Sitosterolemia Presenting as Pseudohomozygous Familial Hypercholesterolemia.

A young girl, age 8.5 years, presented with profound hypercholesterolemia and early xanthomatosis, suggesting homozygous familial (or type II) hypercholesterolemia. The patient's low density lipoprotein (LDL) receptor function and parental lipoprotein profiles were determined to be normal, prompting revision of the initial diagnosis to pseudohomozygous familial hypercholesterolemia. When she subsequently presented with giant platelets, the case was presented to colleagues on an electronic mailing list. It was recommended that plasma and sterol analysis be performed, which led to a diagnosis of sitosterolemia. The presentation of profound hypercholesterolomia in childhood that ultimately is not attributed as due to homozygous or compound heterozygous defects in the LDL receptor gene has been termed pseudohomozygous familial (or type II) hypercholesterolemia (PHT2HC). Patients diagnosed with PHT2HC subsequently confirmed to have sitosterolemia have been previously reported only rarely. The challenge of achieving accurate specific diagnosis and appropriate workup for these conditions in children is discussed in the context of this rare case and review of the historical literature concerning these conditions.

Biomechanical evaluation of 4-strand flexor tendon repair techniques, including a combined Kessler-Tsuge approach.

PURPOSE: To test the ultimate tensile strength and stiffness of 3 flexor tendon repair techniques using looped suture material. METHODS: Seventeen fresh porcine flexor tendons were randomized to a single-throw, 4-strand Kessler technique with a looped structure, a double-throw, 4-strand Tsuge technique with 2 looped structures, or a single-throw, 4-strand Kessler-Tsuge technique with a looped structure. Thirty additional fresh porcine flexor tendons were randomized to the same techniques but with a running epitendinous repair. We measured ultimate tensile strength to failure and stiffness and recorded the cause of failure. RESULTS: The Tsuge technique had the highest mean ultimate tensile strength at 75 N (SD, 14 N) versus 63 N (SD, 13 N) for the Kessler-Tsuge method and 46 N (SD, 11 N) for the Kessler technique. Differences between the Tsuge and Kessler-Tsuge, the Kessler-Tsuge and Kessler, and the Tsuge and Kessler techniques were significant. Mean suture stiffness was 6.8 N/mm for the Tsuge technique, 5.7 N/mm for the Kessler-Tsuge technique, and 4.6 N/mm for the Kessler technique. The difference between the Tsuge and Kessler techniques was significant. Analyzing the tests with or without an epitendinous suture separately did not affect the significance of the differences. CONCLUSIONS: The modified double-throw, 4-strand Tsuge was the strongest suture technique in this study. It may be a clinically acceptable, albeit slightly weaker alternative to the more complicated Tsuge method. CLINICAL RELEVANCE: A combined Kessler-Tsuge approach might be an option for flexor tendon repair.

Comparison of BMI and HbA1c changes before and during the COVID-19 pandemic in type 1 diabetes: a longitudinal population-based study.

Purpose: To compare the changes in body weight and glycemic control before and during the COVID-19 pandemic in people with type 1 diabetes (T1D). Methods: In 47,065 individuals with T1D from the German Diabetes Prospective Follow-up Registry (DPV), we compared the adjusted mean changes in BMI-Z-scores and HbA1c as well as the distribution of individual changes between four periods from March 2018 to February 2022, by sex and age group (4- < 11, 11- < 16, 16-50 years). Results: At population level, the only significant pandemic effects were a slight increase in BMI Z-score in prepubertal children (girls: + 0.03 in the first COVID year vs. before, P < 0.01; boys: + 0.04, P < 0.01) as well as a stabilization of HbA1c in all subgroups or even improvement in women (- 0.08%, P < 0.01). At individual level, however, heterogeneity increased significantly (p < 0.01), especially in children. More prepubertal children gained weight (girls: 45% vs. 35% before COVID; boys: 39% vs. 33%). More pubertal girls lost weight (30% vs. 21%) and fewer gained weight (43% vs. 54%). More children had a decreasing HbA1c (prepubertal group: 29% vs. 22%; pubertal girls: 33% vs. 28%; pubertal boys: 32% vs. 25%) and fewer had increasing values. More women had stable HbA1c and fewer had increasing values (30% vs. 37%). In men, no significant changes were observed. Conclusion: This real-world analysis shows no detrimental consequences of the two first COVID years on weight and HbA1c in T1D on average, but reveals, beyond the mean trends, a greater variability at the individual level.

Treatment of fractures of the fifth metatarsal with the XS-nail retrospective study and comparison with tension-band wiring.

