Pharyngeal airway changes in pre-pubertal children with Class II malocclusion after Fränkel-2 treatment.

AIM: To evaluate two-dimensional changes in pharyngeal airway dimensions in pre-pubertal children with a Class II malocclusion treated with a Fränkel-2 appliance compared to a matched non-treated control sample. MATERIALS AND METHODS: Lateral cephalograms obtained from 28 consecutively treated pre-pubertal children before (T0) and after (T1) a one-year Fränkel-2 treatment were analysed. Fränkel-2 appliance was used for at least 18 hr/day during 12 months. The control group was matched as closely as possible. All the cases presented normal facial growth pattern. Sagittal and vertical cephalometric measurements assessing maxillary and mandibular skeletal positions, as well as sagittal pharyngeal airway dimensions, were calculated. Intraclass correlation coefficient was calculated in order to determine reliability. Differences based on age for all the outcome variables at T0 were compared with an independent t-test. A MANOVA was used thereafter to determine if any factors and their interactions were associated with changes in the outcome variables. Differences between T1 and T0 were evaluated with either a t-student test or a Mann Whitney U test. RESULTS: At T0 differences between groups were noted for several variables. These differences were considered during the follow-up statistical analysis. Changes between groups after treatment (T1-T0) were noted for SNB, PNS to Ba, McNamara Low and Middle to S (increase in treatment group), and ANB and AD1 to Ba (decrease in treatment group). CONCLUSIONS: Some pharyngeal two-dimensional airway dimensions changed in Class II malocclusion pre-pubertal patients during a one-year treatment with Fränkel-2 appliances.

Capillary electrophoresis of supercoiled DNA molecules: parameters governing the resolution of topoisomers and their separation from open forms.

We describe the separation of covalently closed and open circular DNA forms with capillary electrophoresis. This technique is expected to be applied in the research of novel anticancer molecules targeting the activity of topoisomerase I. The separation of a plasmid mixture containing fully supercoiled molecules, single topoisomers, and their relaxed and open circular forms was tested in an electric field of 200 V/cm using Tris/borate buffer with the addition of magnesium ions at low concentrations and various sieving polymers. The resulting separation is quite simple to achieve and is clearly comparable to that obtained in agarose gels run at low voltage, but with an improved resolution, a higher quantitativity, and a higher speed of analysis. We identified three main parameters that influence the separation: (I) Low concentrations of MgCl2 in the separation buffer are required for a good resolution of topoisomers. (II) Cellulose derivatives can be used as sieving polymers; in our hands, HPMC and HEC worked best. (III) High molecular mass forms of sieving polymers allow the best separations.

Purification and characterization of two recombinant human granulocyte colony-stimulating factor glycoforms. Pharmacokinetic and activity studies of single-dose administration in mice.

Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line. The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133. The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GalNAc. The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM). In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli. The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively. The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination. The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life. Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c. route either singularly or as a mixture. Detectable levels of rhG-CSF persisted for more than 8 h in the i.v. and more than 24 h in the s.c. route of administration. All three substances induced early neutrophilia in mice. All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection. Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v. and s.c. administration, respectively.

Overproduction of soluble, extracellular cytotoxin alpha-sarcin in Escherichia coli.

The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin alpha-sarcin to be used for immunoconjugate production. alpha-Sarcin cDNA was isolated from Aspergillus giganteus strain MDH 18,894 and its expression in Escherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant alpha-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernatant. A variant form of alpha-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.

Immunoconjugates made of an anti-EGF receptor monoclonal antibody and type 1 ribosome-inactivating proteins from Saponaria ocymoides or Vaccaria pyramidata.

The present paper describes two immunoconjugates consisting of an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), named Mint5, covalently linked to the type 1 ribosome-inactivating proteins (RIPs) ocymoidine (Ocy) and pyramidatine (Pyra) from Saponaria ocymoides and Vaccaria pyramidata respectively. Both antibody and toxins are shown to retain their respective biological properties upon chemical conjugation. The immunoconjugates exert specific inhibition of EGFR expressing target cell proliferation and protein synthesis in in vitro assays and also inhibit the growth of grafted human tumour cells in nude mice.

Expression and characterization of a mouse/human chimeric antibody specific for EGF receptor.

Murine antibodies which recognize the epidermal growth factor receptor (EGF-r) are good candidates for therapy and diagnosis of tumors overexpressing this receptor. Here we report the isolation of the variable regions from a murine monoclonal antibody anti-EGF-r (Mint5), the procedure to obtain the mouse/human chimeric antibody (chMint5) and its expression in COS, NS0 and CHO cells. The approach followed to construct chMint5 is based on the use of consensus primers specific for the ends of the variable regions. The sequence imposed by the primers did not affect the targeting potential of the antibody. In fact, the affinity of the chimeric antibody for EGF-r was nearly the same as that of the parental murine antibody. Based on previous in vitro and in vivo animal studies. Mint5 was shown to be a good candidate for the targeting of EGF-r overexpressing tumours. chMint5 is expected to be less immunogenic than murine antibody and therefore, could be useful for human treatment.

