Through-thickness patterns of shear strain evolve in early osteoarthritis.

OBJECTIVE: Given the structural changes associated with the progression of Osteoarthritis (OA), we hypothesized that patterns of through-thickness, large-strain shear evolve with early-stage OA. We therefore aimed to determine whether and how patterns of shear strains change during early-stage OA to 1) gain insight into the progression of OA by quantifying changes in local deformations; 2) gauge the potential of patterns in shear strain to serve as image-based biomarkers of early-stage OA; and 3) provide high-resolution, through-thickness data for proposing, fitting, and validating constitutive models for cartilage. DESIGN: We completed displacement-driven, large-strain shear tests (5, 10, 15%) on 44 specimens of variably advanced osteoarthritic human articular cartilage as determined by both Osteoarthritis Research Society International (OARSI) grade and PLM-CO score. We recorded the through-thickness deformations with a stereo-camera system and processed these data using digital image correlation (DIC) to determine full-thickness patterns of strains and relative zonal recruitments, i.e., the average shear strain in a through-thickness zone weighted by its relative thickness and normalized by the applied strain. RESULTS: We observed three general shapes for the curves of averaged through-thickness, Green-Lagrange shear strains during progression of OA. We also observed that during the progression of OA only the deep zone is recruited differently under shear in a statistically significant way. CONCLUSIONS: We propose that changes in through-thickness patterns of shear strain could provide sensitive biomarkers for early clinical detection of OA. The relative zonal recruitment of the deep zone decreases with progressing OA (OARSI grade) and microstructural remodeling (PLM-CO score), which do not consistently affect recruitment of the superficial and middle zones.

The evolving large-strain shear responses of progressively osteoarthritic human cartilage.

OBJECTIVE: The composition and structure of articular cartilage evolves during the development and progression of osteoarthritis (OA) resulting in changing mechanical responses. We aimed to assess the evolution of the intrinsic, large-strain mechanics of human articular cartilage-governed by collagen and proteoglycan and their interactions-during the progression of OA. DESIGN: We completed quasi-static, large-strain shear tests on 64 specimens from ten donors undergoing total knee arthroplasty (TKA), and quantified the corresponding state of OA (OARSI grade), structural integrity (PLM score), and composition (glycosaminoglycan and collagen content). RESULTS: We observed nonlinear stress-strain relationships with distinct hystereses for all magnitudes of applied strain where stiffnesses, nonlinearities, and hystereses all reduced as OA advanced. We found a reduction in energy dissipation density up to 80% in severely degenerated (OARSI grade 4, OA-4) vs normal (OA-1) cartilage, and more importantly, we found that even cartilage with a normal appearance in structure and composition (OA-1) dissipated 50% less energy than healthy (control) load-bearing cartilage (HL0). Changes in stresses and stiffnesses were in general less pronounced and did not allow us to distinguish between healthy load-bearing controls and very early-stage OA (OA-1), or to distinguish consistently among different levels of degeneration, i.e., OARSI grades. CONCLUSIONS: Our results suggest that reductions in energy dissipation density can be detected by bulk-tissue testing, and that these reductions precede visible signs of degeneration. We highlight the potential of energy dissipation, as opposed to stress- or stiffness-based measures, as a marker to diagnose early-stage OA.

Characterization of a gene conferring bialaphos resistance in Streptomyces coelicolor A3(2).

A gene (bar) was identified adjacent to the hrdD sigma factor gene in Streptomyces coelicolor A3(2). The predicted bar product showed 32.2% and 30.4% identity to those of the pat and bar genes of the bialaphos (Bp) producers Streptomyces viridochromogenes and Streptomyces hygroscopicus, respectively; these genes encode phosphinothricin (PPT) N-acetyltransferases that function as enzymes in the Bp biosynthetic pathway and as resistance determinants. The S. coelicolor bar gene conferred high-level resistance to Bp when cloned in S. coelicolor on a high-copy-number vector. Enzymic assay showed that the S. coelicolor bar gene product inactivates PPT by transfer of acetyl groups from acetyl CoA. The S. coelicolor bar gene appears to be expressed from two promoters (p1 and p2) and is divergently transcribed with respect to hrdD. The downstream (barp2) transcript overlaps the hrdDp1 transcript and the upstream (barp1) transcript overlaps both the hrdDp1 and hrdDp2 transcripts. Inactivation of hrdD did not prevent transcription from either bar promoter, indicating that sigma hrdD is not essential for recognition of these sequences.

