Search

The synthesis and characterization of a new photolabile protecting group (caging group) for carboxylic acids, the 2-(dimethylamino)-5-nitrophenyl (DANP) group, is described. This compound has a major absorption band in the visible wavelength region with a maximum near 400 nm (epsilon400 = 9077 M(-1) cm(-1) at pH 7.4 and 21 degrees C). The caging group is attached through an ester linkage to the carboxyl functionality of beta-alanine, which activates the inhibitory glycine receptor in the mammalian central nervous system. Such caged compounds play an important role in transient kinetic investigations of fast cellular processes. Upon photolysis of DANP-caged beta-alanine, the caging group is released within 5 micros. Quantum yields of 0.03 and 0.002 were obtained in the UV region (308 and 360 nm) and the visible region (450 nm), respectively. Laser-pulse photolysis experiments, using 337 or 360 nm light, were performed with the caged compound equilibrated with HEK 293 cells transiently transfected with cDNA encoding the alpha1 homomeric, wild-type glycine receptor. The experiments demonstrated that neither DANP-caged beta-alanine nor its byproducts inhibit or activate the glycine receptors on the cell surface. Under physiological conditions, the DANP-caged beta-alanine is water-soluble and stable and can be used for transient kinetic measurements.

The challenge of meeting human development needs while protecting the earth's life support systems confronts scientists, technologists, policy makers, and communities from local to global levels. Many believe that science and technology (S&T) must play a more central role in sustainable development, yet little systematic scholarship exists on how to create institutions that effectively harness S&T for sustainability. This study suggests that efforts to mobilize S&T for sustainability are more likely to be effective when they manage boundaries between knowledge and action in ways that simultaneously enhance the salience, credibility, and legitimacy of the information they produce. Effective systems apply a variety of institutional mechanisms that facilitate communication, translation and mediation across boundaries.

Several crystal structures of the hepatitis C virus NS5B protein (genotype-1b, strain J4) complexed with metal ions, single-stranded RNA or nucleoside-triphosphates have been determined. These complexes illustrate how conserved amino acid side-chains, together with essential structural features within the active site, control nucleotide binding and likely mediate de-novo initiation. The incoming nucleotide interacts with several basic residues from an extension on the NS5B fingers domain, a beta-hairpin from the NS5B thumb domain and the C-terminal arm. The modular, bi-partite fingers domain carries a long binding groove which guides the template towards the catalytic site. The apo-polymerase structure provides unprecedented insights into potential non-nucleoside inhibitor binding sites located between palm and thumb near motif E, which is unique to RNA polymerases and reverse transcriptases.

It has been recently reported that the endogenous expression of HIV-1 Nef in human monocyte/macrophages induces the release of chemokines and other as yet unidentified soluble factors leading to multiple effects of pathogenic significance, such as the recruitment and activation of quiescent lymphocytes. However, the description of underlying molecular mechanisms remained elusive. We recently demonstrated that human monocyte-derived macrophages (MDM) efficiently internalize soluble rNef, thereby inducing effects largely resembling those observed in cells endogenously expressing Nef. By exploiting the rNef/MDM model, we sought to gain more insights on the molecular mechanisms underlying the response of MDM to Nef. Array analysis for the detection of transcripts from a large number of monokines, chemokines, cytokines, and receptors thereof showed that MDM promptly responded to rNef treatment by increasing the transcription of genes for several inflammatory factors. Analysis of supernatants revealed that rNef treatment induced the release of macrophage inflammatory proteins 1alpha and 1beta, IL-1beta, IL-6, and TNF-alpha. Conversely, rNefs mutated in domains critical for the interaction with the endocytotic machinery (i.e., EE155-156QQ, and DD174-175AA) were ineffective. Interestingly, we found that the Nef-dependent release of inflammatory factors correlated with the activation of the NF-kappaB transcription factor, mainly in its p50/p50 homodimeric form, and in a de novo protein synthesis-independent manner. Our data add new hints supporting the idea that the presence of Nef is per se heavily detrimental for monocyte/macrophages and relative cross-talking cell types.