INTRODUCTION: Many different surgical methods and implants for the treatment of fifth metatarsal fractures have been established yet. A high rate of complications, such as nonunion, fragment dislocation, refracture, implant deformation and irritation are widely occurring due to the insufficient ability of the implants to compensate the tension applied to the proximal fragment through the peroneal tendon combined with an impaired blood supply at the fracture zone. Therefore, the search for improved surgical solutions is thoroughly understandable. Thus, we have introduced the XS-nail as an intramedullary nail system that bears the ability to provide a compression to the fracture zone through a grub screw. In this work, we have analyzed the position of the XS-nail in relationship to other methods with special regard to the tension-band wiring. METHOD: In a retrospective analysis, we examined 77 cases, where a proximal fifth metatarsal fracture has been treated with the XS-nail. As a comparison group, we collected data from 47 patients who had been treated with tension-band wiring for the same indication in our hospital. Altogether, we included 124 patients, representing the largest study population of surgically treated cases of proximal fifth metatarsal fractures as compared to the actual literature. RESULTS: When compared with the tension-band wiring group, we found in mean a shorter duration of the surgery, a lower necessity of an open reduction (18 vs. 100%), fewer postsurgical weight-bearing restrictions (54 vs. 100%) and a shorter duration of rehabilitation (48 vs. 71 days). Especially, the fracture compression was distinctively higher in the XS-nail group (postsurgical lateral dislocation was 0-59%). The advantages of the tension-band wiring were found in the fixation of small fragments and an easier implant removal. Generally, nonunion and refracture were not seen in both methods. When compared with the results from literature, we found positive results regarding the hospitalization duration, the weight-bearing ability, the rehabilitation course and the patients' satisfaction. CONCLUSION: Thus, the XS-nail proved to be an effective and technical optimized implant for the treatment of proximal fifth metatarsal fractures that provides a rapid full-weight-bearing mobilization and shows good long-term results.

Natural and artificial cystine knots for assembly of homo- and heterotrimeric collagen models.

Native collagens are molecules that are difficult to handle because of their high tendency towards aggregation and denaturation. It was discovered early on that synthetic collagenous peptides are more amenable to conformational characterization and thus can serve as useful models for structural and functional studies. Single-stranded collagenous peptides of high propensity to self-associate into triple-helical trimers were used for this purpose as well as interchain-crosslinked homotrimers assembled on synthetic scaffolds. With the growing knowledge of the biosynthetic pathways of natural collagens and the importance of their interchain disulfide crosslinks, which stabilize the triple-helical structure, native as well as de novo designed cystine knots have gained increasing attention in the assembly of triple-stranded collagen peptides. In addition, natural sequences of collagens were incorporated in order to biophysically characterize their functional epitopes. This review is focused on the methods developed over the years, and future perspectives for the production of collagen-mimicking synthetic and recombinant triple-helical homo- and heterotrimers.

A noncovalent switch for lysozyme.

A new concept for the external control of protein activity is presented and demonstrated on the example of an artificial Lysozyme switch. Radical copolymerization of selected methacrylamide-based comonomer units tailored for amino acid residues surrounding the active site furnishes polymeric protein hosts that are able to inhibit enzymatic activity in a highly efficient dose-dependent manner (IC50 approximately 1.0 equiv approximately 0.7 microM). All binding site types on the polymer work cooperatively, using electrostatic attraction, hydrophobic forces, and substrate mimicry. In a native gel electrophoresis, the well-defined 2:1 complex (polymer/protein) migrates to the anode. Even at 250 mM NaCl, a 10-fold polymer excess is able to shut down bacterial cell wall degradation completely. A kinetic investigation points to a competitive mechanism (Lineweaver-Burk plots). CD spectra of pure Lysozyme and its polymer complex are indistinguishable; together with a total lack of preincubation time for maximum inhibition, experimental evidence is thus produced for a preserved tertiary enzyme structure-no denaturation occurs. Addition of the superior complexing agent polyarginine to the enzyme-polymer complex mildly detaches the inhibitor from the protein surface and leads to 90% recovery of enzymatic activity. Thus, Lysozyme could be turned off, on, and off again by consecutive addition of the polymeric inhibitor, polyarginine, and polymer again.

Macrocyclic statine-based inhibitors of BACE-1.

Minimal sequence requirements for binding of substrate-derived statine peptides to the aspartyl enzyme were established on the basis of the X-ray cocrystal structure of the hydroxyethylene-octapeptide OM00-3 in complexation with BACE-1. With this information to hand, macrocyclic compounds that conformationally restrict and preorganize the peptide backbone for an entropically favoured binding to the enzyme active site cleft were designed. By means of a side chain-to-side chain ring closure between two aspartyl residues in the P2 and P3' positions through phenylene-1,3-dimethanamine, a 23-membered ring structure was obtained; this structure retained an extended conformation of the peptide backbone, including the transition state analogue statine for tight interactions with the two aspartyl residues of the active centre. The conformational preorganization of the inhibitor molecule was verified by NMR structural analysis and was then confirmed by the crystal structure of the BACE-1/inhibitor complex. Detailed insights into the binding mode of this macrocyclic inhibitor explained its moderate binding affinity in cell-free assays (K(i)=2.5 microM) and yielded precious information for possible structural optimization in view of the lack of steric clashes of the macrocycle with the flap domain of the enzyme.