Characterization of the rpoC gene of Streptomyces coelicolor A3(2) and its use to develop a simple and rapid method for the purification of RNA polymerase.

The Streptomyces coelicolor rpoC gene, that encodes the beta' subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparison of the predicted amino acid sequence of the S. coelicolor beta' subunit to those characterized from other bacteria revealed three distinct subfamilies of beta' subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor beta' subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoCHIS, was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel-NTA agarose affinity chromatography followed by heparin-sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoCHIS allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB (rifA), conferring rifampin-resistance, was isolated in S. coelicolor to provide a genetic marker to follow transfer of the rpoCHIS allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoCHIS allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus.

Allopurinol, an inhibitor of xanthine oxidase, reduces uric acid levels and modifies the signs associated with copper deficiency in rats fed fructose.

This study was designed to focus on the potential stress that xanthine oxidase could produce in copper-deficient rats fed fructose. Fructose consumption results in an excess production of uric acid due to an increased degradation of nucleotides. The enzyme xanthine oxidase catalyzes the oxidation of both hypoxanthine and xanthine. During the oxidation process free radicals are generated, which in turn, induce lipid peroxidation and premature death. Allopurinol -- a competitive inhibitor of xanthine oxidase -- could alleviate the combined effects of fructose feeding and copper deficiency. Twenty-five male rats were fed for 4 weeks from weaning a copper-deficient or adequate diet containing fructose. Twelve rats were given a daily oral dose of 5 mg allopurinol/100 g b.wt. Two copper-deficient rats that were not treated with allopurinol died prematurely during the fourth week of the study. No mortality occurred in the group of copper-deficient rats that had been treated with allopurinol. Anemia was alleviated by allopurinol, which in turn, could be responsible for improved growth rate. Allopurinol was effective in inhibiting xanthine oxidase activity in vivo as measured by the dramatic reduction of uric acid production. Lipid peroxidation, however, was not affected by allopurinol. It is concluded that the beneficial effects of allopurinol in copper deficiency do not appear to be related to prevention of oxygen radicals, but rather, to the protection against the catabolic destruction of purines, which in turn, increases nucleotide pool.

Postcaesarean section uterovesical fistula lined by persistent intermediate trophoblast.

We report a unique case of a postcaesarean section uterovesical fistula lined by intermediate trophoblast. The patient, a 42-year-old woman, presented with cyclical haematuria, and she had had a lower segment caesarean section 1 year previously. She was found to have uterovesical fistula, which was excised. On microscopic examination the fistula tract was lined by intermediate trophoblast characterised by cells with abundant eosinophilic cytoplasm, occasional multiclefted nuclei, and cytoplasmic immunoreactivity for epithelial cytokeratins and human placental lactogen. This finding has not, to our knowledge, been previously described and indicates that the survival of third trimester intermediate trophoblast is independent of pregnancy.

Hepatic iron overload may contribute to hypertriglyceridemia and hypercholesterolemia in copper-deficient rats.

The present study was conducted in order to determine whether hepatic iron retention in rats fed a copper-deficient diet containing fructose is associated with hypertriglyceridemia and hypercholesterolemia, and whether a reduction of iron intake will prevent elevation of blood triglycerides and cholesterol. Rats were fed from weaning either a copper-deficient (0.6 microgram Cu/g) or copper-adequate (6.0 micrograms Cu/g) diet for 4 weeks. Half the rats consumed either an adequate level of iron (50 micrograms Fe/g) or a low level (17 micrograms Fe/g). Reduction of iron intake reduced blood levels of both triglycerides and cholesterol in rats fed a copper-deficient diet containing fructose. In addition, hepatic lipid peroxidation was also decreased. The combination of high iron, low copper, and fructose may be responsible for increased levels of risk-factor metabolites associated with heart disease.

Cholesterol-lowering nature of unsaturated fat in rats may be due to its inability to increase hepatic iron.