DNA polymerase beta (pol beta) offers a simple system to examine the role of polymerase structure in the fidelity of DNA synthesis. In this study, the M282L variant of pol beta (M282Lbeta) was identified using an in vivo genetic screen. Met282, which does not contact the DNA template or the incoming deoxynucleoside triphosphate (dNTP) substrate, is located on alpha-helix N of pol beta. This mutant enzyme demonstrates increased mutagenesis in both in vivo and in vitro assays. M282Lbeta has a 7.5-fold higher mutation frequency than wild-type pol beta; M282Lbeta commits a variety of base substitution and frameshift errors. Transient-state kinetic methods were used to investigate the mechanism of intrinsic mutator activity of M282Lbeta. Results show an 11-fold decrease in dNTP substrate discrimination at the level of ground-state binding. However, during the protein conformational change and/or phosphodiester bond formation, the nucleotide discrimination is improved. X-ray crystallography was utilized to gain insights into the structural basis of the decreased DNA synthesis fidelity. Most of the structural changes are localized to site 282 and the surrounding region in the C-terminal part of the 31-kDa domain. Repositioning of mostly hydrophobic amino acid residues in the core of the C-terminal portion generates a protein with enhanced stability. The combination of structural and equilibrium unfolding data suggests that the mechanism of nucleotide discrimination is possibly affected by the compacting of the hydrophobic core around residue Leu282. Subsequent movement of an adjacent surface residue, Arg283, produces a slight increase in volume of the pocket that may accommodate the incoming correct base pair. The structural changes of M282Lbeta ultimately lead to an overall reduction in polymerase fidelity.

Antisense RNAs interact with their complementary target RNAs as folded structures. The formation of early binding intermediates is the most important step in determining the overall rates of stable complex formation in vitro and the efficiency of control in vivo. In the case of CopA and CopT (antisense/target RNA pair of plasmid R1), recent studies have identified a four-way junction structure as the major binding intermediate. Previously, the kinetics of antisense/target RNA interaction was studied by indirect methods. Here we have used surface plasmon resonance to follow the binding of CopI (a truncated variant of CopA) to CopT in real time. A protocol was developed that permitted the determination of association and dissociation rate constants for wild-type and mutant CopI-CopT pairs. The K(D)-values calculated from these rate constants were in good agreement with the results obtained by indirect methods. In comparison to earlier model studies of interactions between simple complementary nucleic acids, we observe a different temperature dependence for dissociation rate constants. This may be indicative of the complexity of the steps required for interacting folded RNAs; intramolecular structure competes with intermolecular helix progression during complex formation. The association rate constants were not significantly dependent on temperature. The analysis presented shows that the stability of a kissing complex is not the primary determinant of the rate of stable CopA/CopT complex formation.

Green transparent single crystals of alpha-Ca3[Al2N4] (monoclinic, P2(1)/c, No. 14, a = 957.2(3) pm, b = 580.2(3) pm, c = 956.3(5) pm, beta = 111.62(3) degrees; Z = 4) were obtained from reactions of mixtures of the representative metals with nitrogen above temperatures of 1000 degrees C. beta-Ca3[Al2N4] (monoclinic, C2/c, No. 15, a = 1060.6(2) pm, b = 826.0(2) pm, c = 551.7(1) pm, beta = 92.1(1) degrees; Z = 4) was formed as a byproduct of a reaction of calcium with alumina under nitrogen at T = 930 degrees C in form of colorless crystals. The crystal structures of the two polymorphs contain edge- and corner-sharing AlN4 tetrahedra, leading to different layered anionic partial structures: infinity 2[AlN2/2N2/3)2(AlNN2/2N1/3)6/3(12-)] in the alpha-phase and infinity 2[Al2N2N4/2(6-)] in the beta-polymorph.

