bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeny within the Chondracanthidae (Poecilostomatoida, Copepoda)
Authors: Østergaard Pia , Boxshall Geoff A. , Quicke Donald L. J.
Published: June 2003

The existing systematics of the Chondracanthidae is based predominantly on female characters and divides them into two subfamilies: Chondracanthinae and Lernentominae. Phylogenetic analyses using maximum parsimony were performed using 186 male and female characters. Different trees were generated when male and female characters were analysed separately. Differential weighting showed that the female characters were dominant but not to a great extent and subsequent analyses were run with both partitions combined. Different trees were generated depending on the character setting (unordered, ordered and irreversible‐up). Interestingly, a basal backbone comprising the same nine ingroup taxa was present in all of the trees, although the sequence of those taxa could differ. Constraining the two subfamilies to be monophyletic caused the tree length to be increased and the Templeton and Kishino–Hasegawa tests showed the constrained tree to be significantly different from the unconstrained. The two subfamilies are considered invalid and Lernentominae Oakley, 1927 is formally synonymized with Chondracanthinae Milne Edwards, 1840. The validity of the Pharodidae was tested similarly. Pharodes tortugensis, representing the family Pharodidae, was always recovered nested deep within Chondracanthidae. The Pharodidae Illg, 1948 is therefore synonymized with the Chondracanthidae Milne Edwards, 1840.
Crystallization environment of Kazakhstan microdiamond: evidence from nanometric inclusions and mineral associations
Authors: Dobrzhinetskaya L. F. , Green H. W. , Bozhilov K. N. , Mitchell T. E. , Dickerson R. M.
Published: June 2003

Nanometric solid inclusions in diamond incorporated in garnet and zircon from felsic gneiss of the Kokchetav massif, Kazakhstan, have been examined utilizing electron microscopy and focused ion beam techniques. Host garnet and zircon contain numerous pockets of multiple inclusions, which consist of 1–3 diamond crystals intergrown with quartz, phengite, phlogopite, albite, K‐feldspar, rutile, apatite, titanite, biotite, chlorite and graphite in various combinations. Recalculation of the average chemical composition of the entrapped fluid represented by multiple inclusion pockets indicates that such fluid contained a low wt% of SiO2, suggesting a relatively low‐temperature fluid rather than a melt. Transmission electron microscopy revealed that the diamond contains abundant nanocrystalline inclusions of oxides, rare carbonates and silicates. Within the 15 diamond crystals studied, abundant inclusions were found of SiO2, TiO2, FexOy, Cr2O3, ZrSiO4, and single grains of ThxOy, BaSO4, MgCO3, FeCr2O4 and a stoichiometric Fe‐rich pyroxene. The diversity of trace elements within inclusions of essentially the same stoichiometry suggests that the Kokchetav diamond crystallized from a fluid containing variable amounts of Si, Fe, Ti, Cr, Zr, Ba, Mg and Th and other minor components such as K, Na, P, S, Pb, Zn, Nb, Al, Ca, Cl. Most of the components in crystals included in diamond appear to have their origin in the subducted metasediments, but some of them probably originate from the mantle. It is concluded that Kokchetav diamond most likely crystallized from a COH‐rich multicomponent supercritical fluid at a relatively low temperature (hence the apparently low content of rock‐forming elements), and that the diversity of major and minor components suggests interactions between subducted metasediments and mantle components.
Isobaric cooling and anti‐clockwise P–T paths in the Variscan Odenwald Crystalline Complex, Germany
Authors: Will T. M. , Schmädicke E.
Published: June 2003

Mineral assemblages in Al2O3‐rich, SiO2‐ and K2O‐poor metapelitic rocks from the western Odenwald Crystalline Complex (Variscan Mid‐German Crystalline Rise, southern Germany) include corundum, spinel, cordierite, sillimanite, garnet and staurolite. Quartz is absent from almost all samples. Therefore, the applicability of conventional geothermobarometry is very limited or even impossible. Detailed petrographic investigation on selected samples permits inference of the sequence of appearance and disappearance of several mineral assemblages. The recognition of such partial re‐equilibration stages and their associated mineral assemblages, together with mineral stabilities predicted from KFMASH pseudosections, enables the determination of the pressure‐temperature (P–T) trajectories experienced by these rocks during the Variscan metamorphism. The rocks were metamorphosed under low‐P/high‐T conditions and underwent an anti‐clockwise P–T evolution. A pressure increase from about 2 kbar to 4 ± 0.5 kbar was accompanied by heating. Peak metamorphic conditions were reached at pressures of 4 ± 0.5 kbar and temperatures of at least 640 °C, probably even higher. The retrograde evolution is characterised by near‐isobaric cooling from ≥ 640 °C to approximately 550 °C. The rocks underwent the anti‐clockwise evolution in a subduction‐related magmatic arc setting. The close spatial association of the low‐P/high‐T rocks with recently discovered metabasic eclogites in the eastern part of the Odenwald Crystalline Complex may indicate a fossil paired metamorphic belt in the Central European Variscides.
Metamorphism of pelites in NKFMASH – a new petrogenetic grid with implications for the preservation of high‐pressure mineral assemblages during exhumation
Authors: Proyer A
Published: June 2003

