Isolation of cDNA clones from an osteosarcoma-ROS17/2.8 library by differential hybridization

We have used differential hybridization to isolate and characterize two novel cDNAs expressed in chondrocytes and some osteoblastic cells. A rat osteosarcoma ROS17/2.8 cDNA library was screened and cDNA clones hybridizing strongly to radiolabeled porcine calvaria cDNA but weakly to a control radiolabeled cDNA were isolated. Two clones were obtained--p.6.1 and p.10.15. A radiolabeled probe of p10.15 was shown to hybridize specifically to a 2.3 Kb message RNA from a chondrogenic clonal cell population from rat calvaria-RCJ 3.1C5.18, and the mRNA was downregulated by 1,25 (OH)2D3, which inhibits chondrogenesis in these cells. The other clone, p6.1, was found to hybridize to a 0.95 Kb message that is expressed in rat liver, kidney, lung, muscle, and brain, but not expressed in spleen and expressed only in low levels in thymus.     

Histopathology of the liver in children with chronic hepatitis C viral infection

Although the epidemiology, natural history, and pathological aspects of chronic hepatitis C are well-defined in the adult population, little is known about the characteristics of chronic hepatitis C infection in children. Reports on the histological features and progression of hepatitis C in children are scarce, and consist primarily of multicenter studies in Japanese and European children. Given the geographic variations in viral genotype and the association of pathology with genotype, whether the Japanese and European studies can be extended to the North American populations is unclear. We report the histopathology of the liver in 40 children with chronic hepatitis C infection treated in a single North American institution. The children included 19 males and 21 females ranging in age from 2.0 to 18.6 years at the time of liver biopsy (mean +/- SD: 11.4 +/- 4.3 years). Our findings indicate that the characteristic histopathological lesions of chronic hepatitis C infection, including sinusoidal lymphocytosis, steatosis, portal lymphoid aggregates/follicles, and bile duct epithelial damage, occur with approximately the same frequencies in children as have been reported in adults. Necroinflammatory activity was generally mild. Portal fibrosis was present in 78% of the specimens, including fibrous portal expansion (26%), bridging fibrosis (22%), bridging fibrosis with architectural distortion (22%), and cirrhosis (8%). Centrilobular pericellular fibrosis, which has not been previously reported in the context of chronic hepatitis C infection in adults or children, was also a prominent feature in our series, occurring with a similar frequency as steatosis or portal lymphoid aggregates/follicles. Our data suggest that in spite of mild histological necroinflammatory activity in general, the stage of fibrosis in children can be severe in spite of relatively short duration of infection.     

The internal workings of a DNA polymerase clamp-loading machine

Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins. The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader. We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme. The gamma complex uses ATP to open the beta clamp and assemble it onto DNA. Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp. The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it. The delta' subunit acts as a modulator of the interaction between delta and beta. On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface. The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA. Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex.     

A candidate recombination modifier gene for Zea mays L

Maize meiotic mutant desynaptic (dy) was tested as a candidate recombination modifier gene because its effect is manifested in prophase I. Recombination rates for desynaptic (dy) and its wild type were compared in two ways: (1) segregation analysis using six linked molecular markers on chromosome 1L and (2) cytogenetic analysis using fluorescence in situ hybridization (FISH)-aided meiotic configurations observed in metaphase I. Chromosome 1L map lengths among the six linked markers were 45-63 cM for five F2 dy/dy plants, significantly lower than the wild-type F2 map distance of 72 cM. Chromosomes 2 and 6 were marked with rDNA FISH probes, and their map lengths were estimated from FISH-adorned meiotic configurations using the expectation-maximization algorithm. Chiasma frequencies for dy/dy plants were significantly reduced for both arms of chromosome 2, for chromosome arm 6L, and for eight unidentified chromosomes. There was a notable exception for the nucleolus-organizing region-bearing arm chromosome arm 6S, where dy increased chiasma frequency. Maize meiotic mutant desynaptic is a recombination modifier gene based on cytogenetic and segregation analyses.     

