Human cervicovaginal mucus contains an activity that hinders HIV-1 movement.

Cervical and vaginal epithelia are primary barriers against HIV type I (HIV-1) entry during male-to-female transmission. Cervical mucus (CM) is produced by the endocervix and forms a layer locally as well as in the vaginal compartment in the form of cervicovaginal mucus (CVM). To study the potential barrier function of each mucus type during HIV-1 transmission, we quantified HIV-1 mobility in CM and CVM ex vivo using fluorescent microscopy. Virions and 200-nm PEGylated beads were digitally tracked and mean-squared displacement was calculated. The mobility of beads increased significantly in CVM compared with CM, consistent with the known decreased mucin concentration of CVM. Unexpectedly, HIV-1 diffusion was significantly hindered in the same CVM samples in which bead diffusion was unhindered. Inhibition of virus transport was envelope-independent. Our results reveal a previously unknown activity in CVM that is capable of impeding HIV-1 mobility to enhance mucosal barrier function.

Bidirectional regulation of Munc13-3 protein expression by age and dark rearing during the critical period in mouse visual cortex.

Rearing in darkness slows the time course of the visual cortical critical period, such that at 5 weeks of age normal cats are more plastic than dark-reared cats, while at 20 weeks dark-reared cats are more plastic [Mower GD (1991) The effect of dark rearing on the time course of the critical period in cat visual cortex. Dev Brain Res 58:151-158]. Thus, genes that are important for visual cortical plasticity should show differences in expression between normal and dark-reared visual cortex that are of opposite direction in young versus older animals. Previously, we showed by differential display polymerase chain reaction and northern blotting that mRNA for Munc13-3, a mammalian homologue of the C. elegans uncoordinated (unc) gene, shows such bidirectional regulation in cat visual cortex [Yang CB, Zheng YT, Li GY, Mower GD (2002) Identification of Munc13-3 as a candidate gene for critical period neuroplasticity in visual cortex. J Neurosci 22:8614-8618]. Here, the analysis is extended to Munc13-3 protein in mouse visual cortex, which will provide the basis for gene manipulation analysis. In mice, Munc13-3 protein was elevated 2.3-fold in dark-reared compared with normal visual cortex at 3.5 weeks and 2.0-fold in normal compared with dark-reared visual cortex at 9.5 weeks. Analysis of variance of protein levels showed a significant interaction, indicating that the effect of dark rearing depended on age. This bidirectional regulation was restricted to visual cortex and did not occur in frontal cortex. Bidirectional regulation was also specific to Munc13-3 and was not found for other Munc13 family members. Munc13 proteins serve a central priming function in synaptic vesicle exocytosis at glutamatergic and GABAergic synapses and this work contributes to the growing evidence indicating a role of Munc13 genes in synaptic plasticity.

The effect of posterior tibial tendon dysfunction on hindfoot kinematics.

This biomechanical study investigated the functional role of the posterior tibial tendon (PTT) in acquired flatfoot mechanics. Acquired flatfoot deformity has been attributed to PTT dysfunction; however, the progression from acute dysfunction to end-stage deformity has not been fully demonstrated. Eight human cadaver lower leg and foot specimens were used in two phases of experimental testing. In Phase 1, intact (normal) specimens were loaded to simulate (a) heel strike, (b) stance, and (c) heel rise both with and without PTT function. Then, each specimen was subjected to a procedure designed to create a simulated flatfoot deformity. The resulting flattened feet were used in Phase 2 to examine the effect of restoring PTT function to a flatfoot model. During both phases of testing, the 3-D kinematic orientation of the hindfoot complex was recorded. Small but statistically significant changes in the angular orientation of the hindfoot complex were observed, during both Phase 1 and 2 testing, when comparing the effects of a functional and dysfunctional PTT. The greatest angular changes were recorded during heel rise. For the normal foot, the small changes observed in the orientation of the hindfoot complex following release of the PTT load suggest that the intact osteo-ligamentous structure of the hindfoot is initially able to maintain normal alignment following acute PTT dysfunction. Once the soft tissues have been weakened, as in our flatfoot model, the PTT had little effect in overcoming the soft tissue laxity to correct the position of the foot.

