Microbial metabolism of quinoline and related compounds. XIV. Purification and properties of 1H-3-hydroxy-4-oxoquinoline oxygenase, a new extradiol cleavage enzyme from Pseudomonas putida strain 33/1.

1H-3-Hydroxy-4-oxoquinoline oxygenase was purified to apparent homogeneity from Pseudomonas putida strain 33/1 which can use 1H-4-oxoquinoline as sole source of carbon. The molecular mass of the enzyme was determined to 26,000 Da by gel chromatography and by SDS polyacrylamide gel electrophoresis. The enzyme is labile at temperatures above 30 degrees C and has a pH optimum of 8.0. It requires oxygen for the reaction and is significantly inhibited by metal ions like Cu2+, Zn2+, Hg2+ and by 4-chloromercuribenzoate. The enzyme is specific only for 1H-3-Hydroxy-4-oxoquinoline; the apparent Km value for this substrate is 24 microM.


Oligopeptidases, and the emergence of the prolyl oligopeptidase family.

Oligopeptidases are endopeptidases that are not proteinases in the strict sense, since they do not hydrolyse peptide bonds in proteins, but act only on smaller polypeptides or oligopeptides. These enzymes apparently perform important, specialized biological functions that include the modification or destruction of peptide messenger molecules. Oligopeptidases have few naturally occurring inhibitors, and their distinctive specificity prevents them from interacting with alpha 2-macroglobulin, unlike the great majority of endopeptidases. The specificity of these specialized endopeptidases doubtless depends upon the three-dimensional structure of the active site, but no crystallographic structure is yet available for an oligopeptidase. Study of the primary structure of prolyl oligopeptidase has recently shown that it is a member of a new family of serine-type peptidases most of which are exopeptidases. The alignment of the sequences leads to the identification of some catalytic triad residues that have not yet been elucidated experimentally.


The primary structure and tissue distribution of cathepsin C.

A cDNA for rat cathepsin C (dipeptidylaminopeptidase I) was isolated. The encoded protein is composed of the signal peptide of 28 residues, the propeptide of 201 residues and the mature enzyme region of 233 residues. The amino acid sequence of the mature enzyme region has 39.5 to 30.5% identity to other papain family proteinases. Cathepsin C is, therefore, belongs to papain family, although its propeptide region is much longer than those of other cysteine proteinases and show no significant sequence similarity to any other cysteine proteinase. The mRNA and protein for cathepsin C are broadly distributed in rat tissues, but the relative proportions of cathepsin C and other cysteine proteinases are found to vary from tissue to tissue.


Basic amino acids preferring broad specificity aminopeptidase from human erythrocytes.

An aminopeptidase hydrolyzing 2-naphthylamides of Lys, Arg, Leu, Met, Phe and Tyr, as well as different di- to tridecapeptides, was purified from the cytosol of human erythrocytes. The enzyme showed preference for Lys and Arg at N-terminus, as proline and D-amino acids were nonpermissive at P1' site. Higher affinity for oligopeptides than for aminoacyl naphthylamides was observed. Among the substrates were Lys-bradykinin, angiotensin III, thymopentin and enkephalins. Aminopeptidase was shown to be a monomeric protein of M(r) approximately 110000 and of pI approximately 4.8, activated by Co2+ and inhibited by EDTA, pHMB, amastatin, bestatin and puromycin. The isolated enzyme could be classified as cytosolic, Lys(Arg) preferring, broad specificity aminopeptidase.


A new family of proteinases is defined by several snake venom metalloproteinases.

Recently, the complete amino acid sequences have been determined for several snake venom metalloproteinases from the genera Crotalus, Trimeresurus and Lachesis of the Crotalidae family. Among these are both hemorrhagic and nonhemorrhagic metalloproteinases. Despite differences in their molecular weights and activities, they all appear to be related through a single ancestral gene, as observed by the comparison of their amino acid sequences. None of these proteins bear significant similarity to any other known protein, with the exception of the zinc binding site demonstrated in thermolysin and other metalloproteinases. Thus we propose that these proteins define a new family of zinc metalloproteinases which is likely to consist of (but is not necessarily limited to) many of the zinc metalloproteinases found in snake venoms.


Pancreatic procarboxypeptidases: their activation processes related to the structural features of the zymogens and activation segments.

The molecular events leading to the complete activation of pancreatic procarboxypeptidases A and B have been investigated. For both proteins the activation process follows a similar general scheme: trypsin is responsible for the first cleavage that separates the active enzyme from the activation segment, the degradation of the activation segment proceeds only from its C-terminal end, and activity release can be correlated with the disappearance of the long forms of the activation segment. In both systems, trypsin and the released carboxypeptidase participate in the trimming of the severed activation regions. However, the rate of enzymatic activation is much faster in the case of procarboxypeptidase B. This phenomenon may be explained by some structural differences in the connecting region which acts as a linker between the globular domain of the activation segment and the N-terminal end of carboxypeptidases and also by the higher efficiency of carboxypeptidase B for the digestion of its own activation segment. It is not due to unfolding of the activation domain, since the isolated activation domain retains its globular conformation in solution.


The characteristics of cysteine proteinases of parasitic protozoa.

Cysteine proteinases have now been detected in most of the important species of parasitic protozoa. Characterization of the enzymes and sequence determinations have revealed that the enzymes are related to papain and the mammalian cathepsins. All of the protozoan enzymes analyzed to date are members of the cathepsin L/cathepsin H/papain branch of the papain superfamily and are more distantly related to cathepsin B. They thus share some characteristics with the cysteine proteinases of their hosts. Individual enzymes, however, are likely to have sufficient novel features to be potential targets for specific antiprotozoal drugs, and a number of proteinase inhibitors and substrates are currently being tested as possible chemotherapeutic agents.


Bovine cathepsins S and L: isolation and amino acid sequences.

The purification procedure of cathepsin S includes acid activation of spleen homogenate, incubation at 37 degrees C, precipitation with (NH4)2SO4 in H2O/tert-butanol medium, gel chromatography, chromatofocusing, covalent chromatography and cation chromatography of FPLC system. Cathepsin S has a M(r) of about 24,000 Da with pI of 6.5 and 6.8. The mixture of both forms gave a single sequence. Cathepsin L was purified from bovine kidney by acid treatment and incubation of 37 degrees C, precipitation by (NH4)2SO4, two ion exchange chromatographies on CM-Sephadex, gel chromatography and ion exchange chromatography on FPLC system. Cathepsin L exists in multiple forms with pI 5.3-5.7 and M(r) of about 29,000 Da. N-terminal amino acid sequence confirms that cathepsin L and cathepsin S are different enzymes.


Investigation of structure function relationships in cathepsin B.

Previous suggestions from sequence alignment studies and examination of the recently determined X-ray crystal structures of cathepsin B point to roles for several specific residues in substrate binding and catalysis. The role of these groups is being examined by studying cathepsin B mutants produced using a yeast expression system. The substitutions Gly198Asp, Arg202Ala, His111Gln and Glu245Gln provide a mechanistic basis for the exopeptidase activity of cathepsin B and the ability of this cysteine proteinase to accept an arginine residue in the S2 subsite.


Characterisation of the activity and stability of single-chain cathepsin L and of proteolytically active cathepsin L/cystatin complexes.

The activity of single-chain cathepsin L was found to be markedly dependent on cysteine concentration, while a covalent, proteolytically active cathepsin L/cystatin complex was less cysteine-dependent. Cysteine levels and ionic strength did not affect the stability of either enzyme form and both enzyme forms were found to be stable for significant periods of time at or near physiological pH.


Antibodies to rat procathepsin B recognize the active mature enzyme.

Use of mature cathepsin B for immunization invariably yields antisera that react with the denatured protein but not with the native enzyme. This is thought to be due to spontaneous denaturation of the immunizing antigen on introduction into the animal. Recombinant rat procathepsin B has been expressed in yeast as a secreted product. A procathepsin B mutant (Cys29Ser), where autoprocessing is prevented, has been purified and used to raise a rabbit polyclonal antiserum. Both immunodiffusion analysis and an activity depletion assay demonstrated that this antibody recognized native mature cathepsin B. It appears that conformational epitopes existing on the active enzyme are lost on denaturation. The stability of the proenzyme however permits their presentation for antibody generation.


Membrane permeable fluorogenic rhodamine substrates for selective determination of cathepsin L.

The dipeptidyl rhodamine diamide substrates (Z-Phe-Arg)2-R110 and (Z-Arg-Arg)2-R110 are 820- and 360-fold more selective for cathepsin L than for cathepsin B allowing a sensitive determination of cathepsin L activity in the presence of high activity of cathepsin B. The results obtained with cell lysates suggest that the cysteine proteinase activity of vital macrophages detected by flow cytometry with these substrates is mainly due to cathepsin L.


Isolation and characterization of bovine stefin B.

A new cysteine proteinase inhibitor (CPI) was isolated from bovine thymus. According to the amino acid sequence it belongs to the stefin family. It appears as a monomer and a dimer with monomer M(r) of 11,178 and pI values 5.6 for the monomer and 5.2 and 5.6 for the dimer. Ki for the interaction with papain was determined to be 0.12 nM. The most interesting feature of bovine stefin B is the replacement of the highly conserved QVVAG region in stefins with the QLVAG sequence without interfering its inhibitory properties.


Conformation and fluctuations of free stefin B: a molecular dynamics study.

Molecular dynamics study was performed on the cysteine proteinase inhibitor stefin B. Structure of inhibitor from the complex with papain was used as a starting point. Amino terminal "trunk" of the inhibitor which lies extended along the cleft of the enzyme in the complex, folded onto the body of inhibitor during MD simulation, thereby reducing the total and particularly hydrophobic surface exposed to the solvent. This effect counterbalances hydrophobic contribution of the "trunk" and explains why its deletion in stefin B and related inhibitors doesn't reduce the dissociation constant. The rest of stefin B conformation is conserved together with main chain hydrogen bonds. Fluctuations of C alpha atoms resembles crystallographic B factors with exception of residues in contact with enzyme.


Denaturation of stefin B by GuHCl, pH and heat; evidence for molten globule intermediates.

GuHCl, pH and thermal denaturation of the recombinant stefin B was followed by circular dichroism (CD) and size-exclusion chromatography (SEC). CD at 277 nm was taken as an indicator of integral tertiary structure and CD at 222 nm as an indicator of the secondary structure. Compactness was expressed by the volumes of elution on a SEC (Superose 12) column. Data on equilibrium denaturation were recalculated to the fractions of the native state (fN). The results have shown that equilibrium intermediates of the molten globule type exist under conditions of low pH, high temperature or medium GuHCl concentrations, namely A, T and G. Recent findings on structure and energetics of molten globule intermediates are reviewed.


Characterization and structure of pineapple stem inhibitor of cysteine proteinases.

