Mental health and well-being in parents of excessively crying infants: Prospective evaluation of a support package.

BACKGROUND: During the first 4 months of age, approximately 20% of infants cry a lot without an apparent reason. Most research has targeted the crying, but the impact of the crying on parents, and subsequent outcomes, need to receive equal attention. This study reports the findings from a prospective evaluation of a package of materials designed to support the well-being and mental health of parents who judge their infant to be crying excessively. The resulting "Surviving Crying" package comprised a website, printed materials, and programme of Cognitive Behaviour Therapy-based support sessions delivered to parents by a qualified practitioner. It was designed to be suitable for United Kingdom (UK) National Health Service (NHS) use. METHODS: Parents were referred to the study by 12 NHS Health Visitor/Community Public Health Nurse teams in one UK East Midlands NHS Trust. Fifty-two of 57 parents of excessively crying babies received the support package and completed the Edinburgh Postnatal Depression Scale and Generalized Anxiety Disorder-7 anxiety questionnaire, as well as other measures, before receiving the support package and afterwards. RESULTS: Significant reductions in depression and anxiety were found, with numbers of parents meeting clinical criteria for depression or anxiety halving between baseline and outcome. These improvements were not explained by reductions in infant crying. Reductions also occurred in the number of parents reporting the crying to be a large or severe problem (from 28 to 3 parents) or feeling very or extremely frustrated by the crying (from 31 to 1 parent). Other findings included increases in parents' confidence, knowledge of infant crying, and improvements in parents' sleep. CONCLUSIONS: The findings suggest that the Surviving Crying package may be effective in supporting the well-being and mental health of parents of excessively crying babies. Further, large-scale controlled trials of the package in NHS settings are warranted.

Septin crystallization for structural analysis.

Septins are filament-forming proteins found in many eukaryotes. Despite being important components of the cytoskeleton, only recently details of their macromolecular assemblies and crystal structures have started to appear in the literature. These are of fundamental importance to the understanding of cytoskeleton dynamics, membrane barrier formation, and bacterial caging, as well as essential cellular processes such as cell division, exocytosis, and vesicle trafficking. However, obtaining this data is frequently hindered by several experimental difficulties common to the majority of septin samples. Here we provide an overview of the current approaches to circumvent or minimize the experimental complications observed in septin crystallography focusing mainly, but not exclusively, on the choice of the septin construct and how to best prepare the sample itself.

Effects of exercise training on neuromuscular junction morphology and pre- to post-synaptic coupling in young and aged rats.

The objective of this study was to determine whether pre- to post-synaptic coupling of the neuromuscular junction (NMJ) could be maintained in the face of significant morphological remodeling brought about by exercise training, and whether aging altered this capacity. Eighteen young adult (8 mo) and eighteen aged (24 mo) Fischer 344 rats were randomly assigned to either endurance trained (treadmill running) or untrained control conditions resulting in four groups (N=9/group). After the 10-week intervention rats were euthanized and hindlimb muscles were surgically removed, quickly frozen at approximate resting length and stored at -85°C. The plantaris and EDL muscles were selected for study as they have different functions (ankle extensor and ankle flexor, respectively) but both are similarly and overwhelmingly comprised of fast-twitch myofibers. NMJs were stained with immunofluorescent procedures and images were collected with confocal microscopy. Each variable of interest was analyzed with a 2-way ANOVA with main effects of age and endurance training; in all cases significance was set at P⩽0.05. Results showed that no main effects of aging were detected in NMJs of either the plantaris or the EDL. Similarly, endurance training failed to alter any synaptic parameters of EDL muscles. The same exercise stimulus in the plantaris however, resulted in significant pre- and post-synaptic remodeling, but without altering pre- to post-synaptic coupling of the NMJs. Myofiber profiles of the same plantaris and EDL muscles were also analyzed. Unlike NMJs, myofibers displayed significant age-related atrophy in both the plantaris and EDL muscles. Overall, these results confirm that despite significant training-induced reconfiguration of NMJs, pre- to post-synaptic coupling remains intact underscoring the importance of maintaining proper apposition of neurotransmitter release and binding sites so that effective nerve to muscle communication is assured.

Crystallization and preliminary structural analysis of the giant haemoglobin from Glossoscolex paulistus at 3.2 Å.

