Impaired nucleotide excision repair pathway as a possible factor in pathogenesis of head and neck cancer.

Tobacco smoking is one of the major risk factors in pathogenesis of head and neck squamous cell carcinomas (HNSCC). Many of the chemical compounds present in tobacco are well-known carcinogens which form adducts with DNA. Cells remove these adducts mainly by the nucleotide excision repair pathway (NER). NER also eliminates a broad spectrum of pyrimidine dimers (CPD) and photo-products (6-4PP) induced by UV-radiation or DNA cross-links after cisplatin anti-cancer treatment. In this study DNA damage and repair was examined in peripheral blood lymphocytes obtained from 20 HNSCC patients and 20 healthy controls as well as HTB-43 larynx and SSC-25 tongue cancer cell lines. DNA repair kinetics in the examined cells after cisplatin or UV-radiation treatment were investigated using alkaline comet assay during 240min of post-treatment incubation. MTT assay was used to analyse cell viability and the Annexin V-FITC kit specific for kinase-3 was employed to determine apoptosis after treating the cells with UV-radiation at dose range from 0.5 to 60J/m(2). NER capability was assessed in vitro with cell extracts by the use of a bacterial plasmid irradiated with UV-light as a substrate for the repair. The results show that lymphocytes from HNSCC patients and HTB-43 or SSC-25 cancer cells were more sensitive to genotoxic treatment with UV-radiation and displayed impaired DNA repair. Also evidenced was a higher rate of apoptosis induction after UV-radiation treatment of lymphocytes from the HNSCC patients and the HTB-43 cancer cells than after treatment of those from healthy donors. Finally, our results showed that there was a significant decrease in NER capacity in HTB-43 or SSC-25 cancer cells as well as in peripheral blood lymphocytes of HNSCC patients compared to controls. In conclusion, we suggest that the impaired NER pathway might be a critical factor in pathogenesis of head and neck cancer.

Analysis of postural sway in patients with normal pressure hydrocephalus: effects of shunt implantation.

Poor postural balance is one of the major risk factors for falling in normal pressure hydrocephalus (NPH). Postural instability in the clinic is commonly assessed based upon force platform posturography. In this study we focused on the identification of changes in sway characteristics while standing quiet in patients with NPH before and after shunt implantation. Postural sway area and sway radius were analyzed in a group of 9 patients and 46 controls of both genders. Subject's spontaneous sway was recorded while standing quiet on a force platform for 30-60 s, with eyes open and then closed. Both analyzed sway descriptors identified between-group differences and also an effect of shunt implantation in the NPH group. Sway radius and sway area in patients exhibited very high values compared with those in the control group. Importantly, the effect of eyesight in patients was not observed before shunt implantation and reappeared after the surgical treatment. The study documents that static force platform posturography may be a reliable measure of postural control improvement due to shunt surgery.

Assessment of postural instability in patients with Parkinson's disease.

Postural instability is one of the most disabling features of idiopathic Parkinson's disease (PD). In this study, we focused on postural instability as the main factor predisposing parkinsonians to falls. For this purpose, changes in sway characteristics during quiet stance due to visual feedback exclusion were studied. We searched for postural sway measures that could be potential discriminators for an increased fall risk. A group of 110 subjects: 55 parkinsonians (Hoehn and Yahr: 1-3), and 55 age-matched healthy volunteers participated in the experiment. Their spontaneous sway characteristics while standing quiet with eyes open and eyes closed were analyzed. We found that an increased mediolateral sway and sway area while standing with eyes closed are characteristic of parkinsonian postural instability and may serve to quantify well a tendency to fall. These sway indices significantly correlated with disease severity rated both by the Hoehn and Yahr scale as well as by the Motor Section of the UPDRS. A forward shift of a mean COP position in parkinsonians which reflects their flexed posture was also significantly greater to compare with the elderly subjects and exhibited a high sensitivity to visual conditions. Both groups of postural sway abnormalities identified here may be used as accessible and reliable measures which allow for quantitative assessment of postural instability in Parkinson's disease.

Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage.

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

Gait transitions during unrestrained locomotion in dogs.