The complete amino acid sequence of the inhibitor of cysteine proteinases from pineapple stem acetone powder was determined. The inhibitor consists of 52 amino acids and is composed of two polypeptide chains (41 and 11 amino acids) linked via disulphide bonds. It differs from already known sequences in one to four amino acids. Data from its amino acid sequence analysis clearly show that this inhibitor cannot be a member of the cystatin superfamily. The Ki values for papain, bromelain and cathepsin L were determined.


Cystatin mimicry by synthetic peptides.

Synthetic peptides which tentatively mimic the cystatin inhibitory surface were used to study the mechanism of inhibition of cysteine proteinases by their natural inhibitors. The inhibitory properties of these peptides depend mainly on the presence of the QxVxG consensus sequence. N and C-terminal peptide derivatives bearing large hydrophobic groups showed dramatically improved inhibition. Molecular dynamic studies after energy minimization showed that the non covalent interaction between these hydrophobic groups induced the formation of a loop structure which probably favours inhibition. Antibodies were raised against one of these peptides, which recognized kininogens in the serum of all mammal species tested, but not cystatins from family two.


Demonstration of sequence variations in the promoter region of the human cystatin C gene.

Four point mutations in the promoter region of the human cystatin C gene have been detected by direct sequencing of polymerase chain reaction (PCR) amplified DNA. The four base changes are all localized within a short segment of 85 base pairs. Three cystatin C gene alleles could be defined with respect to these promoter mutations; one with the sequence previously published, one carrying three of the mutations and one with all four base substitutions. Two of the observed mutations are involved in a novel Sst II polymorphism and another generates a new Dde I restriction site. A PCR-based assay for analysis of these Sst II and Dde I sites was designed and used to demonstrate Mendelian inheritance of the polymorphisms as well as to determine the frequencies of the cystatin C gene alleles in the population.


Bovine tryptase: purification and characterization.

Bovine tryptase, a mast cell trypsin-like protease, was isolated from liver capsula and from mast cells obtained from the same tissue. The purification procedure which leads to an increase in tryptase activity of 850 fold, involves high salt extraction, hydrophobic interaction chromatography on octyl-Sepharose and affinity chromatography on heparin-Sepharose. The enzyme is oligomeric, with an apparent M(r) of 360,000 +/- 40,000 (as obtained by gel filtration in high salt). The constituent subunits with M(r) 39,000 and 41,000 Da are both labeled with [3H] diisopropyl fluorophosphate and cross-react with anti-rat tryptase immunoglobulins. Only a single N-terminal sequence was found, identical to that of human, dog and rat tryptases. Tripeptide fluorogenic substrates with basic residues in P1 and P2 positions are preferentially hydrolyzed by this enzyme, suggesting a possible processing role as proposed for other tryptases. Bovine tryptase activity is inhibited by NaCl and is insensitive to high molecular weight inhibitors, such as alpha 1 antitrypsin and soybean trypsin inhibitor, as for human and dog tryptases. However it is inhibited by low molecular weight serine protease inhibitors and, similarly to rat tryptase, by the bovine pancreatic trypsin inhibitor (BPTI or aprotinin), in a pH dependent fashion.


Interactions of thrombin with benzamidine-based inhibitors.

Trypsin and trypsin-like enzymes cleave C-terminal bonds of the basic amino acids Arg and Lys. Inhibitors of these enzymes have been found not only among Arg and Lys derivatives but also with structurally related benzamidines. Especially cyclic amides of 4-amidinophenylalanine were found to be inhibitors of thrombin. The most potent selective thrombin inhibitor of these type is N alpha-(beta-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidine. From the X-ray crystal structures of thrombin and trypsin-inhibitor complexes the thrombin complexes formed with inhibitors derived from amidinophenylalanine have been modeled. These models allow valuable predictions to design inhibitors of improved selection and binding properties. Most recently, also the X-ray crystal structures of complexes of inhibitors with bovine thrombin have been solved.


Heparin interferes with the inhibition of neutrophil elastase by its physiological inhibitors.

Heparin depresses the second-order rate constant kass for the inhibition of neutrophil elastase by alpha 1-proteinase inhibitor. For high and low molecular weight heparin the decrease in kass is 290-fold and 40-fold, respectively. This is due to a tight binding of the polymer to elastase: Kd = 3.3 nM or 89 nM for high or low molecular weight heparin respectively. In contrast heparin increases the rate of inhibition of elastase by mucus proteinase inhibitor. For low molecular weight heparin, there is a 27-fold increase in kass. This is due to a strong binding of the polymer to the inhibitor (Kd = 50 nM) which undergoes a conformational change.


Migration of polymorphonuclear leukocytes through human amnion membrane--a scanning electron microscopic study.

Scanning electron microscopy has been used to examine the changes in the basement membrane structure induced by polymorphonuclear leukocytes during leukodiapedesis. A diapedesis model based on a Boyden chamber fitted with a human amnion membrane simulated inflammatory processes during which leukocytes are stimulated to leave blood vessels and to penetrate basement membranes. In the Boyden chamber, a chemotactic gradient was developed from a solution of 10(-7) M formyl-methionyl-leucyl-phenylalanine, which induced the cells to migrate through the membrane. Our observations suggest that leukocytes, like tumor cells, emigrate in a three-step process. In the first instance, they adhere to the basement membrane. A local partial proteolysis follows, which is caused by secreted metalloproteinases, especially by gelatinase. The partial dissolution of the matrix facilitates cell penetration. Active locomotion and squeezing through the residual membrane matrix allows the cells to penetrate without complete local membrane destruction. The last step involves migration of the cells through the connective tissue barrier to the site of infection. The type I collagen fibres which form this loose stroma tissue are no obstacle and can be pushed aside without proteolytic degradation.


Regulation of proteinase activation in mammalian tissues.

A lot of new results have been published on activation of one or more proteinases in mammalian tissues. Explanation of proteinase activation without invasion of leukocyte during atrophy of mammalian tissues is the subject of the present study. The mechanism of activation process can be divided into two parts. The first one consists of modification of proteinases and their inhibitors by oxidation or reduction including oxygen free radical reactions. The second part of activation is a cascade or cycle including limited proteolysis of proenzymes and/or proteinase inhibitors. The hypothetic system of proteinase activation can give a hope for regulation of proteinase activity in mammalian tissues under pathological- or pathologic-like conditions as during exhausting exercise.


Endocytosis by liver cells during suppression of intralysosomal proteolysis.

The lysosomotropic agent chloroquine is widely used as a specific inhibitor of intralysosomal proteolysis in isolated hepatocytes. It was shown that in vitro chloroquine reversibly inhibited purified cathepsins H, B, L in concentrations less than those observed inside lysosomes in vivo. However, administration of high doses of chloroquine to rats (30-50 mg/kg i.p. as a single or repeated injections) was followed by increased cathepsin D and cysteine proteinase activities, as well as other lysosomal enzymes. Chloroquine administration did not induce any changes of carbon particles phagocytosis by liver cells (macrophages); modifications of fluid-phase (125I-PVP uptake) and receptor-mediated endocytosis (125I-asialo-fetuin uptake) were noted. Chloroquine administered in vivo reproduced some symptoms of lysosomal storage diseases (especially during repeated drug administration).


Proteolytic inactivation of alpha 1-proteinase inhibitor in vivo: detection, characterization and quantitation of the main fragment excreted in the urine of leukemia patients.

During the search for a therapy response parameter in patients with acute myeloid leukemia, we observed the appearance of a 41 kDa glycoprotein band in the urines of these patients under therapy. To investigate the nature of this molecule and to develop a specific detection system, the protein was isolated and antibodies were raised. Urines and sera of patients and healthy subjects were screened for crossreacting proteins by immunoblotting. Only the leukemia patients showed the urinary 41 kDa protein plus a 53 kDa band. In all sera, including those from healthy donors, a 53 kDa protein was intensely stained. Isolation of the plasma protein and sequence analysis of the urinary protein revealed that alpha 1-proteinase inhibitor is the crossreacting plasma protein and that the 41 kDa molecule is proteolytically modified alpha 1-PI, which has lost its antitryptic activity. Cleavage occurred in the N-terminal part as well as in the reactive site loop of the inhibitor. The 41 kDa truncated inhibitor was also found in the leukemic blast cells. A densitometric method is described for the quantitation of the molecule in the nanomolar range.


Cysteine endopeptidases and their inhibitors in malignant progression of rat embryo fibroblasts.

Cathepsins B and L and their endogenous inhibitors were evaluated in rat embryo fibroblast lines which have been developed as a model system for the study of malignant progression and metastatic capability. Three groups of lines were analyzed: 1) immortalized/non-tumorigenic, 2) tumorigenic/metastatic lines transfected with c-Ha-ras, and 3) metastatic revertants transfected with c-Ha-ras+the E1A region of adenovirus type 2. The metastatic revertants are tumorigenic, but non-metastatic. No correlation was seen between tumorigenicity and metastatic potential and the level of expression of cathepsin B or the subcellular distribution of cathepsins B and L. However, cathepsin L activity was increased 2-fold in the 4R metastatic line. Although transfection of aneuploid 3T3 fibroblasts with ras has been shown to increase the expression of cathepsin L and cathepsin B, transfection of the diploid rat embryo fibroblasts with ras did not correlate with increased expression of cathepsin L or cathepsin B. However, ras transfection of the rat embryo fibroblasts was associated with a significant (4-15-fold) decrease in the activity of heat-stable cysteine endopeptidase inhibitors. Thus, in tumorigenic rat embryo fibroblast lines, regulation of the activities of cysteine endopeptidases by their endogenous inhibitors may be compromised, resulting in increased effective activities of the cysteine endopeptidases.


Cystatins and cathepsins in breast carcinoma.

The increased expression of proteolytic systems is one of the characteristics of transformed and malignant cells and their evaluations in whole tumor homogenates were considered as possible diagnostic and/or prognostic factors. Abnormal intracellular distribution, increased activities and secretion of cysteine proteinases (CPs) cathepsin B (Cat B) and L (Cat L), were associated with tumor progression. In the present study of matched pairs of breast carcinoma and normal breast tissue, the activities of Cat B and Cat L in breast carcinoma homogenates were found to be 20 and 50 fold higher, respectively, than in normal tissues. In contrast, a decrease in total inhibitory activity of cysteine proteinase inhibitors (CPIs) was observed but an average ratio between tumor and normal tissues was only 0.75. One of the CPIs, stefin A, was also determined immunochemically. The activities of CPs and CPIs were compared to the increased levels of cathepsin D (Cat D) activities in individual patients, but no statistically significant correlations were found. We correlated CPs and CPIs with morphological and receptor data as well as the axillary lymph node metastases. There was no statistical correlation of CP and CPIs with the number of lymph node metastases. However, highly elevated levels of Cat B and Cat L and lowered CPI activities in tumor cytosols were often associated with poorly differentiated carcinomas and those with negative ER and PR values. We conclude that cysteine-dependent proteolysis may play an important role in breast tumors.


Growth-related proteinases in cultured human tumour cells.

