Search

Burkholderia pseudomallei is an infectious bacterium of clinical and biodefense concern, and is the causative agent of melioidosis. The mortality rate can reach up to 50% and affects 165,000 people per year; however, there is currently no vaccine available. In this study, we examine the antigen-specific immune response to a vaccine formulated with antigens derived from an outer membrane protein in B. pseudomallei, Bucl8. Here, we employed a number of bioinformatic tools to predict Bucl8-derived epitopes that are non-allergenic and non-toxic, but would elicit an immune response. From these data, we formulated a vaccine based on two extracellular components of Bucl8, the β-barrel loops and extended collagen and non-collagen domains. Outbred CD-1 mice were immunized with vaccine formulations—composed of recombinant proteins or conjugated synthetic peptides with adjuvant—to assess the antigen-specific immune responses in mouse sera and lymphoid organs. We found that mice vaccinated with either Bucl8-derived components generated a robust TH2-skewed antibody response when antigen was combined with the adjuvant AddaVax, while the TH1 response was limited. Mice immunized with synthetic loop peptides had a stronger, more consistent antibody response than recombinant protein antigens, based on higher IgG titers and recognition of bacteria. We then compared peptide-based vaccines in an established C57BL/6 inbred mouse model and observed a similar TH2-skewed response. The resulting formulations will be applied in future studies examining the protection of Bucl8-derived vaccines.

Keratinized epidermis constitutes a powerful barrier of the mucosa and skin, effectively preventing bacterial invasion, unless it is wounded and no longer protective. Wound healing involves deposition of distinct extracellular matrix (ECM) proteins enriched in cellular fibronectin (cFn) isoforms containing extra domain A (EDA). The streptococcal collagen-like protein 1 (Scl1) is a surface adhesin of group A Streptococcus (GAS), which contains an N-terminal variable (V) domain and a C-terminally located collagen-like domain. During wound infection, Scl1 selectively binds EDA/cFn isoforms and laminin, as well as low-density lipoprotein (LDL), through its V domain. The trimeric V domain has a six-helical bundle fold composed of three pairs of anti-parallel α-helices interconnected by hypervariable loops, but the roles of these structures in EDA/cFn binding are unclear. Here, using recombinant Scl (rScl) constructs to investigate structure–function determinants of the Scl1–EDA/cFn interaction, we found that full-length rScl1, containing both the globular V and the collagen domains, is necessary for EDA/cFn binding. We established that the surface-exposed loops, interconnecting conserved α-helices, guide recognition and binding of Scl1-V to EDA and binding to laminin and LDL. Moreover, electrostatic surface potential models of the Scl1-V domains pointed to a conserved, negatively charged pocket, surrounded by positively charged and neutral regions, as a determining factor for the binding. In light of these findings, we propose an updated model of EDA/cFn recognition by the Scl1 adhesin from GAS, representing a significant step in understanding the Scl1–ECM interactions within the wound microenvironment that underlie GAS pathogenesis.

Signaling lymphocytic activation molecule–associated protein (SAP), a critical intracellular signaling molecule for T–B lymphocyte interactions, drives T follicular helper (Tfh) cell development in germinal centers (GCs). High-affinity islet autoantibodies predict type 1 diabetes (T1D) but do not cause β cell destruction. This paradox intimates Tfh cells as key pathologic effectors, consistent with an observed Tfh signature in T1D. To understand how fully developed Tfh (GC Tfh) contribute to different autoimmune processes, we investigated the role of SAP in T1D and autoantibody-mediated arthritis. Whereas spontaneous arthritis depended on SAP in the autoantibody-mediated K/BxN model, organized insulitis and diabetes onset were unabated, despite a blocked anti-insulin vaccine response in SAP-deficient NOD mice. GC Tfh and GC B cell development were blocked by loss of SAP in K/BxN mice. In contrast, although GC B cell formation was markedly reduced in SAP-deficient NOD mice, T cells with a GC Tfh phenotype were found at disease sites. CXCR3+ CCR6− (Tfh1) subset bias was observed among GC Tfh cells infiltrating the pancreas of NOD mice, which was enhanced by loss of SAP. NOD T cells override SAP requirement to undergo activation and proliferation in response to Ag presentation, demonstrating the potential for productive cognate T–B lymphocyte interactions in T1D-prone mice. We find that SAP is essential when autoantibody-driven immune complexes promote inflammation but is not required for effective organ-specific autoimmune attack. Thus, Tfh induced in classic GC reactions are dispensable for T1D, but the autoimmune process in the NOD model retains pathogenic Tfh without SAP.

Anti-insulin B lymphocytes (AIBCs) are key drivers of the autoimmune attack in type 1 diabetes (T1D) but their exact functions in T1D pathogenesis, and the point(s) during their development where tolerance is lost, are unknown. In the class-switch-competent anti-insulin B cell receptor transgenic non-obese diabetic (VH125SD.NOD) mice, 1–2% of B lymphocytes are insulin-specific, which accelerates diabetes development despite limited insulin autoantibody (IAA) production. Here we used VH125SD.NOD mice to determine where immune tolerance is lost throughout AIBC class-switch, germinal center entry, and antibody-secreting cell differentiation. We find an increased percentage of AIBCs enter germinal centers relative to non-insulin binding B lymphocytes in the same mice. In addition, an increased percentage of AIBCs are found within the pancreas, class-switch to IgG1, and proliferate relative to non-insulin binding B lymphocytes. We also find that AIBCs are activated in the gut to a greater extent than non-insulin binding B lymphocytes. These data suggest that AIBCs escape anergy and are able to class-switch, enter GCs, and proliferate, but maintain functional silencing preventing spontaneous IAA-production. Moreover, the escape from anergy may occur within the pancreas and/or the gastrointestinal tract. Our data has implications in immunotherapy treatments for T1D, as these GC checkpoints may provide an opportunity for T1D intervention.