Gait transitions during long distance, unrestrained locomotion were studied in 22 mongrel dogs. Spatial and temporal limb movement parameters were collected and the phase relationships between limb movements based upon a 2-dimensional (2-D) gait diagram were computed. During most of the trials, the dogs trotted within a relatively narrow velocity range. Gait transitions were observed during radical changes of the movement velocity. In most cases the gait switches were abrupt and completed within 2 strides of the gait cycle. The dogs walked, depending on the animal size, within the upper velocity range of 0.93-1.21 m/s. Most of the walk-trot transitions were observed within this range. All of them had a typical pattern that involved changes of the phase shift between diagonal limb movements from 0.31 +/- 0.02 (a typical value for a walking dog) down to 0.02 +/- 0.03. These changes appeared abruptly within one stride cycle for each diagonal pair of limbs; therefore, the transition was completed in 2 strides of the gait cycle. The switch involved momentary shortening of the hindlimb amplitudes. During the next gait cycle, all limb movement amplitudes were reduced with a concomitant increase in limb movement frequencies. In contrast to the clear border between the symmetrical gaits, the dogs switched to gallop at any speed within the trot range (most frequently between 1.5-2.6 m/s). The transitions were usually completed within one stride of the diagonal limbs. In most cases, the switch from trot to gallop had a similar pattern; while maintaining synchronous movement of one diagonal pair of limbs, the other pair movement control was modified accordingly. The typical transition pattern involved the shortening of the swing phase in the front limb with simultaneous lengthening of the swing phase in the diagonal hindlimb. These transient modifications had their equivalent in the analogous limb movement amplitude changes. A mirror-image pattern of phase changes was observed in the majority (82%) of the gallop-trot transitions. Three of the dogs, however, besides the typical gallop-trot switch, occasionally employed a second pattern of transition.

Unusual conformational changes in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by X-ray crystallography and NMR.

The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-A resolution with a crystallographic R factor of 0.191. The solution structure of HPPK in complex with Mg(2+) and beta,gamma-methyleneadenosine 5'-triphosphate (MgAMPPCP) has been determined using a simulated annealing protocol with 3,523 experimental NMR restraints. The root mean square deviation of the ensemble of 20 refined conformers that represent the solution structure from the mean coordinate set derived from them is 0.74 +/- 0.26 A for all backbone atoms and 0.49 +/- 0.22 A when residues Pro(14), Pro(44)-Gln(50), and Arg(84)-Pro(91) are excluded. Binding of MgADP causes significant changes in the conformation and dynamical property of three loops of HPPK that are involved in catalysis. A dramatic, unusual conformational change is that loop 3 moves away from the active center significantly with some residues moving by >17 A. The binding of MgADP also stabilizes loop 1 and loop 3 but makes loop 2 more mobile. Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK.MgAMPPCP. The conformational and dynamical changes may play important roles in both substrate binding and product release in the catalytic cycle.

Postural stability and fractal dynamics.

Methods of non-linear dynamics and deterministic chaos may provide us with effective quantitative descriptors of the dynamics of postural control. The goal of this study was to introduce a new measure, which would allow to determine the fractal structure of posturographic signals and to measure the effect of the loss of visual feedback information in postural control. The results of the study show that fractal dimension (Df) is a very useful, reliable and sensitive measure of the complexity of posturographic signals. Therefore Df can be used for the evaluation of postural stability and its changes due to pathology or an age-related decline.

The absorption edge of protein-bound mercury and a double-edge strategy for HgMAD data acquisition.

The L(III) absorption edge of protein-bound mercury (Hg) has been experimentally determined using X-ray data collection from a crystal. This absorption edge is 12 291 eV, 4 eV higher than the theoretical value of elemental Hg. Considering the possible shift of the Hg absorption edge with the chemical environment in different protein crystals, a double-edge strategy for multiwavelength anomalous diffraction (MAD) data collection has been developed. The approach provides a convenient way to optimize the dispersive signal between a remote wavelength and two edge wavelengths separated from each other by 4 eV. The dispersive signals derived from both edges are used, along with anomalous signals, in MAD phasing and phase refinement. This approach has been used in the crystal structure determination of three proteins containing one Hg atom per 186-196 amino-acid residues at 2.0, 2.6 and 2.7 A resolution. A set of four wavelengths is recommended for HgMAD data acquisition: 1.0087 A (12 291 eV, edge1), 1.0084 A (12 295 eV, edge2), 1.0064 A (12 320 eV, peak) and 0.9918 A (12 500 eV, remote). Although it is no longer necessary to determine the L(III) absorption edge of protein-bound Hg experimentally, an initial fluorescence scan on the crystal for data collection is still necessary to verify the existence of Hg in the crystal.

