Quantification of valproic acid and its metabolite 2-propyl-4-pentenoic acid in human plasma using HPLC-MS/MS

A specific and sensitive HPLC-MS/MS method for the quantitative determination of valproic acid (VPA) and its metabolite, 2-propyl-4-pentenoic acid in human plasma has been developed, using VPA-d15 as the internal standard. The method was based on pre-column derivatization using 4-dimethylaminobenzylamine dihydrochloride. The derivatives were separated with a gradient elution and quantified by positive electrospray ionization with multiple reaction monitoring. The assay provides routine quantification limits of 200 ng/mL for VPA and 20 ng/mL for 4-ene VPA with within- and between-day coefficients of variation of <10%. This method has been applied to the analysis of plasma samples obtained from patients treated with this drug.     

Tag retention and survival of juvenile bighead carp implanted with a dummy acoustic tag at three temperatures

Bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix (together, the bigheaded carps) are invasive fishes in North America that have resulted in substantial negative effects on native fish communities and aquatic ecosystems. Movement and behavior of adult bigheaded carps has been studied previously using telemetry, while similar studies with juvenile bigheaded carps have yet to be attempted. Recent technological advances in telemetry transmitters has increased the availability of tags sufficiently small enough to implant in juvenile carps. However, the effects of surgical implantation of telemetry tags on juvenile bigheaded carps have not been evaluated. We determined tag retention and survival associated with surgical implantation of acoustic telemetry tags into juvenile bighead carp (range 128–152 mm total length) at three temperatures (13, 18, and 23°C). In addition, we assessed the effect of surgically implanted transmitters on the fitness, defined as changes in weight or critical swimming speed, of carp implanted with transmitters. Survival was high among tagged fish (85%) with 47% of tags retained at the conclusion of the 45‐day study. No substantial decline in fitness of the fish was observed in tagged fish compared to untagged fish.     

Maxillary reconstruction: functional and aesthetic considerations

Maxillary reconstruction is a challenging endeavor in functional and aesthetic restoration. Given its central location in the midface and its contributions to the key midfacial elements--the orbits, the zygomaticomaxillary complex, the nasal unit, and the stomatognathic complex--the maxilla functions as the keystone of the midface and unifies these elements into a functional and aesthetic unit. Maxillary defects are inherently complex because they generally involve more than one midfacial component. In addition, most maxillary defects are composite in nature, and they often require skin coverage, bony support, and mucosal lining for reconstruction. In the reconstruction of maxillary defects secondary to trauma, ablative tumor surgery, or congenital deformities, the following goals must be met: (1) obliteration of the defect; (2) restoration of essential functions of the midface, such as mastication and speech; (3) provision for adequate structural support to each of the midfacial units; and (4) aesthetic reconstruction of the external features. This review will discuss the pertinent anatomic considerations, the historical approaches to maxillary reconstruction, and the merits of the techniques in use today, with an emphasis on state-of-the-art reconstruction and dental rehabilitation of extensive maxillary defects.     

Ataxia-telangiectasia-like disorder (ATLD)-its clinical presentation and molecular basis

Comparison of the clinical and cellular phenotypes of different genomic instability syndromes provides new insights into functional links in the complex network of the DNA damage response. A prominent example of this principle is provided by examination of three such disorders: ataxia-telangiectasia (A-T) caused by lack or inactivation of the ATM protein kinase, which mobilises the cellular response to double strand breaks in the DNA; ataxia-telangiectasia-like disease (ATLD), a result of deficiency of the human Mre11 protein; and the Nijmegen breakage syndrome (NBS), which represents defective Nbs1 protein. Mre11 and Nbs1 are members of the Mre11/Rad50/Nbs1 (MRN) protein complex. MRN and its individual components are involved in different responses to cellular damage induced by ionising radiation and radiomimetic chemicals, including complexing with chromatin and with other damage response proteins, formation of radiation-induced foci, and the induction of different cell cycle checkpoints. The phosphorylation of Nbs1 by ATM would indicate that ATM acts upstream of the MRN complex. Consistent with this were the suggestions that ATM could be activated in the absence of fully functional Nbs1 protein. In contrast, the regulation of some ATM target proteins, e.g. Smc1 requires the MRN complex as well as ATM. Nbs1 may, therefore, be both a substrate for ATM and a mediator of ATM function. Recent studies that indicate a requirement of the MRN complex for proper ATM activation suggest that the relationship between ATM and the MRN complex in the DNA damage response is yet to be fully determined. Despite the fact that both Mre11 and Nbs1 are part of the same MRN complex, deficiency in either protein in humans does not lead to the same clinical picture. This suggests that components of the complex may also act separately.     