Clavin, a type-1 ribosome-inactivating protein from Aspergillus clavatus IFO 8605. cDNA isolation, heterologous expression, biochemical and biological characterization of the recombinant protein.

We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the alpha-sarcin family of ribosome-inactivating proteins (RIPs). The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell-free and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (t1/2 beta) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates.

Isolation and characterization of a novel collagen-binding protein from Streptococcus pyogenes strain 6414.

In this report we have analyzed the binding of collagen to Streptococcus pyogenes strain 6414. This binding was rapid, specific, and involved a limited number of receptor molecules (11,600 copies per cell). When the proteins in a streptococcal lysate were blotted onto a nitrocellulose filter and probed with 125I-labeled collagen, a prominent collagen-binding protein of 57 kDa was identified as well as minor 130-150-kDa components. The major 57-kDa protein was isolated by affinity chromatography on collagen-Sepharose followed by gel filtration chromatography. The 57-kDa protein purified from S. pyogenes was used to raise a monospecific antibody which also reacted with a collagen-binding protein of similar molecular size isolated from Streptococcus zooepidemicus. The two collagen-binding proteins from streptococci have a similar amino acid composition and isoelectric points. Isolated collagen-binding protein was specifically recognized by 125I-collagen in a solid-phase binding assay and displayed an affinity for the ligand quite similar to that exhibited by intact bacteria (Kd = 3.1 versus 3.5 x 10(-9) M, respectively). Surface-labeled bacteria attached to microtiter wells coated with different collagen types and the 57-kDa protein blocked the adhesion to collagen substrate. We propose that the 57-kDa protein is an adhesin involved in the attachment of streptococci to host tissues.

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability.

The alcohol dehydrogenase gene from the thermophilic archaeum Sulfolobus solfataricus has been subcloned and expressed in Escherichia coli under the control of the T7 inducible promoter. The recombinant protein shows properties analogous to those of the native enzyme, including thermostability, despite the fact that E.coli does not post-translationally modify two lysine residues which are N-epsilon-methylated in the native enzyme. We constructed a 3-D model of the S.solfataricus alcohol dehydrogenase using the known structure of its isozyme from horse liver as a template. Our analysis of the structural zinc binding site suggested that this site is present and functional in the S.solfataricus enzyme and that a glutamate ligand can contribute to thermostability by influencing electrostatic interactions around the metal centre. To investigate this hypothesis, we constructed, expressed and characterized a mutant where the glutamate is replaced by a cysteine, thus restoring the zinc binding site of mesophilic alcohol dehydrogenases. The mutant shows the same activity but a reduced thermostability with respect to the wild-type recombinant protein, as suggested by our model.

Farm workers' occupational allergy to Tetranychus urticae: clinical and immunologic aspects.

A group of 46 farm workers (32 men), affected by a recurrent "occupational disease of undetermined origin", underwent an immunologic investigation with a Tetranychus urticae (TU) whole-body extract (TU-WBE) prepared in our laboratory. The patients suffered from seasonal attacks of rhinitis, during the summer and autumn periods, when working in open fields (30 subjects) or in greenhouse flower cultivation (16 subjects). In most patients, rhinitis was associated with bronchial asthma (16 subjects), urticaria (14 subjects), or both (three subjects). Allergic alveolitis or other common allergic diseases had been excluded, and a diagnosis of "occupational disease of undetermined origin" had been made before by other medical centers. Ten healthy farm workers and 10 atopic townsmen were chosen as control groups. An in vivo and in vitro diagnostic trial by skin prick testing (SPT) and serum specific IgE dosage with TU-WBE were done in all subjects. Thirty-six patients (78%) were found to be positive to both SPT and the IgE enzyme allergosorbent test (EAST), with a good correlation between IgE serum levels and cutaneous wheal size. Control groups did not show any reaction. The IgE-EAST homologous inhibition test was positive. The IgE-EAST cross-inhibition test excluded cross-reactivity between TU and Dermatophagoides pteronyssinus. The TU-exposure test was positive for the 36 patients with TU-WBE-specific IgE. Three patients who were negative for TU-WBE-specific IgE reacted to the TU-exposure test; in these patients, the scratch-chamber test (Finn chamber) with eggs and droppings from TU was positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibronectin receptors from Streptococcus dysgalactiae and Staphylococcus aureus. Involvement of conserved residues in ligand binding.