The antisense RNA, CopA, regulates the replication frequency of plasmid R1 through inhibition of RepA translation by rapid and specific binding to its target RNA (CopT). The stable CopA-CopT complex is characterized by a four-way junction structure and a side-by-side alignment of two long intramolecular helices. The significance of this structure for binding in vitro and control in vivo was tested by mutations in both CopA and CopT. High rates of stable complex formation in vitro and efficient inhibition in vivo required initial loop-loop complexes to be rapidly converted to extended interactions. These interactions involve asymmetric helix progression and melting of the upper stems of both RNAs to promote the formation of two intermolecular helices. Data presented here delineate the boundaries of these helices and emphasize the need for unimpeded helix propagation. This process is directional, i.e. one of the two intermolecular helices (B) must form first to allow formation of the other (B'). A binding pathway, characterized by a hierarchy of intermediates leading to an irreversible and inhibitory RNA-RNA complex, is proposed.

The four genes required for selenocysteine incorporation were isolated from the gram-positive, amino acid-fermenting anaerobe Eubacterium acidaminophilum, which expresses various selenoproteins of different functions. The sel genes were located in an unique organization on a continuous fragment of genomic DNA in the order selD1 (selenophosphate synthetase 1), selA (selenocysteine synthase), selB (selenocysteine-specific elongation factor), and selC (selenocysteine-specific tRNA). A second gene copy, encoding selenophosphate synthetase 2 (selD2), was present on a separate fragment of genomic DNA. SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, selD1 and selD2, contained an in-frame UGA codon encoding selenocysteine, which corresponds to Cys-17 of Escherichia coli SelD. The function of selA, selB, and selC from E. acidaminophilum was investigated by complementation of the respective E. coli deletion mutant strains and determined as the benzyl viologen-dependent formate dehydrogenase activity in these strains after anaerobic growth in the presence of formate. selA and selC from E. acidaminophilum were functional and complemented the respective mutant strains to 83% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid. Complementation of the E. coli selB mutant was only observed when both selB and selC from E. acidaminophilum were present. Under these conditions, the specific activity of formate dehydrogenase was 55% of that of the wild type. Transformation of this selB mutant with selB alone was not sufficient to restore formate dehydrogenase activity.

The synthesis and characterization of a new photolabile precursor of glycine (caged glycine) is described. The alpha-carboxyl group of glycine is covalently coupled to the alpha-carboxy-2-nitrobenzyl (alphaCNB) protecting group. Photolysis of the caged glycine with UV light produces free glycine. At 308 nm, the compound photolyzes with a quantum yield of 0.38. The absorption spectrum and the pH dependence of a transient absorption produced after laser-flash illumination are typical for aci-nitro intermediates of alphaCNB-protected compounds. The time constant for the major component of the aci-nitro intermediate decay ( approximately 84% of the total aci-nitro absorbance) was determined to be 7 micros at physiological pH. A minor component ( approximately 16%) decays with a rate constant of 170 micros. The compound does not activate or inhibit the alpha(1)-homomeric glycine receptor transiently expressed in HEK293 cells. After photolysis with a 10 ns pulse of 325 nm laser light, the glycine released from the caged compound activates glycine-mediated whole-cell currents in the same cells. The rise of these currents can be measured in a time-resolved fashion and occurs on a millisecond to sub-millisecond time scale. It can be described with a single-exponential function over >85% of the total current. The rate constant of the current rise is about 2 orders of magnitude slower than the rate constant of caged glycine photolysis. Thermal hydrolysis of the alphaCNB-caged glycine takes place with a half-life of 15.6 h at physiological pH. The new caged glycine is the first in a series of photoprotected glycine derivatives that has the required properties for use with chemical kinetic methods for investigation of glycine-activated cell surface receptors. Photolysis is rapid and efficient with respect to the receptor reactions to be studied; hydrolysis in aqueous solution is sufficiently slow, and the compound is biologically inert. It will, therefore, be a useful tool for investigation of the processes leading to channel opening of glycine receptor channels and the effects of mutations of the glycine receptor and of inhibitors on these processes.