A petrogenetic grid for metapelites in the system NKFMASH is presented. The P–T range is investigated in three sections: (1) The high‐ and ultrahigh‐pressure range is discussed in the system NFMASH because phengite is the only stable potassic phase. (2) The transition region is characterised by four NKFMASH‐invariant points that separate high‐pressure glaucophane‐bearing from medium‐pressure biotite‐bearing metapelites. (3) The medium‐pressure range contains the fifth NKFMASH‐invariant point. The univariant reactions of this point terminate the stability range of paragonite, which breaks down to form staurolite or kyanite and plagioclase during decompression and/or heating. As the growth of albitic plagioclase by decomposition of paragonite via continuous reactions may be conspicuous already before these staurolite‐ or kyanite‐producing reactions are reached, such albite porphyroblast schists are typical indicators of a former high‐pressure metamorphic history.

Considering the preservation of high‐pressure metapelitic assemblages, those crossing the NKFMASH‐transition region during exhumation commonly dehydrate and preservation is unlikely. Three types of metapelites have a fairly good survival potential: (1) low‐temperature metapelites (up to c. 540 °C) with an exhumation path back into the chlorite + albite stability field, (2) assemblages with chloritoid + glaucophane, and (3) the relatively high‐temperature glaucophane + kyanite and jadeite + kyanite bearing parageneses, that are relatively dry at the onset of exhumation. A comparison with data from the literature shows that these rock types are the most abundant in nature.
Impact of Migraine on the Family
Authors: Smith Robert
Published: June 2003

The extensive literature on pain and the family contains little on migraine and the family.

A national sample of migraine sufferers, interviewed by telephone, was questioned on how they perceived migraine affected their family life.

Sixty percent believed that their families were significantly affected. Most stated that their families were understanding, but a considerable number reported that family members took a negative attitude. This was particularly so with younger children deprived of parental care during migraine attacks. Spousal relationships also suffered. Frequency and quality of sexual relationships were affected; and in a small number of cases, divorce was the outcome. The significance of these findings in migraine management is discussed.
Migraine Chronobiology
Authors: Fox Anthony W. , Davis Randy L.
Published: June 2003

This study was undertaken to determine whether migraine attacks exhibit circadian, menstrual, or seasonal variations in frequency and, thus, to characterize more precisely this relapsing, remittent pleomorphic disease. An analysis of 3582 well‐documented migraine attacks in 1698 adults was undertaken. The demographics of the study population accurately represented the known epidemiology of the disease. Migraine attacks started more frequently between 4 am and 9 am and within the first few days after onset of menses; this migraine periodicity is strongest amongst women not using oral contraceptives. Seasonal periodicity, if any, is clearly weaker than circadian or menstrual. These chronobiological features may assist in the differential diagnosis of migraine from premenstrual headache and fibromyalgia.
Cognitions, Emotions, and Behavior of Patients With Migraine When Taking Medication During an Attack
Authors: Passchier J. , Mourik J. , Brienen J.A. , Hunfeld J.A.M.
Published: June 2003

Fifty‐three patients with migraine, recruited from the Dutch Society of Migraine Patients and a general practice, were investigated regarding pain, moods, thoughts, and functioning during their most recent migraine attack, using a semistructured interview. Salient findings were: the high pain intensity the patients endured before they took analgesic medication, concerns about medication damaging their health, overoptimism regarding the effect of analgesic medication, and the relatively large proportion of patients (43%) who took medication primarily to be able to continue their activities. We recommend that future clinical trials on the effects of medication on migraine should not only include the measurement of pain during the attack, but also emotions, concerns about potential side effects, and the ability to continue or resume work. Futhermore, it is important to provide patients with information about the side effects of medication and to apply cognitive‐behavioral techniques for improvement of their mood during the attack.
Cytokines and Migraine: Increase of IL‐5 and IL‐4 Plasma Levels
Authors: Munno I. , Centonze V. , Marinaro M. , Bassi A. , Lacedra G. , Causarano V. , Nardelli P. , Cassiano M. A. , Albano O.
Published: June 2003