The proliferation of normal cells, and of early-passage cells from tumours, is inhibited as a consequence of inhibition of a cell-surface proteinase activity. In contrast, established tumour cell lines are usually resistant to these effects. It has previously been suggested that the change from sensitivity to resistance may be a consequence of tumour progression, but experiments with an additional three human tumour cell cultures show that this change is not inevitable.


Tumor-associated urokinase-type plasminogen activator: biological and clinical significance.

Evidence has accumulated that invasion and metastasis in solid tumors require the action of tumor-associated proteases, which promote the dissolution of the surrounding tumor matrix and the basement membranes. Receptor-bound urokinase-type plasminogen activator (uPA) appears to play a key role in these events. uPA converts plasminogen into plasmin and thus mediates pericellular proteolysis during cell migration and tissue remodeling under physiological and pathophysiological conditions. uPA is secreted as an enzymatically inactive proenzyme (pro-uPA) by tumor cells and stroma cells. uPA exerts its proteolytic function on normal cells and tumor cells as an ectoenzyme after having bound to a high-affinity cell surface receptor. After binding, pro-uPA is activated by serine proteases (e.g. plasmin, trypsin or plasma kallikrein) and by the cysteine proteases cathepsin B or L, resp. Receptor-bound enzymatically active uPA converts plasminogen to plasmin which is bound to a different low-affinity receptor on tumor cells. Plasmin then degrades components of the tumor stroma (e.g. fibrin, fibronectin, proteoglycans, laminin) and may activate procollagenase type IV which degrades collagen type IV, a major part of the basement membrane. Hence receptor-bound uPA will promote plasminogen activation and thus the dissolution of the tumor matrix and the basement membrane which is a prerequisite for invasion and metastasis. Tissues of primary cancer and/or metastases of the breast, ovary, prostate, cervix uteri, bladder, lung and of the gastrointestinal tract contain elevated levels of uPA compared to benign tissues. In breast cancer uPA and PAI-1 antigen in tumor tissue extracts are independent prognostic factors for relapse-free and overall survival.


Hydrophobic properties of an amyloidogenic kappa I-Bence Jones protein fragment in charge shift electrophoresis.

Increase in hydrophobicity of amyloid-A protein as compared to the serum amyloid-A precursor protein has been demonstrated with charge shift electrophoresis. Charge shift electrophoresis was applied to the kappa-Bence Jones protein MEV, to amyloid-like fibrils generated in vitro by limited proteolysis of MEV and to an isolated, naturally occurring isolated A lambda (ULI) amyloid fibril protein, to see whether an increase in hydrophobicity is also found in to the immunoglobulin-type amyloid. The results also demonstrate here an increase in hydrophobicity during amyloidogenesis of immunoglobulin-derived amyloid fibril proteins, here.


Plasma lactoferrin content in neutropenic patients: effects of treatment with recombinant granulocyte-macrophage colony-stimulating factor.

Plasma lactoferrin content was measured before and after therapy with recombinant granulocyte-macrophage colony-stimulating factor in five patients with aplastic anaemia, six with myelodysplasia, and three with prolonged, severe, chemotherapy-induced neutropenia. Before therapy plasma lactoferrin content was uniformly low. However, patients with aplastic anemia and those with chemotherapy-induced neutropenia had a normal lactoferrin:neutrophil ratio. The low levels of plasma lactoferrin thus reflected the low granulocyte mass. On the other hand, patients with myelodysplasia also had reduced lactoferrin:neutrophil ratios, suggesting qualitative/quantitative abnormalities of neutrophil lactoferrin production. After treatment with granulocyte-macrophage colony-stimulating factor, plasma lactoferrin levels increased in patients with aplastic anemia and in those with chemotherapy-induced neutropenia who showed a neutrophil response to treatment. In these patients, the lactoferrin:neutrophil ratio became elevated, suggesting increased synthesis/release of lactoferrin from neutrophils. However, patients with myelodysplasia continued to show depressed lactoferrin:neutrophil ratios, even when there had been an increase in granulocyte count, suggesting persistent abnormalities of neutrophil lactoferrin production/release. The implications of these findings for treatment of neutropenic patients with granulocyte-macrophage colony-stimulating factors are discussed.


Biologic and therapeutic efficacy of mafosfamide in patients with metastatic renal cell carcinoma.

It is well known that oxazaphosphorines [e.g., cyclophosphamide and 4-hydroperoxycyclophosphamide (mafosfamide)] are potent immunosuppressive agents. Under the proper conditions, they can potentiate immune responses as well. Immunomodulation represents a major breakthrough in the management of chemotherapy-resistant tumors. Thus, we evaluated the clinical and laboratory sequelae of low to intermediate doses (100-1000 mg/m2) of mafosfamide administered to 16 patients. Four weeks after therapy, one patient had a complete remission, eight patients presented with stable disease, and seven patients did not respond. Clinical and laboratory toxicity was mild and totally reversible, and therapy was well tolerated in all patients. Analyses of phenotypic cell surface antigens on circulating peripheral blood mononuclear cells showed inconsistent alterations of the CD4/CD8 ratio, initial depletion with later rebound of CD8+ cells, increase of CD20+ cells, and a mafosfamide dose-dependent regulation of natural killer-like cells as characterized by CD16 and CD56 positivity. Cell-mediated cytotoxicity against K562 target cells peaked 1 day after therapy and was most pronounced in patients who had received 300 mg/m2 mafosfamide, whereas cytotoxicity against Daudi targets was essentially unchanged, consistent with an increase in natural killing activity without augmentation of lymphokine activated killing. We conclude that mafosfamide administration at low to intermediate doses can be performed with good safety and tolerance; immunophenotypic analyses and cytotoxicity assays showed most pronounced alterations in patients receiving low doses of mafosfamide. These observations support the use of mafosfamide in the attempt to augment antitumor immune responses.


Interleukin-2 increases the antibody response in patients receiving autologous intralymphatic tumor cell vaccine immunotherapy.

The production of tumor-binding antibodies was studied in a group of cancer patients undergoing active specific immunotherapy with irradiated, cholesterol-treated, cell culture-derived autologous tumor cells injected by the intralymphatic route. Fifteen patients were analyzed: nine patients (four melanoma, one breast, one sarcoma, one colon, and one undifferentiated cancer) received three injections of 10 to 15 x 10(6) tumor cells, spaced 2 weeks apart, and six patients (two melanoma, two renal, one breast, and one colon cancer) received tumor cells admixed with 3 x 10(6) U recombinant interleukin-2 (IL-2) (Proleukin, Cetus, Emeryville, CA, USA) plus a 10-day intravenous infusion of 15 x 10(6) U/kg/day IL-2 after each immunization. Serum antibody binding to autologous tumor cells was measured at 2 and 4 weeks after initiation of therapy using an enzyme-linked immunosorbent assay with patient serum being added to adherent tumor cells bound to 96-well microtiter plates. After 4 weeks, we found a significant difference (0.02 less than P less than 0.04) in serum titer in the group receiving IL-2 (33% mean increase) compared with the non-IL-2 group (8% mean increase). Although neither group showed clinical improvement in response to the therapy, the results clearly demonstrated the efficacy of IL-2 in augmenting patient antibody response to autologous intralymphatic tumor cell immunization.


Genetic engineering of anticolorectal carcinoma Fv molecule in myeloma cells.

The anti-TAG72 Fv molecule composed of a heterodimer of both heavy- and light-chain variable domains was produced by the construction of the expression vector mpSV2neo-EP1-Fv72.3. This vector contained the neo gene as a selection marker, the murine immunoglobulin heavy chain promoter (P1), enhancer (E), the SV40 polyadenylation signal region, and the murine cDNA fragments of VH and VL regions amplified and cloned directly from the B72.3 hybridoma RNA by the polymerase chain reaction technique. Termination codons were introduced into the 3' end of both VH and VL regions. The expression vector was transfected into the SP2/0 cell line. The Fv72.3 molecules were purified by the rabbit anti-B72.3 idiotype antibody affinity column, and retained the binding reactivity for the TAG72 antigen. The small size of Fv72.3 molecule (25 kD) makes it attractive for structural studies and immunodetection of cancers.


Modulation of hematologic and immunologic effects of high dose chemotherapy by interleukin-2 in a murine tumor model.

To determine if intensive chemotherapy consisting of cyclophosphamide (C), etoposide (E), and cisplatin (P) (CEP) may be usefully combined with recombinant human interleukin-2 (rhIL-2), we examined a murine tumor model designed to approximate a common clinical situation: macroscopic, drug-resistant cancer. Using C57BL/6 mice with extensive tumor burden 10 days after intravenous B16 melanoma cell injection, we observed (1) C, E, and P synergize to enhance survival but do not cure mice at the highest tolerable dose (C = 200 mg/kg, E = 60 mg/kg, and P = 3 mg/kg); (2) rhIL-2 at 3 x 10(5) U (subcutaneously) daily for 4 days administered 10-18 days after B16 injection significantly improves survival; (3) CEP plus rhIL-2 is more effective than CEP alone only when rhIL-2 is administered before CEP; (4) CEP suppresses IL-2-induced lymphokine-activated killer cell activity in the spleen; and (5) rhIL-2 protects mice incompletely from the immunologic and hematologic suppression of CEP. Our results suggest that intensive chemotherapy combined with rhIL-2 may be beneficial. The success of any such combination may be schedule dependent.


Augmentation of human lymphokine-activated killer cell activity in splenic lymphocytes by the combination of low-dose interleukin 2 plus interleukin 3.

The effect of interleukin 3 (IL-3) on lymphokine-activated killer cell (LAK) generation in splenic lymphocytes was examined in patients with gastric cancer or idiopathic thrombocytopenic purpura (ITP). IL-3 alone did not induce any significant LAK activity from splenic lymphocytes. However, IL-3 addition to the culture with low-dose IL-2 significantly augmented the activity of LAK cells. Spleen cells precultured with IL-3 for 2 days and then added to IL-2 became more potent LAK cells than the spleen cells cultured with the same doses of IL-3 plus IL-2. Phenotypic analysis using flow cytometry demonstrated that IL-2 alone increased in cells expressing CD2+, -11+, and -16+ cells, whereas IL-3 plus IL-2 induced the expansion of CD3+ and CD8+ cells in addition to CD2+, -11+, and -16+ cells. These results suggest that IL-3 plus IL-2 phenotypically induces not only natural killer-like LAK cells (CD2+, -11+, and -16+) but T cell-like LAK cells (CD3+ and -8+). We are now investigating the characteristics of immature T cell populations in the spleen responsive to IL-3 using T-cell receptor antibody.


Modulation of viral infections by cytokines.

The survival of a host challenged by viral infection depends on many factors. Some are specific, such as antiviral T-cell and B-cell responses. Others are nonspecific, such as intrinsic cellular resistance, macrophage activation, and activation of humoral protective mechanisms (e.g., complement, coagulation, etc.). Cytokines are important mediators and regulators of both types of host response. Furthermore, orchestration of specific humoral and cellular immune responses requires the participation of many cytokines. Details of the complex and overlapping roles that cytokines play in specific immune responses is beyond the scope of this review. Instead, this review will focus on the nonspecific antiviral effects of cytokines.


