purity on tensile properties of Ni-Ti alloy castings.

The influence of composition and purity of titanium on the mechanical properties and the transformation temperatures of Ni-Ti alloy dental castings was investigated by tensile testing and differential scanning calorimetry (DSC). The compositions of the ingots were 49.0-49.2 at% of titanium content. Three grades of titanium of relatively high purity were used as starting materials. The result showed Ni-49.0Ti to be a somewhat brittle property, and Ni-49.2Ti to have low apparent proof strength and large elongation. Residual strain increased with increasing titanium content. Even small reductions of titanium purity influenced the tensile properties and the transformation temperatures, causing high apparent proof strength, low residual strain and low elongation because of the reduction in transformation temperatures.


Compressive creep and recovery of composite resins with various filler contents in water.

The compressive creep and the recovery of composites in water were examined. The creep strain of the composites after a 500h test linearly decreased with the increase of their filler content. Their water sorption during testing decreased with the increase of applied stress on the composites containing low filler contents. In the composites containing high filler content, the water sorption was almost constant for some stress levels. The recovery of the composites after testing was significant immediately after testing. From the measurement of the water sorption of the composites, the microcrack or the craze should occur at the filler/matrix interface or in the matrix itself during the creep test, and water could diffuse to the microcrack or the craze after testing in unstressed conditions. This would explain the increase in the water absorbed in the composites in the recovery period.


Residual monomer and pendant methacryloyl group in light-cured composite resins.

Microareas (100 x 100 microns 2) of thin visible light-cured composite sections irradiated for 10-60 s were analyzed with a Fourier transform infrared spectrometer equipped with a microscopic unit before and after extraction of residual monomers to determine the percent unreacted double bonds (UDB) and percent pendant double bonds (PDB). The percent eluted double bonds (EDB) was calculated using these percents. Analysis was conducted at various depths along the midline from the irradiated surface to the base. There were significant differences among the tested materials at each depth in UDB, PDB and EDB percents. The percent PDB of each material remained almost constant (25-40%) to a certain depth. This depth was equivalent to 70-80% of the depth of cure for each material. The range of depths at which percent PDB was constant indicated greater than 35-40% degree of conversion. At these locations, the composites may polymerize with a high rate of crosslinking.


Improved power control during microwave heating in biological applications.

The heating performance of prototype variable power control units attached to two domestic microwave ovens, 500 W and 700 W, was investigated. The units employed the phase control method where rated voltage was obtained by switching AC supply at a particular angular position on the sinusoidal voltage. In order to create experimental conditions employed in a previous study, Sydney tap water (50 mL) was heated at a 10% power level in the 500 W oven. Water boiled at 10 min in both experiments. However, the precision and control of the heating was greatly improved with addition of the control unit. A disinfecting solution (50 mL) and a tissue fixative (10 mL) were included for experiments with the 700 W oven. A power level of only 1% was sufficient to maintain the temperature of the fixative at low temperatures (30-40 degrees C) where many biological reactions occur. The present results indicate that microwave heating power can be controlled by the variable power control method. This will make temperature control possible through the provision of an electronic feedback loop which links a thermocouple with the power control unit.


Thermal coefficients of paste-paste type pulp capping cements.

Thermal coefficients of four kinds of commercially available paste-paste type pulp capping cements were examined. Control reference samples were made of dentin. A thermal coefficient analyzer was used, heating specimens for a few nanoseconds by a xenon flash bulb and measuring thermal changes by using a thermocouple. Thermal coefficients were examined by this non-steady state method. Thermal conductivities of all cements were almost the same or lower than that of dentin. Therefore, when each cement was heated, the penetrating energy was almost the same or lower than that of dentin. The thickness of the cements was converted into that of the dentin by using the obtained thermal conductivity. The 1-mm thickness of the examined cements were equal to between 0.97-mm and 2.10-mm thicknesses of lost dentin. The use of a pulp capping cement provided better pulp protection from thermal stimuli than did the same thickness of dentin.


[The preliminary study of monoamine oxidase activity in platelets in patients with schizophrenia and affective disorder].

