Alterations in quadriceps muscle cellular and molecular properties in adults with moderate knee osteoarthritis.

OBJECTIVE: Quadriceps muscle weakness is common in knee osteoarthritis (OA). While pain, disuse, and atrophy are commonly cited causes for muscle weakness in OA, emerging evidence suggests changes in muscle quality also occur. Alterations in muscle quality are not well understood, but likely include both cellular and morphologic adaptions. The purpose of this study was to conduct the first cellular-level analysis of the vastus lateralis in adults with moderate knee OA. METHODS: Vastus lateralis biopsies were obtained from 24 subjects with moderate knee OA and 15 healthy controls. Quadriceps strength, muscle fiber cross sectional area (CSA), fiber type distribution, extracellular matrix (ECM) content, satellite cell abundance, and profibrotic gene expression were assessed. RESULTS: Relative to controls, quadriceps strength was significantly lower in OA subjects (OA 62.23, 50.67-73.8 Nm vs 91.46, 75.91-107.0 Nm, P = 0.003) despite no difference in fiber CSA. OA subjects had significantly fewer Type I fibers (OA 41.51, 35.56-47.47% vs 53.07, 44.86-61.29%, P = 0.022) and more hybrid IIa/x fibers (OA 24.61, 20.61-28.61% vs 16.4, 11.60-21.20%, P = 0.009). Significantly greater ECM content, lower satellite cell density, and higher profibrotic gene expression was observed with OA, and muscle collagen content was inversely correlated to strength and satellite cell (SC) density. CONCLUSION: Lower quadriceps function with moderate OA may not result from fiber size impairments, but is associated with ECM expansion. Impaired satellite cell density, high profibrotic gene expression, and a slow-to-fast fiber type transition may contribute to reduced muscle quality in OA. These findings can help guide therapeutic interventions to enhance muscle function with OA.

A rat resistance exercise regimen attenuates losses of musculoskeletal mass during hindlimb suspension.

Exposure to microgravity and/or spaceflight causes dramatic losses in both muscle and bone mass. In normal gravity, resistance exercise has been effectively used to increase muscle and bone mass. We tested a novel form of resistance exercise training using flywheel technology as a countermeasure to offset the loss of musculoskeletal mass during 4 weeks of adult rat hindlimb suspension (HS), an unloading model of microgravity. Male, Sprague-Dawley rats (6-month old) were operantly conditioned to perform resistance exercise, and then randomly assigned to groups of sedentary control (CON), HS, and HS with resistance exercise training (HSRT; 2 sets of approximately 21 repetitions, 3 days week(-1) for 4 weeks during suspension). In soleus, HS resulted in lower (P < 0.05) muscle mass to body mass ratio (approximately 50% of controls) and rates of protein synthesis. HSRT significantly attenuated the loss of muscle mass in soleus muscle, and rates of protein synthesis for soleus were similar for HSRT and controls. There were no differences among groups for mass or rates of protein synthesis in extensor digitorum longus. In cancellous regions of the distal femur, HS resulted in significant reductions of bone mineral density (BMD), but this was restored to control levels with HSRT. Cortical regions of the femur were not different among HS, HSRT or control groups. Together, these data suggest that resistance training using flywheel technology may be a promising tool to attenuate losses of the musculoskeletal system during periods of hindlimb unloading.

Frequencies of plasmodesmata in Allium cepa L. roots: implications for solute transport pathways.