Metastases to skeletal muscles and interferon treatment.

Blood-borne metastases to a skeletal muscle are rare and may originate in various primary tumors. Recurrent solitary metastasis of renal cell carcinoma, and metastatic lesion as part of disseminated malignant melanoma in skeletal muscles are reported in two patients. In both cases interferon treatment with or without chemotherapy failed in arresting the disease.


Structure of RG-12561 dichloromethane solvate and a diastereomer.

(III): [4 alpha,6 beta(E)]-(+-)-6-(2-[2-(4-Fluoro-3-methylphenyl)- 4,4,6,6-tetramethyl-1-cyclohexen-1-yl]ethenyl)-4-hydroxytetrahy dro pyran-2-one (RG-12561) dichloromethane solvate, 2C24H31FO3.CH2-Cl2, Mr = 857.94, triclinic, Pl, a = 11.7413 (5), b = 13.0279 (5), c = 16.2332 (9) A, alpha = 99.456 (4), beta = 94.217 (4), gamma = 101.893 (4) degrees, V = 2381.9 (4) A3, Z = 4 [four molecules of (III)+two molecules of solvent per unit cell], Dx = 1.195 g cm-3, Cu K alpha, lambda = 1.54178 A, mu = 16.69 cm-1, F(000) = 912, T = 293 K, final R = 0.053, wR = 0.060 for 4031 reflections with I greater than 3 sigma(I). (IV): [4 beta,6 alpha(E)]-(+-)-6-(2-[2- (4-Fluoro-3-methylphenyl)-4,4,6,6-tetramethyl-1-cyclo-hexen-1-yl]e thenyl)-4- hydroxytetrahydropyran-2-one, C24H31FO3, Mr = 386.51, triclinic, Pl, a = 6.054 (2), b = 12.931 (2), c = 14.838 (3) A, alpha = 67.70 (2), beta = 85.75 (2), gamma = 82.85 (2) degrees, V = 1066.0 (8) A3, Z = 2, Dx = 1.203 g cm-3, Cu K alpha, lambda = 1.54178 A, mu = 6.86 cm-1, F(000) = 414, T = 293 K, final R = 0.073, wR = 0.081 for 1588 reflections with I greater than 3 sigma(I). (III) is a potent HMG-CoA reductase inhibitor and has the potential to function as a superior hypocholesterolemic agent; (IV) lacks this activity. (III) and (IV) have different conformations and molecular-model calculations suggest that crystal-packing effects are primarily responsible for the overall conformation of (IV). The principal intermolecular contacts are hydrogen bonds of the type O-H...O = C.


Structures of methyl 7-phenyldibenz[a,j]anthracene-14-carboxylate and methyl 7-phenylbenzo[1,2-h:5,4-h']diquinoline-14-carboxylate: twisted aromatic spacers containing bay-region esters.

C30H20O2, (1), Mr = 412.49, triclinic, P1 (Ci1), a = 11.301 (7), b = 12.069 (3), c = 9.777 (2) A, alpha = 96.12 (2), beta = 111.59 (4), gamma = 114.68 (3) degrees, V = 1072 (2) A3, Z = 2, Dx = 1.278 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 0.73 cm-1, F(000) = 432, T = 299 K, final R = 0.065 for 1231 observed reflections. C28H18N2O2, (2), Mr = 414.46, monoclinic, I2/a (C2h6), a = 20.039 (10), b = 11.762 (8), c = 19.850 (5) A, beta = 117.61 (3) degrees, V = 4146 (8) A3, Z = 8, Dx = 1.328 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 0.79 cm-1, F(000) = 1728, T = 298 K, final R = 0.048 for 1924 observed reflections. Aromatic system (1) is highly twisted (43 degrees from end to end) in a helical sense. Half of aromatic system (2) is nearly planar with the ester C atom, whereas the other half is twisted by ca 27 degrees for planarity.


Structure of N-acetyl-L-leucyl-L-prolinamide monohydrate.

C13H23N3O3.H2O, Mr = 287.36, orthorhombic, P2(1)2(1)2(1), a = 6.3863 (8), b = 14.346 (2), c = 17.247 (2) A, V = 1580.1 (6) A3, Z = 4, Dx = 1.208 g cm-3, lambda(Cu K alpha) = 1.54178 A, mu = 7.04 cm-1, F(000) = 624, room temperature, final R = 0.037 for 1693 reflections with I greater than or equal to 2.5 sigma (I) and 257 variables. The peptide linkage is in a trans conformation. The pyrrolidine ring adopts a puckered C gamma-endo conformation and the leucine side chain is in a twisted [g-(tg-)] conformation. The crystal packing is stabilized by a three-dimensional network of hydrogen bonds which involve all the donor groups. The water molecule plays an important part in the crystal packing, being engaged in three hydrogen bonds with different N-acetylamide molecules.


Synthesis and structure of new bronchospasmolytic agents. I.

The crystal structures of two phenylethanolamines showing bronchospasmolytic activity have been determined at room temperature [293 (2) K]. Crystal data are as follows: 11-morpholinotricyclo[6.3.0.0(2,7)]undeca-2,4,6-trien-l-ol (3), C15H19NO2, M(r) = 245.3, triclinic, P1, a = 10.360 (5), b = 12.169 (5), c = 12.488 (4) A, alpha = 95.14 (10), beta = 108.49 (12), gamma = 114.69 (5) degrees, V = 1311 (2) A3, Z = 4, Dx = 1.243 Mg m-3, Cu K alpha 1 radiation, lambda = 1.540562 A, mu = 0.618 mm-1, F(000) = 528, R = 0.0537 for 3009 observed reflections; 4-morpholino-1,2-benzocyclononen-3-ol monohydrate (5), C17H25NO2.H2O, M(r) = 293.4, monoclinic, P2(1)/c, a = 10.063 (9), b = 19.398 (5), c = 8.670 (5) A, beta = 110.56 (1) degree, V = 1585 (2) A3, Z = 4, Dx = 1.230 Mg m-3, Mo K alpha 1 radiation, lambda = 0.709300 A, mu = 0.0778 mm-1, F(000) = 640, R = 0.0376 for 1407 observed reflections. The stereochemistry of compound (3) is found to be 'all cis', which allows the mechanism of formation of these compounds to be interpreted. The various aspects of the conformations of these molecules are discussed.


Structure of ribonuclease T1 complexed with zinc(II) at 1.8 A resolution: a Zn2+.6H2O.carboxylate clathrate.

In order to study the inhibitory effect of Zn2+ on ribonuclease T1 [RNase T1; Itaya & Inoue (1982). Biochem. J. 207, 357-362], the enzyme was cocrystallized with 2 mM Zn2+, pH 5.2, from a solution containing 55% (v/v) 2-methyl-2,4-pentanediol. The crystals are orthorhombic, P2(1)2(1)2(1), a = 48.71 (1), b = 46.51 (1), c = 41.14 (1) A, Z = 4, V = 93203 A3. The crystal structure was determined by molecular replacement and refined by restrained least-squares methods based on Fhkl for 8291 unique reflections with Fo greater than or equal to 1 sigma (Fo) in the resolution range 10 to 1.8 A and converged at a crystallographic R factor of 0.140. The Zn2+ is not bonded to the active site of RNase T1, probably because the His40 and His92 side chains are protonated. Zn2+ occupies the same site as Ca2+ in a series of crystal structures of free and nucleotide-complexed RNase T1. It is coordinated to Asp15 carboxylate and to six water molecules forming a dodecahedron of square antiprismatic form. The Zn2+...O distances are approximately 2.5 A, suggesting that Zn2+ is clathrated and not coordinated, which would require distances of 2.0 A.


Structure determination and refinement of homotetrameric hemoglobin from Urechis caupo at 2.5 A resolution.

A 5 A resolution multiple isomorphous replacement solution for hemoglobin isolated from Urechis caupo revealed a previously unobserved quaternary structure for tetrameric hemoglobin [Kolatkar, Meador, Stanfield & Hackert (1988). J. Biol. Chem. 263(7), 3462-3465]. We report here the structure of Urechis hemoglobin in the cyanomet state refined to 2.5 A resolution by simulated annealing yielding R = 0.148 for reflections F greater than 3 sigma between 5.0 and 2.5 A resolution. The starting model was fitted to a map originally derived from multiple-wavelength anomalous-dispersion phases to 3 A resolution that was then subjected to cyclic twofold molecular averaging and solvent flattening. Structural analysis of the resultant model shows that the unique quaternary assemblage is possible due to several favorable interactions between subunits, including salt links, hydrophobic pockets and interactions mediated by bound water. The tetramer is stabilized by subunit-subunit interactions between the G/H turns and D helices within the crystallographic dimer, and the A/B turn regions and E helices between subunits related by a molecular twofold axis. Interestingly, each subunit has one cysteine residue (Cys21) located in the A/B turn. These twofold-related cysteinyl residues are near enough to one another to form a disulfide bridge but do not.


Structure determination of turkey egg-white lysozyme using Laue diffraction data.

The three-dimensional structure of turkey egg-white lysozyme (TEWL) has been solved and refined at 2.5 A resolution using X-ray data collected by the Laue method. This is the first protein structure determination undertaken using Laue diffraction data. A re-examination of the existing structure of TEWL was necessary when attempts to refine an atomic model based on the C alpha positions in the Protein Data Bank (entry 1LZ2) failed. The correct orientation and position of the turkey lysozyme molecules within the crystallographic unit cell were determined by molecular replacement using a refined model of the homologous hen egg-white lysozyme crystal structure. After modification of the model to reflect the differences in amino-acid sequence between the chicken and turkey enzymes, the structure was subjected to crystallographic refinement using the simulated-annealing refinement technique and conventional least-squares refinement. This yielded a final residual of R = 20.7%. This crystal form is of potential interest for time-resolved crystallographic studies since the amino-acid residues involved in catalysis (Asp52 and Glu35) are accessible to solvent and not blocked by crystal contacts.


Relationship of three-dimensional structure of muscarinic antagonists to antimuscarinic activity: structure of thiodeacylaprophen hydrochloride.

C20H28NS+.Cl-, 2-(diethylamino)ethyl 1,1-diphenylethyl sulfide hydrochloride (thiodeacylaprophen hydrochloride), M(r) = 349.9, orthorhombic, P2(1)2(1)2(1), a = 8.933 (2), b = 11.710 (3), c = 18.934 (4) A, V = 1980.6 (7) A3, Z = 4, Dx = 1.173 g cm-3, Cu K alpha, lambda = 1.54178 A, mu = 26.70 cm-1, F(000) = 752, room temperature, final R = 4.1% for 1417 reflections with /Fo/ greater than 3 sigma (F). Thiodeacylaprophen crystallized as a tertiary amine hydrochloride salt. The S--C--C--N+ segment adopts a trans configuration as does one of the Cphenyl--C--S--C segments. A comparison of the structure of thiodeacylaprophen with the crystal structures of potent antimuscarinic agents suggests that the relatively weak antimuscarinic activity of thiodeacylaprophen compared to atropine and aprophen may be substantially due to the short intramolecular S...N+ distance of 4.106 (6) A. Other contributing structural factors may include the direction of the N+--H bond and restricted accessibility of the sulfur atom for interatomic interactions.