This paper has studied the MAO activity in platelets of 52 schizophrenic patients and 44 affective disorder patients. The results indicate that the MAO activity in platelets of schizophrenic patients is lower than that of normal group, especially the chronic schizophrenic patients. It's no difference between the acute schizophrenic patients and normal group. The MAO activity in platelets of bipolar affective disorder patients is much lower than that of normal group. Platelet MAO activity in male bipolar affective disorder is lower than that of female's, that of bipolar-manic effective disorder is lower than that of bipolar-depressive effective disorder. The MAO activity in platelets of unipolar depression is even lower than that of normal group.


[A double-blind study of metoclopramide in the treatment of schizophrenia and determination of prolactin].

This paper reported that 40 schizophrenic patients treated with metoclopramide and haloperidol respectively with double blind observation. Dosage of these two drugs were 450 = 133 mg and 32 +/- 9.2 mg everyday respectively. The effect of metoclopramide were that: 67.5% (27/40) of patients had marked improvement and 85% (34/40) improvement. And the effect of haloperidol were 65% (26/40) and 80% (32/40) respectively. Both of these two drugs had no significant difference. Their main side effect was extrapyramidal reaction, and metoclopramide was milder in this. Detecting serum prolactin before and after treatment in 6 cases of metoclopramide therapy and 5 cases of haloperidol therapy. The ratio of serum levels of prolactin before and after treatment was 3.58 and 4.86 in both drugs therapy respectively.


[A control study on memory and related factors in patients with depression].

The results of an analysis of memory function and related factors in 107 patients with depression and in 76 normal controls revealed that obvious disturbance of memory level and various functions including long-term memory, short-term memory and immediate memory, were seen in observation group. The disturbance of memory function was related to sex, age, education and occupation. No relationship in EGG and depression was found.


[Application of ischemic score of Hachinski in differentiation of multi-infarct dementia].

The Beijing Geriatric Mental Health Center has admitted 46 cases of dementia of the aged in recent more than two years. For them many routine examinations as well as rating scale related and CT scanning were carried out. Their final diagnosis at discharge were of 4 categories: (1) Multi-infarct dementia (MID); (2) Alzheimer's disease(AD); (3) MID+AD, mixed type; and (4) dementia of other causes. We applied Ischemic Score of Hachinski for the 14 cases of MID. The score was over 7 points in all the 14 cases. And the CT pictures in 11 cases showed multiple infarctions, only 3 cases gave evidence of cerebral atrophy. Therefore, the Ischemic Score and CT Scanning were very significant in the diagnosis of MID. The role of each item of the total 13 items of the ischemic score in the diagnosis of MID was discussed according to the clinical materials. It is pointed out that the "stepwise deterioration", "fluctuating course", "history of strokes" and "focal neurological signs" were more important in the diagnosis of MID.


[A clinical and follow-up study in 33 cases of rapid cyclic affective psychosis].

Three hundred and fifty one cases of manic-depressive psychosis in our hospital were reviewed. Of these, 33 cases were rapid cyclothymic type (9.4%). Their clinical features, including sex, age, personality characteristics, phenomena and clinical types were analysed. The results suggested that: the major precipitating factors are psychosocial factors and antidepressants; the better therapy is the administration of the lithium combined with carbamazepine or neuroleptics; and more ideal prognosis occurs in any patient persisting in receiving the therapy.


[Clinical analysis of senile and non-senile affective psychosis].

This article analysed 101 cases of affective psychosis in accordance with CCMD-2 diagnosis criteria. There were 42 cases over and 59 under 60 in age. The results indicated that the patients of the senile affective psychotics accounted for 30.47% of the hospitalized psychotics in the same time. 78.57% of the old group were psychically and somatically precipitated. The proportion was much higher than the non-senile. The degree of the senile group was much higher than the non-senile in manic attack, while it was much lower in depressive attack. The dosages and the plasma level of Li in the former were markedly decreased in comparison to the latter group.


[The study of occurrence mechanism in acute and chronic cerebral vasospasm].

Using radioimmunoassay, fluorescence and transmittal++ electronmicroscopy, we studied the of prostaglandin(PG), lipid peroxidation(LPO) and ultrastructure. Fresh arterial blood (imaging acute spasm) produced basilar arterial spasm strongly(+ + +) and vacuole deterioration in vascular walls but there were no changes in 6-keto-PGF1 alpha and TXB2 and PGE2 and LPO. Incubated arterial blood (imaging chronic spasm) produced basilar arterial spasm markedly (+ + +) also. Vacuole deterioration in vascular walls either, and decreased the level of 6-keto-PGF1 alpha apparently (P < 0.01) and elevated the content of LPO significantly (P < 0.01) and there were no changes in the level of TXB2 and PGE alpha. This experiment suggests that the mechanism of acute spasm and chronic spasm is different.