The size difference between leishmania major friedlin chromosome one homologues is localized to sub-telomeric repeats at one chromosomal end.

Leishmania species are members of the evolutionarily ancient protozoan order Kinetoplastidae and are important human pathogens. The Leishmania genome is relatively small (approximately 34 Mbp) and is distributed among 36 chromosome pairs, ranging in size from 0.3 to 2.5 Mbp. The smallest chromosome of Leishmania major Friedlin, chrl, consists of three homologues which differ in size by approximately 29 kb. Previous sequence and Southern analyses of all three homologues reveal a conserved chromosomal core, consisting of coding and adjacent 'non-informational' sequence. Here we show the size difference between homologues is largely restricted to variation in both the number and content of several sub-telomeric repetitive elements localized on one chromosomal end. These repetitive elements also occur on other chromosomes, but some are more dispersed in the Leishmania genome than others.

Lipid-coated microgels for the triggered release of doxorubicin.

We have systematically engineered a polymeric, multi-component drug delivery system composed of a lipid-coated hydrogel microparticle (microgel). The design of this delivery system was motivated by the recent elucidation of the mechanism of regulated secretion from the secretory granule and the compositional and structural features that underlie its ability to store and release endogenous drug-like compounds. The present work describes the assembly and response of a prototype construct which displays several important features of the secretory granule, including its high drug loading capacity, and triggered microgel swelling, resulting in the burst release of drug. To achieve this, ionic microgels were synthesized, and loaded with doxorubicin via ion exchange. These microgels were then coated with a lipid bilayer, and the release of doxorubicin was triggered from the gels using either lipid-solubilizing surfactants or electroporation. The use of a microanalytical technique is featured utilizing micropipette manipulation that allows the study of the behavior of individual microparticles. The lipid-coated microgels were electroporated in saline solution; they swelled and disrupted their bilayer coating over a period of several seconds and exchanged doxorubicin with the external plasma saline over a period of several minutes. It is envisioned that this system will ultimately find utility in drug delivery systems that are designed to release chemotherapeutic agents and peptides by the application of a triggering signal.

Residual oil fly ash and charged polymers activate epithelial cells and nociceptive sensory neurons.

Residual oil fly ash (ROFA) is an industrial pollutant that contains metals, acids, and unknown materials complexed to a particulate core. The heterogeneous composition of ROFA hampers finding the mechanism(s) by which it and other particulate pollutants cause airway toxicity. To distinguish culpable factors contributing to the effects of ROFA, synthetic polymer microsphere (SPM) analogs were synthesized that resembled ROFA in particle size (2 and 6 microm in diameter) and zeta potential (-29 mV). BEAS-2B human bronchial epithelial cells and dorsal root ganglion neurons responded to both ROFA and charged SPMs with an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the release of the proinflammatory cytokine interleukin-6, whereas neutral SPMs bound with polyethylene glycol (0-mV zeta potential) were relatively ineffective. In dorsal root ganglion neurons, the SPM-induced increases in [Ca(2+)](i) were correlated with the presence of acid- and/or capsaicin-sensitive pathways. We hypothesized that the acidic microenvironment associated with negatively charged colloids like ROFA and SPMs activate irritant receptors in airway target cells. This causes subsequent cytokine release, which mediates the pathophysiology of neurogenic airway inflammation.

Leishmania major Friedlin chromosome 1 has an unusual distribution of protein-coding genes.