Glossoscolex paulistus is a free-living earthworm encountered in south-east Brazil. Its oxygen transport requirements are undertaken by a giant extracellular haemoglobin, or erythrocruorin (HbGp), which has an approximate molecular mass of 3.6 MDa and, by analogy with its homologue from Lumbricus terrestris (HbLt), is believed to be composed of a total of 180 polypeptide chains. In the present work the full 3.6 MDa particle in its cyanomet state was purified and crystallized using sodium citrate or PEG8000 as precipitant. The crystals contain one-quarter of the full particle in the asymmetric unit of the I222 cell and have parameters of a = 270.8 Å, b = 320.3 Å and c = 332.4 Å. Diffraction data were collected to 3.15 Å using synchrotron radiation on beamline X29A at the Brookhaven National Laboratory and represent the highest resolution data described to date for similar erythrocruorins. The structure was solved by molecular replacement using a search model corresponding to one-twelfth of its homologue from HbLt. This revealed that HbGp belongs to the type I class of erythrocruorins and provided an interpretable initial electron density map in which many features including the haem groups and disulfide bonds could be identified.

Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1.

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 A resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 A. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

Crystal structure of calf spleen purine nucleoside phosphorylase complexed to a novel purine analogue.

The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.

Getting the most out of X-ray home sources.

The structures of a 14 kDa phospholipase, an 18 kDa proteinase inhibitor and a novel glycoside hydrolase with molecular weight 60 kDa were solved using the SAD technique and the effects of the amount of anomalous signal, completeness and redundancy of data on heavy-atom substructure determination, phasing and model building were analyzed. All diffraction data sets were collected on a Cu-anode X-ray home source. The structure of the phospholipase was obtained using the anomalous scattering contribution from its 16 S atoms. Three-dimensional models for the other two macromolecules were obtained using the anomalous contribution of I atoms rapidly incorporated into the crystal through the quick cryo-soaking method of derivatization. These results were used to discuss the application of sulfur- and iodine-SAD approaches in combination with X-ray home sources for high-throughput protein crystal structure solution. The estimates of the anomalous signal from S atoms in the gene products of four genomes are given and the prospects for increasing the anomalous contribution using longer wavelengths (e.g. from a chromium home source) and quick cryo-soaking derivatization are discussed. The possibility of rapidly preparing tangible home-source isomorphous derivatives suggests that this approach might become a valuable tool in the future of post-genomic projects.

Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose.

Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date.

Crystal structure of human phosphoglucose isomerase and analysis of the initial catalytic steps.

The second enzyme in the glycolytic pathway, phosphoglucose isomerase (PGI), catalyses an intracellular aldose-ketose isomerization. Here we describe the human recombinant PGI structure (hPGI) solved in the absence of active site ligands. Crystals isomorphous to those previously reported were used to collect a 94% complete data set to a limiting resolution of 2.1 A. From the comparison between the free active site hPGI structure and the available human and rabbit PGI (rPGI) structures, a mechanism for protein initial catalytic steps is proposed. Binding of the phosphate moiety of the substrate to two distinct elements of the active site is responsible for driving a series of structural changes resulting in the polarisation of the active site histidine, priming it for the initial ring-opening step of catalysis.

Expression and preliminary X-ray diffraction studies of cytosolic Cu,Zn superoxide dismutase from Schistosoma mansoni.

Active cytosolic (CT) Cu,Zn superoxide dismutase from Schistosoma mansoni (SmCTSOD) was recovered after thrombin cleavage of a glutathione-S-transferase linked fusion protein (GST-SmCTSOD) expressed in the presence of the active-site metals. Crystals have been obtained in two space groups, P2(1)2(1)2(1) and P2(1). The former have unit-cell parameters a = 74.64, b = 78.24, c = 95.18 A and typically diffract to 2.2 A. The monoclinic crystals have unit-cell parameters a = 39.27, b = 95.08, c = 78.41 A, beta = 103.55 degrees and diffract to at least 1.55 A. The calculated solvent content of the crystals is compatible with two dimers of SmCTSOD in the asymmetric unit in both cases. Molecular-replacement solutions have been obtained for both crystal forms and show that slight distortions in the crystal packing relate one form to the other.

Crystallization and preliminary X-ray analysis of jararhagin, a metalloproteinase/disintegrin from Bothrops jararaca snake venom.