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing beta cells. An essential role for B lymphocytes in the disease process and molecular signatures for T follicular helper cells (Tfhs) in T1D point to the importance of T-B lymphocyte interactions in the pathological process. To understand mechanisms that underpin these T-B interactions, we introduced deficiency of the signaling lymphocyte activation molecular (SLAM)-associated protein (SAP) into VH125SD.NOD mice. In VH125SD.NOD mice a targeted anti-insulin VH gene generates a small population of anti-insulin B lymphocytes that are highly diabetogenic yet are functionally silent for autoantibody production. We find that SAPko dramatically eliminates germinal centers in spleen, pancreatic draining lymph nodes and pancreas. However, insulitis was similar between SAP-sufficient and SAPko VH125SD.NOD mice early in T1D development (8–12 weeks old) but differed later in disease onset (13–17 weeks old). Strikingly, SAP is essential for T1D development, as SAPko VH125SD.NOD mice did not develop T1D. Despite elimination of germinal centers, the numbers of Tfh cells were largely unaffected in the absence of SAP. These findings suggest that prolonged T-B lymphocyte conjugates maintained by SAP are not required for retaining Tfhs; however, these interactions maybe essential for the genesis of pathogenic Tfhs when functionally silent B lymphocytes recognize a beta cell autoantigen.

The ultraviolet and visible spectra of organic compounds are associated with transitions between electronic energy levels in which an electron from a low-energy orbital in the ground state is promoted into a higher-energy orbital. Normally, the transition occurs from a filled to a formerly empty orbital (Fig. 2.1) to create a singlet excited state. The wavelength of the absorption is a measure of the separation of the energies of the ground and excited states, related to but not the same as the energy separation E in Fig. 2.1 of the energy levels of the orbitals concerned.

The conventional NMR spectrum is called a one-dimensional spectrum because it has one frequency dimension—the chemical shift and the coupling are displayed on the same axis with intensity plotted in the second dimension. In two-dimensional NMR spectra we have two frequency dimensions, displayed on orthogonal axes, with intensity as the third dimension. They are used to establish connections, coupling or spatial, between the nuclei giving the signals displayed on one axis and the nuclei giving the signals on the other axis. If both axes have spectra derived from the same element, the spectra are said to be auto-correlated. If the spectra on the two axes are from different elements they are said to be cross-correlated.

Some atomic nuclei have a nuclear spin (I), and the presence of a spin makes these nuclei behave like bar magnets. In the presence of an applied magnetic field the nuclear magnets can orient themselves in 2I + 1 ways. Those nuclei with an odd mass number have nuclear spins of 1/2, or 3/2, or 5/2, …, etc. (given in the first table at the end of this chapter). In the applications of NMR spectroscopy in organic chemistry, the five most important nuclei are 1H and 13C, followed by 19F, 29Si and 31P, all of which have spins of 1/2. These nuclei, therefore, can take up one of only two orientations, a low-energy orientation aligned with the applied field and a high-energy orientation opposed to the applied field. The difference in energy between these two orientations is given by Eq. (4.1), the Boltzmann distribution by Eq. (4.2), and the frequency ν in Hz corresponding to the difference in energy by Eq. (4.3):where γ is the magnetogyric ratio (also called the gyromagnetic ratio), which is a measure of the relative strength of the nuclear magnet, and is different for each element and for each isotope of each element, B0 is the strength of the applied magnetic field, Nα is the number of nuclei in the low-energy state, and Nβ the number in the high-energy state. A radio-frequency signal applied to the system changes the Boltzmann distribution when the radio frequency matches the frequency ν. It is called the resonance frequency or Larmor frequency for that particular nucleus. The effect is to promote nuclei from the low-energy Nα level to the high-energy level Nβ (Fig. 4.1).

The infrared spectra of organic compounds are associated with transitions between vibrational energy levels. Molecular vibrations may be detected and measured either in an infrared spectrum or indirectly in a Raman spectrum. The most useful vibrations, from the point of view of the organic chemist, occur in the narrower range of 3.5–16 μm (1 μm = 10−6 m). The position of an absorption band in the spectrum may be expressed in microns (μm), but standard practice uses a frequency scale in the form of wavenumbers, which are the reciprocals of the wavelength, cm−1. The useful range of the infrared for an organic chemist is between 4000 cm−1 at the high-frequency end and 600 cm−1 at the low-frequency end.