Leishmania are evolutionarily ancient protozoans (Kinetoplastidae) and important human pathogens that cause a spectrum of diseases ranging from the asymptomatic to the lethal. The Leishmania genome is relatively small [ approximately 34 megabases (Mb)], lacks substantial repetitive DNA, and is distributed among 36 chromosomes pairs ranging in size from 0.3 Mb to 2.5 Mb, making it a useful candidate for complete genome sequence determination. We report here the nucleotide sequence of the smallest chromosome, chr1. The sequence of chr1 has a 257-kilobase region that is densely packed with 79 protein-coding genes. This region is flanked by telomeric and subtelomeric repetitive elements that vary in number and content among the chr1 homologs, resulting in an approximately 27.5-kilobase size difference. Strikingly, the first 29 genes are all encoded on one DNA strand, whereas the remaining 50 genes are encoded on the opposite strand. Based on the gene density of chr1, we predict a total of approximately 9,800 genes in Leishmania, of which 40% may encode unknown proteins.

Support of the talus: a biomechanical investigation of the contributions of the talonavicular and talocalcaneal joints, and the superomedial calcaneonavicular ligament.

The goal of this study was to determine the magnitude of force transmission to the talus by its inferior articulations to provide insight into mechanisms involving acquired deformities of the hindfoot. Cadaver feet were mounted in a loading apparatus that applied axial force through the tibia and fibula as well as tensile loading of the tendons of extrinsic musculature. This also permitted positioning of the tibia in the sagittal plane. Eighteen specimens were tested in three selected positions of the gait cycle. In one series, pressure-sensitive film was inserted into the posterior and anteromedial facets of the talocalcaneal joint as well as into the talonavicular joint. In a second series, film was inserted between the talar head and the superomedial calcaneonavicular ligament. In stance position, the specimens were also tested without posterior tibial tendon (PTT) tension. Contact areas and force transmitted across the articulations were greatest in near toe-off position, in the posterior facet of the talocalcaneal joint. The talonavicular joint, the anteromedial facet of the talocalcaneal joint, and the calcaneonavicular ligament articulation showed sequentially decreasing amounts of contact area and force transmission. Mean pressures were similar across all articulations, except in the posterior facet in near toe-off position. From heel-strike to stance, to near toe-off, a trend to increasing contact area and force was noted. No difference in contact characteristics was found in the calcaneonavicular ligament articulation after PTT release. The contact force of the calcaneonavicular ligament against the talus was found to be much smaller than those of other talar articulations; however, its medially oriented direction must contribute to stabilization of the head of the talus against medial displacement. Loss of PTT tension was not found to alter the contact forces acting at the talar head in this model, which might indicate that it shares its talar stabilizing function with other structures.

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of rat somatosensory barrel cortex.

The postnatal development of glutamic acid decarboxylase (GAD; GAD67 and GAD65) expression was studied in the rat somatosensory cortex. Delineation of barrels in layer IV by GAD67 immunoreactivity occurred between postnatal days P3 and P6 and remained evident into adulthood. At birth, a band of GAD67-positive elements was already present in superficial layer V. This band was prominent until P6 and gradually disappeared after P9. In parallel, there was a gradual appearance of GAD67-immunoreactive cells neuropil and puncta, which began in layer VI/subplate at P1 and achieved the adult laminar pattern by about P13. This later GAD67 immunoreactivity was responsible for the demarcation of barrels in layer IV. Development of GAD65 immunoreactivity was delayed relative to GAD67. GAD65 immunoreactivity, which was in little evidence before P6, increased markedly in density and in delineation of cell bodies over the next several weeks. During this prolonged developmental process, GAD65 first showed a negative image of the barrels compared with the septae and the surrounding cortex. Subsequently, there was a filling in of the barrels resulting in rather uniform GAD65 immunoreactivity across the barrel field and surrounding cortex. These results suggest that the development of the gamma-aminobutyric acid (GABA) synthetic system in the barrel cortex involves several processes: the disappearance of a precocious GAD67 system in layer V, the temporally overlapping maturation of the mature GAD67 system in an inside-outside manner, and the delayed and prolonged development of the GAD65 system.