X-ray studies on anti-tumour triazenes. Structures of 1-(4-carbamoylphenyl)-3,3-dimethyltriazene 1-oxide and 3,3-dimethyl-1-(4-nitrophenyl)triazene.

The molecular structures of the title compounds have been determined by X-ray crystallographic methods. The analyses revealed differences in the geometry, and by inference the bond delocalization in these two triazenes owing to the presence in the 1-oxide structure of an N-O bond. The geometries are compared to the crystal structures of the isomeric 3-oxides [Kuroda & Wilman (1985). Acta Cryst. C41, 1543-1545; Neidle, Webster, Kuroda & Wilman (1987). Acta Cryst. C43, 674-676] which shows the dominance of an alternative tautomeric equilibrium for the triazene groups.


Electrostatic properties of 1-methyluracil from diffraction data.

1-Methyluracil (1-methyl-2,4-dioxopyrimidine), C5H6-N2O2, M(r) = 126.12, orthorhombic, Ibam, a = 13.188 (6), b = 13.175 (5), c = 6.214 (3) A, V = 1079.7 (8) A3, Z = 8, Dx = 1.552 g cm-3, lambda (Mo K alpha) = 0.7107 A, mu = 1.317 cm-1, F(000) = 528, T = 123 K, R(F2) = 0.068 for 2996 reflections with sin theta/lambda less than 1.08 A-1. The electronic charge-density distribution has been analyzed in terms of Stewart's rigid pseudoatom model, using restricted Slater radial functions and angular multipole terms extending to octapoles for C, N and O, and quadrupoles for H pseudoatoms. Three different structure refinements have been carried out based on two X-ray diffraction data sets from different crystals collected at temperatures differing by about 20 K. The molecular dipole moment is 6.4 (27) D. Maps of the electrostatic potential for a molecule isolated from the crystal show that atoms O2 and O4 confer overall electro-negativity on one side of the molecule while the CH groups and the C1 methyl group confer electropositivity on the other. For the centrosymmetric hydrogen-bonded dimer (N3-H3...O4'; H...O distances 1.77 A) the electrostatic potential shows electropositive bridges between the molecules. These features are lacking for the C-H...O interactions (distances H6...O2, 2.37; H11...O4, 2.34 A). The electron density and its Laplacian have been determined at the intramolecular bond-critical points and also for the intermolecular H...O interactions. Values for the former are characteristic of covalent bonds. Values of the electron density and Laplacian for the C-H...O interactions are very small and have little or no significance in terms of their e.s.d.'s. The electrostatic energy of interaction for the N-H...O hydrogen-bonded dimer is -10 (12) kJ mol-1. The attractive electrostatic energy increases to -67 (33) kJ mol-1 for a centrosymmetric planar tetramer in which the C-H...O interactions are also formed.


Crystallization and preliminary X-ray analysis of human endothelin.

Endothelin, a potent regulator of vasoconstriction and hypertension, is a naturally produced peptide of 21 amino acids containing two disulfide bonds. We have crystallized endothelin from humans using the vapor-diffusion technique, characterized the crystals by X-ray diffraction analysis, and have collected the X-ray intensities to a resolution of 1.8 A. The crystals, which demonstrate physical properties similar to most protein crystals and have a comparable solvent content, are hexagonal prisms that frequently grow to lengths of 400 microns and widths of 150 microns. The space group of the crystals is P6(1)22 (or P6(5)22), with a = 27.4, c = 79.6 A. There is one molecule of endothelin in the asymmetric unit of the crystals.


Crystallization studies of the catalytic subunit of cAMP-dependent protein kinase: crystals of murine recombinant catalytic subunit and a mutant, Cys 343----Ser, diffract to 2.7 A resolution.

The recombinant mouse catalytic subunit of cAMP-dependent protein kinase, expressed and purified from E. coli, has been successfully cocrystallized as a binary complex with an inhibitor peptide and as a ternary complex with an inhibitor peptide and MgATP. In contrast to the catalytic subunit obtained from porcine heart, the recombinant catalytic subunit lacks a myristoyl group at the amino terminus and differs in sequence at nine positions out of 350 amino acids. The catalytic activities of the two enzymes, however, are nearly identical. Both enzymes cocrystallized with a 20-amino-acid inhibitor and MgATP; however, the porcine-heart enzyme crystallized in a hexagonal space group (P6(1)22) while the recombinant murine catalytic subunit crystallized in an orthorhombic space group (P2(1)2(1)2(1), a = 73.70, b = 76.26, c = 80.74 A). The orthorhombic crystals of the recombinant catalytic subunit exhibit the best diffraction characteristics of all catalytic subunit crystals obtained so far: 2.7 A resolution. Unlike the mammalian porcine-heart enzyme, no crystals of the recombinant apo-enzyme were obtained under the same crystallization conditions. These results are consistent with earlier conclusions that the catalytic subunit exists in at least two distinct conformational states and furthermore suggests that the inhibitor peptide alone is sufficient to induce the major conformational changes that distinguish the two states. A mutant form of the catalytic subunit where Cys343 was replaced with Ser was also cocrystallized with the 20-amino-acid peptide inhibitor and MgATP, and resulted in an orthorhombic crystal isomorphous to crystals of the unmutated enzyme with a similar diffraction of 2.7 A.


Immunosensors in medical diagnostics--major hurdles to commercial success.

In the last few years the format of commercial immunoassays has dramatically changed. Automated instruments running up to 180 samples per hour and dry strip or film immunochemistry products are now available. These changes have been precipitated by the need for immunoassays that require less time and skill to perform. To date, no immunosensor technology has been successfully commercialized although a few are purported to be on the brink of being released for sale. Here, B. Manning and T. Maley evaluate the major factors affecting the success of immunosensors.


A quartz crystal biosensor for measurement in liquids.

The detection of anti-human immunodeficiency virus (HIV) antibodies by means of synthetic HIV peptide immobilized on a piezoelectric quartz sensor is demonstrated. The measurement set-up consists of an oscillator circuit, a suitably modified AT-cut thickness-shear-mode quartz crystal with gold electrodes, which is housed in a special reaction vessel, and a computer-controlled frequency counter for the registration of the measured frequency values. The quartz crystal is adapted for a steady operation in liquids at a frequency of 20 MHz. In phosphate-buffered saline solution the oscillator reaches a stability of about 0.5 Hz within a few seconds, of about 2 Hz within 10 min and about 30 Hz within 1 h. The frequency shift due to the adsorption of various proteins to the uncoated sensor surface has been investigated. It can be shown that a stable adsorptive binding of proteins to an oscillating gold surface is feasible and can be used for the immobilization of a receptor layer (e.g. HIV peptide). Specific binding of the anti-HIV monoclonal antibody to the HIV peptide immobilized on the quartz sensor is demonstrated. Control experiments show, however, additional unspecific binding. According to the experiments, the Sauerbrey formula gives a sufficiently accurate value for the decrease of the resonant frequency due to adsorption or binding of macromolecular proteins on the quartz crystal surface.


A surface plasmon resonance immunosensor based on the streptavidin-biotin complex.

It is shown that a streptavidin monolayer immobilized onto an evaporated gold film with biotin forms the basis of a highly specific sensing element. As an example, we show that by immobilizing the biotinylated antibody sex hormone binding globulin (alpha-SHBG) to the bound streptavidin monolayer a specific sensor for the antigen SHBG is readily fabricated. The interaction between immobilized antibody and corresponding antigen is monitored by surface plasmon resonance spectroscopy and is shown to follow a classic Langmuir isotherm. Detection of SHBG at nanomolar concentrations is demonstrated.


A piezoelectric immunobiosensor for atrazine in drinking water.

A piezoelectric crystal immunobiosensor has been developed for the assay of atrazine herbicides in drinking water. Determinations from 0.03-100 micrograms l-1 (parts per billion) of atrazine can be made with a relative SD of about +/- 8%. Atrazine antibodies (polyclonal from sheep) are layered onto the gold electrode of 10 MHz piezoelectric crystals, which are precoated with protein A. The sensor is reversible, being reusable for about eight or nine assays.


Comparison of colloidal gold electrode fabrication methods: the preparation of a horseradish peroxidase enzyme electrode.

In order to prepare biosensing electrodes which respond to hydrogen peroxide, horseradish peroxidase has been adsorbed to colloidal gold sols and electrodes prepared by deposition of these enzyme-gold sols onto glassy carbon using three methods: evaporation, electrodeposition and electrolyte deposition. In the latter method the enzyme-gold sol is applied to the surface of a glassy carbon disk electrode followed by an equal volume of 2 mM CaCl2. The electrolyte causes the sol to precipitate on the electrode surface, producing an immobilized enzyme electrode. Satisfactory electrodes which gave an electrochemical response to hydrogen peroxide in the presence of the electron transfer mediator ferrocenecarboxylic acid were produced by all three methods. Evaporation of horseradish peroxidase-gold sols produced electrodes with the best reproducibility and the widest linear amperometric response range. These electrodes can also easily be stored in a dry state. Although not as good as evaporation, electrodeposition also produced satisfactory electrodes. Electro-deposition provides the added advantage that it lends itself to the preparation of multi-enzyme/multi-analyte electrodes by the adsorption of different enzymes to separate gold sols, followed by sequential electrodeposition onto discrete areas of a multichannel electrode.


A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum).

The chilling responses of two differentially cold tolerant cultivars of tomato were monitored through in vivo labelling of polypeptides with [35S]methionine, both during a gradual temperature decrease (2 degrees C/day) and also during a rapid cold shock (4 degrees C). The polypeptides were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed by fluorography. Both cultivars showed changes in the polypeptide profiles resulting from either chilling treatment. During the gradual temperature decrease, there were few differences exhibited between the two cultivars. However, during cold shock both cultivars showed the altered synthesis of several unique polypeptides. Both cultivars showed the appearance of a 35-kDa polypeptide during the gradual temperature decrease and also during the cold shock. The appearance of three high relative mass polypeptides was found in both cultivars only during the gradual temperature decrease. Treatments with cycloheximide and chloramphenicol suggested that cold-shock polypeptides are both nuclear and organelle encoded. The cold-shock response in roots was different from the response in leaves and between cultivars. A comparison of the two cultivars showed a number of differences in polypeptide synthesis which may be related to increased cold tolerance.


A simple method using PCR for direct sequencing of genomic DNA from frozen tumor tissue embedded in optimal cutting temperature compound.

