Maturation of the posterolateral spinal fusion and its effect on load-sharing of spinal instrumentation. An in vivo sheep model

We investigated the temporal relationship among the biomechanical, radiographic, and histological properties of a posterolateral spinal fusion mass to elucidate the changes in load-sharing of the spinal instrumentation and that of the fusion mass throughout the healing process. Destabilization of the posterior spinal column and transpedicular screw fixation at the segments between the third and fourth and the fifth and sixth lumbar vertebrae was performed in twenty-four sheep. A posterolateral spinal arthrodesis with use of autologous corticocancellous bone graft was done randomly at one of the two segments; the other segment (without bone graft) served as the instrumented control. Six animals each were killed at four, eight, twelve, and sixteen weeks postoperatively. Biomechanical testing showed that the posterolateral fusion mass had increased mechanical stiffness after the fourth week. The strain on the hardware, measured with use of rods instrumented with strain-gauges, decreased significantly (p < 0.01) beginning at eight weeks. Radiographically, three independent observations of each of the six animals at each time-period showed that, although all of the fusion masses were considered solid unions at sixteen weeks, bridging of trabecular bone was noted during only ten of eighteen observations at twelve weeks, three of eighteen observations at eight weeks, and none of eighteen observations at four weeks. Computerized tomography and histomorphometric analyses demonstrated that mineralization in the fusion mass increased in a linear fashion even after eight weeks. Histologically, the fusion mass consisted predominantly of woven bone at eight weeks; thereafter, it was gradually trabeculated. CLINICAL RELEVANCE: We found a great discrepancy between biomechanical stability and histological maturation of the posterolateral fusion mass. The biomechanical properties of a stable spinal fusion preceded the radiographic appearance of a solid fusion by at least eight weeks, suggesting that immature woven bone provided substantial stiffness to the fusion mass. The spinal instrumentation was subjected predominantly to bending stress rather than to axial stress, and the load-sharing of the spinal instrumentation decreased concurrently with the development of the spinal fusion.     

Characterization of Y3 receptor-mediated synaptic inhibition by chimeric neuropeptide Y-peptide YY peptides in the rat brainstem

Archival material from primary and metastatic renal clear cell carcinomas of 25 patients was studied by comparative genomic hybridization. Copy number changes of entire chromosomes or chromosomal subregions were detected in 22 primary and 21 metastatic tumors. Copy number changes affected the following chromosomes in at least 20% of the 25 primary tumors (minimal common region given in parentheses): gains were noted for chromosomes 1 (1q21-->q23), 5 (5q31-->q34), 7 (7p), 8 (8q), 16 (16p), 17 (17q12-->qter), 19, and 22 (22q12-->qter); losses were revealed for chromosomes 3 (3p21-->pter), 8 (8p23-->pter), 14(14q21-->qter), and Y. The same chromosomal regions that were involved in primary renal clear cell carcinomas were also found in the respective metastatic tumors but with strikingly different frequencies for a few regions. Metastatic tumors showed a significantly higher frequency of complete or partial gains of the long arm of chromosome 1, in particular at 1q21-->q23 than primary tumors (16 cases versus 6 cases; P < 0.005). These data suggest a correlation of metastatic events in renal clear cell carcinomas with an increase in the copy number of genes located at 1q, in particular at 1q21-->q23. In contrast, the entire or partial loss of the short arm of chromosome 3 was significantly less frequent in metastatic tumors (8 cases versus 15 cases; P < 0.025). The validity of 1q and 3p copy number changes detected by comparative genomic hybridization was confirmed by interphase cytogenetics with region-specific yeast artificial chromosomes to paraffin-embedded tumor tissue sections.     

Killing of Chlamydia trachomatis by novel antimicrobial lipids adapted from compounds in human breast milk

