Obtaining an Ent35-MccV derivative with mutated hinge region that exhibits increased activity against Listeria monocytogenes and Escherichia coli.

The present paper describes the generation of derivatives from the hybrid peptide called Ent35-MccV, active against Gram-positive and Gram-negative bacteria. This peptide has a triple glycine hinge region between enterocin CRL35 and microcin V. In order to obtain variants of Ent35-MccV with greater biotechnological potential, a saturation mutagenesis was carried out in the hinge region. As a result, we obtained a bank of E. coli strains expressing different mutated hybrid bacteriocins in the central position of the hinge region. From all these variants, we found that the one bearing a tyrosine in the central region of the hinge (Ent35-GYG-MccV) is 2-fold more active against E. coli and 4-fold more active against Listeria than the original peptide Ent35-MccV. This derivative was purified and characterized. The development and evaluation of alternative hinges for Ent35-MccV represents a step forward in the bioengineering of antimicrobial peptides. This approach fosters the rational design of peptides with enhanced antimicrobial activity.

Degradation of ß-casomorphin-7 through in vitro gastrointestinal and jejunal brush border membrane digestion.

This work aimed to study the opioid peptide ß-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.

Driving h-osteoblast adhesion and proliferation on titania: peptide hydrogels decorated with growth factors and adhesive conjugates.

Hydrogels from self-assembling ionic complementary peptides have been receiving much interest from the scientific community as mimetics of the extracellular matrix that can offer three-dimensional support for cell growth or become vehicles for the delivery of stem cells or drugs. These scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In order to develop a novel bioactive titanium implant, we propose the introduction of a layer of ionic-complementary self-assembling peptides (EAbuK) on Ti whose surface has been previously sandblasted and acid etched. The peptide layer is anchored to the metal by covalent functionalization of titania with self-assembling sequences. The peptide layer has also been enriched by the insulin-like growth factor-1 incorporated to the layer and/or a conjugate obtained by chemoselective ligation between EAbuK and a sequence of 25 residues containing four GRGDSP motifs per chain. X-ray photoelectron spectroscopy studies confirmed a change in the surface composition in agreement with the proposed decorations. An evaluation of the contact angle showed a substantial change in wettability induced by the peptide layer. The human osteoblast adhesion and proliferation assays showed an increase in adhesion for the surfaces enriched with conjugate at a concentration of 3.8 × 10(-7)m and an enhanced proliferation for samples enriched with insulin-like growth factor-1 at the highest concentration tested (2.1 × 10(-5)m).

Characterization and genetic study of the ovine alphaS2-casein (CSN1S2) allele B.

An interesting and quite complex protein pattern has been described at ovine milk proteins but the genetic control of the variation observed was assessed only in few cases. The aim of this work was to characterize the ovine alpha ( s2 )-casein (CSN1S2) B variant, first observed in the Italian Gentile di Puglia, a fine-wooled ovine breed, and to investigate its occurrence in two further breeds, the Sarda and Camosciata, which are the most widespread dairy breeds in Italy. The B variant differs from the most common form A with two amino acid exchanges: Asp(75) --> Tyr(75) and Ile(105) --> Val(105). The first substitution, resulting in a loss of a negative charge, is responsible for the higher isoelectric point of the B protein variant, which allows its detection by isoelectric focusing electrophoresis (IEF). The occurrence of CSN1S2*B in Sarda and Comisana was demonstrated. Since the Asp(75) --> Tyr(75) substitution modifies the protein electric charge, milk properties may result affected to some extent.

Application of capillary electrophoresis to determine the technological properties of wheat flours by a glutenin index.

