Biomedical subjects

R Valluzzi

Publications and source records attributed to R Valluzzi.

8 recordsLinked to original sources

PubMed2007

A comparison of the performance of two penicillin reagent kits in the diagnosis of beta-lactam hypersensitivity.

BACKGROUND: Skin testing with penicilloyl polylysine (PPL) and minor determinant mixture (MDM) represents the first-line method for diagnosing beta-lactam hypersensitivity. However, in 2004, Allergopharma and Hollister-Stier announced their decision to stop the production of penicillin reagents (Allergopen and PrePen, respectively) within 1 year. Therefore, we decided to compare PPL and MDM from Allergopharma (Allergopen) with those from Diater (DAP). METHODS: We skin-tested 195 subjects with both Allergopen and DAP reagents, as well as with other beta-lactams; 74 (group A) had had immediate reactions to beta-lactams and 74 (group B) nonimmediate reactions, while 47 (group C) underwent prophylactic tests. RESULTS: One hundred two patients (52.3%) had positive skin tests; 29 (14.9%) were positive to PPL and/or MDM. Of the 102 skin-test-positive patients, 44 belonged to group A, 57 to B and 1 to C; the last was positive only to Allergopen PPL (PPL-A) and tolerated the benzylpenicillin challenge. Minor determinant mixture reagents produced identical results in the 148 patients of groups A and B, 22 (14.9%) of which were positive. Both PPL reagents produced negative results in 139 of these 148 patients and positive ones in 5; one subject was positive to DAP PPL (PPL-D) and negative to PPL-A, while three patients were positive to PPL-A and negative to PPL-D; two of the latter tolerated benzylpenicillin challenges. CONCLUSIONS: Minor determinant mixture reagents produced identical results in all 195 patients. Results of skin testing with PPL reagents were concordant in 190 (97.4%) of them. Therefore, DAP reagents are a reliable alternative to Allergopen ones.

Adolescent↗

PubMed2000

Sequence-specific liquid crystallinity of collagen model peptides. I. Transmission electron microscopy studies of interfacial collagen gels.

The conformation, crystal structure and self-assembly behavior of three peptides with collagen-like repetitive sequences [(1) peptide GAPGPP: (Glu)(5)(Gly-Ala-Pro-Gly-Pro-Pro)(6)(Glu)(5); (2) peptide GVPGPP: (Glu)(5)(Gly-Val-Pro-Gly-Pro-Pro)(6)(Glu)(5); and (3) peptide GAPGPA: (Glu)(5)(Gly-Ala-Pro-Gly-Pro-Ala)(6)(Glu)(5)] were compared. The peptides were characterized using transmission electron microscopy, electron diffraction, environmental scanning electron microscopy, and Fourier transform ir spectroscopy in order to determine how the molecular geometry dictated by each sequence affects the spontaneous generation of long-range ordered structures. Samples of each peptide, at ambient temperature and at 5 degrees C, were examined as films dried from aqueous solution, air-water interfacial films, and chloroform-water interfacial films. Peptide GAPGPP prepared at 5 degrees C and dried from bulk solution was found to have a collagen-like triple-helical structure. A sinusoidally textured gel, suggestive of cholesteric behavior was observed for peptides GAPGPP and GVPGPP at the aqueous chloroform interface at 5 degrees C. Peptide GAPGPA also formed a gel, but less reproducibly and the sinusoidal texture was not as well defined. The periodicities of the sinusoidal textures were reproducibly 10 microm for peptide GAPGPP, 7 microm for peptide GVPGPP, and 6 microm for peptide GAPGPA. The differences in the periodicity of the banded structure and in the crystallization behavior of the three peptides is attributed to differences in the symmetry of the preferred packing arrangement for each peptide, as evidenced by electron diffraction from crystallites that coexist with the sinusoidal gel. These differences are believed to be a measure of the effective symmetry and shape of the molecular cross section.

Amino Acid Sequence↗

PubMed2000

Conformational transitions in model silk peptides.