The development of new methods for prevention of sexually transmitted Chlamydia trachomatis infection is a top public health priority. Topical self-administered vaginal microbicides represent one such approach in which the organism is eradicated at the time of initial exposure. To this end, we examined the activity of five synthetic lipids adapted from naturally occurring compounds found in human breast milk. C. trachomatis serovar D or F elementary bodies were added to serial dilutions of the lipids and incubated for various times. Aliquots were then cultured in monolayers of McCoy cells, and inclusions were counted. A 7.5 mM concentration of 2-O-octyl-sn-glycerol completely prevented growth of C. trachomatis after 120 min of contact with the organism. The remaining lipids, 1-O-octyl-, 1-O-heptyl-, 2-O-hexyl-, and 1-O-hexyl-sn-glycerol, showed less activity. On electron microscopic examination, the lipids were shown to have disrupted the chlamydial inner membrane, allowing leakage of the cytoplasmic contents from the cell. Lipid activity was unaffected by the presence of 10% human blood or alterations in pH from 4.0 to 8.0, conditions reflecting those sometimes found in the vagina. Our results suggest that these lipids, especially 2-O-octyl-sn-glycerol, may be effective as topical microbicides in preventing the transmission of C. trachomatis. Further efficacy and toxicity studies with these lipids and assessment of their activity against other sexually transmitted disease pathogens are in progress.     

BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation

Secretory proteins are cotranslationally translocated across the mammalian ER membrane through an aqueous pore in the translocon while the permeability barrier is maintained by a tight ribosome-membrane junction. The lumenal end of the pore is also blocked early in translocation. Extraction of soluble lumenal proteins from microsomes and reconstitution with purified proteins demonstrate, by fluorescence collisional quenching, that BiP seals the lumenal end of this pore. BiP also seals translocons that are assembled but are not engaged in translocation. These ribosome-free translocons have smaller pores (9-15 A diameter versus 40-60 A in functioning translocons) and are generated when ribosomes dissociate from functioning translocons with large pores. BiP therefore maintains the permeability barrier by sealing both nontranslocating and newly targeted translocons.     

Actions of A-131701, a novel, selective antagonist for alpha-1A compared with alpha-1B adrenoceptors on intraurethral and blood pressure responses in conscious dogs and a pharmacodynamic assessment of in vivo prostatic selectivity

A-131701 (3-[2-((3aR,9bR)-cis-6-methoxy-2,3,3a,4,5,9b, hexahydro-[1H]-benz[e]isoindol-2-yl)ethyl]pyrido [3',4': 4,5]thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione) is a novel compound previously shown to be selective for alpha-1a sites compared with alpha-1b adrenoceptors in radioligand binding studies and isolated tissue bioassays and to block canine urethral pressure (IUP) responses to exogenous alpha-1 adrenergic agonists to a greater extent than blood pressure responses. In conscious dogs in which IUP and mean arterial blood pressure (MABP) responses were measured periodically up to 24 hr, A-131701 blocked phenylephrine (PHE)-induced increases in IUP to a greater extent than MABP responses, and the blockade of the IUP effects of PHE was significantly different from control for up to 12 hr after doses greater than 0.3 mg/kg p.o., whereas blood pressure effects were of a lesser extent and duration. In addition to the weak antagonism of PHE-induced blood pressure responses, A-131701 also exhibited minimal effects on basal blood pressure in the dog, unlike terazosin, doxazosin or tamsulosin. Pharmacokinetic analysis of plasma samples from dogs indicated that A-131701 had a half-life of 0.4 to 0.8 hr and a bioavailability of 30 to 50% in dogs. Somewhat longer half-lives were observed in rat and monkey, with bioavailability values in the 25 to 30% range. Evidence of nonlinearity of pharmacokinetics was obtained in dogs and monkeys. Pharmacodynamic analysis revealed differences between A-131701 and nonselective alpha-1 adrenoceptor antagonists in selectivity for prostatic versus vascular alpha-1 adrenoceptors based on either extent or duration of blockade, which were either similar to or superior to compounds such as tamsulosin or REC 15/2739. These data demonstrate that A-131701 selectively blocks canine prostatic alpha-1 adrenoceptors for prolonged periods compared with MABP responses in vivo. Therefore, A-131701 should have clinical utility in the pharmacotherapy of benign prostatic hyperplasia.     

Sorting human beta-cells consequent to targeted expression of green fluorescent protein