[Changes in gerbil brain tissue following cerebral ischemia and postischemic reperfusion and studies of the effects of the Chinese drugs].

With the animal model of cerebral ischemia and reperfusion, we conducted experiments on such model to study the effects of Ligustrazine(LZ) and Salvia Miltirrhizae(SM). The results obtained are as follows: (1) The ischemic brain showed hyperperfusion (congestion period) after 10 min reperfusion following 50 min of ischemia, and then entered a delayed hypoperfusion period after 60 minutes reperfusion and afterward the hypoperfusion was remained till the end of 120 min reperfusion. (2) Following 50 min of ischemic insult, ATP and glucose contents in brain tissue were almost depleted and much of lactate accumulated. Although rapid recovery of energy metabolism occurred within 60 min of reperfusion, a secondary deterioration emerged at 120 min of reperfusion. (3) Apparent brain edema occurred after cerebral ischemia and its further development was observed at the early stage of reperfusion owing to congestive response. Despite the degree of brain edema alleviated obviously after 60 min of reperfusion, the condition become worse at 120 min of reperfusion, which was accompanied by secondary metabolic deterioration. (4) Experimental results showed that LZ and SM could significantly elevate rCBF during the delayed hypoperfusion period, and limit the development of secondary deterioration in energy metabolism and brain edema after 120 min of reperfusion. (5) Notably, LZ and SM had no significant effect on MABP when these two Chinese drugs manifested their therapeutic actions in the animal model of cerebral ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)


[A study on the activity of three antioxidant enzymes in the brain of experimental acute cerebral ischemia].

The activities of Superoxide Dismutase (SOD), Glutathione Peroxidase (GSH-Px) and Catalase (CAT) in the ischemic cerebral tissue following the unilateral middle cerebral artery occlusion of rats were assessed. In comparison with the sham-operated rats, both SOD and GSH-Px activity in the ischemic area (striatum and fronto-parietal cortex) were significantly reduced by 30 min. of ischemia, GSH-Px activity in the peri-ischemic area (parieto-parasagittal) was significantly reduced as well. It was shown that in the striatum the GSH-Px activity was much higher than that in the cortex. According to our data, it was suggested that in the ischemic condition, cerebral Superoxide (O2-) and Hydrogen Peroxide (H2O2) were accumulated, and thus the polyunsaturated fatty acids in the neuronal membrane were trapped by these free radical. And such a process resulted in neuronal damage. It implicated that the oxygen free radical might be involved in the neuronal damage induced by Dopamine, since the O2- and H2O2 were excessively generated during the oxidative deamination of Dopamine and the free radical scavengers, SOD and GSH-Px were decreased concomitantly in the cerebral ischemic tissue.


[Treatment of experimental ischemic cerebral lactic acidosis in rats with dichloroacetate].

In the cerebral ischemic pathophysiologic mechanism, lactic acidosis is a important factor to exacerbate cerebral damage. Our research showed that the lactic level of cerebral cortex in rats increased rapidly after the focal cerebral ischemia or during blood reperfusion after cerebral ischemia, 26.99 +/- 5.89 and 28.63 +/- 5.08 mumol/g brain wight respectively, it exacerbated significantly brain edema and pathological damage. The lactic level decreased rapidly to treat with dichloroacetate (50 mg/kg body weight) after cerebral ischemia or during blood reperfusion, 14.11 +/- 2.06 and 13.23 +/- 1.71 mumol/g brain wight respectively, brain edema and pathology improved significantly. It suggested that dichloroacetate might across blood-brain barrier into the cerebral ischemic region and lowered lactic level, improved brain internal environment, relieved cerebral damage after focal cerebral ischemia or during blood reperfusion. It may improve the prognosis of patient with ischemic cerebral vascular disease to be treated with dichloroacetate early.


[Research on myasthenia gravis in children with HLA supratype].

38 cases of MG Children's patients were typed for HLA-I, II and III antigens. It shows that MG significantly associated with HLA-A2, Bw46 and DRw9. Their relative risk were 3.60, 8.18. 4.60, Pc < 0.05 or 0.001 respectively. The association with C4 and Bf were not found. Aw19 is seemed as a protection factor of this disease. These results are similar with the research on Chinese MG patients in Hong Kong but are quite different with those of caucasian data.