The streptococcal collagen-like proteins 1 and 2 (Scl1 and Scl2) are major surface adhesins that are ubiquitous among group A Streptococcus (GAS). Invasive M3-type strains, however, have evolved two unique conserved features in the scl1 locus: (i) an IS1548 element insertion in the scl1 promoter region and (ii) a nonsense mutation within the scl1 coding sequence. The scl1 transcript is drastically reduced in M3-type GAS, contrasting with a high transcription level of scl1 allele in invasive M1-type GAS. This leads to a lack of Scl1 expression in M3 strains. In contrast, while scl2 transcription and Scl2 production are elevated in M3 strains, M1 GAS lack Scl2 surface expression. M3-type strains were shown to have reduced biofilm formation on inanimate surfaces coated with cellular fibronectin and laminin, and in human skin equivalents. Repair of the nonsense mutation and restoration of Scl1 expression on M3-GAS cells, restores biofilm formation on cellular fibronectin and laminin coatings. Inactivation of scl1 in biofilm-capable M28 and M41 strains results in larger skin lesions in a mouse model, indicating that lack of Scl1 adhesin promotes bacterial spread over localized infection. These studies suggest the uniquely evolved scl1 locus in the M3-type strains, which prevents surface expression of the major Scl1 adhesin, contributed to the emergence of the invasive M3-type strains. Furthermore these studies provide insight into the molecular mechanisms mediating colonization, biofilm formation, and pathogenesis of group A streptococci.

Keratinized epidermis constitutes a powerful barrier of the mucosa and skin, effectively preventing bacterial invasion, unless it is wounded and no longer protective. Wound healing involves deposition of distinct extracellular matrix (ECM) proteins enriched in cellular fibronectin (cFn) isoforms containing extra domain A (EDA). The streptococcal collagen-like protein 1 (Scl1) is a surface adhesin of group A Streptococcus (GAS), which contains an N-terminal variable (V) domain and a C-terminally located collagen-like domain. During wound infection, Scl1 selectively binds EDA/cFn isoforms and laminin, as well as low-density lipoprotein (LDL), through its V domain. The trimeric V domain has a six-helical bundle fold composed of three pairs of anti-parallel α-helices interconnected by hypervariable loops, but the roles of these structures in EDA/cFn binding are unclear. Here, using recombinant Scl (rScl) constructs to investigate structure–function determinants of the Scl1–EDA/cFn interaction, we found that full-length rScl1, containing both the globular V and the collagen domains, is necessary for EDA/cFn binding. We established that the surface-exposed loops, interconnecting conserved α-helices, guide recognition and binding of Scl1-V to EDA and binding to laminin and LDL. Moreover, electrostatic surface potential models of the Scl1-V domains pointed to a conserved, negatively charged pocket, surrounded by positively charged and neutral regions, as a determining factor for the binding. In light of these findings, we propose an updated model of EDA/cFn recognition by the Scl1 adhesin from GAS, representing a significant step in understanding the Scl1–ECM interactions within the wound microenvironment that underlie GAS pathogenesis.

To assess whether the type of surgical approach to the sella (sublabial transseptal vs transnasal) affects surgical outcome among patients with Cushing disease.Both procedures were performed at our institution from January 1, 1995, through January 31, 2003. From a total of 106 patients with Cushing disease who had had surgery, we identified 42 adults undergoing an initial surgery, with microadenoma (1 cm) determined by magnetic resonance imaging and a minimal follow-up of 3 months.We identified 21 patients (4 male, 17 female) for sublabial transseptal transsphenoidal microsurgery. Mean+/-SD age was 45.0+/-12.9 years (range, 27.0-69.0 years); median duration of symptoms was 2.5 years (range, 1.5-7.5 years). Median follow-up was 1.0 year (range, 0.3-11.0 years). We identified another 21 patients (5 male, 16 female) for endoscopic transsphenoidal microsurgery whose mean+/-SD age was 43.8+/-14.1 years (range, 19.0-70.0 years); median duration of symptoms was 2.4 years (range, 0.2-6.0 years). Median follow-up was 2.5 years (range, 0.3-8.7 years). Complications (cerebrospinal fluid leak and transient diabetes insipidus) and cure (86% initial cure; 76% relapse-included cure) were equivalent between the 2 procedures. However, the endoscopic approach was associated with shorter anesthesia time, less blood loss, and shorter hospital stays.The endoscopic transnasal surgical approach led to shorter total anesthesia time, less blood loss, and shorter hospital stay when compared with the sublabial transseptal approach. However, we found no difference between the 2 surgical procedures with respect to cure or complications, suggesting that outcome is more closely related to the experience of the operating team.

Sentinel lymph node biopsy in women with operable breast cancer is routinely used in some countries for staging the axilla despite limited data from randomized trials on morbidity and mortality outcomes. We conducted a multicenter randomized trial to compare quality-of-life outcomes between patients with clinically node-negative invasive breast cancer who received sentinel lymph node biopsy and patients who received standard axillary treatment.The primary outcome measures were arm and shoulder morbidity and quality of life. From November 1999 to October 2003, 1031 patients were randomly assigned to undergo sentinel lymph node biopsy (n = 515) or standard axillary surgery (n = 516). Patients with sentinel lymph node metastases proceeded to delayed axillary clearance or received axillary radiotherapy (depending on the protocol at the treating institution). Intention-to-treat analyses of data at 1, 3, 6, and 12 months after surgery are presented. All statistical tests were two-sided.The relative risks of any lymphedema and sensory loss for the sentinel lymph node biopsy group compared with the standard axillary treatment group at 12 months were 0.37 (95% confidence interval [CI] = 0.23 to 0.60; absolute rates: 5% versus 13%) and 0.37 (95% CI = 0.27 to 0.50; absolute rates: 11% versus 31%), respectively. Drain usage, length of hospital stay, and time to resumption of normal day-to-day activities after surgery were statistically significantly lower in the sentinel lymph node biopsy group (all P.001), and axillary operative time was reduced (P = .055). Overall patient-recorded quality of life and arm functioning scores were statistically significantly better in the sentinel lymph node biopsy group throughout (all Por = .003). These benefits were seen with no increase in anxiety levels in the sentinel lymph node biopsy group (P.05).Sentinel lymph node biopsy is associated with reduced arm morbidity and better quality of life than standard axillary treatment and should be the treatment of choice for patients who have early-stage breast cancer with clinically negative nodes.