Thirty‐two patients suffering from migraine without aura were assessed during the interictal period to evaluate the contribution of cytokines to the pathophysiology of migraine. To this end, plasma levels of IFN‐γ, IL‐4, IL‐5, and IL‐10 were measured by enzyme‐linked immunosorbent assay (ELlSA) techniques. Plasma levels of both IFN‐γ and IL‐10 were not increased in the patients and did not differ significantly from healthy controls. Of interest, we observed a strong increase of IL‐5 levels in 84.3% as well as increased IL‐4 levels in 37.5% of patients with migraine without aura. These results suggest a preferential enhancement of some Th2‐type cytokines and may support the growing arguments of an immunoallergic mechanism in the pathophysiology of migraine.
Botulinum Toxin A, Adjunctive Therapy for Refractory Headaches Associated With Pericranial Muscle Tension
Authors: Wheeler Anthony H.
Published: June 2003

Pericranial muscle tension may contribute to the development of facial discomfort, chronic daily headache, and migraine‐type headache. Elimination of pericranial muscle tension may reduce associated myalgia and counteract influences that can trigger secondary headaches which fall within the migraine continuum. Four patients with chronic, predominantly tension‐type headaches and associated pericranial muscles tension failed prolonged conventional treatment and, therefore, symptomatic areas were treated with botulinum toxin A. This alleviated myalgia and reduced the severity and frequency of migraine‐type headaches with a concomitant reduction in subsequent medical and physical therapy interventions. Judicious use of botulinum toxin A into defined areas of pericranial muscle tension may be useful for reducing primary myalgia and secondary headache.
Multiple Intracerebral Hemorrhages and Vasospasm Following Antimigrainous Drug Abuse
Authors: Nighoghossian N. , Derex L. , Trouillas P.
Published: June 2003

The occurrence of intracerebral hemorrhage during an acute attack of migraine remains questionable. A normotensive migrainous woman experienced multiple intracerebral hemorrhages. No drug abuse was recorded, but bleeding occurred after the use of several antimigrainous drugs. Angiography revealed severe vasospasm of both anterior cerebral arteries. Subsequent brain MRI and MRI angiography failed to show any vascular abnormalities. Echocardiography and an exhaustive biological evaluation ruled out an alternative condition favoring intracerebral hemorrhage. Multiple intracerebral hemorrhages in migraine may exceptionally be related to vasospasm leading to arterial wall injury. Intracranial arterial vasospasm could be precipitated by excessive vasoactive therapy.
Headache Secondary to Deep Brain Implantation
Authors: Veloso Felix , Kumar Krishna , Toth Cory
Published: June 2003

This article examines the headaches that can develop with deep brain stimulation. We identified 15 (23.4%) of 64 patients who developed chronic headaches following implantation of deep brain stimulation electrodes for relief of chronic pain syndromes. Electrodes were implanted in the periaqueductal gray, sensory thalamus, and internal capsule. None of these 15 patients reported similar headaches prior to the implantation. Although 5 patients were intermittent headache sufferers prior to implantation, they reported post‐implantation headaches to be completely different in nature. Our experience suggests that headache syndromes may arise due to disturbance of serotonergic neurotransmission within deep brain structures.
Antidepressant Treatment of Chronic Tension‐Type Headache: A Comparison Between Fluoxetine and Desipramine
Authors: Walker Zuzana , Walker Rodney W. H. , Robertson Mary M. , Stansfeld Stephen
Published: June 2003

Amitriptyline, which is a noradrenaline reuptake and 5‐HT reuptake inhibitor, has an established role in the management of chronic tension‐type headaches.

In a single‐blind study, patients with chronic tension‐type headache were randomized to either fluoxetine 20 mg (a selective 5‐HT reuptake inhibitor) or desipramine 75 mg (a selective noradrenaline reuptake inhibitor) and followed for 12 weeks to compare the effectiveness of the two drugs in improving headache, and to assess whether pain control is related to changes in depression.

Patients were evaluated at weekly intervals on an analog pain‐rating scale and at 4‐weekly intervals on the Montgomery and Asberg Depression Rating Scale (MADRS), the MOS general health status questionnaire (SF36), the Hospital Anxiety and Depression Scale (HADS), and a side effects checklist.

Eighteen patients were randomized to take fluoxetine and 19 to take desipramine. Of the 25 patients who completed the trial, 12 were on fluoxetine and 13 were on desipramine. There was no significant difference between the two groups at baseline nor in change of pain; reduction in use of analgesic medication; nor change in the HADS, MADRS, or SF36 scores at 12 weeks, but 72% of patients who completed the study improved, and this improvement almost exactly mirrored the improvement on the MADRS.