Herpes simplex virus type 1 glycoprotein D (gD) is essential for virus infectivity and is responsible for binding to cellular membrane proteins and subsequently promoting fusion between the virus envelope and the cell. No structural data are available for gD or for any other herpesvirus envelope protein. Here we present a three-dimensional model for the baculovirus-expressed truncated protein gD1(306t) based on electron microscopic data. We demonstrate that gD1(306t) appears as a homotetramer containing a pronounced pocket in the center of the molecule. Monoclonal antibody binding demonstrates that the molecule is oriented such that the pocket protrudes away from the virus envelope.

Underpinned by a database of more than a dozen different crystal structures, an increasingly complete and coherent picture of polymerase structure and function is emerging. Recently determined structures of DNA and RNA polymerases have revealed some of the molecular features and structural changes governing catalysis, oligomerization, processivity and fidelity. Despite having minimal similarities in sequence and protein topology, the polymerases all display a functionally analogous set of subdomains that bind the primer, template and nucleotide substrates in similar though not identical fashions. The two-metal-ion mechanism for nucleotide incorporation, however, is shared even by nonhomologous polymerases.

Electron transfer reactions for the reduction of glycine in Eubacterium acidaminophilum involve many selenocysteine (U)- and thiol-containing proteins, as shown by biochemical and molecular analysis. These include an unusual thioredoxin system (-CXXC-), protein A (-CXXU-) and the substrate-specific protein B of glycine reductase (-UXXCXXC-). Most probably a selenoether is formed at protein B by splitting the C-N-bond after binding of the substrate. The carboxymethyl group is then transferred to the selenocysteine of protein A containing a conserved motif. The latter protein acts as a carbon and electron donor by giving rise to a protein C-bound acetyl-thioester and a mixed selenide-sulfide bond at protein A that will be reduced by the thioredoxin system. The dithiothreitol-dependent D-proline reductase of Clostridium sticklandii exhibits many similarities to protein B of glycine reductase including the motif containing selenocysteine. In both cases proprotein processing at a cysteine residue gives rise to a blocked N-terminus, most probably a pyruvoyl group. Formate dehydrogenase and some other proteins from E. acidaminophilum contain selenocysteine, e.g., a 22 kDa protein showing an extensive homology to peroxiredoxins involved in the detoxification of peroxides.

The kinetics of the main phase transition of dimyristoylphosphatidyl choline (DMPC) unilamellar vesicles were investigated in the time range from microseconds to seconds. Iodine laser-temperature jump (ILTJ) experiments showed three discrete relaxation phenomena. Time resolved cryo-electron microscopy (CEM) was applied to produce images of intermediate states typical for the relaxation times of lipid vesicles in the micro- to millisecond time window. A careful measurement of the rate of temperature decrease observed during the production of vitrified lamellae of aqueous samples on a copper grid was performed. The best conditions resulted in average rates of cooling of 3 x 10(4) K/s. By comparing the images from CEM of DMPC vesicle samples vitrified above, at, and below the phase transition temperature a structural model was designed, which explains the temperature jump relaxation times in the micro- to millisecond time range by the formation and disappearance of coexisting clusters of crystalline, intermediate, and fluid lipid areas inside the DMPC bilayers.

Chicken mitochondrial and Escherichia coli aspartate aminotransferases K258H, in which the active site lysine residue has been exchanged for a histidine residue, retain partial catalytic competence [Ziak et al. (1993) Eur. J. Biochem. 211, 475-484]. Mutant PLP and PMP holoenzymes and the complexes of the latter (E. coli enzyme) with sulfate and 2-oxoglutarate, as well as complexes of the mitochondrial apoenzyme with N-(5'-phosphopyridoxyl)-L-aspartate or N-(5'-phosphopyridoxyl)-L-glutamate, were crystallized and analyzed by means of X-ray crystallography in order to examine how the side chain of histidine 258 can substitute as a general acid/base catalyst of the aldimine-ketimine tautomerization in enzymic transamination. The structures have been solved and refined at resolutions between 2.1 and 2.8 A. Both the closed and the open conformations, identical to those of the wild-type enzyme, were observed, indicating that the mutant enzymes of both species exhibit the same conformational flexibility as the wild-type enzymes, although in AspAT K258H the equilibrium is somewhat shifted toward the open conformation. The replacement of the active site K258 by a histidine residue resulted only in local structural adaptations necessary to accommodate the imidazole ring. The catalytic competence of the mutant enzyme, which in the forward half-reaction is 0.1% of that of the wild-type enzyme, suggests that the imidazole group is involved in the aldimine-ketimine tautomerization. However, the imidazole ring of H258 is too far away from C alpha and C4' of the coenzyme-substrate adduct for direct proton transfer, suggesting that the 1,3-prototropic shift is mediated by a water molecule. Although there is enough space for a water molecule in this area, it has not been detected. Dynamic fluctuations of the protein matrix might transiently open a channel, giving a water molecule fleeting access to the active site.