The nucleotide sequence of two genes encoding fibronectin (Fn) receptors FnBA and FnBB of Streptococcus dysgalactiae S2 revealed the presence of repeated motifs (called RA1-A3 and RB1-B3, respectively) which encode Fn binding activity (Lindgren, P.-E., McGavin, M. J., Signäs, C., Guss, B., Gurusiddappa, S., Höök, M., and Lindberg, M. (1993) Eur. J. Biochem. 214, 819-827). Synthetic peptides of 32-37 amino acids, corresponding to individual repeated motifs, were assayed for the ability to inhibit Fn binding to cells of S. dysgalactiae. Within the RA motifs, peptide A2 was 10-fold more active than either A1 or A3, while in the RB motifs, only B3 was active. The same level of activity is observed when these synthetic peptides were assayed for inhibition of Fn binding to cells of Staphylococcus aureus. Likewise, synthetic peptides corresponding to the RD1-D3 motifs, which comprise a ligand binding domain in a Fn receptor from S. aureus, inhibit binding of Fn to both S. aureus and S. dysgalactiae. Assays of chemically modified peptides and peptide fragments derived from chemical or proteolytic cleavage suggest that a conserved core sequence, defined as ED(T/S) (X9,10)GG(X3,4)(I/V)DF, within a 30-amino acid-long segment is present in the active RA and RD motifs. Analyses of the importance of individual residues of this core sequence indicate that the ED(T/S) motif is nonessential, whereas the GG and the (I/V)DF together with additional acidic residues in the C-terminal half of the peptide are required for activity.

[The epidemiology of adverse reactions to nonsteroidal anti-inflammatory drugs. Nimesulide is tolerated by patients with adverse reactions to NSAIDs and modulates in man the skin response to histamine and codeine]. / Epidemiologia delle reazioni avverse ai farmaci antiinfiammatori non steroidei. La nimesulide è tollerata dai pazienti con reazioni avverse ai FANS e modula nell'uomo la risposta cutanea all'istamina ed alla codeina.

We evaluated the tolerance to NIM in patients with adverse reactions to NSAIDs, expressed by urticaria-angioedema, bronchial asthma or erythema polymorphous on 92 patients among which 32 atopics affected by asthma, rhinitis or contact dermatitis. Challenge test has been performed with increasing amounts of NIM per os every 2 hrs to a maximum of 100 mg/dose. The last dosage was repeated every 12 hrs for 4 times more. No reactions were observed in 86 patients (93.4%). Moreover we evaluated the efficacy of NIM in modulating pomphoid cutaneous reaction to histamine (HIS) and codeine (COD). Three subjects were prick tested with HIS and COD solutions at different concentrations (0.1 to 10 mg/ml), before and after a 5 days therapy with NIM at different dosages/kg/die. At NIM dosages of 3.7 and 2.7 mg/kg/die we observed a strong reduction of pomphoid activity of both HIS and COD. No clear effects were detected at 2.2 mg/kg/die dosage. We assume a dose-related in vivo inhibitory effect of NIM on cutaneous mast cells releasability or a direct anti-histaminic effect. We point out the possible therapeutic role of NIM in the treatment of allergic flogosis at steroid and cromon's side.

Fibronectin binding determinants of the Staphylococcus aureus fibronectin receptor.

Synthetic peptide analogs mimicking a repeated motif within the Staphylococcus aureus fibronectin receptor inhibit binding of the bacteria to fibronectin (Signäs, C., Raucci, G., Jonsson, K., Lindgren, P. E., Anantharamaiah, G. M., Höök, M., and Lindberg, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 699-703). In this study, we have further localized the fibronectin-binding determinant within the 37 amino acid D3 peptide. Chemical modification of the carboxyl side chains of the glutamic and aspartic residues in D3 abolished fibronectin-binding activity, whereas modifications of lysine or tyrosine residues had little effect. An active peptide encompassing residues 15-36 was isolated from a trypsin digest of D3, and a synthetic peptide S16-36 had activity comparable with that of intact D3. Scrambling the amino acid sequence of S16-36 or replacing the aspartic and glutamic residues with asparagine and glutamine resulted in loss of activity. Therefore, one or more of the acidic residues are essential for activity. However, additional sequence is required. Reduction in the size of S16-36 from either the N- or C-terminal end resulted in peptides with greatly diminished activity. These data suggest that the amino acids essential for binding fibronectin are contained within residues 21-33 of the D3 peptide and that the flanking N- and C-terminal amino acids are necessary for the peptide to acquire a conformation that is favorable for fibronectin binding.