The results from this trial are compatible with the notion that the beneficial effect of antidepressants in chronic tension‐type headache is indirect, mediated by an effect on depression, and not more,dependent on serotonin reuptake inhibition than noradrenaline reuptake inhibition.
Plasticity of 5‐HT2A Serotonin Receptor in Patients With Analgesic‐Induced Transformed Migraine
Authors: Srikiatkhachorn Anan , Puangniyom Supaporn , Govitrapong Piyarat
Published: June 2003

Chronic drug exposure can induce a significant change in neurotransmitter receptor systems and is possibly involved in the pathogenesis of drug‐induced neurological disorders. Abuse of analgesics is known to induce deterioration in headache status in patients with primary headaches, especially migraine. To assess the possibility of 5‐HT2A serotonin receptor plasticity in this condition, we investigated receptor binding on the platelet membrane in patients with analgesic‐induced transformed migraine, patients with migraine, and nonheadache controls. Various concentrations of [3H]‐spiperone (0.4 to 12 nmol) was used as a radioligand, and ketanserin was used to determine nonspecific binding. A lower maximal number of receptors (Bmax) was observed in patients with migraine as compared to patients with transformed migraine, and controls (467 ± 58, 708 ± 36, and 786 ± 64 fmol/mg protein, respectively, P<0.01); whereas the value of the dissociation equilibrium constant (Kd) remained unchanged (1.72 ± 0.16, 1.41 ± 0.13, and 1.25 ± 0.21 nmol for patients with migraine, patients with transformed migraine, and nonheadache controls, respectively). A significant decrease in Bmax value was observed in patients with transformed migraine after 4 weeks of analgesic withdrawal (770 ± 25 and 345 ± 31 fmol/mg protein, P<0.001), whilst no significant change in Kd value was observed (1.95 ± 0.12 and 2.47 ± 0.30 nmol, respectively). These findings indicate that 5‐HT2A serotonin receptor system is altered in patients with transformed migraine with analgesic overuse. Such receptor plasticity may be an important step in the pathogenic mechanism of transformed migraine.
Long‐term Evolution of a Laughing Headache Associated With Chiari Type 1 Malformation
Authors: Morales‐Asín F. , Mauri J.A. , Iñiguez C. , Larrode M.P. , Mostacero E.
Published: June 2003

We describe an acute‐onset, sharp, short‐lasting (few seconds) headache at the vertex, that first occurred 10 years ago in a 44‐year‐old woman. Attacks were triggered by vigorous laughing. A magnetic resonance imaging study showed a Chiari type 1 malformation.

To our knowledge, laugh‐induced headache with long‐term evolution in association with Chiari type 1 malformation has not been previously reported.
Crystal structure of soybean 11S globulin: Glycinin A3B4 homohexamer
Authors: Adachi Motoyasu , Kanamori Jiro , Masuda Taro , Yagasaki Kazuhiro , Kitamura Keisuke , Mikami Bunzo , Utsumi Shigeru
Published: June 2003

Most plant seeds contain 11S globulins as major storage proteins for their nutrition. Soybean glycinin belongs to the 11S globulin family and consists of five kinds of subunits. We determined the crystal structure of a homohexamer of the glycinin A3B4 subunit at 2.1-Å resolution. The crystal structure shows that the hexamer has 32-point group symmetry formed by face-to-face stacking of two trimers. The interface buries the highly conserved interchain disulfide. Based on the structure, we propose that an ingenious face-to-face mechanism controls the hexamer formation of the 11S globulin by movement of a mobile disordered region to the side of the trimer after posttranslational processing. Electrostatic analysis of the faces suggests that the interchain disulfide-containing face has high positive potential at acidic pH, which induces dissociation of the hexamer into trimers that may be susceptible to proteinases after seed imbibition. This dissociation might result in the degradation and mobilization of 11S globulins as storage proteins in embryos during germination and seedling growth.
A ligand-binding pocket in the dengue virus envelope glycoprotein
Authors: Modis Yorgo , Ogata Steven , Clements David , Harrison Stephen C.
Published: June 2003

Dengue virus is an emerging global health threat. Its major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. A crystal structure of the soluble ectodomain of E from dengue virus type 2 reveals a hydrophobic pocket lined by residues that influence the pH threshold for fusion. The pocket, which accepts a hydrophobic ligand, opens and closes through a conformational shift in a β-hairpin at the interface between two domains. These features point to a structural pathway for the fusion-activating transition and suggest a strategy for finding small-molecule inhibitors of dengue and other flaviviruses.
NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site
Authors: Hoffmann Bernd , Mitchell G. Thomas , Gendron Patrick , Major François , Andersen Angela A. , Collins Richard A. , Legault Pascale
Published: June 2003

Substrate cleavage by the
Neurospora
Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg
2
+
binding sites in the active internal loop. One of the Mg
2
+
binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program
mc
-
search
, we identified the active internal loop fold in other RNA structures. In
Thermus thermophilus
16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the
Neurospora
VS ribozyme.
Architecture of initiation-competent 12-subunit RNA polymerase II
Authors: Armache Karim-Jean , Kettenberger Hubert , Cramer Patrick
Published: June 2003