The three-dimensional (3D) structure of the giant hemoglobin of the common earthworm Lumbricus terrestris was determined from cryomicroscopical images of a vitrified molecular solution. From about 5000 molecular images, the best-fit 3D structure was calculated using recently developed analysis techniques. Multivariate statistical analysis data compression and automatic classification were used to find the characteristic projection images of the oligomer. The angular reconstitution approach was then applied to find the Euler angle orientations of these characteristic views. The Lumbricus hemoglobin molecule has an overall D6 (622) point-group symmetry and is found to have a local threefold symmetry axis within the 1/12th subunit. This additional symmetry appears to predict the presence of 36 abcd globin tetramers in the Lumbricus hemoglobin or a total of 144 heme chains for the whole oligomer. A distinct doughnut-shaped structure is elucidated in the center of the molecule. This central subunit, representing around 10% of the protein volume, may consist of non-heme-containing linker chains found generally in giant annelid hemoglobins.

Background: Reverse transcriptase (RT) from HIV-1 is responsible for replicating the single-stranded RNA genome to double-stranded DNA. The three-dimensional structure of RT shows that it is a strikingly asymmetric heterodimer consisting of two differently folded subunits (molecular weights 66 kDa and 51 kDa) with identical amino-terminal amino acid sequences (residues 1-428). The large active site cleft is composed of subdomains named 'finger', 'palm' and 'thumb'. There is also an RNAse H domain., Results: We have compared four RT structures. The structures of two independent RT heterodimers comprising the asymmetric unit of an orthorhombic crystal form have been determined by molecular replacement and are noticeably different from each other. Comparison of the molecules in this crystal form with the two previously reported RT structures shows a related pattern of variations in relative sub domain positions. The structural differences can be described as a molecular twist between the polymerase active site located on the finger and palm domains of p66 and the rest of the molecule. This twist occurs around an axis which runs from the p66 palm domain through the p66/p51 connection domain interface and which exits below the RNAse H domain., Conclusions: From the differences in the four RT structures we infer that the molecule has a specific flexibility that allows rotation of the polymerase active site relative to the rest of the molecule. The observed swivelling motion of RT may allow the polymerase to accommodate the rotational and translational movements of the growing nucleic acid duplex, which present an especial problem for RT because it uses an asymmetric molecule (tRNA(Ly3)) as a primer for first strand synthesis.