Tetrameric rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) binds successively four molecules of its cofactor (NAD + ) with affinities of ca 10 11 M −1 , 10 9 M −1 , 10 7 M −1 , and 10 5 M −1 . The reduction in the dynamics of the protein is greatest upon binding the first NAD + molecule. Smaller reductions then occur upon binding the second and third NAD + molecules, and the fourth NAD + molecule binds without dynamic change. Reduction of the GAPDH dynamics, with consequent improvements in its internal bonding, can account for the increase in NAD + binding affinity from 10 5 M −1 to 10 11 M −1 . Evidence is provided that comparable fractions of the binding energy of other ligands, and of the catalytic efficiency of enzymes, may be derived in the same way.

Nichtkovalente Wechselwirkungen werden bisweilen als additiv behandelt. Mit diesem Verfahren lassen sich brauchbare mittlere Bindungsenergien fur typische Wechselwirkungen in wassriger Losung ableiten, der Ansatz scheitert jedoch in vielen Fallen, da sich nichtkovalente Wechselwirkungen oft gegenseitig verstarken (positiv kooperativ sind) oder abschwachen (negativ kooperativ sind). Bindungsenergie wird gewonnen, wenn ein Ligand die innere Beweglichkeit seines Rezeptors einschrankt (positiv kooperative Bindung). Auf ahnliche Weise wird bei enzymatisch katalysierten Reaktionen Energie fur die Bindung des Ubergangszustands gewonnen, wenn das Substrat im Ubergangszustand innere Bewegungen des Enzyms einschrankt. Liganden und Substrate erhohen auf diese Weise ihre Affinitaten fur Proteine. Diese Organisation geht einher mit einem Gewinn an Bindungsenthalpie und einer Einschrankung der Dynamik (einem Entropieaufwand), erfordert aber nicht, dass neue nichtkovalente Wechselwirkungen entstehen, sondern lediglich, dass die bereits existierenden Wechselwirkungen gestarkt werden. Negativ kooperative Effekte wirken sich umgekehrt aus: Die Packungsdichte wird verringert, und Enthalpie wird aufgewendet, wahrend Entropie gewonnen wird.

Recent hydrogen-deuterium exchange experiments have highlighted tightening and loosening of protein structures upon ligand binding, with changes in bonding (DeltaH) and order (DeltaS) which contribute to the overall thermodynamics of ligand binding. Tightening and loosening show that ligand binding respectively stabilises or destabilises the internal structure of the protein, i.e. it shows positive or negative cooperativity between ligand binding and the receptor structure. In the case of membrane-bound receptors, such as G protein-coupled receptors (GPCRs) and ligand gated ion channel receptors (LGICRs), most binding studies have focussed on association/dissociation constants. Where these have been broken down into enthalpic and entropic contributions, the phenomenon of "thermodynamic discrimination" between antagonists and agonists has often been noted; e.g. for a receptor where agonist binding is predominantly enthalpy driven, antagonist binding is predominantly entropy driven and vice versa. These data have not previously been considered in terms of the tightening, or loosening, of receptor structures that respectively occurs upon positively, or negatively, cooperative binding of ligand. Nor have they been considered in light of the homo- and hetero-oligomerisation of GPCRs and the possibility of ligand-induced changes in oligomerisation. Here, we argue that analysis of the DeltaH and DeltaS of ligand binding may give useful information on ligand-induced changes in membrane-bound receptor oligomers, relevant to the differing effects of agonists and antagonists.

Object. Selective peripheral denervation is currently the primary surgical treatment for intractable cervical dystonia. The authors assessed preoperative factors to determine which, if any, correlated with outcomes in patients with torticollis who had undergone this procedure. Methods. The records of 168 consecutive patients who had undergone selective peripheral denervation for cervical dystonia between 1988 and 1996 at the Mayo Clinic were reviewed. There were 89 women (53%) and 79 men (47%) with a mean age of 53.4 years. Selection of muscles for denervation was based on the patient's clinical presentation and electromyography mapping results. The most common torticollis vectors were rotational in 141 patients (84%) and laterocollis in 59 (35%). Seventy patients (42%) presented with combined vectors. The technique used to remedy both conditions involved denervation of the ipsilateral posterior cervical paraspinal and splenius capitis muscles. Denervation of the sternocleidomastoid muscle was performed on the contralateral side for rotational torticollis and on the ipsilateral side for laterocollis. A rigorous physical therapy program followed surgery. At the 3-month postoperative evaluation, 125 patients (77%) of the 162 who were available for follow up had moderate to excellent improvement in their head position, and pain was moderately to markedly improved in 131 patients (81%). The long-term follow up lasted a mean of 3.4 years and was undertaken in 130 patients. The original level of moderate to excellent improvement in head position and pain was retained in at least 71 patients (70%). Outcome was not predicted by preoperative head position, severity of abnormal posture of head, symptom duration, presence of tremor or phasic dystonic movements, or failure to respond to botulinum toxin treatment. Five patients recovered from postoperative complications including one myocardial infarction, one pulmonary embolism, and three respiratory failures. Three patients suffered from persistent C-2 distribution dysesthesias and three from slight shoulder weakness; one had a wound infection, and one died of respiratory arrest. Conclusions. Selective peripheral denervation is an effective method of achieving lasting improvement of dystonia in most patients with intractable torticollis.