RNA polymerase (Pol) II consists of a 10-polypeptide catalytic core and the two-subunit Rpb4/7 complex that is required for transcription initiation. Previous structures of the Pol II core revealed a “clamp,” which binds the DNA template strand via three “switch regions,” and a flexible “linker” to the C-terminal repeat domain (CTD). Here we derived a model of the complete Pol II by fitting structures of the core and Rpb4/7 to a 4.2-Å crystallographic electron density map. Rpb4/7 protrudes from the polymerase “upstream face,” on which initiation factors assemble for promoter DNA loading. Rpb7 forms a wedge between the clamp and the linker, restricting the clamp to a closed position. The wedge allosterically prevents entry of the promoter DNA duplex into the active center cleft and induces in two switch regions a conformation poised for template-strand binding. Interaction of Rpb4/7 with the linker explains Rpb4-mediated recruitment of the CTD phosphatase to the CTD during Pol II recycling. The core–Rpb7 interaction and some functions of Rpb4/7 are apparently conserved in all eukaryotic and archaeal RNA polymerases but not in the bacterial enzyme.
Sympatric speciation through intraspecific social parasitism
Authors: Savolainen Riitta , Vepsäläinen Kari
Published: June 2003

Sympatric speciation through intraspecific social parasitism has been proposed for the evolution of Hymenopteran workerless parasites. Such inquilines exploit related host taxa to produce their own sexual offspring. The relatedness of inquilines to their hosts has been generalized in Emery's rule, suggesting that social parasites are close or the closest relatives to their host species. If the closest relative of each parasite is its host, then multiple independent origins of the parasite species are implied even within a single genus, probably through sympatric speciation. To test the plausibility of sympatric speciation in inquilines, we conducted a mitochondrial DNA phylogenetic analysis in three inquiline–host pairs ofMyrmicaant species. We show that congeneric inquilines have originated independently several times. We also show that two of the inqulines are more closely related to their hosts than to any other species. Our results suggest sympatric speciation ofMyrmicainquilines. Sympatric speciation is probably facilitated by the social biology and ecology ofMyrmica, with polygyny as a prerequisite for the evolution of intraspecific parasitism.
The BglF sensor recruits the BglG transcription regulator to the membrane and releases it on stimulation
Authors: Lopian Livnat , Nussbaum-Shochat Anat , O'Day-Kerstein Kathryn , Wright Andrew , Amster-Choder Orna
Published: June 2003

The
Escherichia coli
BglF protein is a sugar-sensor that controls the activity of the transcriptional antiterminator BglG by reversibly phosphorylating it, depending on β-glucoside availability. BglF is a membrane-bound protein, whereas BglG is a soluble protein, and they are both present in the cell in minute amounts. How do BglF and BglG find each other to initiate signal transduction efficiently? Using bacterial two-hybrid systems and the Far-Western technique, we demonstrated unequivocally that BglG binds to BglF and to its active site-containing domain
in vivo
and
in vitro
. Measurements by surface plasmon resonance corroborated that the affinity between these proteins is high enough to enable their stable binding. To visualize the subcellular localization of BglG, we used fluorescence microscopy. In cells lacking BglF, the BglG-GFP fusion protein was evenly distributed throughout the cytoplasm. In contrast, in cells producing BglF, BglG-GFP was localized to the membrane. On addition of β-glucoside, BglG-GFP was released from the membrane, becoming evenly distributed throughout the cell. Using mutant proteins and genetic backgrounds that impede phosphorylation of the Bgl proteins, we demonstrated that BglG-BglF binding and recruitment of BglG to the membrane sensor requires phosphorylation but does not depend on the individual phosphorylation sites of the Bgl proteins. We suggest a mechanism for rapid response to environmental changes by preassembly of signaling complexes, which contain transcription regulators recruited by their cognate sensors-kinases, under nonstimulating conditions, and release of the regulators to the cytoplasm on stimulation. This mechanism might be applicable to signaling cascades in prokaryotes and eukaryotes.
Complete, 12-subunit RNA polymerase II at 4.1-Å resolution: Implications for the initiation of transcription
Authors: Bushnell David A. , Kornberg Roger D.
Published: June 2003

The x-ray structure of complete RNA polymerase II from
Saccharomyces cerevisiae
has been determined, including a heterodimer of subunits Rpb4 and Rpb7 not present in previous “core” polymerase II structures. The heterodimer maintains the polymerase in the conformation of a transcribing complex, may bind RNA as it emerges from the enzyme, and is in a position to interact with general transcription factors and the Mediator of transcriptional regulation.
Targeted mutation of the outer membrane protein P66 disrupts attachment of the Lyme disease agent,
Borrelia burgdorferi
, to integrin α
v
β
3
Authors: Coburn Jenifer , Cugini Carla
Published: June 2003