Described here is a three-day protocol that directly yields DNA sequence after isolating and PCR amplifying genomic DNA from a small sample of frozen nasopharyngeal carcinoma tissue embedded in optimal cutting temperature (OCT) compound. The method is consistently successful, reproducible and will facilitate the rapid analysis of DNA sequence from very small samples.


Detergents inhibit chloramphenicol acetyl transferase.

Potent inhibition of chloramphenicol acetyl transferase (CAT) by Triton X-100 and Nonidet P-40 was observed. The CAT activity was also moderately inhibited by sodium deoxycholate and sodium dodecyl sulfate, and least by Tween 20. Detergents should, therefore, not be used for cell lysate preparation when CAT activity is used as the reporter in a transient expression experiment.


Essential fatty acid deficiency lowers the activity of the acetylated low density lipoprotein receptor of rat peritoneal macrophages.

We compared phospholipid fatty acid composition, cholesterol ester accumulation, and receptor-mediated binding, internalization, and degradation of acetylated low-density lipoprotein (acetyl-LDL) in cultured peritoneal macrophages from rats fed an essential fatty acid deficient or control diet. The deficient diet increased the 5,8,11-eicosatrienoic acid and decreased the omega 6 fatty acid content of macrophage phospholipid relative to control. The deficient diet did not affect macrophage uptake of [1-14C]oleate; however, it lowered the accumulation of intracellular labelled cholesteroyl oleate to 66% of the control. This effect was attributed to a diminution of the specific binding of acetyl-LDL, and not to acetyl-LDL internalization nor to degradation. The results demonstrate the sensitivity of the acetyl-LDL receptor to changes in its membrane environment, brought about through dietary means.


A useful modification of cDNA that enhances purification of recombinant protein.

With the availability of new and improved DNA vectors for producing large amounts of proteins by recombinant DNA technology, there is a growing need for efficient recovery of the desired protein, often from liters of culture medium. In this report, we describe a relatively simple modification of recombinant cDNA, that, in combination with immobilized metal affinity chromatography, is a rapid, inexpensive and highly effective method for enriching or purifying a desired protein.


The Jeanne Manery-Fisher Memorial Lecture 1991. Maturation of reticulocytes: formation of exosomes as a mechanism for shedding membrane proteins.

The transferrin receptor is a member of a group of reticulocyte surface proteins that disappear from the membranes of reticulocytes as the cells mature to the erythrocyte stage. The selective loss of membrane proteins appears to be preceded by the formation of multivesicular bodies (MVBs). At the reticulocyte stage, many species of mammalian red cells including man, and one nucleated avian species (chicken), contain these intracellular structures in both natural and induced anemias. Also characteristic of blood containing reticulocytes is the presence of circulating vesicles (exosomes), which contain proteins and lipids characteristic of the plasma membrane. These exosomes appear to arise from the contents of the MVBs, after the fusion of MVBs with the plasma membrane. The proteins in the exosomes are those frequently lost during red cell maturation (e.g., transferrin receptor). The major transmembrane proteins (such as the anion transporter) are fully retained into the mature red cell, indicating a highly selective mechanism of recognition of a specific group of proteins. The exosomes are largely devoid of soluble proteins and proteins associated with lysozomes or mitochondria. A speculative model is proposed which addresses the questions of the maturation-induced structural changes in a class of membrane proteins, their recognition and selective loss involving exosome formation, and the release of exosomes to the circulation.


Properties of phosphorylated protein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

The phosphohydrolysis properties of the following phosphoprotein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) were investigated: enzyme I, HPr, and the IIAGlc domain of the glucose enzyme II of Bacillus subtilis; and IIAGlc (fast and slow forms) of Escherichia coli. The phosphohydrolysis properties were also studied for the site-directed mutant H68A of B. subtilis IIA Glc. Several conclusions were reached. (i) The phosphohydrolysis properties of the homologous phosphoprotein intermediates of B. subtilis and E. coli are similar. (ii) These properties deviate from those of isolated N delta 1- and N epsilon 2-phosphohistidine indicating the participation of neighbouring residues at the active sites of these proteins. (iii) The rates of phosphohydrolysis of the H68A mutant of B. subtilis IIAGlc were reduced compared with the wild-type protein, suggesting that both His-83 and His-68 are present at the active site of wild-type IIAGlc. (iv) The removal of seven N-terminal residues of E. coli IIAGlc reduced the rates of phosphohydrolysis between pH 5 and 8.


Rapid high resolution western blotting: from gel to image in a single day.

A streamlined protocol is described that allows high sensitivity antigen detection by Western blotting in a single day. The choice of membrane blotting matrix, as well as blocking reagents, has been optimized in order to allow rapid development of the blot with chemiluminescent reagents. The entire process, from gel to blot to a permanent, hard copy image on x-ray film, can be accomplished within six hours.


Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants.

Paralyzed flagellar mutants pf-1, pf-2, pf-7, and pf-18 of the green alga Chlamydomonas reinhardtii (Dangeard) were shown to store a significantly greater amount of starch than the motile wild type 137c+. The increase in starch storage was significant relative to protein, chlorophyll, and cell number. Analysis of average cell size revealed that the paralyzed mutants were larger than the wild type. This increase in storage molecule accumulation supports an inverse relationship between chemical energy storage and energy utilization for biomechanical/motile cellular functions. Chlamydomonas reinhardtii provides a useful model for studies of the role of cytoskeletal activity in the energy relationship and balance of organisms.


Production of polyclonal antibodies in rabbits is simplified using perforated plastic golf balls.

Production of polyclonal antibodies in the lumen of a perforated golf ball implanted surgically under the skin of a rabbit offers advantages over conventional techniques. Less stress is placed on the rabbit because bleeding is eliminated, complete adjuvants are not used and animal handling is minimized. The technique also offers the advantage that large amounts of antibody-containing fluid can be removed easily from the ball. In this report we describe the surgical protocol and demonstrate use of this technique to produce high-titered antibodies to plant and plant viral proteins.


The effect of epidermal growth factor on liver lipid peroxidation and glutathione levels in lobectomized rats.

Epidermal growth factor (EGF) is a growth-promoting polypeptide which is found in highest levels in male mice in the submaxillary gland. It may also be a key factor in regeneration of the liver. We performed experiments with 18 male Wistar rats, divided into three groups. Hepatic left lobectomy (%30) was performed on the first group of rats. This group received an intraperitoneal injection of EGF for 7 days. The second group was the control group into which normal saline was injected for 7 days. The third group was sham-operated. On days 5 and 7 tomographic studies of liver were performed. On day 7 EGF levels, lipid peroxidation, and glutathione in liver were measured in all of the rats. While serum EGF levels did not show any significant change, the levels of lipid peroxide were decreased and glutathione was increased. Tomographic measurements indicated that administration of EGF increased the amount of regeneration.


N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, a general reducing agent for 5-, 12-, and 15-lipoxygenases and a substrate for their pseudoperoxidase activities.

Lipoxygenases contain a nonheme iron that undergoes oxidation and reduction during the catalytic cycle. The conversion from the Fe3+ enzyme form to the Fe2+ form can be achieved using reducing inhibitors, a reaction that can be reversed with lipid hydroperoxides. The present study describes the properties of N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea (CPHU), which functions as a reducing agent for various lipoxygenases and stimulates the degradation of lipid hydroperoxide catalyzed by these enzymes (pseudoperoxidase activity). CPHU was a substrate for the pseudoperoxidase reaction of purified soybean lipoxygenase-1 with apparent Km values for CPHU and 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE) of 14 and 15 microM, respectively. CPHU was converted during the pseudoperoxidase reaction to a mixture of products that can be resolved by reverse-phase high pressure liquid chromatography. By comparison with the chemical reaction of CPHU and potassium nitrosodisulfonate, the major enzymatic reaction product was tentatively identified as a one-electron oxidation product of CPHU. At low concentrations (50 microM), dithiothreitol completely protected against the degradation of hydroxyurea without inhibiting the pseudoperoxidase reaction. Under these conditions, the rate of the pseudoperoxidase reaction with CPHU as a substrate can be quantitated by the change in absorbance at 234 nm owing to the consumption of 13-HpODE. In addition to soybean lipoxygenase-1, CPHU was found to be a substrate for the pseudoperoxidase activities of purified recombinant human 5-lipoxygenase and porcine leukocyte 12-lipoxygenase. The results are consistent with CPHU reacting with lipoxygenase by a one-electron oxidation to generate the ferrous enzyme form and the nitroxide radical, which could be reduced back to CPHU by DTT.(ABSTRACT TRUNCATED AT 250 WORDS)


DH11S: an Escherichia coli strain for preparation of single-stranded DNA from phagemid vectors.

A new E. coli strain DH11S [mcrA delta(mrr-hsdRMS-mcrBC) delta(lac-proAB) delta(rec1398) deoR rpsL srl- thi-/F'proAB+ lacIqZ delta M 15] has been constructed. Transformation of DH11S competent cells with any of several different phagemid vectors [pSPORT1, pBluescript II SK(+), pGEM11Zf(+)] results in the production of highly purified single-stranded DNAs upon the addition of M13KO7 helper phage. Contamination by double-stranded DNAs was observed with all the other studied strains (XL1-Blue, JM109, DH5 alpha F'IQ). The optimal yield of single-stranded DNA production was obtained when glycerol stocks made from stationary phase cells or single colonies from overnight ampicillin plates of DH11S containing the phagemid vector were infected with M13KO7 helper phage using a wide range (1 to 100) of multiplicities of infection. Five different pSPORT1 clones containing cDNA inserts of various lengths (0.3 kb to 2.0 kb) were compared using these four different bacterial strains. The use of strain DH11S results in the best yields and quality of single-stranded DNA. Therefore, DH11S appears to be the best all-around host for various applications that require single-stranded DNA such as DNA sequencing, in vitro mutagenesis and construction of subtractive cDNA libraries.


Producing a P1 bacteriophage library from pine: isolation and cloning of very high molecular weight DNA.

We have generated a genomic P1 bacteriophage library using Monterey pine (Pinus radiata) DNA. We first developed a method for isolating from pine tissue the very high molecular weight DNA necessary for the preparation of libraries requiring large inserts. The method involves protoplasting the cells, isolating nuclei and lysis in a high concentration of detergent. Fragments of greater than two megabases in size are produced in solution. Modifications introduced to the protocol for library preparation and for P1 plasmid isolation are described.


Analysis of the T cell repertoire using the PCR and specific oligonucleotide primers.

Applications of PCR have revolutionized the field of immunogenetics particularly in studies of human leukocyte antigen class II polymorphism and more recently in the analysis of T cell receptor usage. The diversity of the variable region of the T cell receptor, however, has made it difficult to amplify the complete repertoire of T cell receptor transcripts. We have chosen to address this problem through the design of oligonucleotide primers specific for each of the known V alpha- and V beta-region T cell receptor families in order to characterize the T cell receptor repertoire. Using nonradioactive probes labeled with horse radish peroxidase, the system presented here allows for the rapid elucidation of the T cell receptor repertoire expressed in cells or tissue samples, such as those derived from autoimmune lesions. The identification of the T cell receptor repertoire involved in a pathogenic process can have therapeutic implications given the success of reversing experimental autoimmune disorders by directing specific forms of immunotherapy against V region gene products.