Some small molecules bind to their receptors, and transition states to enzymes, so strongly as to defy current understanding. We show that in the binding of biotin to streptavidin, the streptavidin structure becomes better packed. We conclude that this contraction of the streptavidin structure promotes biotin binding. The improved packing is associated with positively cooperative binding, occurring with a benefit in enthalpy and a cost in entropy. Evidence indicating that catalytic efficiency can also originate via improved packing in some enzyme transition states, derived from the work of others, is presented. Negatively cooperative ligand binding is concluded to induce converse effects (less efficient packing, a cost in enthalpy, and a benefit in entropy). It applies to the binding of O2 to haemoglobin, which indeed occurs with a hitherto unreported loosening of the amide backbones of the haemoglobin monomers.

A wide range of dimerisation constants (Kdimca. 101–106 M−1) for various glycopeptide antibiotics have been determined. We consider these dimerisation constants in the light of the published X-ray structures of the antibiotics, in particular, the relationship between Kdim and the length of a specified distance at the dimer interface. In the crystals, we find that this distance is smaller for strongly dimerising antibiotics and larger for weakly dimerising antibiotics. Thus, the dimerisation constant is correlated with tightness at the dimer interface. Despite the crystal-packing forces exerted between adjacent dimer molecules in the crystals, the noncovalent bond distances at the dimer interface are correlated with the distances in solution (inferred from solution NMR data). These observations can account for the benefits in enthalpy, and costs in entropy, associated with positively cooperative binding.

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein βγ subunits (Gβγs). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P2, and the Gβγs are localized at the plasma membrane. We have addressed the mechanism by which Gβγs regulate the PI3K using an in vitro approach. We used sedimentation assays and surface plasmon resonance to determine association of type IB PI3K with lipid monolayers and vesicles of varying compositions, some of which had Gβγs incorporated. Association and dissociation rate constants were determined. Our results indicated that in an assay situation in vitro the majority of PI3K will be associated with lipid vesicles, irrespective of the presence or absence of Gβγs. In line with this, a constitutively active membrane-targeted PI3K construct could still be activated substantially by Gβγs in vitro. We conclude that Gβγs activate type IB PI3K by a mechanism other than translocation to the plasma membrane.

LY154989 is a vancomycin group antibiotic closely related in structure to teicoplanin aglycone. In view of the clinical importance of teicoplanin, the dimerisation, aggregation, and binding of bacterial cell wall analogues by LY154989 are of interest. These properties have been studied by proton NMR spectroscopy. LY154989 has been shown to form concentration-dependent aggregates in aqueous solution, similar to those of teicoplanin, even though it does not possess a C11 acyl chain, which was hitherto thought to be the cause of this aggregation. The aggregation can be disrupted by the addition of bacterial cell wall precursor analogues such as Ac2-KDADA, Ac-DADA or Ac-DA. Thus, growing bacteria may disrupt aggregates of teicoplanin and LY154989. LY154989 dimerises weakly in aqueous solution and the dimerisation is weakly cooperative with ligand binding.

The biosynthesis of a fungal secondary metabolite, chlorofusin, which disrupts the interaction between the proteins p53 and MDM2, has been investigated; the acetogenic origin of the chromophore backbone as well as of an aminodecanoic acid residue is demonstrated.

Objective: To assess the reliability, validity, and sensitivity of outcome measures that might be used in a clinical trial of surgery for the treatment of severe tremor associated with MS (MS tremor). Methods: Nine patients with MS tremor were evaluated before and 3 and 12 months after thalamic surgery (six thalamotomy, three deep brain stimulation). A clinical tremor rating scale (CTRS), a novel quantitative movement analysis technique (QMA), and a variety of measures of disability, neurologic impairment, and quality of life was utilized. Results: Both the CTRS and QMA were reliable measures of tremor and both were sensitive to the improvement in tremor following surgery. However, QMA correlated with disability measures and corresponded better to patient and examiner assessment of surgical results. The disability scales used were insensitive to functional improvements that may follow surgery. The box and blocks test clearly separated three patients who had excellent results from three who had poor results. Baseline QMA values predicted improvement on the box and blocks test. Conclusions: 1) QMA is a reliable, objective and valid measure of MS tremor that could be used in a clinical trial. 2) The box and blocks test can detect the improvement in prehensile function that follows surgery, but standard disability scales are poorly responsive to this change. 3) Preoperative QMA values may predict which patients are most amenable to functional improvement after surgery.

Attempts to quantify binding interactions of noncovalent complexes in aqueous solution have been stymied by complications arising from enthalpy-entropy compensation and cooperativity. We have extended work detailing the relationship between noncovalent structure and free energy of binding to include the roles of enthalpy and entropy of association. On the basis of van't Hoff measurements of the dimerization of vancomycin type antibiotics, we demonstrate that positive cooperativity manifests itself in a more favorable enthalpy of association and a partially compensating less favorable entropy of association. Finally, we extend these results to rationalize thermodynamic observations in unrelated systems.