[Clinical analysis of 30 cases of stiff-man syndrome].

Thirty cases of SMS, including 22 cases reported previously in China, were analysed clinically manifested by involvement of neck and facial muscles, slurred speech, dysphagia or dyspnea, exaggerated tendon reflexes, ankle clonus and/or Hoffmann's sign. Half of them had past history of infection. Five out of 18 CSF examined showed elevation of protein content, immunoglobulin and white cell count, suggesting the presence of inflammation or demyelination changes in CNS. The pathology of the syndrome is probably located in the spinal cord or brain stem. The criteria for diagnosis are proposed. For treatment, the dosage and way of administration of diazepam should be judged according to the severeness of the disease; nitrazepam and clonazepam are effective, too.


[A study of the P300 cognitive potential on mental deficiency in children].

The study of 36 cases of P300 Cognitive Potential (P300 CP) of mental deficiency children have been made. The results of examination were abnormal in 31 cases (85%). In the 31 cases the latency of P300 the cognitive waves (P300 CW) were prolonged, the amplitude were reduced and the P300 CW disordered. After discussion of the relation among the P300 CW, the intelligence quotient (IQ) and the EEG, it were pointed out that P300 CP is as sensitive, objective, valuable indexes for checking and testing intelligence of mental deficiency children.


Comparison between postoperative astigmatism after classic extracapsular lens extraction and after phacoemulsification with implantation of a Pearce tripod or Pearce vaulted Y-loop intraocular lens.

We compared the surgically induced astigmatism after standard extracapsular cataract extraction (ECCE) with the astigmatism following cataract extraction by phacoemulsification. The surgically induced corneal astigmatism was assessed on several occasions, ranging from one day to one year postoperatively. After phacoemulsification, this astigmatism was considerably slighter than after ECCE on day 1 postoperatively and after two and six weeks. However, one year postoperatively, this difference was less clear. We then measured slight against-the-rule astigmatism for both surgical techniques.


Recurrent hemangiopericytoma and brachytherapy.

A 42-year-old woman had two recurrences following excision of a hemangiopericytoma of the left orbit. A third local excision was followed by postoperative brachytherapy (55 Gy in 100 hours). One year later there is no sign of recurrence and the side-effects are minimal.


Pseudophakic endophthalmitis.

We performed a retrospective study on 45 patients admitted to the Rotterdam Eye Hospital and the Ophthalmic Department of the Academic Medical Center in Amsterdam with pseudophakic endophthalmitis. Vitreous loss during cataract extraction was associated with a significantly increased risk of postoperative endophthalmitis when compared with uncomplicated cataract extraction (p < 0.0001). The incidence of pseudophakic endophthalmitis in diabetic patients was not significantly higher as compared to non-diabetic patients. Vitrectomy in the treatment of postoperative endophthalmitis did not improve the final visual acuity, probably because of selection bias. Methicillin and cephazolin, used intravitreally against gram-positive organisms, failed to provide a good coverage for endophthalmitis due to Staphylococcus epidermidis.


Endophthalmitis: incidence, therapy and visual outcome in the period 1983-1992 in the Rotterdam Eye Hospital.

From 1983 to 1992, 134 patients were treated for clinically suspected endophthalmitis. 61% of this endophthalmitis population consisted of cases that were referred to our clinic. In this nine year period antibiotic treatment was carried out according to three consecutively used guidelines. These three treatment schemes differed in antibiotic spectrum and mode of antibiotic delivery. In 68 patients we performed vitrectomy on account of clinical deterioration under antibiotic treatment. We did not find significant differences in visual outcome between the three treatment groups. The incidence of endophthalmitis following cataract or vitreous surgery did not change throughout the study period. There was however a dramatic decrease in incidence of post-traumatic endophthalmitis following the introduction of a prophylactic antibiotic treatment scheme consisting of fortified gentamicin and cefazolin eyedrops, and intravenously and subconjunctivally administered gentamicin, cefazolin, and clindamycin. In 55 of 68 cases in which vitrectomy was performed in conjunction with intravitreal antibiotics, a vitreous or anterior chamber specimen was cultured. 36 patients had a positive culture result. In the group with positive culture result 42% had better visual acuity in the post-treatment period than before treatment. In the group with a sterile culture result 79% had better vision after treatment.


