A simple multipurpose double-beam optical image analyzer.

In the paper we present a low cost optical device which splits the light in the focal plane into two separate optical paths and collimates it back into a single image plane, and where a selective information processing can be carried out. The optical system is straightforward and easily implementable as it consists of only three lenses and two mirrors. The system is dedicated for imaging in low-light-level conditions in which widely used optical devices, based on beam splitters or dichroic mirrors, suffer from light loss. We expose examples of applications of our device, using a prototype model. The proposed optical system may be employed for: monitoring the objects located at different distances from observer (1), creating regions of different magnification within a single image plane (2), high dynamic range photometry (3), or imaging in two wavelength bands simultaneously (4).

Quantum telescope: feasibility and constraints.

The quantum telescope is a recent idea aimed at beating the diffraction limit of spaceborne telescopes and possibly other distant target imaging systems. There is no agreement yet on the best setup of such devices, but some configurations have already been proposed. In this Letter we characterize the predicted performance of quantum telescopes and their possible limitations. Our extensive simulations confirm that the presented model of such instruments is feasible and the device can provide considerable gains in the angular resolution of imaging in the UV, optical, and infrared bands. We argue that it is generally possible to construct and manufacture such instruments using the latest or soon to be available technology. We refer to the latest literature to discuss the feasibility of the proposed QT system design.

Identification of the peptide-binding site in the heat shock chaperone/tumor rejection antigen gp96 (Grp94).

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624-630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by alpha-helices and lies on a surface consisting of antiparallel beta-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.

Expression and properties of recombinant HbA2 (alpha2delta2) and hybrids containing delta-beta sequences.

Hemoglobin A2 (alpha2delta2), which is present at low concentration (1-2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O2 binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A2 and systematically studying its functional properties. The construct expressing HbA2 contains only alpha and delta genes so that the extensive purification required to isolate natural HbA2 is circumvented. Although natural hemoglobin A2 is expressed at low levels in vivo, the amount of recombinant alpha2delta2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the alpha + beta or the alpha + gamma genes, respectively, are present on the construct. Recombinant HbA2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA2 are described.

The N-terminal sequence affects distant helix interactions in hemoglobin. Implications for mutant proteins from studies on recombinant hemoglobin felix.

The N-terminal 18-amino acid sequence of the beta-chain of hemoglobin, as far as the end of the A helix, has been replaced by the corresponding sequence of the gamma-chain of fetal hemoglobin with the remaining sequence of the beta-chain retained (helices B through H). The gamma-beta-chain had the correct mass, and its entire sequence was established by mass spectrometric analysis of its tryptic peptides; the alpha-chain also had the correct mass. This recombinant hemoglobin (named Hb Felix) retains cooperativity and has an oxygen affinity like that of HbA both in the presence and absence of the allosteric regulators, 2,3-diphosphoglycerate or chloride but differs from HbF in its 2,3-diphosphoglycerate response. However, Hb Felix has some features that resemble fetal hemoglobin, i.e. its significantly decreased tetramer-dimer dissociation and its circular dichroism spectrum, which measure the strength of the tetramer-dimer interface in the oxy conformation and its rearrangement to the deoxy conformation, respectively. Even though Hb Felix contains the HbA amino acids at its tetramer-dimer interface, which is located at a distance from the substitution sites, its interface properties resemble those of HbF. Therefore, the N-terminal sequence and not just those amino acids directly involved at the subunit interface contacts with alpha-chains must have a strong influence on this region of the molecule. The results reinforce the concept of fluid long range relationships among various parts of the hemoglobin tetramer (Dumoulin, A., Manning, L. R., Jenkins, W. T., Winslow, R. M., and Manning, J. M. (1997) J. Biol. Chem. 272, 31326-31332) and demonstrate the importance of the N-terminal sequence, especially in some mutant hemoglobins, in influencing its overall structure by affecting the relationship between helices.

Recombinant sickle hemoglobin containing a lysine substitution at Asp-85(alpha): expression in yeast, functional properties, and participation in gel formation.