Vasopeptidase inhibitors: incorporation of geminal and spirocyclic substituted azepinones in mercaptoacyl dipeptides

A series of 7-(di)alkyl and spirocyclic substituted azepinones were generated and incorporated as conformationally restricted dipeptide surrogates in mercaptoacyl dipeptides. Clear structure-activity relationships with respect to both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) activity in vitro were observed. The best in this series, compound 1g, a geminally dimethylated C-7-substituted azepinone, demonstrated excellent blood pressure lowering in animal models. Compound 1g (BMS-189921) is characterized by a good duration of activity and excellent oral efficacy in models relevant to ACE or NEP inhibition, and its activity is comparable to that of the clinically efficacious agent omapatrilat. Consequently this inhibitor has been advanced clinically for the treatment of hypertension and congestive heart failure.     

Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: a novel multihemic cytochrome bc, a new complex III

Rhodothermus marinus, a thermohalophilic bacterium, has a unique electron-transfer chain, containing, besides a cbb3 and a caa3 terminal oxidases, a novel cytochrome bc complex [Pereira, M. M., Carita, J. N., and Teixeira, M. (1999) Biochemistry 38, 1268-1275]. The membrane-bound iron-sulfur centers of this bacterium were studied by electron paramagnetic resonance (EPR) spectroscopy, leading to the identification of its main electron-transfer complexes. The resonances typical for the Rieske-type centers are not detected. Clusters S1 and S3 from succinate dehydrogenase were identified; interestingly, center S3 is shown to be present in two different conformations, with g values at 2.035, 2.009, and 2.001 and at 2.025, 2.002, and 2.000. Upon addition of NADH and dithionite, EPR signals assigned to resonances characteristic of binuclear and tetranuclear clusters develop and are attributed to the iron-sulfur centers of complexes I and II. A high-potential iron-sulfur protein- (HiPIP-) type center previously detected in the membranes of this bacterium [Pereira et al. (1994) FEBS Lett. 352, 327-330] is shown to belong indeed to a canonical HiPIP. This protein was purified and extensively characterized. It is a small water-soluble protein of approximately 10 kDa, containing a single [4Fe-4S]3+/2+ cluster. The reduction potential, determined by EPR redox titrations in intact and detergent-solubilized membranes as well as by cyclic voltammetry in solution, has a pH-independent value of 260 +/- 20 mV, in the range 6-9. In vitro reconstitution of the R. marinus electron-transfer chain shows that the HiPIP plays a fundamental role in the chain, as the electron shuttle between R. marinus cytochrome bc complex and the caa3 terminal oxidase, being thus simultaneously identified a HiPIP reductase and a HiPIP oxidase.     

In vitro and in vivo effects of phenolic antioxidants against cisplatin-induced nephrotoxicity

We have investigated the effect of phenolic antioxidants on cisplatin-induced cytotoxicity in vero (African Green Monkey Kidney) cells and in rat renal cortical slices in vitro, and on cisplatin-induced nephrotoxicity in rats in vivo. Incubation of cisplatin with vero cells resulted in time- and concentration-dependent cytotoxicity, as characterized by decreased tryphan blue exclusion (TBE) and increased release of lactate dehydrogenase (LDH) into the medium. Cisplatin also caused reduction of glutathione (GSH) in a concentration-dependent manner. In the rat renal cortical slices model, incubation of cisplatin for 120 min caused an increase in malondialdehyde (MDA), a decrease in GSH and inhibited p-aminohippurate (PAH) uptake in a concentration-dependent manner. Among phenolic antioxidants, isoeugenol (IG) was found to be more active against cisplatin-induced cytotoxicity in vero cells as well as in rat renal cortical slices than eugenol (EG) and dehydrozingerone (DZ). However none of the test compounds were able to arrest the reduction of the GSH content induced by cisplatin in either the vero cells or the renal cortical slice model. Administration of cisplatin (3 mg/kg) i.p. to rats resulted in significant reduction of body weight, and elevation of blood urea nitrogen (BUN) and serum creatinine. Treatment with IG 10 mg/kg i.p. 1 h before cisplatin resulted in partial but significant protection against the cisplatin-induced reduction of body weight, and elevation of BUN and serum creatinine, the protection being 34, 46, and 62%, respectively. EG and DZ (10 mg/kg, i.p.) were found to be inactive in vivo. Because IG is a potent free radical scavenger and protects against cisplatin-induced toxicitiy, the present results have many clinical implications in chemotherapy and thus warrants further investigation.     