Preparative peptide purification by cation-exchange and reversed-phase perfusion chromatography.

Cation exchange was compared to reversed-phase chromatography for the preparative purification of a 28-residue peptide (vasoactive intestinal polypeptide) on the 100-mg scale. Optimized high-speed, high-resolution methods were developed for both chromatographic modes on POROS Perfusion Chromatography flow-through particle chromatography columns. While both methods appeared to provide similar purity, the cation exchange column had approximately ten times the loading capacity per unit column volume as the reversed-phase column. Five-minute methods for desalting the cation exchange-purified peptide and analysis of fractions were developed using small reversed-phase columns. The cation-exchange method was scaled up to process 95 mg of crude peptide in a 12-min run.


Improved chemiluminescent western blotting procedure.

A chemiluminescent Western blotting procedure and its application in assays for human transferrin and human immunodeficiency virus-I antibodies are described. The procedure is based on a chemiluminescent substrate, adamantyl 1,2-dioxetane aryl phosphate and alkaline phosphatase-labeled detection antibodies. Different membranes (polyvinylidene fluoride, nitrocellulose, nylon) and a proprietary membrane treatment agent (Nitro-Block) have been studied. This sensitive blotting procedure utilizing AMPPD, a polyclonal rabbit anti-transferrin:goat anti-rabbit IgG-alkaline phosphatase detection complex, and a PVDF membrane blocked with Nitro-Block permits the detection of 125 pg (1.6 fmol) of human transferrin. A novel 1,2-dioxetane substrate, CSPD, has also been evaluated.


Changes in protein synthesis pattern during in vitro maturation of goat oocytes.

The regulation of meiotic events of goat oocytes from prophase I to metaphase II was studied by inhibiting protein synthesis at different times of the transition and by analyzing the changes in the protein synthesis pattern during maturation. Protein synthesis was required for germinal vesicle breakdown (GVBD). Nevertheless, the concomitant event to the rupture of germinal vesicle, i.e., chromosome condensation, took place even in a cycloheximide-containing medium. The transition from metaphase I to metaphase II was also protein synthesis dependent as evidenced by experiments using this protein synthesis inhibitor. The inhibition was partly reversible, i.e., after removal of the drug, oocytes were able to progress until metaphase I but could not proceed beyond this stage. Changes in the protein synthesis pattern were studied by radiolabelling of oocytes with [35S]methionine. These changes were correlated with the nuclear status of the oocyte: At GVBD, a polypeptide of 25 kD disappeared, while one of 27 kD appeared. At the same time, a polypeptide of 33 kD appeared, whereas concomitantly one of 34 kD became barely detectable and finally disappeared as the maturation progressed. During maturation, the synthesis of a 67 kD polypeptide increased and became predominant at the end of the maturation process. The synthesis of actin decreased after 18 hr of culture from a very high to a low level of synthesis.


Role of a germ cell-specific sulfolipid-immobilizing protein (SLIP1) in mouse in vivo fertilization.

Sulfolipid-immobilizing protein 1 (SLIP1) is a germ cell plasma membrane protein that binds specifically to sulfogalactosylglycerolipid, a sulfoglycolipid found preferentially in mammalian male germ cells (Lingwood, Can. J. Biochem. Cell. Biol. 63:1077-1085, 1985b). SLIP1 in mouse and rat sperm exists on the periacrosomal membrane, where sperm initially bind to eggs. Using the in vitro mouse sperm-egg binding assay with in vitro-capacitated sperm, we obtained results previously suggesting that sperm SLIP1 is involved in mouse sperm-zona pellucida interaction. In this study, using the in vitro sperm-egg binding assay, we showed that SLIP1 in uterine sperm was similarly engaged in this process. Involvement of mouse sperm SLIP1 was also shown to be important in the in vivo fertilization process. Superovulated females inseminated with caudal epidididymal and vas deferens sperm preexposed to anti-SLIP1 IgG yielded only 20% fertilized zygotes, while 80% fertilization was observed in females inseminated with sperm preincubated with preimmune serum IgG. The lower fertilization rate was not due to changes in the sperm capacitation rate as assessed by chlortetracycline staining.


Assessment of Pisum sativum agglutinin in identifying acrosomal damage in stallion spermatozoa.

The use of fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) was evaluated for its ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosome-intact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostral portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozen-thawed spermatozoa in a sample) of FITC-PSA binding. The binding of FITC-PSA increased in samples as the portion of frozen-thawed (acrosome-damaged) to fresh (acrosome-intact) spermatozoa increased. A positive correlation existed (r = 0.98, P less than 0.05) between the percentage of FITC-PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome-damaged spermatozoa, determined by electron microscopy using gold-conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC-PSA binding may be useful in determining acrosomal integrity of fresh and frozen-thawed stallion spermatozoa.


Release of tissue-type plasminogen activator by activated rat eggs and its possible role in the zona reaction.

The resumption of meiosis results in synthesis of tissue-type plasminogen activator (tPA) in the rat and mouse oocytes (Haurte et al., Cell 43:551-558, 1985). The present study demonstrates that freshly ovulated rat oocytes released their tPA into the surrounding medium upon in vitro activation by sperm penetration or treatment with a calcium ionophore. The presence of a neutralizing monoclonal anti-tPA antibody during in vitro activation by the calcium ionophore inhibited the activation-induced zona hardening and also preserved the ability of the oocyte to be penetrated by sperm subsequent to activation. Rat oocytes undergo zona hardening during in vitro maturation in the absence of serum, presumably as a result of spontaneous cortical granule release, based on findings in mice and hamsters. In the present study, the anti-tPA antibody prevented the zona hardening and enhanced partition by spermatozoa of rat oocytes that were matured in vitro without serum. Collectively, the observations reported have suggest a possible role of tPA released during the cortical granule reaction in the zona reaction, which contributes to the block to polyspermy.


A transient rise in intracellular Ca2+ is a precursor reaction to the zona pellucida-induced acrosome reaction in mouse sperm and is blocked by the induced acrosome reaction inhibitor 3-quinuclidinyl benzilate.

The acrosome reaction induced by the zona pellucida in mouse sperm has been shown to proceed in two stages experimentally distinguishable by the fluorescent probe chlortetracycline. Entry into the first stage of sperm bound to isolated, structurally intact zonae pellucidae is blocked by the compound 3-quinuclidinyl benzilate. In this study, we show, utilizing the fluorescent Ca2+ indicator fluo-3, that the first stage of the zona-induced acrosome reaction is characterized by an increase in intracellular Ca2+, followed by a decrease as the acrosome reaction proceeds. This calcium transient is completely suppressed by 3-quinuclidinyl benzilate. We conclude that the Ca2+ transient is induced by the zona pellucida and is required for the zona-induced acrosome reaction. Blockage of this sperm intracellular Ca2+ transient provides a mechanism for the inhibitory action of 3-quinuclidinyl benzilate on the zona-induced acrosome reaction in mouse sperm.


Natural degenerating mitochondria in ovarian follicles of a cyprinodontidae fish, Epiplatys spilargyreus (teleost).

This study examines the evolution of mitochondria in the follicular cells during the development of the ovarian follicle in the teleostean fish Epiplatys spilargyreus. The mitochondria are few in number until the end of previtellogenesis; their matrix is dense, and their cristae are well developed. They proliferate during vitellogenesis and then are modified by deterioration of their matrix. Multilamellar structures are organized in the vacuolized mitochondria. During postvitellogenesis, these modifications become more advanced. The mitochondria degenerate, leaving vacuoles that contain heterogeneous structures, which will be released into the intercellular spaces. At the end of these mitochondrial transformations, the follicular cells degenerate. They release the elements which will participate in forming the secondary envelope.


Clusterin (SGP-2) in epididymal luminal fluid and its association with epididymal spermatozoa in androgen-deprived rats.

Clusterin is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells and epididymal epithelium. The goal of this study was to determine the presence of clusterin in the luminal fluid of the cauda epididymides and its association with the membranes of developing spermatozoa in the presence and absence of androgen. We have previously demonstrated by two-dimensional (2-D) Western blot probing for clusterin that in epididymal fluid the amounts of clusterin were: caput greater than corpus greater than cauda. Luminal fluid from cauda epididymides was collected from control and orchiectomized rats (6 and 12 days) and orchiectomized animals that received testosterone implants. Equal volumes of fluid were analyzed by 2-D Western blot probing for clusterin. Following orchiectomy, there was an increase in clusterin in the luminal fluid after 6 days and maximal amount after 12 days compared with control cauda fluid. Orchiectomized animals which received testosterone treatment showed levels of clusterin comparable to that of controls. Serum clusterin was detected in fluid of orchiectomized animals with and without testosterone. Western blots of cauda sperm membrane extracts of control animals and orchiectomized animals treated with testosterone had a very low level of epididymal clusterin, whereas extracts collected from orchiectomized animals revealed high levels of clusterin. We suggest that, in the normal animal, clusterin is secreted into the lumen of the proximal epididymis where it binds to the sperm membrane. In the distal epididymis, clusterin dissociates from sperm and is processed (proteolysis/endocytosis). We hypothesize that, in the absence of androgen, the processing and regulation of clusterin is disrupted.


Tight junctions and cavitation in the human pre-embryo.

In the human morula, tight junctions are found between all cell pairs, at all levels of cellular apposition, associated with underlying masses of microfilaments. In cavitating morula, lanthanum tracer gained access to the intracellular spaces, except at the intersections with nascent extracellular cavities, marking the first assembly of zonulae occludentes. Presumptive trophectoderm cells contained vacuoles and larger cavities often associated with secondary lysosome-like bodies. Since the vacuoles and intracellular and extracellular cavities contain electron-dense polygranules of about 23 nm diameter, they may have common origins. In trophectoderm cells of the early blastocytes, the large intracellular vacuoles and cavities were absent, and the zonulae occludentes were located apically. Mechanisms for nascent blastocoele formation are discussed.


Intracellular Ca2+ response of rabbit oocytes to electrical stimulation.

