Human tissue-type plasminogen activator synthesized by using a baculovirus vector in insect cells compared with human plasminogen activator produced in mouse cells.

A cDNA fragment encoding the human tissue-type plasminogen activator was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream from the polyhedrin promoter. The induction kinetics of t-PA was followed, after infection of Spodoptera frugiperda cells, at both mRNA and protein levels. Fibrinolytically active plasminogen activator accumulated in the culture medium and reached 2.5 micrograms/ml after 120 h. The protein was compared with recombinant plasminogen activator produced in mouse cells and was found to be slightly smaller. This difference in size was found to be caused by N-linked oligosaccharides which are shorter in the recombinant activator obtained from insect cells. The molecules produced in such cells contain at least two different types of N-linked glycans, since only one out of three oligosaccharides is sensitive to endoglycosidase H. However, all glycan structures bind strongly to concanavalin A-Sepharose.

PCR differential display of immune gene expression in Trichoplusia ni.

The immune state of insects is defined by a set of proteins that is absent in the naive state. To explore the immune system of Trichoplusia ni in more detail we have employed a PCR differential display technique to compare the mRNA population of untreated last instar larvae to that of immunized animals. In the primary display, more than one hundred bands seemed induced upon bacterial challenge. When they were used as probes in Northern blots, 35% of these probes detected inducible mRNA species. Such probes were used to screen a cDNA library from immunized larvae. We isolated clones for T. ni homologs of cecropin A, lysozyme and attacin. One differentially expressed band hybridized to clones for BJHSP1, a hemacy-anin-related protein which is hormonally up-regulated in last instar larvae; this induction is probably not related to the bacterial infection. Still other probes recognized inducible mRNAs of 1.6 and 1.0 kb. The corresponding cDNA clones did not show strong sequence homology to any known proteins. We have demonstrated the potential of this PCR technique to display both known and unknown genes specific for the immune state of whole insects against a background of genes involved in larval development.

Food-holding and -biting behavior in human subjects lacking periodontal receptors.

Previous studies have suggested that information provided by periodontal mechanoreceptors is particularly important for the fine motor control of the mandible, i.e., when humans hold and carefully manipulate food particles between the teeth with low biting forces. In the present study, we further evaluated this hypothesis by comparing the performance of three age- and gender-matched groups of subjects for which the integrity of the periodontal sensory apparatus differed. Specifically, the subjects had either natural teeth (natural group), dental prostheses supported by oral mucosa (denture group), or dental prostheses supported by osseointegrated implants (implant group). Each subject was instructed to hold half a peanut between the upper and lower central incisors for ca. 3 sec, and then to split it. The force applied by the anterior teeth was continuously monitored by a transducer-equipped bar on which the morsel rested. While the peanut was held, the force generated by subjects in the denture and implant groups was more variable and averaged four times that generated by subjects in the natural group. The peanut was split by a distinct, rapid ramp-increase in force that was similar for all three groups. In subjects lacking periodontal receptors, the morsel frequently escaped from the incisal edges during both phases of the task. The results demonstrate a marked disturbance in the control of precisely directed, low biting forces in subjects lacking periodontal receptors and suggest that the receptors play a significant role in the specification of the level, direction, and point of attack of forces used to hold and manipulate food between the anterior teeth. Moreover, other types of mechanoreceptors can not fully compensate for the loss of periodontal receptors.

PR-39, a proline-rich peptide antibiotic from pig, and FALL-39, a tentative human counterpart.

The peptide antibiotic PR-39 was originally isolated from the upper part of pig intestine. It has antibacterial activity against Gram negative bacteria at concentrations comparable with tetracycline. Studies of the mechanism of action showed that PR-39 inhibits both DNA and protein synthesis. Recently, PR-39 was found in wound fluid and was shown to have inductive activity on matrix components as part of the wound repair process. We have now sequenced the complete gene and possible mediators of its expression will be discussed. Our attempts to characterize the human counterpart of PR-39 by probing for the well conserved prepro-part led to a different peptide antibiotic. A clone containing the coding information for this new peptide was isolated from a human bone marrow cDNA library. The putative human peptide antibiotic was designated FALL-39 after the first four residues and the total number of residues. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family with three disulfide bridges, while FALL-39 lacks cysteine. The clone for the prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the peptide. In the presence of the basal medium E, synthetic FALL-39 was highly active against Escherichia coli D21 and Bacillus megaterium Bm11. Residues 13-34 in FALL-39 can be predicted to form a perfect amphipatic helix and CD spectra showed that medium E induced 30% helix formation in FALL-39. By Northern blot analyses the transcript was located in bone marrow and testis. The structure of the gene and the chromosomal location is under investigation.