Protein structural transitions and beta-sheet formation are a common problem both in vivo and in vitro and are of critical relevance in disparate areas such as protein processing and beta-amyloid and prion behavior. Silks provide a "databank" of well-characterized polymorphic sequences, acting as a window onto structural transitions. Peptides with conformationally polymorphic silk-like sequences, expected to exhibit an intractable beta-sheet form, were characterized using Fourier transform infrared spectroscopy, circular dichroism, and electron diffraction. Polymorphs resembling the silk I, silk II (beta-sheet), and silk III (threefold polyglycine II-like helix) crystal structures were identified for the peptide fibroin C (GAGAGS repetitive sequence). Two peptides based on silk amorphous sequences, fibroin A (GAGAGY) and fibroin V (GDVGGAGATGGS), crystallized as silk I under most conditions. Methanol treatment of fibroin A resulted in a gradual transition from silk I to silk II, with an intermediate state involving a high proportion of beta-turns. Attenuated total reflectance Fourier transform infrared spectroscopy has been used to observe conformational changes as the peptides adsorb from solution onto a hydrophobic surface. Fibroin C has a beta-strand structure in solution but adopts a silk I-like structure upon adsorption, which when dried on the ZnSe crystal contains silk III crystallites.

Amino Acid Sequence↗

PubMed2000

Reduction-oxidation control of beta-sheet assembly in genetically engineered silk.

Genetically engineered spider dragline silk protein was modified to incorporate methionines flanking the beta-sheet forming polyalanine regions. The methionines could be selectively chemically oxidized and reduced. This chemical change altered the bulkiness and charge of the sulfhydryl groups, and in turn, the beta-sheet forming tendencies of the polyalanine domains and solubility of the protein. The genes encoding these redesigned proteins were constructed, cloned and expressed in Escherichia coli. In the reduced state (beta-mercaptoethanol) the approximately 25 kDa protein behaved similarly to native spider dragline silk, crystallizing into beta-sheets based on diffraction analysis and appearing fibrous by TEM. The addition of the methionines into the consensus dragline silk sequence did not disrupt the normal macromolecular assembly behavior of the protein. In the oxidized state (phenacyl bromide) the protein did not form beta-sheet crystals and appeared morphologically featureless based on TEM. A reduction in beta-strand content was also observed upon oxidation based on FTIR and TEM analysis and confirmed by X-ray diffraction analysis. To further confirm changes in assembly behavior observed for the recombinant protein containing the methionines, a model peptide with the same repeat amino acid sequence was synthesized and characterized. Shifts in molecular weight, observed by MALDI, along with corresponding changes in crystallinity, by electron diffraction, agreed with the changes expected on activation and deactivation of the redox trigger. These results support the use of a redox trigger as a useful feature with which to control the assembly of beta-sheet forming proteins.

Amino Acid Sequence↗

PubMedDate not supplied

Designing recombinant spider silk proteins to control assembly.

The consensus repeat sequence found in the dragline silk from the spider, Nephila clavipes, was redesigned to incorporate a redox trigger flanking the beta-sheet forming polyalanine sequences. The methionine redox trigger, in the oxidized state, was incorporated to prevent the formation of the beta sheets, while in the reduced state would not result in sterical limitations to beta sheet formation. A synthetic gene incorporating the trigger was constructed, cloned and then expressed in Escherichia coli. The purified protein, about 25 kDa, contained the expected amino acid composition and migration behavior on SDS-PAGE. The recombinant protein was analyzed by X-ray diffraction, TEM, electron diffraction and circular dichroism in both oxidized and reduced states. Based on the results, the incorporation of a redox trigger appears to be a powerful experimental strategy to explore the self-assembly of fibrous proteins such as silks.

Amino Acid Sequence↗

PubMedDate not supplied

Orientation of silk III at the air-water interface.