Three crystal structures of wild type E. coli aspartate aminotransferase (E.C.2.6.1.1) in space group P2(1) have been determined at resolution limits between 2.6 and 2.35 A. The unliganded enzyme and its complexes with the substrate analogues maleate and 2-methylaspartate resulted in different conformations. The unit cell parameters of the unliganded and the inhibited enzyme are a = 87.2, b = 79.9, c = 89.8 A and beta = 119.1 degrees, and a = 85.4, b = 79.8, c = 89.5 A and beta = 118.6 degrees, respectively. The crystallographic symmetry is pseudo-C222(1). The liganded enzyme structures were solved by difference Fourier techniques from that of a Val39-->Leu mutant partially refined to an R-factor of 0.22 at 2.85 A. They have a "closed" conformation like the chicken mAATase:maleate complex. The models were refined to R-factors of 0.19 (maleate complex) and 0.18 (2-methylaspartate complex) by molecular dynamics and restrained least squares methods. The unliganded crystal form was solved by molecular replacement and refined to an R-factor of 0.19 at 2.5 A resolution. The structure is in a "half-open" conformation, with the small domain rotated about 6 degrees from the closed conformation. The cofactor pyridoxal phosphate has a more relaxed conformation than in mAATase. Both maleate and 2-methylaspartate are hydrogen-bonded to the active site as in mAATase. The C alpha-CH3 bond of 2-methylaspartate is oriented at right angles to the cofactor pyridine ring, the most productive orientation for alpha-deprotonation of the substrate L-aspartate. Comparisons with earlier determined eAATase structures in space group C222(1) revealed differences that can probably be attributed to the somewhat lower resolution of the orthorhombic structures and/or mutations in the eAATases used in those studies. The present P2(1) structures confirm the justification of extrapolating properties of active site point mutants to the vertebrate isozymes. They will serve as reference in the interpretation of the properties of further site-directed mutants in continued studies of structure-function relationships of this enzyme.

The aspartate and tyrosine aminotransferases from Escherichia coli have 43% sequence identity and nearly identical active sites. Both are equally good enzymes for dicarboxylate substrates, but the latter transaminates aromatic amino acids 1000 times faster. In an attempt to discover the critical residues for this differential substrate specificity, the aspartate aminotransferase mutant V39L has recently been prepared. It showed improved Kcat/Km values for aspartate, glutamate and tyrosine and the corresponding oxo acids, mainly due to two to ten times lower Km values. For example, the Km values of V39L (wild type) for Asp and Glu are 0.12 (1.0) and 0.85 (2.7) mM respectively. The mutant was co-crystallized with 30 mM maleate from both polyethylene glycol and ammonium sulfate. Both structures were solved and refined to R-factors of 0.22 and 0.20 at 2.85 and 2.5 A resolution respectively. They bear strong resemblance to the closed structure of the wild type enzyme complexed with maleate. The unexpected feature is that, for the first time, the closed form was produced in crystals grown from ammonium sulfate. It is concluded that the mutation has shifted the conformational equilibrium towards the closed form, which leads to generally reduced substrate Kms.

The substrate specificity of tyrosine aminotransferase (eTAT) from Escherichia coli has been tested by transferring the critically different residues Leu39, Glu141, and Arg293 into equivalent positions of aspartate aminotransferase (eAAT). These residues are not directly involved in the catalytic process. The single mutant eAAT V39L possesses greater values of kcat/KM not only for tyrosine but also for aspartate and glutamate. In contrast, the double mutant eAAT P141E,A293R and also the triple mutant eAAT V39L,P141E,A293R exhibit smaller changes of kcat/KM. The converse mutants of tyrosine aminotransferase, in which critical residues of eAAT (Val39) and of mitochondrial AAT (Ala39, Val37) were transferred into equivalent positions of eTAT, exhibited generally decreased values of kcat/KM for both dicarboxylic and aromatic substrates. On the basis of the known structures of eAAT and eAAT V39L as well as of a refined model of eTAT, these results indicate that the different substrate specificities of eAAT and eTAT are due to multiple side chain differences and minor rearrangements of the backbone. The generally improved catalytic efficiency of the mutant eAAT V39L appears to be due to an indirect effect, namely, the facilitated closure of the active site upon substrate binding.

If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P. (1990) Eur. J. Biochem. 187, 329-333]. The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site. If aspartate or glutamate was added to the pyridoxal form of the mutant enzyme [lambda max 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (lambda max = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda max 330 nm, positive CD), a putative ketamine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25 degrees C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with [4'-3H]pyridoxamine 5'-phosphate resulted in rapid release of 3H with a first-order rate constant kappa' = 5 x 10(-4) s-1 similar to that of the wild-type enzyme. Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the active-site lysine residue. The imidazole ring of H258, however, seems too distant from C alpha and C4' to act efficiently as proton donor/acceptor in the aldimine-ketamine tautomerization, suggesting that the prototropic shift might be mediated by an intervening water molecule. Transmination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.