Single proline residues can dictate the oxidative folding pathways of cysteine-rich peptides.

The cysteine-rich N and C-terminal domains of minicollagen-1 from Hydra nematocysts fold with excesses of oxidized/reduced glutathione (10:1) into globular structures with distinct cystine frameworks despite their identical cysteine sequence pattern. An additional main difference is the cis conformation of a conserved proline residue in the N-terminal and the trans conformation of this residue in the C-terminal domain. Comparative analysis of the oxidative folding revealed for the C-terminal domain a fast and highly cooperative formation of a single disulfide isomer. Conversely, oxidation of the N-terminal domain proceeds mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. The rate of conversion of the bead-like isomer into the globular end-product is largely dominated by the trans-to-cis isomerization of the critical proline residue as well assessed by its replacement with (4R)- and (4S)-fluoroproline known to exhibit distinct propensities for the trans and cis conformation, respectively. Independently, whether the trans or cis conformation is favored by these substitutions, both analogues retain sufficient sequence-encoded information to fold almost quantitatively into the identical cystine framework and thus spatial structure of the parent peptide with the critical proline residue as cis isomer, but at rates significantly lower for the (4R) than for the (4S)-fluoroproline analogue. Correspondingly, other sequence-encoded structural elements have to act as a driving force for these unidirectional folding pathways despite the rather simple sequence composition consisting only of aliphatic residues, some proline and only one aromatic residue (tyrosine) in the core parts of the C and N-terminal domains. The two cysteine-rich domains of minicollagen-1 may well represent ideal targets for ab initio structure calculations in order to learn more about the elementary information encoded in such primordial molecules.

The configuration of the Cu(2+) binding region in full-length human prion protein compared with the isolated octapeptide.

The cellular prion protein (PrP(C)) is a copper binding protein. The molecular features of the Cu(2+) binding sites have been investigated and characterized by spectroscopic experiments on PrP(C)-derived peptides and the correctly folded human full-length PrP(C) (hPrP-[23-231]). These experiments allowed us to distinguish two different configurations of copper binding. The different copper complexes depend on sequence context, buffer conditions and stoichiometry of copper. The combined information of spectroscopic data from our EXAFS, EPR and ENDOR experiments was used to create models for these two copper complexes. A large number of conformations of these models were calculated using molecular mechanics computations, and the simulated spectra of these structures were compared with our experimental data. Common features and differences of the copper binding motifs are discussed in this paper and it remains for future investigations to study whether different configurations are associated with different functional states of PrP(C).

The two cysteine-rich head domains of minicollagen from Hydra nematocysts differ in their cystine framework and overall fold despite an identical cysteine sequence pattern.

Synthetic replicates of naturally occurring cysteine-rich peptides such as hormones, neurotransmitters, growth factors, enzyme inhibitors, defensins and toxins often can be oxidatively folded in high yields to their native structure in simple redox buffers. Thereby, identical cysteine patterns in the sequence were found to generate identical disulfide connectivities and homologous spatial structures despite significant variability in the non-cysteine positions. Minicollagen-1 from the nematocysts of Hydra is a trimeric protein that contains cysteine-rich domains at the N and C termini, which are involved in the assembly of an intermolecular disulfide network. Determination of the three-dimensional structures of peptides corresponding to the N-terminal and C-terminal domains by NMR spectroscopy revealed a remarkable exception from the general rule. Despite an identical cysteine pattern, the two domains of minicollagen-1 form different disulfide bridges and exhibit distinctly different folds, both of which are not found in the current structural databases. To our knowledge, this is the first case where two relatively short peptides with the abundant cysteine residues in identical sequence positions fold uniquely and with high yields into defined, but differing, structures. Therefore, the cysteine-rich domains of minicollagen constitute ideal model systems for studies of the interplay between folding and oxidation in proteins.

Synthesis of 2-methyl-3-indolylacetic derivatives as anti-inflammatory agents that inhibit preferentially cyclooxygenase 1 without gastric damage.

Novel substituted 2-methyl-3-indolylacetic derivatives were synthesized and evaluated for their activity in vitro and in vivo on COX-1 and COX-2. Active compounds were screened to determine their gastrointestinal tolerability in vivo in the rat. Results showed that 3 and 4 preferentially inhibited COX-1 in vitro and in vivo. MD simulations indicated an induced fit for COX-1 but not for COX-2, probably because of a lower plasticity of the latter.