NK-lysin, structure and function of a novel effector molecule of porcine T and NK cells.

NK-lysin (NKL), a 78-residue antimicrobial peptide, was isolated from pig small intestine. Standard methods identified the peptide as basic, with six half-cystine residues in three intrachain disulphide bonds. The sequence showed 33% identity with a part of a putative gene product (NKG5) from activated T and NK cells, NK-lysin showed antibacterial activity against Escherichia coli and Bacillus megaterium and marked lytic activity against YAC-1, a NK sensitive tumour cell line, while sheep red blood cells were unaffected. The cDNA clone corresponding to NK-lysin has been characterized. We have also analyzed the cell and tissue specific expression and the induction of the gene. A lymphocyte fraction enriched in T and NK cells, stimulated by human interleukin-2 (IL-2), showed a 30-fold increase of the NKL transcript. NK-lysin specific mRNA is also detectable in spleen, bone marrow and colon. Immunostaining showed NKL to be present in different types of lymphocytes. Our results strongly suggest that NK-lysin is involved in the inducible cytotoxicity of T and NK cells.

[Do lactoferrin, lysozyme and the lactoperoxidase-thiocyanate-hydrogen peroxide-system cause negative microbiological results in mastitis secretions?]. / Laktoferrin, Lysozym und das Laktoperoxidase-Thiozyanat-Wasserstoffperoxid-System (LPS) als Ursache negativer mikrobiologischer Befunde aus Mastitissekreten?

Lactoferrin, lysozyme and the lactoperoxidase-thiocyanate-peroxide-system are naturally occurring antimicrobial components of milk. The objective of this study was to examine, whether these components were responsible for negative results, when mastitis milk is cultured microbiologically. Quarter milk samples from 75 cows with clinical mastitis on a dairy farm in Brandenburg were submitted for microbiological culture and analysed for the content and the activities of the three components. Animals from all stages of lactation with clinical mastitis were included in the study. Animals were examined clinically and milk samples were collected prior to first treatment. Secretions from quarters with clinical mastitis were compared to those of neighbouring quarters without clinical mastitis. Secretions with positive cultural results were compared to those with negative results. The concentrations or activities of the three factors were significantly higher in the diseased quarters than in the quarters without clinical signs of mastitis. The concentration of lysozyme increased with severity of the clinical signs (local swelling and changes in secretion). The concentration of lactoferrin was significantly higher in quarters with slight alterations of glandular tissue than in quarters with medium or severe alterations (P < 0.05). LPS-activities did not correlate with the severity of clinical signs. No differences in the concentration of lactoferrin or LPS-activities were seen between mastitis with positive and negative culture results. The concentration of lysozyme was even higher in culturally positive samples than in negative samples (P < 0.05). Results from this study indicate that the three factors examined did not impair the results of microbiological culture of milk samples from quarters with clinical mastitis.

Masticatory efficiency and dental state. A comparison between two methods.

The masticatory efficiency of subjects with all natural teeth, with complete upper and partial lower dentures, and with complete dentures was measured. Two different methods were used. The results showed significant differences among the groups irrespective of the method used. The subjects with dentures compensated for decreasing masticatory efficiency by using more strokes when chewing until swallowing. Great interindividual differences were found within groups with similar dental states. There was no or weak correlation between the two methods. A value from one method corresponded to a large range of values in the other method and vice versa.

The effect of removable partial dentures on mastication and dietary intake.

Masticatory efficiency, the subjective experience of masticatory performance, and dietary intake were evaluated in 19 subjects who were treated with a removable partial denture in the lower jaw. The subjects were tested on three occasions: before treatment, with the dentures when free from symptoms, and about 4 months after the dentures were inserted. Masticatory efficiency and the subjective experience of masticatory performance increased significantly after the subjects were provided with the dentures, but no changes were found in the dietary intake.

Masticatory efficiency. A new method for determination of the breakdown of masticated test material.

One of the purposes of mastication is to break down the food and enlarge the area of food particles. A method is described by which the masticatory efficiency is estimated by calculating the area of a chewed test material, gelatin-hardened by formalin. When test pieces, after being chewed, are placed in a water-soluble dye, the dye will diffuse into the particles, and consequently the dye concentration in the surrounding solution will decrease. The concentration of the dye solution is read from a photometer. A close correlation was found between the gelatin particle area and the reduction of the dye solution concentration. The method will be applied in clinical studies.

Efficient secretion of attacin from insect fat-body cells requires proper processing of the prosequence.