We present a new model for E. coli tyrosine aminotransferase based on the X-ray structures of the wild type and Val39Leu mutant of E. coli aspartate aminotransferase and computer simulation studies. Active site characteristics of the model are correlated with experimental observations on the specificity of these enzymes towards aromatic/dicarboxylic acid substrates.

Many different inhalative substances may cause allergic alveolitis (hypersensitivity pneumonitis). We report on a worker who developed lung fibrosis due to the sensitization to pearl oyster shell dust. High concentrations of IgG antibodies to proteins (conchagens) of the shell which appear to represent the relevant antigens were found in the patient's serum.

In 24 patients suffering from florid pulmonary tuberculosis we found a fairly characteristic cytologic pattern in smears from aspiration material yielded by bronchoscopy. Besides elements of specific granulation tissue, histiocytes, alveolar macrophages and squamous cells of metaplastic origin a high proportion of specimens exhibited a slightly detritic background rich in leucocytes, containing clusters of respiratory epithelial cells with reactive appearance. The significance of this pattern for suspicion of tuberculosis is emphasized. The diagnosis may be proved by evidence of acid fast bacilli in cytologic smears.

In 860 patients 980 bronchoalveolar lavages were performed. The repeated BAL allows conclusions of the effect of treatment and the prognosis of the disease. BAL is together with other clinical findings an important new method in pulmology. Its value in diagnosis of interstitial pulmonary diseases and other pulmonary diseases is described with special view to occupational expositions.

The interaction of gaseous NO2 with solid carrier-adsorbed polycyclic aromatic hydrocarbons was studied under laboratory conditions with a specific attention to factors that might possibly influence this reaction, such as type of carrier lighting conditions, NO2 concentration, exposure time, and temperature. At the NO2 concentration of 1.33 ppm there were detected the following nitro derivatives: nitroanthracene, nitropyrene, nitrochrysene, two mononitro derivatives of benzo/a/pyrene, and dinitrobenzo/a/pyrene. The experimental data suggest that the formation of nitroaromates in atmospheric environment is to be expected, provided that there are present, besides polycyclic aromatic hydrocarbons and nitrogen oxides, also suitable types of sorbents, such as silica gel or fly ash.

Immunocytochemistry using a monoclonal antibody against glycine receptors revealed that these receptors in cat retina are confined to the inner plexiform layer (IPL). The outer half of that layer showed strong patchy labelling with some indication of two bands corresponding to the on- and off-sublamina. High-affinity uptake of [3H]glycine followed by autoradiography labelled amacrine cells, bipolar cells and the outer portion of the IPL. Silver grains in the IPL were patchily distributed. These results indicate in the cat retina a close match between presynaptic glycinergic elements labelled by high-affinity uptake and the postsynaptic receptor sites for glycine revealed by immunocytochemistry.

The effectiveness of transbronchoscopic lung biopsy (TBLB) in combination with bronchoalveolar lavage (BAL) is presented in 487 patients with disseminated lung diseases. The techniques of both methods are described and the results discussed. Comparison of both methods shows that they supply each other. Therefore they should be used in combination in suspected case of disseminated lung disease. Beyond that BAL is important for the control of the disease's course. In the described technique of TBLB we found complications in 7,8%, while with BAL no essential complication was registered.

Habituation of the human vestibulo-ocular reflex by prolonged low-frequency (0.01 and 0.005 Hz) harmonic (sinusoidal) angular acceleration was assessed by measuring the phase advance of slow-phase eye velocity at 0.01 Hz and the time constant of slow-phase eye velocity decay after impulses of angular acceleration. A single half-hour test session produced increases in low-frequency phase advance and decreases in time constant values in all 16 normal subjects. Although all subjects showed increases in low-frequency advance from the beginning to the end of the initial test session, only a few maintained the increases between test sessions. These changes are qualitatively similar to but quantitatively less than those observed with similar testing in monkeys. For clinical testing, to minimize the effect of habituation during harmonic accelerations, the testing time at low frequencies (i.e., less than 0.1 Hz) should be as short as possible.