The chain register in heterotrimeric collagen peptides affects triple helix stability and folding kinetics.

Collagen type IV is a highly specialized form of collagen found only in basement membranes, where it provides mechanical stability and structural integrity to tissues and organs, and binding sites for cell adhesion. In its ubiquitous form, collagen type IV consists of two alpha1 chains and one alpha2 chain, whose internal alignment within the triple helix seems to exert a strong influence on the binding affinity to alpha1beta1 integrin receptor. This has been assessed recently using two synthetic collagen peptides that contain the cell adhesion epitope of collagen type IV and are assembled into the most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. In the present study, the effects of the chain register on the stability of the triple helix and the folding kinetics of these collagen peptides were investigated by CD spectroscopy and microcalorimetry. The results revealed a multi-domain structural organization for both trimers, with an unexpected strong effect of the chain alignment on the conformational stability. Molecular dynamics simulations served to rationalize more properly the experimental results.

New 5-hydroxytryptamine(1A) receptor ligands containing a norbornene nucleus: synthesis and in vitro pharmacological evaluation.

New arylpiperazine derivatives were prepared to identify highly selective and potent ligands for the 5-hydroxytryptamine 1A (5-HT(1A)) receptor as potential pharmacological tools in studies of central nervous system (CNS) disorders. The combination of structural elements (heterocyclic nucleus, oxyalkyl chain, and arylpiperazine) known to introduce 5-HT(1A) receptor affinity and the proper selection of substituents led to compounds with higher receptor specificity and affinity. In binding studies, several molecules showed affinity in the nanomolar and subnanomolar ranges at 5-HT(1A) and moderate to no affinity for other relevant receptors (5-HT(2A), 5-HT(2C), D(1), D(2), alpha(1), and alpha(2)). The 4-[3-[4-(o-methoxyphenyl)piperazin-1-yl]propoxy]-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione, with K(i) = 0.021 nM, was the most active and selective derivative for the 5-HT(1A) receptor with respect to other serotonin receptors, whereas the most selective derivative for dopaminergic and adrenergic receptors was a CF(3)-substituted arylpiperazine. As a general trend, compounds with a piperazinylpropoxy chain showed a preferential affinity for the 5-HT(1A) receptor, suggesting that the alkyl chain length represents a critical structural feature in determining 5-HT(1A) receptor affinity and selectivity, as confirmed by the molecular modeling invoked for explaining the differential binding affinities of the new arylpiperazines.

Photocontrol of cell adhesion processes: model studies with cyclic azobenzene-RGD peptides.

A photoresponsive integrin ligand was synthesized by backbone-cyclization of a heptapeptide containing the integrin binding motif Arg-Gly-Asp (RGD) with 4-(aminomethyl)phenylazobenzoic acid (AMPB). Surface plasmon enhanced fluorescence spectroscopy showed that binding of the azobenzene peptide to alpha(v)beta(3) integrin depends on the photoisomeric state of the peptide chromophore. The higher affinity of the trans isomer could be rationalized by comparing the NMR conformations of the cis and trans isomers with the recently solved X-ray structure of a cyclic RGD-pentapeptide bound to integrin.

Backbone dynamics of the human MIA protein studied by (15)N NMR relaxation: implications for extended interactions of SH3 domains.

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by (15)N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting beta-sheets, such as the RT-loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT-loop, the n-Src loop, for the loop comprising residues 13 to 19, which we refer to as the"disulfide loop", in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte-derived protein (FDP)/MIA-like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain-like fold in solution with expanded ligand interactions.

TMC-95A analogues with endocyclic biphenyl ether group as proteasome inhibitors.

TMC-95A, a cyclic tripeptide metabolite of Apiospora montagnei, is a potent competitive inhibitor of proteasome. Based on the X-ray structure of its complex with yeast proteasome, the synthetically challenging structure of this natural product was simplified in a first generation of analogues by replacing the highly oxidized side-chain biaryl system with a phenyl-oxindole group. In the present study, the TMC-95 biaryl group was substituted with a biphenyl ether with retainment of significant proteasome inhibition. Because of the facile synthetic access of tripeptides containing in i, i+2 positions residues of the isodityrosine type, this new generation of TMC-95 analogues may represent promising lead structures for further optimization of affinity and selectivity of proteasome inhibitors.

Fluoroprolines as Tools for Protein Design and Engineering.

The preference of the peptidyl-fluoroproline amide bond for the cis or trans conformation in the model compounds N-acetyl-4-fluoroproline methyl esters fully correlates with the thermostability of the related mutants of the model protein barstar. Thus, the (4S)-L-FPro mutants show a higher and the(4R)-L-FPro mutants a lower thermal stability than barstar.