[Evaluation of oxygen free radical generation and antioxidative enzymatic activity in blood of patients with carcinoma of the larynx (preliminary report)]. / Ocena generacji anionorodnika ponadtlenkowego oraz antyoksydacyjnej aktywnosci enzymatycznej we krwi obwodowej chorych z rakiem krtani (doniesienie wstepne).

The aim of the study were the evaluation of generation of oxygen free radical and antioxidative enzymatic activity in blood of patients with carcinoma of larynx (7 persons, aged 48-68) in comparison with healthy persons (15, aged 21-28). The superoxide anion generation of blood granulocytes described by the rate of the reducted cytochrom C at rest and after stimulation with opsonized zymosan according to Bellavite et al. were measured. The enzymatic activity of superoxide dismutase in blood (method of Misra and Fridovich), catalase (method of Beers and Sizer) and malonyl dialdehyde (method of Placer et al.). The obtained data pointed at the growth of generation of oxygen free radical in blood (at rest and after stimulation with opsonized zymosan) of patients with carcinoma of larynx in comparison with the healthy ones. At the same patients with carcinoma of larynx observed at significant reduction of antioxidative enzymatic activity against the oxygen species generation in blood and significant growth of peroxidation lipids and disturbances of function of cells the human body.

Effect of type II diabetic peripheral neuropathy on gait termination in the elderly.

AIMS/HYPOTHESIS: Healthy elderly people can have difficulties in precisely terminating gait due to age-related decline. Diabetes mellitus accelerates the neurodegenerative process, which results in an additional decline in motor control. This biomechanical study investigated goal-oriented gait termination in healthy elderly and elderly diabetic subjects. The trajectories of the centre of pressure and the centre of mass during the gait termination process were analysed in particular. It was hypothesised that the pathology results in an unstable gait termination, expressed in larger overshoots of the centre of pressure and the centre of mass than in healthy control subjects. METHODS: A total of 15 subjects with Type II (non-insulin-dependent) diabetes mellitus with impaired foot sensitivity due to polyneuropathy (median, 66 years) were matched according to age, gender and body mass index with 15 healthy elderly subjects (median, 67 years). The participants walked at their own pace along the walkway and stopped in front of a marked stopping line while kinetic and kinematic data were recorded. RESULTS: The diabetic subjects approached the stopping line more slowly (p = 0.002) than the healthy elderly subjects. They also exhibited a weaker maximal braking force (p = 0.011) and a prolonged relative time to develop this force (p = 0.023). Despite this slower motion, the centre of pressure overshoots were larger in the diabetic subjects than in the healthy elderly (p = 0.027). CONCLUSION/INTERPRETATION: The results show differences between healthy elderly and diabetic subjects during easy goal-oriented stopping tasks. Changes in gait termination parameters and the increased overshoots in particular document the pathology-related decline in postural stability.

Structure and dynamics of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in folate pathway, and therefore, is an ideal target for developing novel antimicrobial agents. Because of its small size and high thermal stability, E. coli HPPK is also an excellent model enzyme for studying the mechanisms of enzymatic pyrophosphoryl transfer. We have determined the crystal structures of HPPK in the unligated form and in complex with HP, two Mg2+ ions, and AMPCPP (an ATP analog that inhibits the enzymatic reaction). Comparison of the two crystal structures reveals dramatic conformational changes of three flexible loops and many side chains and possible roles of the active site residues.

Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: synthesis and biochemical and crystallographic studies.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. Like other enzymes in the folate pathway, HPPK is an ideal target for the development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from human and animals. Three bisubstrate analogues have been synthesized for HPPK and characterized by biochemical and X-ray crystallographic analyses. All three bisubstrate analogues consist of a pterin, an adenosine moiety, and a link composed of 2-4 phosphoryl groups. P(1)-(6-Hydroxymethylpterin)-P(2)-(5'-adenosyl)diphosphate (HP(2)A, 5) shows little affinity and inhibitory activity for E. coli HPPK. P(1)-(6-Hydroxymethylpterin)-P(3)-(5'-adenosyl)triphosphate (HP(3)A, 6) shows moderate affinity and inhibitory activity with K(d) = 4.25 microM in the presence of Mg(2+) and IC(50) = 1.27 microM. P(1)-(6-Hydroxymethylpterin)-P(4)-(5'-adenosyl)tetraphosphate (HP(4)A, 7) shows the highest affinity and inhibitory activity with K(d) = 0.47 microM in the presence of Mg(2+) and IC(50) = 0.44 microM. The affinity of MgHP(4)A for HPPK is approximately 116 and 76 times higher than that of MgADP and 6-hydroxymethylpterin, respectively. The crystal structure of HPPK in complex with 7 (HPPK.MgHP(4)A) has been determined at 1.85 A resolution with a crystallographic R factor of 0.185. The crystal structure shows that 7 occupies both HP- and ATP-binding sites and induces significant conformational changes in HPPK. The biochemical and structural studies of the bisubstrate analogues indicate that the bisubstrate analogue approach can produce more potent inhibitors for HPPK and the minimum length of the link for a bisubstrate analogue is approximately 7 A.

Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes.

The crystal structure of guanylate kinase (GK) from yeast (Saccharomyces cerevisiae) with a non-acetylated N terminus has been determined in its unligated form (apo-GK) as well as in complex with GMP (GK.GMP). The structure of apo-GK was solved with multiwavelength anomalous diffraction data and refined to an R-factor of 0.164 (R(free)=0.199) at 2.3 A resolution. The structure of GK.GMP was determined using the crystal structure of GK with an acetylated N terminus as the search model and refined to an R-factor of 0.156 (R(free)=0.245) at 1.9 A. GK belongs to the family of nucleoside monophosphate (NMP) kinases and catalyzes the reversible phosphoryl transfer from ATP to GMP. Like other NMP kinases, GK consists of three dynamic domains: the CORE, LID, and NMP-binding domains. Dramatic movements of the GMP-binding domain and smaller but significant movements of the LID domain have been revealed by comparing the structures of apo-GK and GK.GMP. apo-GK has a much more open conformation than the GK.GMP complex. Systematic analysis of the domain movements using the program DynDom shows that the large movements of the GMP-binding domain involve a rotation around an effective hinge axis approximately parallel with helix 3, which connects the GMP-binding and CORE domains. The C-terminal portion of helix 3, which connects to the CORE domain, has strikingly higher temperature factors in GK.GMP than in apo-GK, indicating that these residues become more mobile upon GMP binding. The results suggest that helix 3 plays an important role in domain movement. Unlike the GMP-binding domain, which moves toward the active center of the enzyme upon GMP binding, the LID domain moves away from the active center and makes the presumed ATP-binding site more open. Therefore, the LID domain movement may facilitate the binding of MgATP. The structure of the recombinant GK.GMP complex superimposes very well with that of the native GK.GMP complex, indicating that N-terminal acetylation does not have significant impact on the three-dimensional structure of GK.

Epidemiology of childhood bacterial meningitis in Poland. Incidence of bacterial meningitis with special reference to Haemophilus influenzae type b among children 0-59 months old in the former Kielce and Bydgoszcz districts in Poland in 1998-1999.

Population based surveillance was undertaken to assess the incidence of meningitis caused by Haemophilus influenzae type b in children 0-59 months old in Kielce and Bydgoszcz districts in Poland in 1998 and 1999. The cases were prospectively identified in pediatric and neuroinfection wards of local hospitals where all cases of children with suspected meningitis are referred in both districts. The mean annual incidence of meningitis caused by Haemophilus influenzae type b in children 0-59 months old in Kielce district during the study period was estimated at 3.1 per 100,000 per year (10.3% of cases of bacterial meningitis with confirmed etiology). In Bydgoszcz district, the annual incidence was 9.7 per 100,000 (50% of confirmed cases). These estimations are lower than reported in most Western European countries before the immunization against Hib was introduced. Small numbers of Hib vaccinations reported from both districts do not seem likely to have influenced the data significantly.