Capillary electrophoresis was used to characterize glutenin proteins from ancient varieties of Southern Italy common wheat and to determine the technological properties of wheat flours based on a glutenin index. Three zones were identified in the electropherograms, indicated as A, B, and C according to electroelution order. The three zones corresponded to the low molecular weight glutenin subunits and to the y- and x-type high molecular weight subunits, respectively. The ratio B/C was correlated to the alveographic parameter P/L. These results indicated that flours resulting in a B/C ratio lower than 2 produced elastic doughs whereas flours resulting in a B/C ratio higher than 2 produced doughs more resistant to extension. This study showed that capillary electrophoresis is useful for determining the types and quantities of gluten proteins in the evaluation of wheat-flour technological properties of a limited number of noncommercial varieties as evidenced by the x-type content which seems to strongly influence the flour technological parameters.

Occurrence of beta-casein fragments in cold-stored and curdled river buffalo (Bubalus bubalis L.) milk.

The safeguard of river buffalo Mozzarella cheese, a Protected Designation of Origin dairy product, has prompted an analytical study to trace the milk and curd used as raw material in cheesemaking. This is to prevent the illegal use of milk or curd from different geographical areas outside of those indicated in the official production protocol. For this purpose, we studied primary proteolysis occurring in fresh and frozen milk and curd to identify a molecular marker that could indicate the raw material used. Whole casein from frozen river buffalo milk was separated using cation-exchange chromatography and sodium dodecyl sulfate-PAGE, and a protein component with an estimated molecular weight of 15.3 kDa was detected. This protein component was revealed in fresh river buffalo milk as a faint electrophoresis band, which drastically increased in intensity in refrigerated and frozen milk as well as in curd and was found to be associated with beta-CN through hydrophobic interaction. By using matrix-assisted laser desorption/ionization-time of flight peptide mass mapping, this component was identified as the C-terminal fragment f(69-209) of beta-CN (expected molecular weight of 15,748.8 Da). beta-Casein f(69-209), originating from the early hydrolysis of Lys(68)-Ser(69) by plasmin, has no counterpart in bovine milk. The increased rate of hydrolysis by plasmin toward the cleavage site Lys(68)-Ser(69) has to be ascribed to the elevated proline content of the peptide 61-73. The favored production of beta-CN f(69-209) has also drawn attention to the complementary proteose peptone beta-CN f(1-68) that is presumed to play a physiological role in inducing milk secretion similar to that of beta-CN f(1-29). The higher in vivo and in vitro production rate, compared with gamma(1)-CN formation, indicates that beta-CN f(69-209) and its complementary fragment are candidate molecular markers to evaluate milk and curd freshness. We suggested [corrected] indirect ELISA analysis based on the determination of remaining nonhydrolyzed beta-CN to perform a quantitative evaluation of proteolysis.

Formation of structured polymers upon controlled denaturation of beta-lactoglobulin with different chaotropes.

Prolonged exposure (>90 days) of bovine beta-lactoglobulin (BLG) to subdenaturing concentrations of either urea or potassium thiocyanate resulted in the formation of ordered polymers in the form of fibrils. The fibrils obtained with each chaotrope showed major differences in morphology, surface properties, thiol accessibility, and stability to dissociating agents as a consequence of the different chemical bonds involved in their stabilization. Hydrophobic interactions between BLG monomers are predominant in thiocyanate-formed fibrils, whereas urea-formed fibrils are stabilized by intermolecular disulfides generated through a thiol-disulfide exchange reaction. The different features of fibrils obtained with each chaotrope relate to the peculiar structural features and chemical properties of the "active" monomers generated by subdenaturing chaotrope concentrations in the early phases of the polymerization process, as detected by spectroscopic and limited proteolysis/mass spectrometry studies in the earliest stages of the action of individual chaotropes. The chaotrope-specific features of these early intermediates in turn affect the polymerization mechanism, whose intermediates were studied by size-exclusion chromatography on the soluble fraction at different times of fibril formation. The potential of these findings for the production of protein-derived nanostructures having different and controlled geometries and chemical properties is also discussed.

Caseinomacropeptide self-association is dependent on whether the peptide is free or restricted in kappa-casein.