The present investigation was conducted to determine whether the cholesterol-raising properties of saturated fat and cholesterol-lowering properties of unsaturated fat are associated with levels of hepatic iron. The magnitude of hepatic iron retention was manipulated by feeding rats diets that were either copper-deficient or -adequate, iron-adequate or -supplemented, and contained either beef tallow or corn oil. Weanling male Sprague-Dawley rats were randomly divided into eight dietary groups according to the type of dietary fat (beef tallow or corn oil) and level of dietary copper (0.74 or 6.9 microg Cu/g diet) or iron (44.4 or 86.7 microg Fe/g diet). Beef tallow and copper deficiency alone increased hepatic iron levels, which in turn were associated with increased plasma cholesterol. When the three dietary factors were combined, ie, iron, beef tallow, and copper deficiency, they induced the highest magnitude of hepatic iron retention, which in turn was associated with the highest concentration of plasma cholesterol. In contrast, when hepatic iron retention was not increased, such as by feeding a diet containing corn oil or by consumption of a copper-adequate diet, plasma cholesterol was not elevated. Based on these data, it is suggested that nutrients that have the ability to increase hepatic iron have the potential to increase plasma cholesterol.

Alcohol consumption aggravates copper deficiency.

Male weanling Sprague-Dawley rats were fed a copper-deficient (0.6 microgram Cu/g) diet containing either fructose or starch. Half of the animals fed the starch diet drank a 20% solution of ethanol in water. Ethanol was chosen as an agent to mimic fructose metabolism with the intention that ethanol will exacerbate the signs of copper deficiency and will negate the protective effect of dietary starch. The consumption of a 20% ethanol drink for 6 weeks by copper-deficient rats fed starch resulted in the exacerbation of the deficiency similar to that exerted by fructose. The signs associated with the deficiency in both alcohol and fructose consumption included anemia, heart hypertrophy with gross abnormalities, and mortality. In contrast, none of the copper-deficient control rats that drank water exhibited anemia or heart abnormalities, and none died of the deficiency. In addition, sorbitol pathway in the kidney and liver was stimulated by the consumption of alcohol and fructose. The data support the contention that the combination of certain metabolic pathways of carbohydrate metabolism with copper deficiency are responsible for the exacerbation of the deficiency.

Antioxidant defense system in lung of male and female rats: interactions with alcohol, copper, and type of dietary carbohydrate.

Male and female rats were used to investigate the effects of type of dietary carbohydrate (CHO), copper, and ethanol consumption on lung antioxidant enzyme activities and levels of phosphorylated compounds in whole blood. Copper-deficient female rats exhibited a greater degree of copper deficiency than males, as assessed by hepatic copper concentration and hepatic copper superoxide dismutase (CuSOD) activity. However, copper-deficient male rats fed fructose-containing diets exhibited greater growth retardation, anemia, and heart hypertrophy than females consuming the same diets and males fed starch. In addition, one of 10 copper-deficient male rats that ate a fructose-based diet and drank water and one of 10 copper-deficient male rats that ate a starch-based diet and drank ethanol died. Copper-deficient, starch-fed males exhibited the highest activities of glutathione peroxidase (GSH-Px) and catalase as compared with fructose-fed rats. Ethanol consumption elevated the activities of GSH-Px and catalase. Copper-deficient female rats exhibited higher catalase but lower GSH-Px activities than males. It is suggested that in copper deficiency, the ability to increase antioxidant enzyme activities in rats consuming starch is greater than in rats consuming fructose. Rats fed starch are provided with a greater degree of protection against oxidative damage than rats fed fructose. In addition, polyphosphorylated compounds in blood were reduced in copper-deficient male rats that consumed fructose-based diets. This may impair supply of oxygen to tissues.

The role of the adrenals in copper deficiency.

The present study was undertaken in order to establish whether (1) a decrease in catecholamines will prevent the heart hypertrophy of copper deficient rats fed fructose, and (2) an increase in hepatic copper concentration will ameliorate the signs associated with copper deficiency when fructose-based diets are consumed. Adrenalectomy resulted in reduced plasma glucocorticoids and a threefold increase in hepatic copper concentration. The signs associated with the deficiency were not ameliorated in rats fed fructose. In addition, the reduction in catecholamine concentration did not protect the copper-deficient rats fed fructose against cardiomegaly and mortality. The data support the contention that the severity of copper deficiency in rats fed fructose is not solely dependent on hepatic copper concentration and/or levels of catecholamines.

Accumulation of sorbitol in copper deficiency: dependency on gender and type of dietary carbohydrate.