Global conformations,hydrodynamics, and X-ray scattering properties of Taq and Escherichia coli DNA polymerases in solution

Escherichia coli polymerase 1 (Pol 1) and Thermus aquaticus Taq polymerase are homologous Type I DNA polymerases, each comprised of a polymerase domain, a proofreading domain (inactive in Taq), and a 5' nuclease domain. "Klenow" and "Klentaq" are the large fragments of Pol 1 and Taq and are functional polymerases lacking the 5' nuclease domain. In the available crystal structures of full-length Taq, the 5' nuclease domain is positioned in two different orientations: in one structure, it is extended out into solution, whereas in the other, it is folded up against the polymerase domain in a more compact structure. Analytical ultracentrifugation experiments report s20,w values of 5.05 for Taq, 4.1 for Klentaq, 5.3 for E. coli Pol 1, and 4.6 for Klenow. Measured partial specific volumes are all quite similar, indicating no significant differences in packing density between the mesophilic and thermophilic proteins. Small angle x-ray scattering studies report radii of gyration of 38.3 A for Taq, 30.7 A for Klentaq, and 30.5 A for Klenow. The hydrodynamic and x-ray scattering properties of the polymerases were also calculated directly from the different crystal structures using the programs HYDROPRO (Garcia De La Torre, J., Huertas, M. L., and Carrasco, B. (2000) Biophys J. 78, 719-730) and CRYSOL (Svergun, D. I., Barberato, C., and Koch, M. H. J. (1995) J. Appl. Crystalogr. 28, 768-773), respectively. The combined experimental and computational characterizations indicate that the full-length polymerases in solution are in a conformation where the 5' nuclease domain is extended into solution. Further, the radius of gyration, and hence the global conformation of Taq polymerase, is not altered by the binding of either matched primer template DNA or ddATP.     

Structure-function analysis of Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase domains

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.     

Single millimeter wave treatment does not impair gastrointestinal transit in mice

Millimeter wave treatment (MWT) is based on those biological effects that develop following skin exposure to low power electromagnetic waves. This method of treatment is in wide clinical use in several Eastern European countries for treatment of a variety of conditions, including pain syndromes. However, most treatment modes of MWT were developed empirically, and certain indications and contraindications for the use of MWT remain to be established. In our previous blind experiments we have shown that the hypoalgesic effect of MWT may be quantitatively evaluated, and most probably mediated by the neural system in general, and the system of endogenous opioids in particular. Taking in consideration a well-known ability of opioids to cause gastrointestinal disturbances, which could limit clinical application of MWT, the main aim of the present study was to investigate whether a single MWT, that can produce opioid-related hypoalgesia, may also retard gut transit and colorectal passage in mice. The charcoal meal test was used to quantitatively evaluate upper gastrointestinal transit, and the glass bead test was employed to examine colonic propulsion in mice. MWT was applied to the nose area of mice. The MWT characteristics were: frequency = 61.22 GHz; incident power density = 15 mW/cm(2); and duration = 15 min. The results obtained have shown that MWT does not significantly change small intestinal or colonic transit in mice, and thus suppression of gastrointestinal motility should not be a setback in the clinical use of MWT.     

Characterization of an A-kinase anchoring protein in human ciliary axonemes

Although protein kinase A (PKA) activation is known to increase ciliary beat frequency in humans the molecular mechanisms involved are unknown. We demonstrate that PKA is associated with ciliary axonemes where it specifically phosphorylates a 23-kDa protein. Because PKA is often localized to subcellular compartments in proximity to its substrate(s) via interactions with A-kinase-anchoring proteins (AKAPs), we investigated whether an AKAP was also associated with ciliary axonemes. This study has identified a novel 28 kDa AKAP (AKAP28)that is highly enriched in airway axonemes. The mRNA for AKAP28 is up-regulated as primary airway cells differentiate and is specifically expressed in tissues containing cilia and/or flagella. Additionally, both Western blot and immunostaining data show that AKAP28 is enriched in airway cilia. These data demonstrate that we have identified the first human axonemal AKAP, a protein that likely plays a role in the signaling necessary for efficient modulation of ciliary beat frequency.     

Total soft-tissue reconstruction of the middle and lower face with multiple simultaneous free flaps in a pediatric patient

A 2-year-old boy sustained a massive facial soft-tissue wound secondary to a dog attack. Essentially all the soft tissues of the face were absent, including innervation and intraoral lining. We describe the reconstruction of this defect with five simultaneous free tissue transfers. To our knowledge, this is the first report of five simultaneous free flaps in any patient.