Peptide analogs to a fibronectin receptor inhibit attachment of Staphylococcus aureus to fibronectin-containing substrates.

Binding of cells of Staphylococcus aureus to fibronectin has been proposed as a mechanism of bacterial adhesion to host tissues. In this study, we have attempted to define the role of a recently identified fibronectin receptor in the adhesion of staphylococcal cells to fibronectin-containing substrates by using different receptor analogs as potential inhibitors of bacterial adherence. The results showed that synthetic peptides D1, D2, and D3, corresponding to variations of a repeated unit in the fibronectin-binding domain of the receptor, and ZZ-FR, a chimeric protein containing the fibronectin-binding domain of the receptor with the D1, D2, and D3 sequences, inhibited the attachment of staphylococcal cells to microtiter wells coated with intact fibronectin or with the 29-kilodalton amino-terminal fragment of fibronectin. The chimeric protein ZZ-FR also partially inhibited the adherence of staphylococci to human plasma clots formed in vitro but had no effect on bacterial adhesion to clots formed from fibronectin-depleted plasma. These data confirm previous reports suggesting that fibronectin may serve as a substrate for adhesion of staphylococcal cells and indicate that bacterial adhesion is mediated by the identified fibronectin receptor. Furthermore, analogs to the fibronectin receptor can be used to inhibit the adhesion of bacterial cells to these model substrates, and these analogs may be of clinical use.

Clinical evaluation of the imipenem/cilastatin combination in the therapy of severe infections in patients with malignant diseases.

Thirty hospitalized patients (23 men and 7 women), aged 42 to 78 years, with impaired host defences for malignant underlying diseases and affected by life-threatening infections, were treated with the imipenem/cilastatin combination at a dosage ranging between 2 and 4 g daily i.v. at 8 or 12 h intervals. The average length of therapy was 9.6 +/- 2.09 days. The isolated organisms were as follows: Pseudomonas aeruginosa [12], Escherichia coli [10], Klebsiella pneumoniae [5], Proteus mirabilis [3], P. vulgaris [2], Serratia marcescens [1], Staphylococcus aureus [3], Streptococcus faecalis [3], Bacteroides fragilis [5]. In fourteen patients a mixed infection was observed. Twenty-three patients (76.66%) were completely cured of infection and 35 out of 44 isolated organisms (79.54%) were eradicated. No important side-effects were observed.

Nucleotide sequence of the gene for a fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides.

Binding of cells of Staphylococcus aureus to fibronectin, which may represent a mechanism of host tissue adherence, involves a fibronectin-receptor protein present on the bacterial surface. Cloning of a gene coding for a staphylococcal fibronectin-binding protein and construction of a fusion protein with fibronectin-binding properties was previously reported from our laboratory. We have now sequenced the gene and deduced a primary sequence of the fibronectin-binding protein. The protein resembles other cell-wall-associated proteins on Gram-positive bacteria in that it (i) appears to be anchored in the cell membrane via its C-terminal end, (ii) contains a proline-rich repeating unit outside the membrane anchor, and (iii) contains a long (36-amino acid) signal sequence at the N terminus. The fibronectin-binding activity has been localized to a domain composed of a 38-amino acid unit repeated completely three times and partially a fourth time; the identity between the three 38-amino acid sequences varies from 42 to 87%. Three synthetic peptides mimicking the structure of each 38-amino acid unit were constructed. All three peptides interacted with fibronectin, as indicated by their ability to inhibit binding of fibronectin to staphylococcal cells, whereas an unrelated 37-amino acid peptide showed no inhibitory activity.

Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus.

The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.

Binding of collagen to Staphylococcus aureus Cowan 1.

Collagen binds to a receptor protein present on the surfaces of Staphylococcus aureus cells. Binding of 125I-labeled type II collagen to its bacterial receptor is reversible, and Scatchard plot analysis indicates the presence of one class of receptor that occurs on an average of 3 X 10(4) copies per cell and binds type II collagen with a Kd of 10(-7) M. Studies on the specificity of collagen cell binding indicate that the receptor does not recognize noncollagenous proteins but binds all of the different collagen types tested (types I to VI). Furthermore, isolated collagen alpha chains and peptides generated by cyanogen bromide cleavage of type I collagen alpha chains are recognized by the receptor as indicated by the ability of these polypeptides to inhibit binding of 125I-labeled type II collagen to staphylococcal cells. Synthetic collagen analogs were tested as inhibitors of type II collagen binding to bacterial cells. The peptides (Pro-Gly-Pro)n, (Pro-Pro-Gly)10, and (Pro-OH-Pro-Gly)10 were recognized by the receptor, whereas the peptides (Pro-Ala-Gly)n and polyproline showed no inhibitory activity.