Borrelia burgdorferi
, the agent of Lyme disease, expresses several adhesion molecules that are probably required for initial establishment of infection in mammalian hosts, and for colonization of various tissues within the host. The
B. burgdorferi
outer membrane protein P66 was previously identified as a ligand for β
3
-chain integrins by using a variety of biochemical approaches. Although the earlier data suggested that P66 is an adhesin that mediates
B. burgdorferi
attachment to β
3
-chain integrins, lack of genetic systems in
B. burgdorferi
precluded definitive demonstration of a role for P66 in β
3
integrin attachment by intact borreliae. Recent advances in the genetic manipulation of
B. burgdorferi
have now made possible the targeted disruption of the
p66
gene. Mutants in
p66
show dramatically reduced attachment to integrin α
v
β
3
. This is, to our knowledge, the first description of the targeted disruption of a candidate
B. burgdorferi
virulence factor with a known biochemical function that can be quantified, and demonstrates the importance of
B. burgdorferi
P66 in the attachment of this pathogenic spirochete to a human cell-surface receptor.
Crystalline monoclonal antibodies for subcutaneous delivery
Authors: Yang Mark X. , Shenoy Bhami , Disttler Matthew , Patel Reena , McGrath Margaret , Pechenov Sergey , Margolin Alexey L.
Published: June 2003

Therapeutic applications for mAbs have increased dramatically in recent years, but the large quantities required for clinical efficacy have limited the options that might be used for administration and thus have placed certain limitations on the use of these agents. We present an approach that allows for s.c. delivery of a small volume of a highly concentrated form of mAbs. Batch crystallization of three Ab-based therapeutics, rituximab, trastuzumab, and infliximab, provided products in high yield, with no detectable alteration to these proteins and with full retention of their biological activity
in vitro
. Administration s.c. of a crystalline preparation resulted in a remarkably long pharmacokinetic serum profile and a dose-dependent inhibition of tumor growth in nude mice bearing BT-474 xenografts (human breast cancer cells)
in vivo
. Overall, this approach of generating high-concentration, low-viscosity crystalline preparations of therapeutic Abs should lead to improved ease of administration and patient compliance, thus providing new opportunities for the biotechnology industry.
Primary hyperoxaluria type 1 in the Canary Islands: A conformational disease due to I244T mutation in the P11L-containing alanine:glyoxylate aminotransferase
Authors: Santana A. , Salido E. , Torres A. , Shapiro L. J.
Published: June 2003

Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 → Thr (I244T) mutation in theAGXTgene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms withinAGXTand regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 → Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed inEscherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation.
Pseudo-cryptic speciation in coccolithophores
Authors: Sáez Alberto G. , Probert Ian , Geisen Markus , Quinn Patrick , Young Jeremy R. , Medlin Linda K.
Published: June 2003

Coccolithophores are a group of calcifying unicellular algae that constitute a major fraction of oceanic primary productivity, play an important role in the global carbon cycle, and are key biostratigraphic marker fossils. Their taxonomy is primarily based on the morphology of the minute calcite plates, or coccoliths, covering the cell. These are diverse and include widespread fine scale variation, of which the biological/taxonomic significance is unknown. Do they represent phenotypic plasticity, genetic polymorphisms, or species-specific characters? Our research on five commonly occurring coccolithophores supports the hypothesis that such variation represents pseudocryptic speciation events, occurring between 0.3 and 12.9 million years ago from a molecular clock estimation. This finding suggests strong stabilizing selection acting on coccolithophorid phenotypes. Our results also provide strong support for the use of fine scale morphological characters of coccoliths in the fossil record to improve biostratigraphic resolution and paleoceanographic data retrieval.
Neural stem cells and cholinergic neurons: Regulation by immunolesion and treatment with mitogens, retinoic acid, and nerve growth factor
Authors: Calzà Laura , Giuliani Alessandro , Fernandez Mercedes , Pirondi Stefania , D'Intino Giulia , Aloe Luigi , Giardino Luciana
Published: June 2003

Degenerative diseases represent a severe problem because of the very limited repair capability of the nervous system. To test the potential of using stem cells in the adult central nervous system as “brain-marrow” for repair purposes, several issues need to be clarified. We are exploring the possibility of influencing,
in vivo
, proliferation, migration, and phenotype lineage of stem cells in the brain of adult animals with selective neural lesions by exogenous administration (alone or in combination) of hormones, cytokines, and neurotrophins. Lesion of the cholinergic system in the basal forebrain was induced in rats by the immunotoxin
192
IgG-saporin. Alzet osmotic minipumps for chronic release (over a period of 14 days) of mitogens [epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF)] were implanted in animals with behavioral and biochemical cholinergic defect and connected to an intracerebroventricular catheter. After 14 days of delivery, these pumps were replaced by others delivering nerve growth factor (NGF) for an additional 14 days. At the same time, retinoic acid was added to the rats' food pellets for one month. Whereas the lesion decreased proliferative activity, EGF and bFGF both increased the number of proliferating cells in the subventricular zone in lesioned and nonlesioned animals. These results are indicated by the widespread distribution of BrdUrd-positive nuclei in the forebrain, including in the cholinergic area. Performance in the water maze test was improved in these animals and choline acetyltransferase activity in the hippocampus was increased. These results suggest that pharmacological control of endogenous neural stem cells can provide an additional opportunity for brain repair. These studies also offer useful information for improving integration of transplanted cells into the mature brain.
Codon bias and frequency-dependent selection on the hemagglutinin epitopes of influenza A virus
Authors: Plotkin Joshua B. , Dushoff Jonathan
Published: June 2003