Wild-type p53 plays a crucial role in the prevention of cancer. Since dysfunction of p53 can be caused by increased levels of the protein MDM2, small molecules which antagonize the interaction between these two proteins have potential in cancer therapy. The discovery and structure determination of a fungal metabolite, chlorofusin, which antagonizes the p53/MDM2 interaction are reported.

Udgivelsesdato: FEB 4 2000 The formation of heterodimers in mixtures of glycopeptide antibiotics has been detected by electrospray ionization mass spectrometry (ESI-MS), and dimerization constants have been determined. By using NMR spectroscopy, it has been shown that these heterodimers indeed exist in aqueous solution. The dimerization constants obtained by NMR spectroscopy are in good agreement with those determined by ESI-MS. Structural information on the heterodimer interface of some of the heterodimers is obtained by using two-dimensional NMR techniques and reveals that these heterodimers are similar in structure to the homodimers.

The formation of the N-terminal beta-hairpin of ubiquitin is thought to be an early event in the folding of this small protein. Previously, we have shown that a peptide corresponding to residues 1-17 of ubiquitin folds autonomously and is likely to have a native-like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U(1-17)T9D, the native conformation is stabilized with respect to the wild-type sequence, so much so that we are able to characterize the structure of the mutant peptide fully by NMR spectroscopy. The data indicate that U(1-17)T9D peptide does indeed form a hairpin with a native-like register and a type I turn with a G1 beta-bulge, as in the full-length protein. The reason for the greater stability of the U(1-17)T9D mutant remains uncertain, but there are nuclear Overhauser effects between the side chains of Asp9 and Lys 11, which may indicate that a charge-charge interaction between these residues is responsible.

Modified phospholipids possessing carboxyl head groups synthesized from phosphatidylethanolamine were incorporated into supported lipid monolayers on top of a thin gold film. A monoclonal antibody was chemically coupled to the modified lipids in these monolayers and the kinetics of antigen binding were determined by surface plasmon resonance. The binding could be analyzed using a conventional 1:1 binding algorithm and the derived kinetic and affinity constants were almost identical to those reported for the same interaction on a dextran hydrogel-based sensor chip. When an antigen was chemically coupled to a modified lipid monolayer, the binding of a monoclonal antibody to this surface was biphasic. A two-step algorithm describing the formation of a 1:2 antibody:antigen complex was developed which accurately described the data and enabled differentiation of the two binding steps. The binding was assayed varying both the concentration of antibody in solution and the density of antigen on the surface. The affinities determined by Scatchard analysis of equilibrium binding levels were similar to those values obtained from an ELISA.

Intracranial infection after neurosurgical intervention most often is caused by bacteria. A rare case of fatal herpes simplex encephalitis after removal of a meningioma is described and similar cases reported in the literature are reviewed. Recent diagnostic tools, including detection of herpes viral DNA sequences by polymerase chain reaction, complement clinical suspicion and facilitate mandatory early diagnosis, because herpes encephalitis, without rapid initiation of treatment, may lead to severe disability or death.

The factors that give rise to binding enhancements when a strongly dimerizing vancomycin-group antibiotic (chloroeremomycin) binds to a model cell surface of vancomycin-resistant enterococci (VRE) have been semiquantitated. The model cell surface is comprised of vesicles to which have been anchored cell wall precursor analogues of vancomycin-resistant bacteria (which terminate in -D-lactate) via a hydrophobic docosanoyl (C-22) chain. Using H-1 and F-19 NMR spectroscopy, a large binding enhancement at the model cell surface (compared to the binding of an analogous ligand in free solution) has been observed. This enhancement can be partitioned into two distinct factors: a simple concentrating factor arising from an-increase in local concentration of ligand when it is located at the vesicle surface and a factor arising from the cooperative interaction of species mutually bound to the membrane surface. The overall enhancement to binding at a surface compared to binding in free solution was found to be a factor of 10(2)-10(3). In contrast, no significant surface binding enhancement was observed for the weakly dimerizing antibiotic vancomycin.

This review is an account of the discoveries in the Cambridge group of the structures and mode of action of the glycopeptide antibiotics of the vancomycin group. These antibiotics are now of enormous clinical importance, for among their members are two (vancomycin and teicoplanin) of the three antibiotics of last resort against resistant bacterial pathogens (particularly methicillin-resistant Staphylococcus aureus (MRSA, or “superbugs”)) in our hospitals, which would otherwise often be lethal. Their combined sales are of the order of US$1 billion per annum. The structure determination in our laboratory started just over 25 years ago. Within ten years the first glycopeptide structures had been determined. This was quickly followed by the determination of the molecular basis of their action, through the making of five hydrogen bonds to a cell wall peptide precursor that terminated in -Lys-d-Ala-d-Ala. In the early 1990s, we established that all the glycopeptides so far examined (other than teicoplanin) form dimers, and shortly after showed that dimerization promotes antibiotic activity. Concurrently, we were able to demonstrate that teicoplanin possesses a membrane anchor that can act to promote antibiotic activity in lieu of dimerization. The devices of dimerization and membrane anchoring, when acting concurrently, appear to be able to account at least in part for the remarkable activity of a new semi-synthetic glycopeptide—developed at Eli Lilly—against bacteria that are resistant even to vancomycin. A full understanding of these devices is important, since occurrences of the “nightmare scenario” that clinicians have feared over recent years—that resistance to vancomycin will appear in new forms of MRSA—have recently been reported.