Jararhagin is a toxic protein, isolated from the venom of the snake Bothrops jararaca, which is composed of a metalloprotease domain coupled to a disintegrin/cysteine-rich domain. It induces local haemorrhage owing to the proteolytic digestion of the basement membrane of capillaries. Jararhagin also cleaves the alpha(2)beta(1) integrin on the surface of platelets, thereby acting as a potent inhibitor of collagen-induced platelet aggregation. Crystals of jararhagin were obtained by the vapour-diffusion technique at 273 K in 200 mM sodium acetate, 100 mM cacodylate buffer pH 6.5 and 30% PEG 8000. Diffraction data have been obtained to a resolution of 2.8 A from a single frozen crystal, which belonged to space group P2(1)2(1)2(1) with unit-cell parameters a = 73.7, b = 100.3, c = 133.4 A. The asymmetric unit contains two jararhagin molecules and has a solvent content of 45%. A molecular-replacement solution has been obtained using a homology-built model based on the crystal structure of acutolysin, a haemorrhagic zinc metalloproteinase from the venom of the snake Agkistrodon acutus; attempts are under way to locate the remaining domains.

Development and evaluation of detection systems for staphylococcal exfoliative toxin A responsible for scalded-skin syndrome.

Staphylococcal scalded-skin syndrome is usually diagnosed clinically by its characteristic exfoliating rash. Isolation of Staphylococcus aureus from the patient further supports the diagnosis. Several detection systems have been developed to determine whether the isolated strain produces exfoliative toxin, but none are routinely available in hospital laboratories. In a novel approach, we used computer models to predict the structure of the exfoliative toxins based on other serine proteases and to identify surface epitopes for the production of antibodies that specifically bound the exfoliative toxin A (ETA) serotype. Several rapid immunologically based diagnostic tests for ETA were developed with these antibodies and compared with existing systems. Our results showed that Western blot analysis using these antibodies was in complete correlation with PCR, which has been validated against the "gold standard" mouse model. On the other hand, the double-antibody enzyme-linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion assay gave unacceptably high false-positive results due to interference by staphylococcal protein A. This problem was successfully overcome by the development of a F(ab')(2) fragment ELISA, which was rapid and reproducible and was as sensitive and specific as PCR and Western blot analysis. The F(ab')(2) fragment ELISA is superior to existing diagnostic systems because it is quantitative, which may be related to the severity of the condition, and can detect amounts of exfoliative toxin in the picogram range directly from serum. This is the first detection system with the potential to confirm the diagnosis of staphylococcal scalded-skin syndrome from a routine blood test within 3 h of presentation.

Crystallization and preliminary crystallographic studies of a phospholipase A2 from the venom of the Brazilian snake Bothrops moojeni.

A phospholipase A(2) purified from the venom of the snake Bothrops moojeni has been crystallized by vapour-diffusion techniques in hanging drops at 291 K. The crystals, which were grown in the absence of Ca(2+), belong to the cubic system, space group P432, with unit-cell parameters a = b = c = 91.86 A, and contain one molecule in the asymmetric unit (V(M) = 2.71 A(3) Da(-1)). X-ray diffraction experiments provide data to 2.35 A resolution collected on a rotating-anode home source at cryogenic temperatures. The structure has been solved via molecular-replacement techniques using a single monomer of the crystallographic structure of the phospholipase from the Western diamondback rattlesnake (Crotalus atrox) as a search model.

Preliminary crystallographic studies of EcTI, a serine proteinase inhibitor from Enterolobium contortisiliquum seeds.

Enterolobium contortisiliquum trypsin inhibitor (EcTI) belongs to the Kunitz family of plant inhibitors, which are widely distributed in nature, especially in plant seeds. EcTI is composed of two polypeptide chains with a total of 174 residues, homologous to other inhibitors from the same family. EcTI crystals, which were obtained with the acupuncture-gel technique, diffract to 2.0 A resolution and belong to space group P2(1), with unit-cell parameters a = 37.12, b = 38.42, c = 54.08 A, beta = 98.08 degrees. Molecular-replacement techniques using Erythrina caffra trypsin inhibitor (PDB code 1tie) as the search model indicate one monomer in the asymmetric unit. The secondary-structure content of EcTI was determined by circular dichroism spectroscopy, yielding values compatible with the expected topology.

Structural basis for low catalytic activity in Lys49 phospholipases A2--a hypothesis: the crystal structure of piratoxin II complexed to fatty acid.