An analysis of the morphological diagnosis is made in all 339 consecutive patients suffering from lung cancer and received in our clinic in 1979. Including the non-endoscopically examined cases the tumour has been proved morphologically in 88% of central carcinomas, in 81% of the intermediary type and in 47% of peripheral ones. As for the endoscopically examined patients 93% of the central tumours and 48% of the peripheral ones were morphologically assured. Coincidence between histological and cytological type was found in 83% out of 58 resected tumours. The diagnostic efficacy of the bronchial secretion and of the material obtained by catheterbiopsy is investigated in dependence on different tumour-localisations. Furthermore the value of transthoracic needle-biopsy in cases of peripheral carcinomas is assessed.

The influence of various parameters and growth conditions in the "overnight culture" of Salmonella typhimurium strains on mutagenicity test results was investigated. A number of factors were first suspected to be of some importance for the quantitative outcome of the mutagenicity test. None of them, however, was found to influence the results to such a marked extent as to be a major source of variability. Only the brand of nutrient broth used for the propagation of the bacteria proved finally to have a certain effect on the number of (spontaneous and induced) revertant colonies, although no precise and quantitative statements can be made with regard to a possible standardization of this experimental segment in the Salmonella mutagenicity test. The occurrence of such unpredictable but noticeable influences is, however, evidence for the importance of an intralaboratory optimization and standardization of all parts of the test procedure.

In some cases of achalasia, lungs may show characteristic granulomatous pneumonia, following non-symptomatic spill-over and aspiration of fat. The lesion is characterised by fat-containing vacuoles, necrosis, granulocytes, macrophages, some of them with lipid content, epithelioid and giant cells and fibrosis rich in lymphocytes. The histological pattern is pathognomonic and represents an entity. These alterations may be mistaken for tuberculosis and be distinguished from mineral-oil pneumonia (paraffinoma). Affected lung regions may be colonised by nontuberculous (atypical) mycobacteria.

Spectral sensitivity was measured in the light adapted state for two individuals (S1 and S2) of Pseudemys scripta elegans using a behavioral training technique. The curves have three pronounced maxima at 450 nm, at 570 nm (S1) or 540 nm (S2), and at 640 nm. The spectral sensitivity functions can be described by the over-envelope of the effective cone sensitivity functions which are determined by the absorption spectra of the cone photopigments (lambda max at 450, 518 and 620 nm) and by the filter properties of the colored oil droplets.

The paper emphasizes the possibility to distinguish types I, II and III (WHO classification) of lung cancer not only by means of single cells, but also of tissue solids in cytological smears. The morphologic criteria of tissue solids and their special patterns in the different main types of lung cancer are described.

In the course of a screening program for lung cancer detection which has been going on for several years, 2432 male risk persons, aged 42-67, were examined cytologically in 1974 and 1975. The annual rate of incidence of lung cancer in this population is unknown. We estimate it at 3-4%. From two sputa of each patient we screened one specimen. 24.7% of the sputa were unsatisfactory. In the remaining preparations there were neither tumor cells nor cells suspicious for cancer. This result is discussed in view of the experiences of other authors, that the sensitivity of cytological screening for lung cancer is 33-38%. Recommendations are given for increasing the number of detected cases.

The differentiation of interstitial lung diseases and disseminated lung tumors only by radiological methods is impossible. Cytological or histological examinations of specimens taken by bronchological or surgical examinations are able to confirm the diagnosis. In two cases of a disseminated process in which other bronchological diagnostic methods failed we found cells of an adenocarcinoma by BAL. We recommend this method for differential diagnostic between interstitial lung diseases and disseminated intraalveolar carcinoma, especially bronchiolo-alveolar carcinoma.

Presentation of morphological findings in a case of intravenous leiomyomatosis of the uterus in a patient, 43 years of age. Criteria for general and differential diagnosis are given with reference to literature.