A synthetic mimic of the secretory granule for drug delivery.

Secretory cells contain submicroscopic granules composed of a polyanionic polymer network that is collapsed owing to the presence of hydronium ions and weak base cations. The network is encapsulated within a lipid membrane, and functions as a vehicle for the osmotically inert storage of a variety of granule-bound endogenous mediator species, such as histamine, serotonin and proteases. These species are excreted from the granule and thence from the cell in response to external biochemical signals. Hydrogels that swell and shrink in response to external stimuli might serve as synthetic analogues of secretory granules. Here we describe the systematic engineering of multi-component, environmentally responsive hydrogel microspheres, coated with a lipid bilayer to mimic more closely the natural secretory granule. These microspheres exhibit pH- and ion-dependent volume phase transitions and ion-sensitive exchange of bound cations when the encapsulating lipid membrane is porated. We stimulated poration electrically in individual microgel particles immobilized and manipulated with a micropipette. This system could find use for the triggered release of encapsulated drugs in the body.

Two reconstructive techniques for flatfoot deformity comparing contact characteristics of the hindfoot joints.

The effect of two different methods of reconstruction of flatfoot deformity and the role of the posterior tibial tendon on the contact characteristics of the hindfoot joints were quantified using pressure-sensitive film. Each of 10 cadaver feet was loaded quasi-statically by an axial compressive force to simulate varying loads. First, a specimen was tested intact, then it was tested after sectioning the spring ligament and loading the specimen cyclically to create one type of flatfoot deformity. It was then tested again after reconstructing the deformity. Reconstructions used were the Dillwyn-Evans procedure (bone graft in osteotomy of the calcaneus) or the calcaneocuboid distraction arthrodesis (CCDA). We found that surgically produced flatfoot deformity altered mainly the talonavicular joint, by decreasing its contact area. The Dillwyn-Evans method had less effect on the talonavicular joint (altering 2 of 6 measured parameters) than the CCDA (3 of 6) and more effect on the anteriomedial facet (altering 3 of 6 parameters) than the CCDA (1 of 6). The Dillwyn-Evans method had more effect on the posterior facet (altering 2 of 6 measured parameters) than the CCDA (1 of 6). Function of the posterior tibial tendon had no effect on contact characteristics of the hindfoot joints after either type of reconstruction. These findings are based on measurements using a quasi-statically-loaded foot model at three selected positions, and results may be different with dynamic loading.

Biomechanical analysis of capitate shortening with capitate hamate fusion in the treatment of Kienböck's disease.

The biomechanical effects of surgical treatment options for Kienböck's disease have been compared. However, no study has included a direct analysis of capitate shortening along with capitate-hamate fusion (CSCHF). To investigate the biomechanical effects of CSCHF, a cadaver model of the upper extremity was used to determine radiocarpal articular pressure changes resulting from this procedure using pressure-sensitive film. Ten specimens were tested by placing each in an apparatus that applied load across the radiocarpal joint through the wrist flexor and extensor tendons. Testing was performed in 3 wrist positions (ulnar deviation, radial deviation, and neutral) combined with 3 forearm positions (pronation, supination, and neutral) and neutral flexion/extension. Radioscaphoid, radiolunate, and mean contact pressures in the entire radiocarpal joint were determined for each of the 9 wrist positions, both intact and after surgery. The radioscaphoid mean pressure increased in 6 of 9 positions and was unchanged in 3 positions. The radiolunate mean pressure decreased in 9 of 9 positions. The radiocarpal mean pressure increased in 2 of 9 positions and was unchanged in 7 positions. These data suggests that CSCHF increases radioscaphoid mean pressure, decreases radiolunate mean pressure, and has little effect on radiocarpal mean pressure.

Two-, four-, and six-strand zone II flexor tendon repairs: an in situ biomechanical comparison using a cadaver model.

