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Recebido em 17/9/03; aceito em 27/11/03; publicado na web em 17/6/04 CHEMICAL AND ENZYMATIC PEPTIDE SYNTHESES: BASIC ASPECTS AND APPLICATIONS. This review begins with a brief discussion of the biological importance and chemical features of peptides. A description of the existing synthetic methods follows with emphasis on the basic aspects of the chemical and enzymatic syntheses. Techniques used to purify and characterize the synthesized peptides are also discussed. Finally, a few applications of the final products in chemistry, biochemistry, immunology and medicine are presented, such as identification and quantification of naturally occurring peptides, inspection of structureactivity relationships, therapeutics, development and/or improvement of analytical techniques and search for new vaccines.Keywords: peptides; synthesis; applications of synthetic peptides. A DIVERSIDADE FUNCIONAL E QUÍMICA DOS PEPTÍDEOSOs peptídeos são biomoléculas que contém de dois a dezenas de resíduos de aminoácidos unidos entre si através de ligações peptídicas. Se comparados às proteínas, são quimicamente mais versáteis, pois podem ser amidados ou esterificados em suas carboxilas terminais, acetilados em seus grupos amino terminais, fosforilados ou sulfatados em um ou mais resíduos (serina, treonina ou tirosina), lineares, semicíclicos (geralmente via uma ou mais ligações dissulfeto intra-ou intercadeias peptídicas) ou cíclicos (via ligação entre os grupos amino e carboxila dos aminoácidos terminais). Muitos contêm um ácido piroglutâmico como resíduo N-terminal, outros apresentam Daminoácidos e outros, ainda, possuem aminoácidos não usuais 1 . Os peptídeos são também extremamente diversificados em termos funcionais. Muitos atuam como hormônios ou fatores liberadores destes, enquanto outros são neuropeptídeos, neurotransmissores, toxinas, antibióticos naturais, adoçantes ou substratos de proteases. Na verdade, vários deles fazem parte de nossas conversas sem que nos apercebamos disto: é o caso do aspartame, da insulina, da ocitocina e de diversas drogas comerciais, que consistem em antagonistas de peptídeos naturais ou em inibidores de enzimas envolvidas na sua produção e liberação no organismo 2-4 . A Tabela 1 fornece alguns exemplos desta diversidade funcional e química. Todo este conhecimento começou a ser acumulado principalmente a partir da década de 50, quando vários peptídeos ativos foram descobertos e tiveram as suas estruturas químicas determinadas. Foi o caso de diversos hormônios que controlam o metabolismo animal (glucagon e insulina, p.ex.) e de outros que desempenham papéis específicos em nosso organismo (ocitocina, vasopressina e o hormônio estimulador de melanócitos, p. ex.) 10 . Estas descobertas geraram um enorme interesse por esta classe de compostos e por metodologias para seu isolamento, análise, purificação, identificação e quantificação, as quais passaram a ser sistematicamente estudadas e aprimoradas. Em paralelo, deparou-se com a necessidade de sintetizar estas moléculas e análogos (derivados com modificações p...

Peptides derived from endogenous hemoglobin play important biological roles in a variety of living systems. In previous works we showed that the fragment 33-61 of bovine alpha-hemoglobin (Hb33-61) and its C-terminus amidated analogue (Hb33-61a) exhibit antimicrobial activity and we determined the 3D structure of Hb33-61a bound to sodium dodecyl sulfate micelles. Here we report that Hb33-61a is lethal to Candida albicans at 6.25 microM probably through disruption of its plasma membrane. In addition, we show that, even when used at 50 microM, Hb33- 61a produces low hemolysis (16% +/- 3.0%). Recognizing that one of the key steps to study new compounds with potential pharmaceutical application is to identify the structural elements essential to express biological activity, we also investigated the anticandidal activity of Hb33- 61a fragments. The results indicated that Hb40-61a exhibits the same minimal inhibitory concentration as Hb33-61a, whereas Hb33-52a and Hb48-61a are significantly less active. Noteworthy, for all the peptides tested, we observed that C-terminus amidation produces a potentiation of their anticandidal activity and we associate that increased biological activity to a preferred structural and spatial organization of the C-terminal region favored by amidation. Finally, the data show that the most active peptides (Hb33-61a and Hb40-61a) are characterized by a central hinge joining the C-terminal region that presents, containing a beta-turn, followed by and a helical element, to the N-terminal region that presents only a beta-turn. We hypothesize that these two structured regions, by fluctuating independently in the lipid environment, may act in a coordinated fashion disrupting the yeast plasma membrane.