A threefold helical crystal structure of Bombyx mori silk fibroin has been observed in films prepared from aqueous silk fibroin solutions using the Langmuir Blodgett (LB) technique. The films were studied using a combination of transmission electron microscopy and electron diffraction techniques. Films prepared at a surface pressure of 16.7 mN/m have a uniaxially oriented crystalline texture, with the helical axis oriented perpendicular to the plane of the LB film. Films obtained from the air-water interface without compression have a different orientation, with the helical axes lying roughly in the plane of the film. In both cases the d-spacings observed in electron diffraction are the same and match a threefold helical model crystal structure, silk III, described in previous publications. Differences in the relative intensities of the observed reflections in both types of oriented samples, as compared to unoriented samples, allows estimations of orientation distributions and the calculations of orientation parameters. The orientation of the fibroin chain axis in the plane of the interfacial film for uncompressed samples is consistent with the amphiphilic behavior previously postulated to drive the formation of the threefold helical silk III conformation.

Air↗

PubMedDate not supplied

Silk I structure in Bombyx mori silk foams.

Single crystals of Bombyx mori silk fibroin in the metastable silk I polymorph have been produced using a new foaming technique. Foams of silk protein are generated by bubbling pure nitrogen gas through an aqueous solution of regenerated silk fibroin. The foamed material is collected, dried, and then sonicated to yield individual crystals which were examined using transmission electron microscopy and electron diffraction. It is found that slightly acidic conditions in the solution from which the foam was generated favor the formation of silk II while neutral to slightly basic solutions favor silk I formation. More dilute solutions favor the formation of silk II while more concentrated solutions (about 7 wt.% or greater) favor the formation of silk I. X-ray powder diffraction patterns from the dried silk I foams displayed features highly indicative of silk I. We also report the first single crystal electron diffraction patterns of silk I. These patterns indicate a large unit cell, possibly 22.66 x 5.70 x 20.82 A. with six chains of six residues, Gly-Ala-Gly-Ala-Gly-Ser. Although we have not fully characterized this complex structure it appears that the chain is nearly fully extended and thus our data is consistent with models possessing general features similar to those proposed by Fossey SA, Nemethy G, Gibson KD, Scheraga HA. (Biopolymers 1991;31:1529-1541).

Animals↗

PubMedDate not supplied

Bombyx mori silk fibroin liquid crystallinity and crystallization at aqueous fibroin-organic solvent interfaces.

A banded morphology has been observed for Bombyx mori silk fibroin films obtained from an aqueous hexane interface; the period of the banding is approximately 1 microm. Morphology and diffraction from different regions of the banded structure suggest that it is a free surface formed by a cholesteric liquid crystal. Truncated hexagonal lamellar crystallites of B. mori silk fibroin have been observed in films formed in the surface excess layer of fibroin at the interface between aqueous fibroin and hexane or chloroform. Based on initial crystallographic evidence, a three-fold helical conformation has been ascribed to the fibroin chains within the crystals. The chain conformation and crystalline habit appear to be similar to the silk III structure previously observed at the air-water interface (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Valluzzi R, Gido S, Zhang W, Muller W, Kaplan D. Macromolecules 1996;29:8606-8614) but the crystalline packing is different. Diffraction data obtained for the crystallites are similar to diffraction behavior for a collagen-like model peptide. Diffraction patterns obtained from crystallized regions of the banded morphology can be indexed using the same unit cell as the hexagonal lamellar crystallites. Surfactancy of fibroin and subsequent aggregation and mesophase formation may help to explain the liquid crystallinity reported for silk, which is long suspected to play a role in the biological silk spinning process (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Willcox, P. J.; Gido, SP, Muller W, Kaplan DL. Macromolecules 1996:29:5106-5110; Magoshi J, Magoshi Y, Nakamura S. In: Kaplan D, Adams W, Farmer B, Viney C, editors, Mechanism of Fiber Formation of Silkworm. Washington, DC: American Chemical Society 1994:292-310; Magoshi J, Magoshi Y, Nakamura S. J Appl Polym Sci Appl Polym Symp 1985;41:187-204; Magoshi J, Magoshi Y, Nakamura S. Polym Commun 1985;26:309.).

Animals↗