The attacins constitute a group of immune proteins induced after bacterial infection in the moth Hyalophora cecropia. They are synthesized as preproproteins and undergo post-translational modification during transport to the hemolymph. The processing and transport rates of attacin were studied in its natural host as a response to infection. Monensin totally inhibited the processing from proattacin to attacin and the radiolabeled proattacin remained intracellular. This observation indicated that the prosequence is removed at or after the trans-Golgi compartment. It is also suggested that the processing of the prosequence does not occur in acidic vesicles, as the process was not inhibited by the weak base chloroquine. To study prosequence function, the attacin gene and genes with mutations in the prosequence were cloned into the baculovirus Autographa californica nuclear polyhedrosis virus. The processing of proattacin and the transport of attacin were studied by pulse-chase experiments with fat body isolated from Trichoplusia ni larvae. The rate of secretion from fat body was lowest for proattacin, which could not be processed to attacin, intermediate for attacins lacking the prosequence and highest for natural attacin. We could not detect any biological activity for proattacin.

Expression of post-translational processing of preprocecropin A using a baculovirus vector.

A cDNA fragment encoding preprocecropin A was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in recombinant-infected last instar larvae of Trichoplusia ni and in diapausing pupae of Hyalophora cecropia. The identity of the recombinant product was established by electrophoresis with detection of antibacterial activity and mass spectrometry. The prepropeptide had been correctly processed including removal of signal peptide and pro-part. Biologically active and amidated cecropin A was exported to the hemolymph. The yield of recombinant protein in H. cecropia reached a level of 600 micrograms/ml hemolymph and about 70% of the material was amidated.

Structure of preproattacin and its processing in insect cells infected with a recombinant baculovirus.

From a cDNA library made from fatbody of immunized Hyalophora cecropia pupae, a full-length clone corresponding to basic attacin was isolated. The corresponding amino acid sequence suggests that basic attacin is synthesized as a 233-residue preproprotein. This was confirmed by cloning the cDNA fragment encoding the basic attacin in the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter and expressing the protein in Spodoptera frugiperda (Sf9) cells. Biologically active attacin was also produced in last instar larvae of Trichoplusia ni after injection of recombinant virus. The concentration obtained was 300-500 times that obtained in cell culture supernatants. In cell culture the induction kinetics of attacin were followed at both transcriptional and translational levels. From the protein processing pattern it was suggested that a protease produced by the Spodoptera cells cleaves attacin at the double arginine residues at positions 45 and 46. The instability of the attacin proteins is rationalized in terms of their random-coil structure which was deduced from circular dichroism measurements.

The effect of new complete dentures on mastication and dietary intake.

Masticatory efficiency, the subjective experience of masticatory performance, and dietary intake were measured for 43 subjects who were provided with new complete dentures. The subjects were tested on three occasions: with the old complete dentures, with the new complete dentures when free from symptoms, and with the new dentures about 4 months after insertion. Masticatory efficiency and the subjective experience of masticatory performance increased significantly when the subjects were provided with new dentures, but no changes were found in the dietary intake. With the new dentures the masticatory efficiency and the subjective experience of masticatory performance were correlated to each other.

Masticatory efficiency of complete denture patients. A clinical examination of potential changes at the transition from old to new denture.

The masticatory efficiency was studied among 19 complete denture wearers with their old and new dentures. The test chewing material was gelatin hardened by formalin. A standardized preparation of the test chewing material and the sieve-system is described. The patients were tested on seven different occasions from the period with the old dentures until about 1.5 years after the insertion of the new dentures. The test pieces were chewed for ten seconds--a practice test--20 seconds and until ready to be swallowed. The chewed material was strained in a sieve-system and masticatory efficiency indices were calculated. The results revealed no statistical significant differences in masticatory efficiency between any of the seven testing occasions with the method used. Thus no significant difference was noticed when patients changed from old to new dentures or during the first 18 months after insertion of new dentures.

Native and mutant 5-lipoxygenase expression in a baculovirus/insect cell system.

Human 5-lipoxygenase (EC 1.13.11.34), the key enzyme involved in the transformation of arachidonic acid to the potent biologically active leukotrienes, has been overexpressed in insect cells using a baculovirus expression system. A recombinant baculovirus (3B6) carrying the human 5-lipoxygenase coding sequence downstream of the strong polyhedrin protein promoter was isolated. Approximately 48 hr after infection of Spodoptera frugiperda cells with the recombinant baculovirus, maximal intracellular enzyme activity and protein levels were detected. The recombinant 5-lipoxygenase in 10,000 x g supernatant fractions was able to synthesize large amounts of 5-hydroperoxy-6,8,10,14-icosatetraenoic acid, together with smaller amounts of the nonenzymatic hydrolysis products of leukotriene A4, and exhibited a dependence on Ca2+ and ATP for maximal activity. Immunoblot analysis of supernatant proteins from human leukocytes and recombinant virus-infected cells indicated the presence of indistinguishable approximately 80-kDa bands. However, 5-lipoxygenase protein in recombinant-infected cells was found to be present in amounts 50-200 times that present in leukocytes on a per-cell basis. Histidine-362 and histidine-372, potential iron-atom ligands within a putative iron-binding domain, were changed to serine residues. Recombinant baculoviruses carrying the mutations were isolated and used to infect insect cells. Although infected cells were able to express mutant 5-lipoxygenase protein, enzyme activity was not substantially altered, suggesting the nonessential nature of these histidines in binding iron at the putative ferric catalytic site.

FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis.

PR-39, a proline/arginine-rich peptide antibiotic, has been purified from pig intestine and later shown to originate in the bone marrow. Intending to isolate a clone for a human counterpart to PR-39, we synthesized a PCR probe derived from the PR-39 gene. However, when this probe was used to screen a human bone marrow cDNA library, eight clones were obtained with information for another putative human peptide antibiotic, designated FALL-39 after the first four residues. FALL-39 is a 39-residue peptide lacking cysteine and tryptophan. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family and contain three disulfide bridges. The clone for prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the putative peptide. In basal medium E, synthetic FALL-39 was highly active against Escherichia coli and Bacillus megaterium. Residues 13-34 in FALL-39 can be predicted to form a perfect amphiphatic helix, and CD spectra showed that medium E induced 30% helix formation in FALL-39. RNA blot analyses disclosed that the gene for FALL-39 is expressed mainly in human bone marrow and testis.

Implications of hemolin glycosylation and Ca2+-binding on homophilic and cellular interactions.

Insects are useful models for the study of innate immune mechanisms because of their lack of antibodies and receptors involved in adaptive immune response. Nevertheless, hemolin cloned from moths is a soluble and membrane associated Ig-related molecule that is up-regulated during immune response [Lanz-Mendoza, H. & Faye, I. (1999) Dev. Comp. Immunol. 23, 359-374]. The hemolin monomeric form has four, pair-wise, interacting Ig-domains, forming a strongly bent horseshoe structure [Su, X.-D., Gastinel, L.N., Vaughn, D.E., Faye, I., Poon, P. & Bjorkman, P. (1998) Science 281, 991-995]. To elucidate the nature of its homophilic and cellular interactions, the glycosylation and Ca2+-binding properties of hemolin were investigated. We used Hyalophora cecropia hemolin isolated from hemolymph of bacteria-injected pupae, or produced as a recombinant protein in a baculovirus/insect cell system. Both types of hemolin contain N-acetylglucosamine and probably sialic acid, as indicated by peptide:N-glycosidase F and neuraminidase digestion and glycosylation detection by Western-blotting analysis. The N-acetylglucosamine residues on hemolin were confirmed with the use of specific lectins. In addition, hemolin was shown to specifically bind calcium when spotted onto nitrocellulose and treated as for 45Ca2+ autoradiography. Earlier studies demonstrated that hemolin can bind to hemocytes and this was tested for its dependence on calcium and carbohydrates, using hemolin-coated fluorescent microspheres. A greater level of attachment of microspheres occurred in the presence of calcium than if calcium was absent. Furthermore, this binding was inhibited by EGTA and N-acetylglucosamine or N-acetylneuraminic acid, implying that carbohydrates and calcium are crucial factors in homophilic binding and cell-adhesion events mediated by this Ig-superfamily molecule.

NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.

Biochemical and antibacterial analysis of human wound and blister fluid.

Fluid from a post-operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1% trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three): reverse-phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti-Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1-11,1-15 and 1-16, intact thymosin beta-4, defensins HNP1, 2 and 3, lysozyme and the peptide antibiotic FALL-39 and its precursor(s). The HPLC-separated blister fluid was extremely rich in anti-Bm activity (mainly defensins) and lysozyme. It may also contain factors not identified before. The plate assays scored 50-fold differences in anti-Bm activities and more than 10-fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.

Effective reconstruction after pharyngolaryngo-oesophagectomy with an ileal autograft.

Total pharyngolaryngo-oesophagectomy is performed for carcinoma of the cervical oesophagus, the piriform sinuses and the postcricoid region. A safe effective way of reconstructing the cervical oesophagus is of the utmost importance in these patients. Use of a revascularized intestinal segment for this purpose is described. Twenty-one patients who underwent reconstruction of the cervical oesophagus with an ileal autograft were studied, with a follow-up period of one to .58 months. The low postoperative mortality and morbidity, quick resumption of oral intake, short hospitalization and good functional results obtained with this method yield benefits for these patients.