A dynamic in vitro model of zone II flexor tendon repair was used to compare gliding resistance, gap formation, and ultimate strength of the 2-, 4-, and 6-strand repair techniques. Each of 12 hands was mounted to a loading frame with 3 flexor tendons attached to individual pneumatic cylinders. A spring attached to a pin through the distal end of each digit provided a 1.25-kg resistance force. The force required to flex each proximal interphalangeal joint to 90 degrees was determined. Following this, the tendons were sectioned and each was repaired using a different technique so that each specimen acted as its own control. The 2- and 4-strand core sutures were placed using a suture interlock technique with radial and ulnar grasping purchase of the tendon on each side of the transverse part of the repair. Each repair was accomplished using a single core stitch with the knot buried between the tendon ends. The 4-strand repair involved an additional horizontal mattress suture with the knot buried. Repair of the dorsal side of the tendon was performed followed by core suture placement. The palmar portion of the peripheral locking suture was completed after core suture placement. Following repair, each hand was remounted on the frame and cycled 1,000 times. After cyclic loading, the resulting gap between the repaired ends of each tendon was measured, the tendons were removed from the hand, and each was loaded to failure in tension. All tendon repairs showed a small, but not statistically significant, increase in gliding resistance after reconstruction. The 2-strand repair had significantly greater gap formation after cyclic loading (mean gap, 2.75 mm) than either the 4-strand (0.30 mm) or 6-strand (0.31 mm) repair. The tensile strength of the 6-strand repair (mean, 78.7 N) was significantly greater than either the 4-strand (means, 43.0 N) or 2-strand (mean, 33.9 N) repair.

Effects of intra-articular distal radius depression on wrist joint contact characteristics.

Twelve cadaver extremities were used to study the effect of scaphoid and lunate facet depressions on the contact characteristics of the radiocarpal joint. Pressure-sensitive film was inserted into radiocarpal joints with varying degrees of depression, and the specimens were loaded statically in neutral position, radial deviation, and ulnar deviation. The film was removed and analyzed for contact area and pressure. The only statistically significant effect of a lunate fossa depression was an increase in scaphoid fossa pressure with a 3-mm step-off and the hand in neutral position. Scaphoid fossa depression had more significant effects. With a 1-mm scaphoid fossa depression, lunate fossa pressures increased in neutral position and in radial deviation. Lunate fossa contact area increased, compared to intact joints, in ulnar and radial deviation with 1-mm scaphoid fossa depressions and in all loading positions with 3-mm scaphoid fossa depression. Therefore, it appears that the most significant effect on radiocarpal joint contact characteristics occurs with a depression of the scaphoid side of the joint. Even with depressions as small as 1 mm, significant changes on the lunate side of the joint were observed.

The scapholunate allograft: a biomechanical feasibility study.

The feasibility of a new method for reconstruction of the isolated scapholunate ligament tear with a scapholunate allograft was studied biomechanically using a fresh cadaver upper extremity model. Seven specimens were first tested intact for wrist range of motion, contact characteristics of the radiocarpal articulation, and relative motion of the scaphoid with respect to the lunate. Then a segment consisting of the radial third of the lunate, the adjoining scapholunate interosseus ligament, and the proximal pole of the scaphoid was resected. This segment was matched to a donor allograft of similar size and geometry, which was then placed into the recipient carpus and secured with one central transverse Kirschner wire and two shorter interfragmentary Kirschner wires placed into the lunate and scaphoid. The allografted specimen was subjected to the same tests as the intact wrist. Results showed that there were no significant differences in wrist range of motion, radiocarpal articular surface contact area and pressure, and relative flexion-extension rotation between the scaphoid and lunate during passive radioulnar deviation between the intact and allografted wrists.

Electrochemistry of anticonvulsants: electron transfer as a possible mode of action.