The present study was designed to examine tissue sorbitol levels in copper-deficient rats consuming dietary fructose as the only source of carbohydrate and to determine if any changes in tissue sorbitol levels are influenced by the sex of the rat. Tissue levels of glucose, sorbitol, fructose, and glyceraldehyde were measured along with the activities of aldose reductase and sorbitol dehydrogenase of male and female rats consuming copper-deficient or adequate diets containing either fructose or starch for 3 weeks. Regardless of copper status, sorbitol accumulated in the livers of males consuming fructose compared to females and to males eating starch. The greatest sorbitol accumulation in the kidney occurred in the copper-deficient male rat consuming the fructose diet. These results strongly suggest that the pathology and complications of copper deficiency in the male rat fed fructose may be due to the increased sorbitol contents of tissues.

The influence of dietary carbohydrate and deferoxamine on developing copper deficiency of rats.

The present investigation was conducted to follow the development of copper deficiency in male rats from weaning (day 0) to day 31 of dietary copper deprivation and to correlate changes in tissue sizes with copper and iron concentrations. Male rats were fed for 31 days from weaning copper-deficient or adequate diets containing fructose or starch. Another copper-deficient group of rats that was fed fructose was treated with deferoxamine. Rats were killed at day 0, 8, 16, 24, and 31 of the study. In general, no correlation could be found between the development of heart hypertrophy, pancreatic and thymic atrophy, and tissue copper concentrations in copper-deficient rats fed fructose. In contrast, in the heart and pancreas a negative correlation existed between tissue size and iron concentration. In addition, anemia preceded heart hypertrophy. Deferoxamine lowered hepatic iron concentrations, ameliorated the anemia, and decreased heart size compared with untreated rats. The data of the present study suggest that tissue atrophy and hypertrophy and the severity of copper deficiency are not solely due to tissue concentrations of iron and/or copper.

Anemia plays a major role in myocardial hypertrophy of copper deficiency.

The present study was undertaken to establish whether anemia plays a role in the cardiomegaly and myocardial pathology of copper deficiency. Fifteen weanling male rats were fed a copper-deficient (0.6 microgram Cu/g) diet for 5 weeks. Six rats were intraperitoneally injected once a week with packed red blood cells (RBC) that were obtained from copper-deficient rats fed starch. The remainder (n = 9) served as controls. The administration of RBC to copper-deficient rats fed fructose prevented the anemia. As a result, none of the injected rats exhibited heart hypertrophy or gross pathology and they all survived. In contrast, all other control, nontreated copper-deficient rats that were fed fructose were anemic and all exhibited severe signs of copper deficiency, which included heart hypertrophy with gross pathology, and four died of the deficiency. The data suggest that the anemia of copper deficiency contributes to heart pathology. Once the anemia is prevented, the copper-deficient rats should be protected against heart pathology and mortality.

Aspirin reduces blood cholesterol in copper-deficient rats: a potential antioxidant agent?

The purpose of this study was to examine whether the hypocholesterolemic effect of aspirin is to due to its antioxidant properties. Oxidative stress was induced in rats by feeding them a copper-deficient diet. Copper deficiency reduced the activity of the enzyme superoxide dismutase (SOD) and lowered liver copper concentration but elevated liver iron. The combination of reduced SOD activity, high liver iron, and low liver copper resulted in an oxidative stress assessed by increased liver lipid peroxidation compared with copper-adequate controls. In addition, copper-deficient rats exhibited elevation of blood cholesterol. The administration of aspirin lowered both liver lipid peroxidation and blood cholesterol. It is suggested that the hypocholesterolemic properties of aspirin could be due to its ability to reduce oxidative stress.

The influence of gender on developing copper deficiency and on free radical generation of rats fed a fructose diet.

The present investigation was conducted to determine whether differences in copper and iron status between male and female rats can be detected during the development of copper deficiency. These differences may explain the protection of the female against the severity of copper deficiency. In addition, the livers of all rats were exposed to electron-spin resonance (ESR) spectroscopy for the presence of free radicals. Male and female rats were fed from weaning either copper-deficient or -adequate diets containing fructose for 31 days. Rats were killed at day 0, 8, 16, 24, and 31 of the study. Throughout the study, copper-deficient males exhibited the same organ copper concentrations as copper-deficient female rats. However, only in the male did copper deficiency cause a reduction in body weight and an increase in liver and heart sizes but a decrease in pancreas size. In contrast, organ iron concentrations were different between males and females. Only copper-deficient males were anemic. Only the livers of copper-deficient males showed the presence of free radicals. Although the livers of copper-deficient female rats exhibited higher concentrations of hepatic iron than their male counterparts, their livers did not show the presence of free radicals. The data of the present study suggest that changes in organ sizes and the severity of copper deficiency are not solely due to the total concentrations of iron and/or copper. The type of iron compound and the presence of free radicals may be involved in the pathology of copper deficiency of the male.