Die letzte Verteidigungslinie gegen „Superbakterien” bilden die Antibiotica der Vancomycin-Gruppe. Wie aus der umfassenden Aufklarung des Wirkungsmechanismus der Antibiotica und eines Resistenzmechanismus gegen diese Wirkstoffe hevorgeht, kann diese Form der Resistenz uberwunden werden, ohne die Bindungsstelle des Antibioticums fur die Zellwandvorstufen pathogener Bakterien direkt zu modifizieren.

The synthesis of bacterial cell-wall precursor analogues, which resemble the naturally occurring precursor (lipid II) more closely than those hitherto used in NMR binding studies with glycopeptide antibiotics, is reported.

The N-terminal 17 residues of ubiquitin have been shown by 1H NMR to fold autonomously into a beta-hairpin structure in aqueous solution. This structure has a specific, native-like register, though side-chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N-terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space sampled, presumably as a consequence of the strongly beta-structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for beta-structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two-state folding model. Using published "random coil" values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the beta-region of conformational space even in the absence of the hairpin structure.

We report the synthesis of novel derivatives of the glycopeptide antibiotic vancomycin, modified at the N-terminus. Binding constants were measured for the association of these derivatives with the tripeptide analogue N,N-diacetyl-L-lysyl-D-alanyl-D-alanine. Replacement of the sp3 centre of the leucine residue of vancomycin with an sp2 centre resulted in weaker binding in all cases. These findings contrast with the relatively strong binding of some of the analogous derivatives previously obtained from ristocetin A. The reduction in the binding affinities of the vancomycin derivatives is attributed to a conformational change in the antibiotic which is not possible in the analogous derivatives of the aglycone of ristocetin.

The reliable estimation of binding constants by computational means for drugs binding to receptors represents one of the major challenges in molecular recognition. Many approaches to this problem have relied on the partitioning of binding free energy according to each of the interactions which are made or broken on binding, e.g., hydrogen bonds, salt bridges, π-π interactions, or the hydrophobic effect. With particular reference to the hydrophobic effect, we illustrate how such partitioning ignores one of the fundamental properties of systems of non-covalent interactions, namely that of cooperativity. This leads to estimates for the free energy benefit of the hydrophobic effect which vary according to the method of determination used, and whether the system in which the measurements are taken exhibits cooperativity. The context dependence of the magnitude of the hydrophobic effect illustrates the problem associated with considering weak interactions in isolation from the system in which they occur. The approach of partitioning binding affinities can be very useful, but until the problem of cooperativity can be better addressed by computational chemists, there remains the possibility that attempts to estimate at least some binding constants will unfortunately be seriously flawed.

In determining structure-activity relationships, it is advantageous if binding constants for a variety of ligands to a given target molecule can be directly obtained from a single aqueous solution containing a mixture of ligands and the target molecule. In this paper further evidence is provided showing that electrospray ionization mass spectrometry (ESI-MS) can be used in the rapid quantitative analysis of mixtures of vancomycin-group antibiotics and their bacterial cell-wall receptors allowing the identification of even subtle differences in binding constants. Differences in affinities are quantified for a mixture of vancomycin antibiotics (vancomycin, dechlorovancomycin and N-demethylvancomycin) and for a mixture of ristocetin A and its pseudoaglycone. Binding constants determined by ESI-MS were found to be in close agreement with those determined by more direct methods in aqueous solution.

Literature data describing the dilution of carboxylic acids in cyclohexane are reinterpreted according to a monomer–dimer equilibrium (higher order oligomers had previously been postulated in order to explain the data). Apparent dimerisation constants for each of the acids were calculated by incorporating a factor to take into account the varying relative permittivity of the solution during the titration. Dilution titrations of propionic acid using diluents of varying relative permittivities were also carried out and apparent dimerisation constants calculated in a similar manner. The dimerisation constants were found to vary according to the relative permittivity of the diluent, and the tightness of the dimers formed (as evidenced by the chemical shift of the carboxy proton) also varied according to the diluent relative permittivity. The results demonstrate that complex stability is directly related to the strength of the hydrogen bonds which go to form that complex. They also clarify a long-standing debate regarding the origin of an anomalous downfield shift of the carboxylic acid proton seen on dilution of simple aliphatic carboxylic acids with cyclohexane.

Pituitary adenomas are members of the family of neuroendocrine cells and tumors which have secretory granules containing chromogranins/secretogranins and other proteins. Pituitary adenomas express the neuroendocrine specific proconvertases PC1 (also known as PC3) and PC2, which are important for the proteolytic processing of chromogranins/secretogranins molecules. We examined the distribution of PC1 and PC2 in primary cultures of 20 pituitary adenomas and analyzed the regulation of the proconvertase mRNAs and proteins by various secretagogues including hypothalamic hormones and phorbol ester to determine the role of PC1 and PC2 in CgA processing in pituitary adenomas. Although PC2 was present in all adenomas, there was a differential distribution of PC1 with PRL adenomas expressing lower levels of PC1 compared to other adenoma types by RT-PCR analysis, in situ hybridization and immunostaining. Treatment of primary cultures of pituitary adenomas with phorbol 12-myristrate 13-acetate (PMA) resulted in an increase in pancreastatin (PST) secretion in most pituitary adenomas and increased PC1 mRNA and protein expression in gonadotroph adenomas, but not in other types of adenomas. Analysis of a human pituitary adenoma cell line, immortalized by recombinant defective adenovirus (HP75), which expressed chromogranin A, FSH, PC1 and PC2 showed that PST was secreted by these immortalized cells. Treatment with TGFβ1 resulted in an increase in PST secretion and in PC1 mRNA and protein. These results indicate that a) there is a differential distribution of PC1 in human pituitary adenomas with PRL adenomas expressing very little PC1 mRNA and protein and b) that PC1 expression in gonadotropin hormone-producing adenomas is regulated by PMA and TGFβ1. These findings support the observation that chromogranin A is a substrate for the endoproteinase PC1 in human pituitary adenoma cells.