[Generation of reactive oxygen species by peripheral blood neutrophils in patients with pulmonary tuberculosis]. / Wytwarzanie reaktywnych form tlenu przez neutrofile krwi obwodowej chorych na gruzlice pluc.

Oxygen metabolism of neutrophils was measured using chemiluminescence method in patient with pulmonary tuberculosis. Patients were included into three groups. Group I--20 patients (10 men, 10 women) aged 24-74 years (mean 48.3 years) with pulmonary tuberculosis BK(+). Group II--20 patients (15 men, 5 women) aged 19-67 years (mean 45.1 years) with pulmonary tuberculosis BK(0). The control group consisted 16 clinically healthy persons (12 men, 4 women) aged 28-59 years (mean 42.5 years). Blood samples (5 ml) were collected for examination from cubital vein early morning before breakfast. A luminometer 1251 coupled with an IBM PC AT compatible computer was used for the measurement of chemiluminescence. The chemiluminescence of non-stimulated neutrophils and those stimulated by a receptor stimulus formyl-Met-Leu-Phe (fMLP) as well as an extrareceptor stimulus phorbol myristate acete (PMA) was measured. The results of our study demonstrated that chemiluminescence of non-stimulated as well as stimulated by fMLP and PMA neutrophils was lower in comparison to the values of chemiluminescence in the control group.

[Estimation of plasma malonyl dialdehyde concentration in patients with pulmonary tuberculosis]. / Ocena stezenia dialdehydu malonowego w osoczu chorych na gruzlice pluc.

Plasma malonyl dialdehyde (MDA) concentration was determined by Placer method in patients with pulmonary tuberculosis before and during treatment period with tuberculostatic drugs. Patients were divided into three groups. Group I comprised 20 patients (10 men and 10 women) aged 24-74 years (mean 48.3 years) with pulmonary tuberculosis BK(+). Group II comprised 20 patients (15 men and 5 women) aged 19-67 years (mean 45.1 years) with pulmonary tuberculosis BK(0). The control group consisted of 16 clinically healthy persons (12 men and 4 women) aged 28-59 years (mean 42.5 years). In patients blood samples (5 ml) were collected for examination from cubital vein before, after 1-month and 2-month treatment period with tuberculostatic drugs. In the control group blood samples were collected from cubital vein once. Results of our study showed that plasma MDA concentrations in patients with pulmonary tuberculosis, both before and during treatment period with tuberculostatic drugs, were significantly higher, in comparison to the control group.

Acoustic startle and open-field behavior in mice bred for magnitude of swim analgesia.

Acoustic startle response (ASR) and open-field activity was examined in the 46th generation of mice that have been selectively bred for high analgesia (HA) and for low analgesia (LA) induced by 3-min swimming in 20 degrees C water. These lines were earlier found to differ in brain opioid receptor density and in the expression of opioid-mediated phenomena, as analgesic sensitivity to opiates and reversibility of swim stress-induced analgesia (SSIA) by naloxone. For comparison, a randomly bred control (C) line was used. To measure the amplitude of ASR, the mice were exposed to 110-dB acoustic stimuli in a Coulbourn apparatus. In saline-injected mice, the ASR force was found significantly lower in the LA than in the HA, as well in the C line, but did not differ between the two last lines. Naltrexone hydrochloride (10 mg/kg IP 30 min before ASR testing) augmented the startle in the opioid receptor-dense HA line, but had no effect in the opioid receptor-deficient LA line, as well in the C line; therefore, the ASR magnitude in naltrexone-injected HA mice was significantly higher compared to the C line. HA mice displayed less activity in an open-field test; that is, they remained immobile longer in the center of the field, and thereafter performed less ambulation and less rearing against the wall compared to the LA line. Naltrexone failed to modify the open-field activity in any line. The results confirm that the pattern of ASR depends on the genetic makeup of the animals. The higher amplitude of ASR, taken together with the lower open-field activity of HA mice, can be interpreted in terms of higher anxiety level, compared to the LA line. It is suggested that the higher ASR in HA mice relies on a nonopioid mechanism, which is tonically inhibited by the opioid system.