A physical approach to reduce nonspecific adhesion in molecular recognition atomic force microscopy

Atomic force microscopy is one of the few techniques that allow analysis of biological recognition processes at the single-molecule level. A major limitation of this approach is the nonspecific interaction between the force sensor and substrate. We have modeled the nonspecific interaction by looking at the interaction potential between a conical Si3N4 tip with a spherical end face and a mica surface in solution, using DLVO (Derjaguin, Landau, Verwey, Overbeek) theory and numerical calculations. Insertion of the tip-sample potential in a simulation of an approach-retract cycle of the cantilever gives the well-known force-distance curve. Simulating a force-distance curve at low salt concentration predicts a discrete hopping of the tip, caused by thermal fluctuations. This hopping behavior was observed experimentally and gave rise to a novel approach to making measurements in adhesion mode that essentially works in the repulsive regime. The distance between tip and sample will still be small enough to allow spacer-involved specific interactions, and the percentage of nonspecific interactions of the bare tip with the mica is minimized. We have validated this physical model by imaging intercellular adhesion molecule 1 (ICAM-1) antigen with a tip functionalized with anti-ICAM-1 antibody. The measurement demonstrated that a significant decrease in the number of nonspecific interactions was realized, and the topographical image quality and the specific bonding capability of the tip were not affected.     

Elevated levels of the IGF-binding protein protease MMP-1 in asthmatic airway smooth muscle

We characterized the changes in nitric oxide (NO) levels in the brain during global forebrain ischemia and reperfusion and tested the ability of the natural flavonoid, quercetin, and a synthetic flavonoid, FB277, to increase the amount of available NO by elimination of the superoxide radicals produced during reperfusion. In Sprague-Dawley rats, we used a four-vessel occlusion model of forebrain ischemia (15 min) and reperfusion (30 min). Brain NO was measured on samples of cerebral cortex and cerebellum ex vivo by electron paramagnetic resonance (EPR) spectroscopy. The spin trap used was diethyldithiocarbamate sodium salt (DETC) associated with ferrous citrate. The complex Fe(DETC)2NO was detected at 77 K as a triplet signal at g = 2.035. Groups of animals were treated with quercetin or FB277 (3-morpholinomethyl-3',4',5,7tetramethoxyflavone) or polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). In control (intact anesthetized animals), the signal was about 3 times greater in the cortex than in the cerebellum. During ischemia, the signal rose to 110% in cortex (NS) and 283% in cerebellum (P < 0.05). In reperfusion, it fell again to 91% of control in cerebellum (NS) and 35% in cortex (P < 0.05). Treatment by quercetin (5 mg/kg i.v.) of intact and ischemia-reperfusion groups did not significantly change the signal amplitude in the cerebellum, but did double it in the cortex (to 76% of control) for the ischemia-reperfusion group (P < 0.05). In contrast, FB277 (3.75 mg/kg i.v.) did not increase the signal in the cortex during ischemia-reperfusion, but did do so in the cerebellum (to 152% of control, P < 0.05). The results obtained for PEG-SOD (10,000 U/kg i.v.) were similar to those for FB277. In separate in vitro measurements, we found that quercetin but not FB277 efficiently scavenged superoxide. We hypothesize that quercetin but not FB277 scavenged superoxide anions released in the cortex during reperfusion, thus diminishing the amount of NO removed by the formation of peroxynitrite. The lack of effect of PEG-SOD may be related to the need for chronic treatment to obtain protection.