Clinical modalities based on inhibition of gelation of HbS are hindered by the lack of quantitative information on the extent of participation of different amino acid residues in the aggregation process. One such site is Asp-85(alpha), which is involved in a parallel interdouble strand ionic interaction with Lys-144(beta) according to the crystal structure of HbS, but electron microscopy does not specifically show Asp-85(alpha) as a contact site for fiber formation. Using a yeast recombinant system, we have substituted this site by Lys to abolish ion pairing and to make a quantitative determination of its participation in aggregation. The purified double mutant was shown to have the expected pI, the calculated molecular weight, correct amino acid composition, and peptide map. The recombinant double mutant has an oxygen affinity of 10 mm Hg, which is identical to that for HbA and HbS under the same conditions; it also has high cooperativity with an average n value of 2.7. The change in P50 in response to chloride ions was about 25% less than that for HbA or HbS and is ascribed to the introduction of a new positive charge near one of the major oxygen-linked chloride binding sites of hemoglobin. The gelation concentration of the double mutant was measured by a new procedure (Bookchin et al, 1994); the maximal amount of soluble hemoglobin (Csat) in the presence of dextran indicated a decreased tendency for gelation with a Csat of 53 mg/mL compared with 34 mg/mL for HbS. This inhibitory effect is smaller than that of the E6V(beta)/L88A(beta) (Csat, 67 mg/mL) and the E6V(beta)/K95I(beta) (Csat, 90 mg/mL) recombinant hemoglobins. Thus, we would classify Asp-85(alpha) as a moderate contributor to the strength of the HbS aggregate. This wide range of gelation values demonstrates that some sites are more important than others in promoting HbS aggregation.

TCR binding differs for a bacterial superantigen (SEE) and a viral superantigen (Mtv-9).

Both superantigens (SAG) and many anti-TCR monoclonal antibodies (mAb) have specificity for the V beta region of the TCR encoded by TCRBV genes. For instance the bacterial SAG staphylococcal enterotoxin E (SEE), the retroviral SAG MTV-9 and the mAb OT145 each react with human T cells expressing BV6S7. This BV gene encodes two common alleles. We found that SEE and the mAb preferentially activate T cells expressing BV6S7*1 as opposed to BV6S7*2, but Mtv-9 activates T cells expressing either allele. Thus binding to the TCR differs between the two SAGs. A mutation in the TCR HVR-4 region of BV6S7*1 (G72E), where the two BV6S7 alleles differ, indicated that HVR-4 is a component of the binding site for SEE and for the mAb OT145. BV6S7*2 has a charged E72 which may result in electrostatic repulsion of SEE, as SEE contains a similarly acidic aspartic acid residue at a TCR interaction site (204D).

Inhibition of the G1-S transition of the cell cycle by inhibitors of deoxyhypusine hydroxylation.

The formation of the unusual amino-acid hypusine in eIF-5A (eukaryotic initiation factor 5A) is associated with cellular proliferation. We used a panel of compounds, including mimosine, to probe the relationship between the exit from the G1 phase of the cell cycle, i.e., the onset of DNA replication, and the formation of hypusine by the enzyme deoxyhypusyl hydroxylase (DOHH). These two parameters displayed the same dose dependency and structure-activity relationship. Only compounds that inhibited DOHH also suppressed proliferation. This effect was observed: (i) in spontaneously proliferating, virally transformed, and mitogen-stimulated cells; (ii) for both anchorage-dependent and anchorage-independent proliferation; and (iii) with normal and malignant cell lines. DOHH reactivation occurred rapidly after inhibitor withdrawal and correlated with synchronized entry into S. The changes in the expression of specific genes during the G1-to-S transition mimicked the physiological pattern. These findings suggest that hypusine formation in eIF-5A which occurs in a specific, invariant sequence motif acquired early in evolution, may be involved in the G1-to-S transition in the eukaryotic cells tested.

Random chemical modification of the oxygen-linked chloride-binding sites of hemoglobin: those in the central dyad axis may influence the transition between deoxy- and oxy-hemoglobin.