Asp49 plays a fundamental role in supporting catalysis by phospholipases A2 by coordinating the calcium ion which aids in the stabilization of the tetrahedral intermediate. In several myotoxins from the venoms of Viperidae snakes, this aspartic acid is substituted by lysine. The loss of calcium binding capacity by these mutants has become regarded as the standard explanation for their greatly reduced or nonexistent phospholipolytic activity. Here we describe the crystal structure of one such Lys49 PLA2, piratoxin-II, in which a fatty acid molecule is observed within the substrate channel. This suggests that such toxins may be active enzymes in which catalysis is interrupted at the stage of substrate release. Comparison of the present structure with other PLA2s, both active and inactive, identifies Lys122 as one of the likely causes of the increased affinity for fatty acid in Lys49 enzymes. Its interaction with the mainchain carbonyl of Cys29 is expected to lead to hyperpolarization of the peptide bond between residues 29 and 30 leading to an increased affinity for the fatty acid headgroup. This strongly bound fatty acid may serve as an anchor to secure the toxin within the membrane thus facilitating its pathological effects.

The midwife as healer.

In ancient times the midwife was often also a healer, serving communities and women and passing knowledge and insights from woman to woman, mother to daughter. Much of this knowledge has been 'lost' or 'forgotten' due to the witch hunts of the Middle Ages and the discrediting of women's knowledge as scientific rationality emerged. There is evidence now that midwives are exploring alternative knowledge in their rejection of the medical model of care and in their quest to provide enhanced and holistic women-centred care. The three areas highlighted--utilization of complementary therapies, exploration of spirituality and the examination of the nature of the mother--midwife relationship--represent exciting evidence that the midwife is beginning to re-embrace the role of healer.

Identification of free fatty acids in maize protein bodies and purified alpha zeins by (13)C and (1)H nuclear magnetic resonance.

Zeins, the maize storage proteins, are the most abundant proteins in the corn endosperm, and are synthesized on the rough endoplasmatic reticulum and deposited in discrete organelles called protein bodies. Several authors, using circular dichroism and optical rotatory dispersion, have concluded that these proteins have a high alpha-helical content in alcoholic solution. In this work we have studied these proteins, within the protein bodies themselves and after extraction from the corn grains with 70% ethanol, using NMR (nuclear magnetic resonance) spectroscopy. We conclusively demonstrate the presence of free fatty acids within both the protein bodies and also in the alcohol extracted alpha zeins. We present evidence for a direct interaction between the free fatty acids and the alpha zein proteins within the protein body and suggest possible mechanisms by which such an association has arisen during the evolution of the maize endosperm.

Influence of the histidine tail on the structure and activity of recombinant chlorocatechol 1,2-dioxygenase.

We present two efficient expression systems for the chlorocatechol 1, 2-dioxygenase (CCD) from Pseudomonas putida. In the first, CCD (encoded by the clcA gene) was expressed in the pETCLCA vector with the addition of an N-terminal histidine tail. After purification, the enzyme (CCD 6xHis) was proteolytically cleaved with thrombin to remove the His tail. The CD spectra of the cleaved and uncleaved enzymes present only minor differences, indicative of correct protein folding. However, the activity of CCD 6xHis, over a wide range of pH, was typically five times lower. This may be the result of steric hindrance caused by the histidine tail. These data are consistent with results obtained using an alternative construct employing a vector which produces a protein product devoid of the His tail. These results suggest that the His tail may induce subtle effects close to the active site which compromise the recovery of full biological activity.

Determination of the three-dimensional structure of toxins by protein crystallography.

Protein crystallography has significantly contributed to the development of many areas of biochemical research, particularly in the understanding of phenomena related to molecular recognition. Examples include the formation of enzyme-substrate complexes (and their subsequent catalysis), host cell invasion by viruses, antigen neutralization and peptide display by proteins of the immune system and many others. More recently, protein crystallography has also proved to be of great value in unraveling the molecular basis of many diseases as well as in the development of new drugs for their treatment. The X-ray diffraction technique in the elucidation of macromolecular structures is situated at the interface between the traditional research fields of biology, biochemistry, chemistry and physics where researchers are united by a common interest in the detailed understanding of macromolecule function and its relationship to three-dimensional structure. The purpose of this review is to describe, without resort to mathematical detail, all of the necessary steps for the complete determination of a three-dimensional structure by X-ray diffraction techniques. The basic procedures used for protein isolation and crystallization, crystallographic data collection and analysis and, finally, structure determination and refinement are all briefly reviewed. As such our efforts are not directed towards the specialist. Rather, it is our hope that the information presented will aid interested readers from other fields in the understanding of more specialized literature and who may wish to employ the information contained therein in the planning of their biological research. We hope that in so doing we will make clear both the power and limitations of the technique.

Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom: preliminary crystallographic data.

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.