Copper deficiency in pregnancy: effect on maternal and fetal polyol metabolites.

The present study was undertaken to determine whether the mortality of the fetus and the neonate of copper-deficient rats consuming fructose during pregnancy is associated with an aberration in carbohydrate metabolism. A total of 84 Sprague-Dawley rats were fed a copper-deficient or a copper-adequate diet containing fructose or starch for 19 or 21 days after conception. The consumption of a fructose-based diet during pregnancy resulted in higher concentrations of maternal blood fructose, sorbitol, triglyceride, and uric acid when compared with a starch diet. The placenta contained more than 10-fold the concentration of glucose and more than double the concentrations of fructose, triglycerides, and sorbitol when fructose was the dietary carbohydrate compared with starch. The livers of fetuses belonging to the fructose dietary group exhibited high concentrations of glucose and sorbitol. In addition, fetal blood contained higher concentrations of glucose, fructose, sorbitol, and triglycerides than the corresponding values from the starch dietary group. The consumption of a copper-deficient diet containing fructose during pregnancy resulted in massive subcutaneous hemorrhages of the fetus. In contrast, this pathology was rare in other dietary groups. The combination of copper deficiency with fructose feeding resulted in more than double the concentration of sorbitol in fetal liver, and higher concentrations of insulin and dopamine of fetal blood compared with the consumption of a copper-deficient diet containing starch.(ABSTRACT TRUNCATED AT 250 WORDS)

The severity of copper deficiency can be ameliorated by deferoxamine.

The present study was undertaken in order to determine whether hepatic iron overload plays a role in the exacerbation of copper deficiency. Weanling male Sprague-Dawley rats were fed a copper-deficient (0.6 microgram Cu/g) diet containing 62% fructose for 5 weeks. Some of the copper-deficient rats were injected daily with deferoxamine (DFX), an iron chelator that has been widely used to reduce iron overload. DFX reduced hepatic iron concentrations, which in turn ameliorated the pathology of copper deficiency when compared with nontreated copper-deficient animals. It is suggested that hepatic iron overload in a reduced environment plays a major role in the exacerbation of copper deficiency. Once the concentration of hepatic iron is reduced, the severity of the deficiency should be improved.

Failure of body mass index or body weight to influence markedly the response to ovarian hyperstimulation in normal cycling women.

A retrospective analysis was performed of 368 normally cycling women treated with a single cycle of a standard ovarian hyperstimulation regime (CC 100 mg days 5 to 9 and hMG 150 IU days 6, 8, and 10) associated with either an IVF or GIFT program. Neither the peak serum E2 level attained nor the number of days of stimulation required bore a relationship to the BMI or the total body weight of these women. Whereas the mean number of oocytes aspirated from women with BMI less than 19.1 was higher (6.4 +/- 3.2) compared with obese women (BMI greater than 27.6, 4.8 +/- 2.6), the rate of fertilization was not different for both BMI extremes. It is concluded that factors other than BMI or total body weight have more important influences on the response to hyperstimulation in normal women.

Effect of changing the type of dietary carbohydrate or copper level of copper-deficient, fructose-fed rats on tissue sorbitol concentrations.

This study was designed to examine the relationship between the fructose-copper interaction and tissue sorbitol concentrations. Weanling male rats were provided with a diet which contained 62.7% fructose and 0.6 microg copper/g (F-Cu) for 4 weeks. At this time, rats were changed to either a fructose diet which contained 6.0 microg copper/g or to a starch diet with or without copper for 2 weeks. When compared with the other dietary groups, it was found that rats fed the F-Cu diet grew poorly; had altered relative liver, pancreatic, heart, and kidney sizes; were anemic; and had higher tissue concentrations of pancreatic and heart glucose, liver, pancreatic, heart, and kidney fructose, and liver, pancreatic, and kidney sorbitol. When rats were changed from the F-Cu diet to one containing copper or to a starch diet with or without copper, weight gain, relative liver, pancreatic and heart sizes, and hematocrit improved significantly. In general, there was a reduction in pancreatic and heart glucose; liver, pancreatic, heart, and kidney fructose; and pancreatic and kidney sorbitol concentrations when rats were changed from the F-Cu diet to any of the other diets. We conclude that the fructose-copper interaction may have a common biochemical basis related to the metabolism of glucose, fructose, and sorbitol.