Although the surface proteins of human influenza A virus evolve rapidly and continually produce antigenic variants, the internal viral genes acquire mutations very gradually. In this paper, we analyze the sequence evolution of three influenza A genes over the past two decades. We study codon usage as a discriminating signature of gene- and even residue-specific diversifying and purifying selection. Nonrandom codon choice can increase or decrease the effective local substitution rate. We demonstrate that the codons of hemagglutinin, particularly those in the antibody-combining regions, are significantly biased toward substitutional point mutations relative to the codons of other influenza virus genes. We discuss the evolutionary interpretation and implications of these biases for hemagglutinin's antigenic evolution. We also introduce information-theoretic methods that use sequence data to detect regions of recent positive selection and potential protein conformational changes.
Apparent homology of expressed genes from wood-forming tissues of loblolly pine (
Pinus taeda
L.) with
Arabidopsis thaliana
Authors: Kirst Matias , Johnson Arthur F. , Baucom Christie , Ulrich Erin , Hubbard Kristy , Staggs Rod , Paule Charles , Retzel Ernest , Whetten Ross , Sederoff Ronald
Published: June 2003

Pinus taeda
L. (loblolly pine) and
Arabidopsis thaliana
differ greatly in form, ecological niche, evolutionary history, and genome size.
Arabidopsis
is a small, herbaceous, annual dicotyledon, whereas pines are large, long-lived, coniferous forest trees. Such diverse plants might be expected to differ in a large number of functional genes. We have obtained and analyzed 59,797 expressed sequence tags (ESTs) from wood-forming tissues of loblolly pine and compared them to the gene sequences inferred from the complete sequence of the
Arabidopsis
genome. Approximately 50% of pine ESTs have no apparent homologs in
Arabidopsis
or any other angiosperm in public databases. When evaluated by using contigs containing long, high-quality sequences, we find a higher level of apparent homology between the inferred genes of these two species. For those contigs 1,100 bp or longer, ≈90% have an apparent
Arabidopsis
homolog (
E
value < 10
-
10
). Pines and
Arabidopsis
last shared a common ancestor ≈300 million years ago. Few genes would be expected to retain high sequence similarity for this time if they did not have essential functions. These observations suggest substantial conservation of gene sequence in seed plants.
Glycerol replacement corrects defective skin hydration, elasticity, and barrier function in aquaporin-3-deficient mice
Authors: Hara Mariko , Verkman A. S.
Published: June 2003

Mice deficient in the epidermal water/glycerol transporter aquaporin-3 (AQP3) have reduced stratum corneum (SC) hydration and skin elasticity, and impaired barrier recovery after SC removal. SC glycerol content is reduced 3-fold in AQP3 null mice, whereas SC structure, protein/lipid composition, and ion/osmolyte content are not changed. We show here that glycerol replacement corrects each of the defects in AQP3 null mice. SC water content, measured by skin conductance and3H2O accumulation, was 3-fold lower in AQP3 null vs. wild-type mice, but became similar after topical or systemic administration of glycerol in quantities that normalized SC glycerol content. SC water content was not corrected by glycerol-like osmolytes such as xylitol, erythritol, and propanediol. Orally administered glycerol fully corrected the reduced skin elasticity in AQP3 null mice as measured by the kinetics of skin displacement after suction, and the delayed barrier recovery as measured by transepidermal water loss after tape-stripping. Analysis of [14C]glycerol kinetics indicated reduced blood-to-SC transport of glycerol in AQP3 null mice, resulting in slowed lipid biosynthesis. These data provide functional evidence for a physiological role of glycerol transport by an aquaglyceroporin, and indicate that glycerol is a major determinant of SC water retention, and mechanical and biosynthetic functions. Our findings establish a scientific basis for the >200-yr-old empirical practice of including glycerol in cosmetic and medicinal skin formulations.
Direct observation of photolysis-induced tertiary structural changes in hemoglobin
Authors: Adachi Shin-ichi , Park Sam-Yong , Tame Jeremy R. H. , Shiro Yoshitsugu , Shibayama Naoya
Published: June 2003