Reduction potentials were determined for various anticonvulsants, including progabide, SL 75.102, CGS 9896, pyridazines, zonisamide, 1,2,3-triazoles, and copper complexes. The values generally were in the range of about -0.1 to -0.6 V for the protonated drugs and the metal complexes. Reduction potentials provide information on the feasibility of electron transfer (ET) in vivo. If the value is relatively positive (greater than about -0.6 V), the agent can act catalytically as an electron acceptor from an appropriate cellular donor. A concomitant favorable influence on abnormal neuronal processes associated with epilepsy could occur. We describe ET as a possible mode of action of anticonvulsants as well as some antiepileptic agents with no electrochemical data based on this hypothetical ET approach.

Electrochemistry of Cu(I) bipyridyl complexes with alkene, alkyne, and nitrile ligands. Implications for plant hormone action of ethylene.

The redox behavior was evaluated for several (BIPY)Cu(I) complexes (BIPY = 2,2'-bipyridyl) with unsaturated ligands by means of cyclic voltammetry in CH2Cl2 at reduced temperatures (-78 degrees, -23 degrees, 0 degree C). The complexes studied are [Cu(I)(BIPY)(C2H4)]PF6, [Cu(I)(BIPY)(3-hexyne)] PF6, [Cu(I)(BIPY)(DEAD)]PF6, ([Cu(I)(BIPY)]2 DEAD)[PF6]2 (DEAD = diethyl acetylene dicarboxylate) and [Cu(I)(BIPY)(CH3CN)]PF6. The oxidations are quasi-reversible at -78 degrees C for scan rates of 20 to 200 mV/sec. The reductions were irreversible on the CV time scale. Evidence is presented in support of a role for an electron transfer mechanism in the case of the plant hormone ethylene. Related literature data are also discussed.

Are reduction potentials of antifungal agents relevant to activity?

Cyclic voltammetry data were obtained for several categories of fungicidal agents including quinones (akrobomycin, podosporin A), iminium ions and precursors (pyridazines, 15-azahomosterol, griseofulvin-4'-oxime), and metal derivatives of chelators (pyridine-2-aldehyde thiosemicarbazones). The reductions usually occurred in the range of -0.7 to +0.3 V. Reduction potentials provide information on the feasibility of electron transfer in vivo. Catalytic production of oxidative stress from redox cycling is a possible mode of action. Alternatively, there may be interference with normal electron transport chains.

Theoretical calculations on calcium channel drugs: is electron transfer involved mechanistically?

Theoretical studies were done on calcium channel drugs in order to gain insight into the mode of action. Empirical force field calculations with nifedipine, a calcium channel antagonist, indicate that the E-conformation at the ring juncture is lower in energy than the Z-conformation. This energy difference is only 0.2 kcal/mol when the esters in the 3- and 5-positions of the dihydropyridine (DHP) ring are both synperiplanar (sp, sp). Molecular orbital calculations on the ground and excited states in the Z-conformation with the esters in the (ap, sp) conformation show a low lying excited state with substantial intramolecular electron transfer (ET) character. This excited state is only 1.8 eV higher in energy than the ground state and corresponds to a transfer of approximately 0.3 electron from the DHP ring to the nitrobenzene moiety. We suggest that ET may play an important role in the mechanism of action, either intramolecular or, as previously proposed, intermolecular, along with lipophilicity and steric effects.

Transient pulmonary perfusion scintigraphic abnormalities in pulmonary air embolism.

A critically ill man suffered a respiratory arrest due to pulmonary air embolism after the exchange of central venous catheters over a guidewire. A pulmonary perfusion lung scan performed 90 min later demonstrated extensive perfusion defects which were interpreted as "high probability" for PTE. Pulmonary angiography 4.5 h later was normal. A second perfusion lung scan performed 24 h after the respiratory arrest was normal. Pulmonary air embolism can produce segmental (and larger) perfusion defects which may be indistinguishable from those caused by PTE. The rapid (24 h) resolution of the perfusion defects may help differentiate the two disorders.