(An)aerobic bacteria found in secondary-cataract material. A SEM/TEM study.

Twenty four patients, who had marked reduction of vision due to secondary-cataract developed after an ECCE, were treated by surgical cleaning of the posterior lens capsule. During this procedure globular secondary-cataract material was removed and collected for morphological examination by SEM and TEM. Fragments of various sizes and shapes, including some with a 'golf ball' structure, were seen; these closely resembled particles frequently found in cataractous lenses. In addition, in 18 patients micro-organisms were found: rod-shaped bacteria, cocci, and in 2 cases yeasts. These findings were the more remarkable because these were clinically quiet eyes with no signs of intra-ocular inflammation and cultures have been persistently negative. We imagine that these bacteria must have entered the eye during the cataract extraction and have settled there without causing an infection.


Discrepancies between single stimulus and multiple stimulus visual field examinations with the Peritest semi-automated perimeter in glaucoma patients.

In a total of 24 randomly selected eyes of glaucoma patients, tested with the automated (single stimulus) and the manual (multiple stimulus) modes of the semi-automated Peritest perimeter, the numbers and depths of defects were compared as estimated with these two different measurement strategies. It was found that with the manual technique considerably less defects were detected than with the automated strategy.


Serotonin (5-HT) and the rat's eye. Some pilot studies.

Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine which has a multitude of more or less clearly established effects on peripheral vessels. It influences blood viscosity, platelet aggregation, and vasoconstruction and -dilatation, it enhances capillary permeability, it is the precursor of melatonin (a hormone with diurnal production in the eye). Because of these actions a role for serotonin in the development of glaucoma and diabetic retinopathy might be suspected. In a series of pilot studies on rats the effects of serotonin on the anterior and posterior segments of the eye were studied. Serotonin had marked influence on the retinal and choroidal vasculature. The optic disk seemed to be very sensitive to serotonin. Possibly it had an influence on the blood-retinal barrier. It caused transient cataracts, probably by decreasing the production of aqueous. It blocked tropicamin-induced mydriasis. The techniques and provisional results of measurement of serotonin in human aqueous are also described.


The Visual Advice Centre, Eindhoven, The Netherlands. An intervenient evaluation.

On referral by ophthalmologists, the Visual Advice Centre provides the partially sighted with advice and prescriptions for illumination and visual aids. In this paper the multidisciplinary structure of the centre is presented and the results obtained in the first 18 months of its existence are discussed. Two hundred and ninety-eight patients were referred to the centre in this period. The majority of these patients (79.2%) were older than 60 years. The main cause of visual impairment was macular disease (45.3% of the patients). An interesting finding is that, although reading is an important need, reading problems only constituted 35.9% of the total number of demands for help. From an inquiry into the situation of 125 patients 6 months after prescription, it appears that more than 90% of the aids prescribed are used regularly. The conclusion is drawn that patients referred to the Visual Advice Centre benefit from the multi-disciplinary approach to their problems.


Study of the substructure of the Morgagni and Brunescens cataract with the TAO non-coating technique. Part 1: Morgagni cataract.

Lens tissue from a Morgagni cataract was examined by SEM and TEM. For SEM, after prefixation with glutaraldehyde and postfixation with the tannic acid/arginine/OsO4 non-coating (TAO) technique, and for TEM, after prefixation with glutaraldehyde, postfixation with OsO4/K4Fe(CN)6 and poststaining with uranyl acetate/lead citrate. The TAO technique seems to be a particularly suitable postfixation method for the SEM investigation of cataract tissue because of the presence of the protein structures present. The cortical region showed areas of radially, instead of concentrically, arranged lens fibres, degenerated lens fibres with holes (vacuoles), broken ball and socket connections between the lens fibres, and oval or spherical structures varying in size from 0.5-20 microns, the largest resembling a golfball, arising from the cytoplasm of degenerating lens fibres. The smallest, 0.2-0.5 microns, appear to have been expelled from the furrowed lens epithelium.


Study of the substructure of the Morgagni and Brunescens cataract with the TAO non-coating technique. Part 2: Brunescens cataract.