The Bacillus thuringiensis Cry1Ac δ-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein–protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles.

The synthesis of covalent dimers of vancomycin which are linked from C- to N-terminus (head-to-tail linkage) is described. Such dimers have the potential to exploit additional cooperative interactions when binding to bacterial cell-wall precursors at a surface.

Background: The emergence of bacteria that are resistant to vancomycin, the drug of choice against methicillin-resistant Staphylococcus aureus , has made the study of the binding characteristics of glycopeptides to biologically relevant depsipeptides important. These depsipeptides, terminating in -d-alanyl-d-lactate, mimic the cell-wall precursors of resistant bacteria. Results: The use of 19 F-labelled ligands in the study of the therapeutically important vancomycin series of antibiotics is demonstrated. The substantial simplification of spectra that occurs when such labelled ligands are employed is used in the measurement of binding affinities of depsipeptides to chloroeremomycin (CE). Large enhancements of binding affinities are found at a model bacterial cell-wall surface (constituted from depsipetides that are anchored into vesicles) relative to those measured in free solution. Conclusions: Surface-enhanced binding, previously shown for strongly dimerising glycopeptide antibiotics to normal -d-alanyl-d-alanine-terminating cell-wall precursors, is now demonstrated for CE to the surface of models of VanA- and VanB-resistant bacteria. The effect of depsipeptide chain length is shown to be critically important in producing and maximising this enhancement.

The cooperativity between binding of cell wall precursor analogs (ligands) to and antibiotic dimerization of the clinically important vancomycin group antibiotics was investigated by nuclear magnetic resonance. When dimerization was weak in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the dimer interface. When dimerization was strong in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the ligand-antibiotic interface. These results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein can derive in part from changes occurring within the protein.

The binding of a ligand to the asymmetric homodimer of ristocetin A gives rise to a substantial reduction in dimerisation constant. This reduction is due to a higher binding affinity of ligand for monomer than for average dimer. The process can be clearly and usefully monitored by 19F NMR spectroscopy.

The dimerisation constant of the vancomycin group antibiotic ristocetin A has previously been shown to be lower when it is fully bound by ligand (analogues of bacterial cell wall precursors terminating in –Lys-D-Ala-D-Ala) than in its absence, i.e. dimerisation is anticooperative with ligand binding. A derivative of ristocetin A, desrhamno-ristocetin, has now been produced by enzymatic degradation, and the dimerisation constant of this derivative has been measured in the absence and presence of the bacterial cell wall precursor analogue N-acetyl-D-Ala-D-Ala. The dimerisation constant is shown to be greater in the presence of the ligand than in its absence, i.e. dimerisation is cooperative with ligand binding. This change in behaviour from anticooperativity to cooperativity is postulated to be associated with the partial equalisation of the binding affinities of the two sides of the dimer for ligand. It is therefore energetically more favourable for two ligand molecules to bind to the two halves of a desrhamno-ristocetin dimer than to two monomers.

Non-covalent complexes between the glycopeptide antibiotic vancomycin and the cell-wall precursor analog N-acetyl-D-Ala-D-Ala, as well as its deuterated enantiomer N-acetyl-L-Ala-L-Ala-d3 were probed using electrospray ionization mass spectrometry. In solution, the D,D-peptide interacts strongly with the antibiotic, whereas the L,L-peptide shows no observable complexation. Using a competition strategy, mass spectra were obtained from solutions containing vancomycin, N-acetyl-D-Ala-D-Ala, and N-acetyl-L-Ala-L-Ala-d3. With acetonitrile-water as the solvent, the ratio of free vancomycin to vancomycin complex was much higher than expected from solution interactions and the specific D,D-peptide complex and the non-specific L,L-peptide complex were detected in a 1.7 : 1 ratio. In contrast, sprayed from an ammonium acetate buffer solution both the amount and specificity of observed complex reflected solution interactions (only D,D-peptide complex was found). Thus, our results demonstrate that in electrospray mass spectrometry the conditions must carefully be chosen and control experiments must be carried out in order to exclude non-specific aggregation as a possible cause of complex formation.

The self-regulation of biological signalling receptors via homodimerization is discussed in relation to the symmetry changes occurring when these receptors bind their target ligand. The idea of positive and negative cooperativity between dimerization and ligand binding, mediated by changes in the symmetry of the system, as a source of signalling control is considered; and an analogy made with the homodimerization of a glycopeptide antibiotic, ristocetin A, which displays negative cooperativity. Finally, the regulation of the bacterial aspartate receptor and the human growth hormone receptor is discussed as a function of ligand-induced asymmetry. Chirality 10:14–23, 1998. © 1998 Wiley-Liss, Inc.