Electrical stimulation is known to cause activation in mammalian oocytes, possibly by eliciting an elevation in intracellular calcium (Ca2+). This study reports intracellular Ca2+ concentrations in mature rabbit oocytes using the Ca2+ indicator fura-2. Calcium levels were determined prior to, during, and after the administration of an electrical pulse (3.6 kV/cm for 60 microseconds). Baseline Ca2+ levels ranged from 30 to 90 nM. The intracellular Ca2+ transient evoked by a pulse, peaked at 11 sec, was highly variable in amplitude (40-300 nM) and returned to prepulse levels within 300 sec. Electrically stimulated oocytes did not exhibit repetitive Ca2+ transients. The size of the cytoplasmic Ca2+ rise was influenced by the duration of the pulse, the field strength and the concentrations of external Ca2+ rise was influenced by the duration of the pulse, the field strength and the concentrations of external Ca2+ (P less than 0.05). Oocytes electrically stimulated in the presence of 100 microM CaCl2, which evoked Ca2+ transients with a mean magnitude of 120 nM, activated at a higher rate (P less than 0.05) than oocytes stimulated in the presence of either higher or lower levels of external Ca2+. Although oocytes electrically shocked at 16-18 hr after administration of human chorionic gonadotropin (hphCG) activated at a lower rate than oocytes stimulated at 22-24 hphCG (P less than 0.05), their intracellular Ca2+ response to the pulse was similar (P less than 0.05). These results indicate that electrical pulse parameters and extracellular Ca2+ concentrations can be used to modulate intracellular Ca2+ levels and optimize oocyte activation rates.(ABSTRACT TRUNCATED AT 250 WORDS)


Murine monoclonal antibodies against staphylococcal enterotoxin B: production and characterization.

A group of 14 monoclonal antibodies (mAbs) to staphylococcal enterotoxin B (SEB) were obtained by fusion of Sp2/O myeloma cells with spleen cells from female BALB/c mice immunized with commercial SEB. The antibodies belonged to IgG1 and IgG2b subclasses. We evaluated the anti-SEB titres, competition assays and sensitivity of detection by indirect ELISA. Reactivity and cross-reactivity were also studied by indirect ELISA and confirmed by immunoblotting. All the mAbs reacted with SEB and with a second band which had a different electrophoretic mobility and probably represents an aggregate of SEB or SEB bound to membranes. Three mAbs reacted only with SEB and the rest showed cross-reactions with SEC1. No reactions were observed against any other serovar (SEA, SED and SEE) or other proteins.


Susceptibility of isolates of Acinetobacter anitratus and Acinetobacter lwoffii to the bactericidal activity of normal human serum.

The bactericidal activity of normal human serum against the Gram-negative coccobacilli Acinetobacter anitratus and Acinetobacter lwoffii was studied; 12% and 84%, respectively, of the tested strains appeared to be sensitive. Thus, serum resistance may be an important factor contributing to the pathogenic potential of A. anitratus strains. Three types of bactericidal action were shown. In the first, the strains were killed when the alternative complement pathway was activated. In the second, some strains required both the classical and alternative pathways. In the third variant, the strains needed either the alternative or classical activation pathway.


Antibodies to outer membrane proteins of Vibrio cholerae induce protection by inhibition of intestinal colonization of vibrios.

An antiserum was raised against the outer membrane (OM) preparation of a Vibrio cholerae 01 strain (Classical, Ogawa) and rendered specific for the outer membrane proteins (OMPs) by absorption with its lipopolysaccharide (LPS). The anti-OMP serum showed reactivity against OM preparations of other 01 and non-01 V. cholerae strains in enzyme-linked immunosorbent assay. The antiserum also induced significant protection against V. cholerae challenge in the suckling mouse model. This protection was found to be independent of biotype, serotype as well as serovar of the challenge organism and was demonstrable even at subagglutinating dilutions of antiserum. The Fab (IgG) fragment, prepared from the anti-OMP serum, also induced passive protection in similar experiments. Further studies demonstrated that the anti-OMP serum as well as its Fab (IgG) fragment markedly inhibited the intestinal colonization of a highly colonizing V. cholerae 01 strain A17 (El Tor, Ogawa). These results highlight the importance of V. cholerae OMPs in immunoprophylaxis against cholera.


Intracellular growth of Mycobacterium avium in human macrophages is linked to the increased synthesis of prostaglandin E2 and inhibition of the phagosome-lysosome fusions.

A virulent strain of Mycobacterium avium grew actively inside human adherent peripheral blood monocyte-derived macrophages. Bacteria were always confined to the phagosome compartment and were encapsulated. Cytochemical labeling of acid phosphatase using transmission electron microscopy showed a strong inhibition of the phagosome-lysosome fusions (PLF) in macrophages as not more than 25-30% bacteria containing phagosome at any time effectively fused with lysosomes. In case of a positive fusion event, the bacterial capsule prevented the diffusion of the lysosomal contents to the bacterial surface. Moreover, the infection of macrophages both by living and gamma-killed M. avium was linked to an increased synthesis of prostaglandin E2 (PGE2); however the total amount of PGE2 synthesized in the latter case was significantly lower than that observed with viable organisms. Our results suggest that the inability of human macrophages to control M. avium infection is linked to immunosuppressive pathways, e.g. enhanced synthesis of PGE2 and also to an impairment of normal microbicidal functions of the infected macrophages.


Effect of indomethacin on the modulation of Mycobacterium avium growth in human macrophages by interferon gamma, retinoic acid and 1,25(OH)2-vitamin D3.

A virulent strain of Mycobacterium avium was grown actively inside human adherent peripheral blood monocyte-derived macrophages with enhanced synthesis of prostaglandin E2 (PGE2). We therefore decided to investigate if the inability of human macrophages to control M. avium infection could be reversed using various immunomodulators, i.e. retinoic acid (RA), 1,25 dihydroxyvitamin D3 (D3) and interferon gamma (IFN gamma) alone or in combination, and whether this reversal was further potentiated by the addition of indomethacin (IND), a potent inhibitor of PGE2 biosynthesis. Among the various immunomodulators employed, only RA alone or in association with D3 or both D3 and IFN gamma were able to induce a clear mycobacteriostatic effect, which was further potentiated by IND. Our data suggest that immunosuppressive pathways induced in macrophages infected by M. avium result partly from an increased synthesis of PGE2 occurring soon after infection.


Immunodominant antigens recognized by the human immune response to infection by organisms of the species Leptospira interrogans serogroup Australis.

Serum samples from patients infected by organisms of Leptospira interrogans serogroup Australis were tested by Western blot to determine the nature of major antigens that are involved in the immune response. Although there was some patient-to-patient variability, immunodominant genus-specific antigens were found to be proteins of apparent molecular ratio 68, 46 and 35-kDa, and lipopolysaccharide (LPS) sub-units in the 35-14-kDa region. Serogroup epitopes specific for Australis were exclusively saccharides of about 32 and 24 kDa: a serovar-specific antigen for serovar lora was of 38-40 kDa and behaved like a protein. Antibodies to the LPS serogroup-specific antigens and to the 38-40 kDa protein were long-lasting and consequently suggest that these immunodominant epitopes are important in resistance to re-infection.


Intestinal, segmented, filamentous bacteria.

Segmented, filamentous bacteria (SFBs) are autochthonous, apathogenic bacteria, occurring in the ileum of mice and rats. Although the application of formal taxonomic criteria is impossible due to the lack of an in vitro technique to culture SFBs, microbes with a similar morphology, found in the intestine of a wide range of vertebrate and invertebrate host species, are considered to be related. SFBs are firmly attached to the epithelial cells of the distal ileal mucosa, their preferential ecological niche being the epithelium covering the Peyer's patches. Electron microscopic studies have demonstrated a considerable morphological diversity of SFBs, which may relate to different stages of a life cycle. Determinants of SFB colonization in vivo are host species, genotypical and phenotypical characteristics of the host, diet composition, environmental stress and antimicrobial drugs. SFBs can survive in vitro incubation, but do not multiply. On the basis of their apathogenic character and intimate relationship with the host, it is suggested that SFBs contribute to development and/or maintenance of host resistance to enteropathogens.


On the relative importance of specific and non-specific approaches to oral microbial adhesion.

In this paper, it is suggested that specificity and non-specificity in (oral) microbial adhesion are different expressions for the same phenomena. It is argued that the same basic, physicochemical forces are responsible for so-called 'non-specific' and 'specific' binding and that from a physico-chemical point of view the distinction between the two is an artificial one. Non-specific interactions arise from Van der Waals and electrostatic forces and hydrogen bonding, and originate from the entire cell. A specific bond consists of a combination of the same type of Van der Waals and electrostatic forces and hydrogen bonding, now originating from highly localized chemical groups, which together form a stereochemical combination. The absence or presence of specific receptor sites on microbial cell surfaces must therefore be reflected in the overall, non-specific surface properties of cells as well. This point is illustrated by showing that glucan-binding lectins on mutans streptococcal strains may determine the pH dependence of the zeta potentials of these cells. When studying microbial adhesion, a non-specific approach may be better suited to explain adhesion to inert substrata, whereas a specific approach may be preferred in case of adhesion to adsorbed protein films. Adhesion is, however, not as important in plaque formation in the human oral cavity as is retention, because low shear force periods, during which adhesion presumably occurs, are followed by high shear force periods, during which adhering cells must withstand these detachment forces. Evidence is provided that such detachment will be through cohesive failure in the pellicle mass, the properties of which are conditioned by the overall, non-specific substratum properties. Therefore, in vivo plaque formation may be more readily explained by a non-specific approach.


Genetics and molecular biology of methanotrophs.

Over the last 20 years or so, the obligate methane-oxidizing bacteria (methanotrophs) have attracted considerable interest. As they grow on a relatively cheap and abundant carbon source, they appeared ideal organisms for the production of bulk chemicals, single-cell protein and for use in biotransformations. More recently their cooxidation properties have been investigated for bioremediation, including the removal of chlorinated compounds such as trichloroethylene from polluted groundwaters. These studies have resulted in a great deal of information on the physiology and biochemistry of methanotrophs but sadly the molecular biology and genetic studies of these organisms have lagged behind. This has been in part due to the obligate nature of the methanotrophs and the refractory nature of such organisms to conventional genetic analysis. However, the more recent availability of broad-host range plasmids coupled with improvements in molecular biology methods have allowed the development of molecular genetic techniques for methanotrophs. The purpose of this review is to summarize what is known about the genetics and molecular biology of methanotrophs and how this information can be used to complement previous and current biochemical studies on the unique property of these bacteria, i.e. the ability to oxidize methane to methanol. Recent developments in molecular ecology techniques that may be applied to these apparently ubiquitous organism are also considered.


Enzymes and pathways of polyamine breakdown in microorganisms.

The information currently available on the breakdown of spermidine and putrescine by microorganisms is reviewed. Two major metabolic routes have been described, one for the free bases via delta 1-pyrroline (4-aminobutyraldehyde), the other via N-acetyl derivatives. In both pathways oxidases or aminotransferases are the key enzymes in removing the nitrogen atoms. The two routes converge at 4-aminobutyrate, which is then metabolized via succinate. The degradation of putrescine in Escherichia coli has been well characterized at both genetic and biochemical levels, but for other bacteria much less information is available. The C3 moiety of spermidine is broken down via beta-alanine, but the metabolism of this compound and its precursors is poorly understood. In yeasts, a catabolic route for spermidine and putrescine via N-acetyl derivatives has been described in Candida boidinii, and the evidence for its occurrence in other species is reviewed. Except for the terminal step of this pathway, the same group of enzymes can metabolize both the C3 and C4 moieties of spermidine. It is likely that other routes of polyamine catabolism also exist in both bacteria and yeasts.