Lens tissue from a Brunescens cataract was prepared for SEM study by prefixation with glutaraldehyde and postfixation with the tannic acid/arginine/OsO4 combination; for TEM study the material was prefixed with glutaraldehyde, postfixed with OsO4/K4Fe(CN)6 and poststained with uranyl acetate/lead citrate. At low magnification, in contrast to the Morgagni cataract, no difference could be seen between the lens fibres in the cortical and nuclear areas. Morphologically, the destruction of the ball and socket system and the development of holes and spherical structures was striking. The latter appeared to have a thin coating and, after fracture, were either empty or showed remnants of material resembling membranes. In sections of the cataractous material, larger vacuoles containing smaller spheres were indistinctly visible.


Comparative anatomy of the vitreous body in rhesus monkeys and man.

In the isolated unfixed vitreous body a structural organization can be visualized by slitlamp microscopy or by an ink-injection technique. We discuss the observations on human and rhesus monkey (Macaca mulatta) vitreous bodies using the ink-injection technique. Advantages and disadvantages of this method compared with slitlamp microscopy are described. Eventual functional implications of vitreal organization are mentioned briefly.


An analysis of colour vision in 10,000 patients.

Examination of a great number of patients resulted in the depth localisation theory. The combination of this theory with the fixation-eccentrisation theory is clinically useful: acquired colour vision defects can be subdivided by the fixation mode and by signs of receptor damage. There are indications that in multiple sclerosis the slowly progressive cases present with more receptor damage than the acute cases.


Ocular lubricants and intraocular stray light.

The question was raised whether ocular lubricants or artificial tears might have adverse effects on optical clarity of the eye. Eight current commercial products were tested on five young subjects, using the Straylight Meter. No adverse effects were found.


Towards a classification of visual impairment.

For the rehabilitation of people with impaired vision, it is essential to have adequate (preferably quantitative) information about their residual visual functions. Special attention is given to the extra information provided by the results of measurement of the contrast sensitivity, especially in combination with the results of other measurements, such as the visual field, the amount of intraocular straylight and the visual acuity. The value of the contrast sensitivity function as a predictor of the extent of dysfunctioning in the visual activities of everyday life, such as outdoor vision, reading and recognition of faces, will be discussed. As far as this is concerned, a comparison is made between the value of visual acuity and contrast sensitivity as measures of the extent of vision and visual dysfunctioning.


Possible relation between HLA and ABO type and prognosis of uveal melanoma.

Not only local parameters but also genetic determinants like the HLA genes may play a role in the development and clinical behavior of malignant tumors. In skin melanoma the presence of HLA-B40 is associated with a poor prognosis. We tested the hypothesis that the clinical behavior of uveal melanoma is influenced by the HLA type of the patient. The HLA types of 44 patients with uveal melanoma had been determined before operation with a view to using the cornea of the enucleated eye for an HLA-matched corneal transplantation. We compared the ABO and HLA types of the patients with the development of metastases and with the ten-year patient survival. An association was observed between the presence of HLA-B40 and death due to metastasis of uveal melanoma. We conclude that the HLA type of the patient may influence the clinical behavior not only of skin melanoma but also of uveal melanoma.


Iris nevi and melanomas: a clinical follow-up study.

A follow-up study of 113 patients with suspicious iris nevi who were referred to our clinic between 1973 and 1991 was carried out by: reviewing their clinical records, fluorescein angiography, obtaining recent data with cooperation of their own or the referring ophthalmologist and contacting patients for reexamination. After examination the diagnoses were: 64 suspicious nevi, 23 melanomas, 15 ciliary body tumors with iris involvement and 11 other pseudomelanomas. In the group of suspicious nevi 86% was localized in the inferior part and 66% in the temporal part of the iris; for the melanoma group these figures were 78% and 75% respectively. The chamber angle was more often involved in the melanoma group, 40% against 17% in the suspicious nevi group. In this group 11 cases (21.6%) showed growth during the follow-up (mean 10.6 years). In three cases the tumor was surgically removed, with as histopathologic diagnosis: 1 xanthogranuloma, 1 neurolemmoma and 1 possible melanoma. In the melanoma group 16 lesions (76%) showed growth during the follow-up (mean 7.2 years), in most cases within 5 years of the initial diagnosis. The lesion was surgically removed in 11 cases. The histopathologic diagnoses were: 8 melanomas, 1 xanthogranuloma, 1 possible melanoma and 1 metastasis of a skin melanoma. Our study shows that periodic ophthalmic check-ups are of great importance in the management of iris lesions suspect for melanoma.