208 circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of ureainduced unfolding. Temperature and urea denaturation experiments show that Tm 143-235 is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of ␤-branched residues located at d positions.The coiled-coil motif mediates the process of oligomerization of many proteins. This structure is a consequence of a heptapeptide repetition (abcdefg) in the chemical nature of the residues in the primary structure of the polypeptide chain (1-3). An ␣-helical conformation places hydrophobic residues at positions a and d on the same side of the helix, creating a nonpolar interface that promotes dimerization through the burial of hydrophobic surface. Residues at positions e and g are often charged and may form salt bridges with residues at positions g and e, respectively, of the other helix. The maximization of favorable ionic attractions and the minimization of unfavorable repulsions probably influence the particular alignment of the ␣-helical chains (4). The folding of synthetic peptides that adopt a coiled-coil structure has been investigated extensively (4 -11). Synthetic peptide models for coiled-coils may not always be taken as representative because they often employ regular patterns of residues at positions a and d and lack i, iϩ3 and i, iϩ4 intrahelical ionic interactions (7,12). Natural coiled-coil sequences, such as tropomyosin (Tm), 1 are more complex, with irregular patterns of intrahelix and interhelix side chain-side chain interactions and a greater variety of residues at the hydrophobic core. Tm can be used as a natural model for the study of the principal interactions that maintain the coiled-coil structure and ␣-helix stability.Muscle ␣-Tm is a symmetric coiled-coil composed of two parallel and in register ϳ410 Å, 284-residue ␣-helices (2, 13, 14). The C-terminal half of the Tm molecule is less thermally stable than the N-terminal half (15,16). A series of studies (17,18) have suggested that Tm fragments with less than 94 residues were unable to form stable secondary structures at low micromolar concentrations. Recently, Holtzer et al. (19) showed that a 65-amino acid fragment (residues 190 -254) presents a significant amount of ␣-helix (ϳ43%) when present in high micromolar concentrations (115 M) at 10°C but is essentially unfolded at 25°C.Microcalorimetric analysis of ␣-Tm thermal denaturation has been interpreted as a multistep process in which spec...

Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.

BACKGROUND Previous reports have suggested an increasing rate of utilization of spinal fusions, but contemporary data have not been analyzed, and there has been little investigation of putative drivers of increased utilization. OBJECTIVE To investigate whether there is an ongoing trend of increased utilization of spinal fusions in recent data, and if there may be associations with an increasing proportion of elderly in the population, changing patterns of payer-types, and changing reimbursement rates. METHODS We analyze 7.1 million cases from the National Inpatient Sample between 1998 and 2014. We measure annual utilization per 100 000 persons and conduct trend analyses with subgroup analysis of the senior (65 + ) population. Spine surgery utilization is compared with nonspine surgical procedures (coronary artery bypass grafting, hernia repair, hip, and knee replacement). We assess trends in charges, payer type, Medicare reimbursement rates, and hospital type. RESULTS There was an 88% increase in the utilization rate of spinal fusion procedures from 1998 to 2014 (from 74 to 139 cases per 100 000 persons) with a significant upward trend (P < .001) that persisted in the 65 + subgroup (P < .001). An increasing proportion of spinal fusions is paid for by public payers, but per-procedure reimbursement for spinal fusions by Medicare has decreased recently (5% reduction from 2014 to 2016). CONCLUSION Utilization of spinal fusions continues to increase and is not explained by increased proportion of elderly in the population, increased utilization of surgeries across specialties, or increased Medicare reimbursement. In fact, increased utilization of spinal fusions temporally correlated with decreasing per-procedure Medicare reimbursement.

The physical therapy modalities based on strategic manual lymphatic drainage, shoulder girdle massage, facial, tongue and neck exercises, compressive therapy at home, and patient education showed reduction of the lymphedema and pain, both of them secondary to head and neck cancer treatment.

Study Design. This is an observational cohort study. Objective. The aim of this study was to compare the effectiveness of PT to an interdisciplinary treatment approach in patients with chronic low back pain (CLBP). Summary of Background Data. CLBP is a costly and potentially disabling condition. Physical therapy (PT), cognitive behavioral therapy, and interdisciplinary pain programs (IPPs) are superior to usual care. Empirical evidence is lacking to clearly support one treatment approach over another in patients with CLBP. Methods. One hundred seventeen adult patients who completed an IPP for individuals with ≥3 months of back pain were compared to 214 adult patients with similar characteristics who completed PT. The Modified Low Back Pain Disability Questionnaire was the primary outcome measure. Additional measures included: PROMIS physical function, global health, social role satisfaction, pain interference, anxiety, fatigue, sleep disturbance, and Patient Health Questionnaire. Patients who completed the IPP were matched by propensity score to a historical control group of patients who completed a course of PT. Change in functional disability was compared between IPP patients and matched controls. Patient-reported outcome measures were assessed pre to post participation in the IPP using paired t test and by calculating the proportion with clinically meaningful improvement. Results. Propensity score matching generated 81 IPP and 81 PT patients. Patients enrolled in the IPP had significantly greater improvement in MDQ scores upon completion compared to patients in PT (15.8 vs. 7.1, P < 0.001). The majority of IPP patients reached the threshold for clinically meaningful change of ≥10 point reduction (60.5%) compared to 34.6% of PT patients, P < 0.01. Patients in the IPP also showed statistically and clinically significant improvement in social role satisfaction, fatigue, and sleep disturbance. Conclusion. CLBP patients in an IPP demonstrated greater functional improvements compared to similar patients participating in PT. Level of Evidence: 3