Pancreatic islets of Langerhans are composed of four major endocrine cell types with a smaller number of nonendocrine cells. To study the molecular constituents and function of just one subpopulation of islet cells, it is necessary to sort them from the other cell types. While rat beta-cells can be sorted by autofluorescence-activated flow cytometry, this has not proved possible on a routine and reproducible basis for human beta-cells. In the present study, we have selectively labeled human beta-cells with green fluorescent protein (GFP), allowing for their sorting by flow cytometry. Human islet cells were infected with replication-defective (attenuated) recombinant adenovirus expressing GFP driven by the rat insulin I promoter (Ad-RIP-GFP) for targeted expression in beta-cells, or beta-galactosidase driven by the promiscuous cytomegalovirus (CMV) promoter (Ad-CMV-beta-gal) as control. Whereas the majority of islet cells can be infected by adenovirus, as shown by control infection with Ad-CMV-beta-gal, increased fluorescence after infection with Ad-RIP-GFP was limited to insulin-containing beta-cells. Infection of islet cells with Ad-RIP-GFP resulted reproducibly in the appearance of a population of intensely fluorescent cells, when analyzed by flow cytometry. These cells were sorted using a fluorescence-activated cell sorter (FACS) and shown by immunofluorescence to consist of >95% beta-cells. The targeted expression of GFP thus allows for preparation of human beta-cells purified close to homogeneity. This method should be readily applicable in any laboratory with FACS capability.     

Dopamine-dependent pathology and trace processes

During recent years, ceramide has received a lot of attention as a possible mediator of the cellular responses to a variety of apoptotic stimuli. In a manner analogous to generation of its sibling diacylglycerol, ceramide is generated by a phospholipase-C-type reaction from its lipid precursor sphingomyelin. Two observations led to the proposal that ceramide plays a role in apoptosis: (1) treatment of cells with tumor necrosis factor or other inducers of apoptosis leads to activation of sphingomyelinases and to an increase in cellular ceramide levels; (2) ectopic generation or administration of ceramide can mimic apoptotic cell death. Recently, several observations have challenged the notion that ceramide is an important cell-death mediator and have prompted a re-evaluation of previously published results.     

Treatment of microfilaraemia in asymptomatic brugian filariasis: the efficacy and safety of the combination of single doses of ivermectin and diethylcarbamazine

BACKGROUND: Nitric oxide (NO) seems to play an important role in modulating tissue injury during reperfusion of the liver. In this study, we have evaluated and compared the effects of FK409 (FK), a potent spontaneous NO releaser, and L-arginine in ischemia-reperfusion injury of the rat liver. METHODS: Male Sprague-Dawley rats underwent 90 min of hepatic ischemia followed by reperfusion. FK or L-arginine was used (intravenously) in two different doses for each drug (group I, 3.2 mg/kg FK; group II, 1.6 mg/kg FK; group IV, 100 mg/kg L-arginine; and group V, 300 mg/kg L-arginine). Saline was used in control animals (group III). Hepatic enzyme status, microcirculation, serum nitrite (NO2-) and nitrate (NO3-) and tissue injury score were evaluated at predetermined times. RESULTS: Serum NO2-/NO3- was elevated immediately by FK treatment dose-dependently but not by L-arginine. However, L-arginine caused late (6-24 hr) elevation of the NO metabolites dose-dependently. The elevation of serum aspartate aminotransferase and alanine aminotransferase was suppressed and hepatic microcirculation was improved in the FK-treated groups dose-dependently. L-Arginine also improved the microcirculation, but hepatic enzymes at 24 hr of reperfusion were significantly higher in group V than in the control group. These findings were well reflected by the extent of tissue injury in respective groups. CONCLUSION: FK treatment in the immediate reperfusion period improves hepatic microcirculation and confers a significant protective effect on hepatic ischemia-reperfusion injury in the rat.     

Use of lectins to characterize plasma membrane preparations from boar spermatozoa: a novel technique for monitoring membrane purity and quantity

The object of this study was to develop a method to quantify the amount of outer acrosomal membrane material in isolated plasma membranes from boar sperm cells. The cells were fractionated by nitrogen cavitation, and plasma membranes were isolated by subsequent differential centrifugation steps. Marker enzyme measurement showed that the plasma membrane isolates were enriched in plasma membrane markers and did not contain nuclei, inner acrosomal membranes, or mitochondria. Since there is no marker enzyme known for the outer acrosomal membrane, lectins were used for the detection of this membrane. The membrane specificity of a number of lectin conjugates was tested with fluorescence microscopy and transmission electron microscopy. Membrane binding of these lectin conjugates was quantified with flow-cytometry and an enzyme-linked lectin binding assay. Wheat germ agglutinin was specific for the plasma membrane while peanut agglutinin was specific for the outer acrosomal membrane. The use of these lectins made it possible for the first time to discriminate between these two membranes. The isolated plasma membrane fraction was enriched more than 10-fold (17-fold after further purification by a sucrose gradient) in plasma membrane material compared to outer acrosomal membrane material. Highly purified sperm plasma membranes should prove to be useful for research on primary sperm-zona interactions.