Human Hb, an α
2
β
2
tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded R-state human Hb at cryogenic temperatures (25–35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the α subunit than in the β subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state β heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.
Correlated motion and the effect of distal mutations in dihydrofolate reductase
Authors: Rod Thomas H. , Radkiewicz Jennifer L. , Brooks Charles L.
Published: June 2003

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme [Rajagopalan, P. T. R, Lutz, S., and Benkovic, S. J. (2002)
Biochemistry
41, 12618–12628]. On the basis of extensive computer simulations for wild-type DHFR from
Escherichia coli
and four mutants (G121S, G121V, M42F, and M42F/G121S), we show that key parameters for catalysis are changed. The parameters we study are relative populations of different conformations sampled and hydrogen bonds. We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme. Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations. We finally examine the role a structural perturbation will have on the hydride transfer step. On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks.
Allele-specific silencing of dominant disease genes
Authors: Miller Victor M. , Xia Haibin , Marrs Ginger L. , Gouvion Cynthia M. , Lee Gloria , Davidson Beverly L. , Paulson Henry L.
Published: June 2003

Small interfering RNA (siRNA) holds therapeutic promise for silencing dominantly acting disease genes, particularly if mutant alleles can be targeted selectively. In mammalian cell models we demonstrate that allele-specific silencing of disease genes with siRNA can be achieved by targeting either a linked single-nucleotide polymorphism (SNP) or the disease mutation directly. For a polyglutamine neurodegenerative disorder in which we first determined that selective targeting of the disease-causing CAG repeat is not possible, we took advantage of an associated SNP to generate siRNA that exclusively silenced the mutant Machado–Joseph disease/spinocerebellar ataxia type 3 allele while sparing expression of the WT allele. Allele-specific suppression was accomplished with all three approaches currently used to deliver siRNA:
in vitro
-synthesized duplexes as well as plasmid and viral expression of short hairpin RNA. We further optimized siRNA to specifically target a missense Tau mutation, V337M, that causes frontotemporal dementia. These studies establish that siRNA can be engineered to silence disease genes differing by a single nucleotide and highlight a key role for SNPs in extending the utility of siRNA in dominantly inherited disorders.
Decorin-binding proteins A and B confer distinct mammalian cell type-specific attachment by
Borrelia burgdorferi
, the Lyme disease spirochete
Authors: Fischer Joshua R. , Parveen Nikhat , Magoun Loranne , Leong John M.
Published: June 2003

Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent,
Borrelia burgdorferi
, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple
B. burgdorferi
surface proteins. We previously showed that
B. burgdorferi
strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. In the current study, we show that the expression of decorin-binding protein (Dbp) A and/or DbpB, two
B. burgdorferi
surface proteins that bind GAGs, is sufficient to convert a high-passage nonadherent
B. burgdorferi
strain into one that efficiently binds 293 epithelial cells. Epithelial cell attachment was mediated by dermatan sulfate, and, consistent with this GAG-binding specificity, these recombinant strains did not bind EA-Hy926 endothelial cells. The GAG-binding properties of bacteria expressing DbpB or DbpA were distinguishable, and DbpB but not DbpA promoted spirochetal attachment to C6 glial cells. Thus, DbpA and DbpB may each play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage
B. burgdorferi
strains may provide a relatively simple genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.
Retinoid activation of retinoic acid receptor but not retinoid X receptor is sufficient to rescue lethal defect in retinoic acid synthesis
Authors: Mic Felix A. , Molotkov Andrei , Benbrook Doris M. , Duester Gregg
Published: June 2003

Two isomers of retinoic acid (RA) may be necessary as ligands for retinoid signaling:
all-trans
-RA for RA receptors (RARs) and 9-
cis
-RA for retinoid X receptors (RXRs). This was explored by using retinaldehyde dehydrogenase (
Raldh)2
-/-
mouse embryos lacking mesodermal RA synthesis that display early growth arrest unless rescued by
all-trans
-RA administration. Because isomerization of
all-trans
-RA to 9-
cis
-RA can occur, it is unclear whether both ligands are needed for rescue. We show here that an RAR-specific ligand can rescue
Raldh2
-/-
embryos as efficiently as
all-trans
-RA, whereas an RXR-specific ligand has no effect. Further, whereas
all-trans
-RA was detected in embryos, 9-
cis
-RA was undetectable unless a supraphysiological dose of
all-trans
-RA was administered, revealing that 9-
cis
-RA is of pharmacological but not physiological significance. Because 9-
cis
-RA is undetectable and unnecessary for
Raldh2
-/-
rescue, and others have shown that 4-oxo-RA is unnecessary for mouse development,
all-trans
-RA emerges as the only ligand clearly necessary for retinoid receptor signaling.