The features of random chemical modification are defined with reference to acetylation of bovine hemoglobin, which has been performed in a random manner so that all of the amino groups that participate in functional chloride binding (i.e., those that are oxygen-linked) could be identified. Random chemical modification, which has objectives different from those of both specific (selective) and extensive chemical modification, has been achieved for bovine hemoglobin with the mild reagent, 14C-methyl acetate phosphate; retention of function was demonstrated by a Hill coefficient of n = 2.2 for the modified hemoglobin. After removal of unmodified Hb chains, the mixture of randomly modified acetylated alpha or beta chains was subjected to tandem treatment with trypsin and chymotrypsin. Peptides were purified by HPLC and identified by amino acid analysis. The amount of radioactivity in the acetylated amino group of a purified peptide was taken as an estimate of the degree of chloride binding. For bovine Hb, two amino groups of the alpha-chain (Val-1 and Lys-99) and three amino groups of the beta-chain (Met-1, Lys-81, and Lys-103) were shown to be oxygen-linked (i.e., to have incorporated significantly more radioactivity in the deoxy conformation compared to the same site in the oxy conformation). Three of these sites were already known chloride-binding sites [i.e., Val-1(alpha), the N-terminus of the alpha-chain, and two sites between the 2 beta-chains of bovine hemoglobin, Met-1(beta) and Lys-81(beta); these findings support the conclusions of the random modification approach. Two other chloride-binding sites, Lys-99(alpha) and Lys-103(beta), align the sides of the central dyad axis connecting the two well-known major chloride-binding sites of bovine Hb. The interrelationship of these five chloride-binding sites was assessed by improved molecular graphics. When viewed through the central dyad axis, the functional chloride-binding sites in the central cavity appear to be symmetrically related and to connect the two major chloride-binding sites. Modifiers or mutants that are directed at these regions in the central dyad axis may favor the deoxy conformation to provide a lower oxygen affinity by preventing the constriction of the central cavity that normally occurs upon oxygenation.

Deficiency of acylpeptide hydrolase in small-cell lung carcinoma cell lines.

During protein biosynthesis, processing of the N terminus of many proteins may occur through acetylation and deacetylation. The enzyme acylpeptide hydrolase is likely involved in deacetylation of nascent peptide chains or of bioactive peptides. The related enzyme, acylase, hydrolyzes the acetyl amino acid product of the acylpeptide hydrolase reaction to acetate and a free amino acid. There is a reciprocal relationship between the substrates for these enzymes (i.e., substrates for one enzyme are competitive inhibitors for the other). In several cultured cell lines, including normal and malignant cells, the ratio of acylpeptide hydrolase to acylase enzyme activities appears to be coordinated and characteristic for a given cell type. Thus, in normal cultured lung cells, hamster ovary cells, hepatoma cells, and lymphocyte cells, nearly equal amounts of these enzymes are expressed, conducive to optimal processing of acetylated N-terminal residues. Four lines of erythroleukemic cell lines were found to express nearly twice as much acylase as acylpeptide hydrolase activity. In the Ehrlich ascites tumor cell line, where 80% of the proteins have been reported to remain acetylated at their N terminus, acylpeptide hydrolase is hardly expressed but acylase activity is not reduced. The 3p21 region of human chromosome 3, which contains the DNF15S2 locus that encodes acylpeptide hydrolase (Jones et al., Proc Natl Acad Sci USA 1991;88:2194), undergoes deletion in some carcinoma cells; the gene that encodes for the acylase is also present on region 3p of the same chromosome. We found that both acylpeptide hydrolase and acylase activities are practically absent in six small-cell lung carcinoma cell lines tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions.

Genetic relationship between acylpeptide hydrolase and acylase, two hydrolytic enzymes with similar binding but different catalytic specificities.

An 87% identity has been found between the reported cDNA sequence that encodes acylpeptide hydrolase (EC 3.4.19.1) [Mitta, M., Asada, K., Uchimura, Y., Kimizuka, F., Kato, I., Sakiyama, F. & Tsunasawa, S. (1989) J. Biochem. 106, 548-551] and a cDNA transcribed from a locus (DNF15S2) on the short arm of human chromosome 3, reported by Naylor et al. [Naylor, S.L., Marshall, A., Hensel, C., Martinez, P.F., Holley, B. & Sakaguchi, A.Y. (1989) Genomics 4, 355-361]; the DNF15S2 locus suffers deletions in small cell lung carcinoma associated with a reduction or loss of acylase activity (EC 3.5.1.14). Acylpeptide hydrolase catalyzes the hydrolysis of the terminal acetylated amino acid preferentially from small acetylated peptides. The acetylamino acid formed by acylpeptide hydrolase is further processed to acetate and a free amino acid by an acylase. The substrates for the acylpeptide hydrolase and the acylase behave in a reciprocal manner since acylpeptide hydrolase binds but does not process acetylamino acids and the acylase binds acetylpeptides but does not hydrolyze them; however, the two enzymes share the same specificity for the acyl group. These findings indicate some common functional features in the protein structures of these two enzymes. Since the gene coding for acylpeptide hydrolase is within the same region of human chromosome 3 (3p21) that codes for the acylase and deletions at this locus are also associated with a decrease in acylase activity, there is a close genetic relationship between the two enzymes. There could also be a relationship between the expression of these two enzymes and acetylated peptide growth factors in some carcinomas.