There is a general agreement that the experimentally determined molecular weight (MW) of caseinomacropeptide (CMP) is greater than the theoretical MW. Some studies suggest that this is due to a pH-dependent aggregation of monomeric CMP. How this aggregation is influenced by pH is not understood. This study was carried out to study the nature of CMP aggregates and to clarify which conditions affect aggregation of CMP. The apparent MW of CMP at different pH values was determined using size-exclusion chromatography. Caseinomacropeptide was further characterized by immunochemical analysis, sodium dodecyl sulfate-PAGE, N-terminal sequencing, and mass spectrometry. The hydrophobicity of CMP was studied by means of 1-anilino-naphthalene-8-sulfonic acid binding experiments. Four CMP products prepared by different methods were studied: CMP produced by enzymatic (chymosin or pepsin) hydrolysis of kappa-casein (CN), and 2 commercial CMP products. Both commercial products and CMP resulting from chymosin-hydrolysis of kappa-CN (at pH 6.6) had elution volumes with a MW corresponding to 35 kDA at pH 8.0 and 3.4. Caseinomacropeptide prepared from pepsin-hydrolysis of kappa-CN (at pH 2.5) eluted as multiple peaks with apparent MW of 35, 18, and 9 kDa, again independently of pH. Hydrolysis of kappa-CN with chymosin or pepsin at different pH values (pH 2.5, 3.4, and 6.6) produced differently sized aggregates of CMP, largely depending on the pH of the hydrolysis. These results indicate that, whereas CMP molecules are irreversibly associated, CMP in kappa-CN may associate reversibly in a pH-dependent manner. We suggest that interactions between para-kappa-CN parts of the kappa-CN molecules may be a requisite for the pH-dependent dissociation/association.

Polypharmacy management in Medicare managed care: changes in prescribing by primary care physicians resulting from a program promoting medication reviews.

OBJECTIVE: To examine the effects of medication reviews by primary care physicians on prescriptions written for elderly members of a Medicare managed care organization who were at risk for polypharmacy. STUDY DESIGN: Prospective study with follow-up survey. PATIENTS AND METHODS: We conducted a study in 1995 to demonstrate the prevalence of polypharmacy (defined as receiving 5 or more prescription medications during the 3-month study period) among elderly members of our managed care organization. Two years later, elderly members identified as being at risk for polypharmacy were sent a letter encouraging them to schedule a medication review with their primary care physician. Each primary care physician was provided with clinical practice guidelines on polypharmacy and patient-specific medication management reports. Patients and physicians were subsequently mailed a survey to assess the impact of the medication review program on prescribing practices. RESULTS: Of 37,372 elderly members screened, 5737 (15%) were at risk for polypharmacy. Of these, 2615 (46%) responded to the follow-up survey. Of the survey respondents, 1087 (42%) had gone to their primary care physician for a medication review. During the review, 96% of patients discussed their prescription medications and 72% discussed nonprescription medications they were taking. Twenty percent reported that their physician discontinued medications, 29% reported that the physician changed the dose of a medication, and 17% informed their physician about a new prescription or nonprescription medication they were taking. Of the 275 primary care physicians surveyed, 56 (20%) returned the questionnaire. Of these, 61% reported that the medication review program was "very" or "somewhat useful." Thirty-five percent reported discontinuing unnecessary medications, and 23% reported decreasing the frequency of dosing. Overall, 45% of physicians reported making at least one change in their prescribing to a member at risk for polypharmacy. CONCLUSIONS: Our program promoting medication reviews between primary care physicians and their elderly patients resulted in significant changes in prescribing by physicians. This type of program is likely to decrease the risk of polypharmacy among older members of a Medicare managed care organization.

How the principles of geriatric assessment are shaping managed care.

In traditional geriatric medicine, comprehensive assessment is considered crucial to the care of frail older patients. The principles of geriatric assessment--identifying high-risk patients and targeting them for preventive interventions--are also practiced by managed care organizations (MCOs). Self-reported health surveys and administrative data are two methods used by MCOs to identify members at high risk for adverse health outcomes and functional decline who may benefit from geriatric case management. For a successful partnership with primary care physicians, it is very important that geriatric care managers should be knowledgeable in the principles of geriatric medicine.