Catalytic center assembly of HPPK as revealed by the crystal structure of a ternary complex at 1.25 A resolution.

BACKGROUND: Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7, 8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate pathway, and therefore is an ideal target for developing novel antimicrobial agents. HPPK from Escherichia coli is a 158-residue thermostable protein that provides a convenient model system for mechanistic studies. Crystal structures have been reported for HPPK without bound ligand, containing an HP analog, and complexed with an HP analog, two Mg(2+) ions, and ATP. RESULTS: We present the 1.25 A crystal structure of HPPK in complex with HP, two Mg(2+) ions, and AMPCPP (an ATP analog that inhibits the enzymatic reaction). This structure demonstrates that the enzyme seals the active center where the reaction occurs. The comparison with unligated HPPK reveals dramatic conformational changes of three flexible loops and many sidechains. The coordination of Mg(2+) ions has been defined and the roles of 26 residues have been derived. CONCLUSIONS: HPPK-HP-MgAMPCPP mimics most closely the natural ternary complex of HPPK and provides details of protein-substrate interactions. The coordination of the two Mg(2+) ions helps create the correct geometry for the one-step reaction of pyrophosphoryl transfer, for which we suggest an in-line single displacement mechanism with some associative character in the transition state. The rigidity of the adenine-binding pocket and hydrogen bonds are responsible for adenosine specificity. The nonconserved residues that interact with the substrate might be responsible for the species-dependent properties of an isozyme.

X-ray and 13C solid-state NMR studies of N-benzoyl-phenylalanine.

A crystalline sample of N-benzoyl-DL-phenylalanine 1 and a polycrystalline sample of N-benzoyl-L-phenylalanine 2 were studied using 13C high-resolution solid-state NMR spectroscopy. The X-ray structure of the DL form was established. Sample 1 crystallizes in a monoclinic form with a P21/c space group, a=11.338(1) A, b=9.185(1) A, c=14.096(2) A, beta=107.53(3) degrees, V=1400(3) A3, Z=4 and R=0.053. The principal elements of the 13C chemical shift tensors deltaii for 1 and 2, selectively 13C (99%) labeled at the carboxyl groups were calculated. On the basis of 13C (delta)ii analysis the hydrogen bonding pattern for sample 2 was deduced. Enriched samples were used to establish the intermolecular distance between chemically equivalent nuclei for 1 and spatial proximity in heterogeneous domain for 2, employing the ODESSA pulse sequence. The consistence of the complementary approach covering X-ray data, analysis of the 13C (delta)ii parameters and ODESSA results is revealed.

Complete crystal structure of monocyte chemotactic protein-2, a CC chemokine that interacts with multiple receptors.

Monocyte chemotactic protein 2 (MCP-2) is a CC chemokine that utilizes multiple cellular receptors to attract and activate human leukocytes. MCP-2 is a potent inhibitor of HIV-1 by virtue of its high-affinity binding to the receptor CCR5, one of the major coreceptors for HIV-1. Although a few structures of CC chemokines have been reported, none of these was determined with the N-terminal pyroglutamic acid residue (pGlu1) and a complete C-terminus. pGlu1 is essential for the chemotactic activity of MCP-2. Recombinant MCP-2 has Gln1 at the N terminus, 12-15% of which cyclizes automatically and forms pGlu1. The chemotactic activity of such MCP-2 mixture, which contains 12-15% pGlu1-form and 85-88% Gln1-form protein, is approximately 10 times lower when compared with that of fully cyclized MCP-2 preparation. Therefore, this chemokine is practically inactive without pGlu1. We have determined the complete crystal structure of MCP-2 that contains both pGlu1 and an intact C-terminus. With the existence of pGlu1, the conformation of the N-terminus allows two additional interactions between the two subunits of MCP-2 dimer: a hydrogen bond between pGlu1 and Asn17 and a salt bridge between Asp3 and Arg18. Consequently, both pGlu1 are anchored and buried, and thereby, both N-terminal regions are protected against protease degradation. We have also observed not previously reported extended helical nature of the C terminal region, which covers residues 58-74.