Plasmodesmatal frequencies (PFs) were analysed in Allium cepa L. roots with a mature exodermis (100 mm from the tip). For all interfaces within the root, the numbers of plasmodesmata (PD) microm(-2) wall surface (Fw) were calculated from measurements of 60 walls on ultrathin sections. For tissues ranging from the epidermis up to the stelar parenchyma, the frequencies were also expressed as total PD numbers mm(-1) root length (Fn), which is most instructive for considering the radial transport of ions and photosynthates (because the tissues were arranged in concentric cylinders). The Fn values were constantly high at the interfaces of exodermis-central cortex, central cortex-endodermis and endodermis-pericycle (4.05x10(5), 5.13x10(5), and 5.64x10(5), respectively). If the plasmodesmata are functional, a considerable symplastic transport pathway exists between the exodermis and pericycle. Two interfaces had especially low PFs: epidermis-exodermis (Fn=8.96x10(4)) and pericycle-stelar parenchyma (Fn=6.44x10(4)). This suggests that there is significant membrane transport across the interface of epidermis-exodermis (through short cells) and direct transfer of ions from pericycle to protoxylem vessels. In the phloem, the highest PF was detected at the metaphloem sieve element-companion cell interface (Fw=0.42), and all other interfaces had much lower PFs (around 0.10). In the pericycle, the radial walls had a high PF (Fw=0.75), a feature that could permit lateral circulation of solutes, thus facilitating ion (inward) and photosynthate (outward) delivery.

Nursing shortage: not a simple problem - no easy answers.

The article examines the issue of nursing workforce planning within the present health system environment and addresses ethical issues related to the shortage. Numerous factors are influencing both the supply of registered nurses as well as the demand for nursing services. Of particular concern is the negative impact that the current nursing practice environment is having on the retention of registered nurses as well as the ability of the profession to recruit students. Other factors driving this present nursing shortage include the increasing age of working nurses and the potential for retirement; the aging of nursing faculty impacting the capacity of nursing schools; and poor wage compensation. The continual swing in the supply of and demand for nursing services will always be present; however, it is time that nursing sat at the policy-making table with the other decision makers to better anticipate the nature of these changes and how to address them.

Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair.

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

Molecular characteristics of aged muscle reflect an altered ability to respond to exercise.

Studies have been performed in humans to identify changes in gene expression that may account for the relatively weak and variable response of aged muscle to resistance exercise. The gene expression profile of skeletal muscle from elderly (62-75 years old) compared to younger (20-30 years old) men demonstrated elevated expression of genes typical of a stress or damage response. The expression of the majority of these genes was unaffected by a single bout of high-intensity resistance exercise in elderly subjects but was altered acutely by exercise in younger subjects so as to approach the pre-exercise levels observed in older subjects. The inability of muscle from elderly subjects to respond to resistance exercise was also apparent in the expression of inflammatory response genes, which increased within 24 hours of the exercise bout only in younger subjects. Othergenes with potentially important roles in the adaptation of muscle to exercise, showed a similar or even more robust response in older compared to younger subjects. Taken together, these results may help to explain the variable hypertrophic response of muscle from older individuals to resistance training.

Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury.

We have shown that cycling exercise combined with fetal spinal cord transplantation restored muscle mass reduced as a result of complete transection of the spinal cord. In this study, mechanisms whereby this combined intervention increased the size of atrophied soleus and plantaris muscles were investigated. Rats were divided into five groups (n = 4, per group): control, nontransected; spinal cord transected at T10 for 8 wk (Tx); spinal cord transected for 8 wk and exercised for the last 4 wk (TxEx); spinal cord transected for 8 wk with transplantation of fetal spinal cord tissue into the lesion site 4 wk prior to death (TxTp); and spinal cord transected for 8 wk, exercised for the last 4 wk combined with transplantation 4 wk prior to death (TxExTp). Tx soleus and plantaris muscles were decreased in size compared with control. Exercise and transplantation alone did not restore muscle size in soleus, but exercise alone minimized atrophy in plantaris. However, the combination of exercise and transplantation resulted in a significant increase in muscle size in soleus and plantaris compared with transection alone. Furthermore, myofiber nuclear number of soleus was decreased by 40% in Tx and was not affected in TxEx or TxTp but was restored in TxExTp. A strong correlation (r = 0.85) between myofiber cross-sectional area and myofiber nuclear number was observed in soleus, but not in plantaris muscle, in which myonuclear number did not change with any of the experimental manipulations. 5'-Bromo-2'-deoxyuridine-positive nuclei inside the myofiber membrane were observed in TxExTp soleus muscles, indicating that satellite cells had divided and subsequently fused into myofibers, contributing to the increase in myonuclear number. The increase in satellite cell activity did not appear to be controlled by the insulin-like growth factors (IGF), as IGF-I and IGF-II mRNA abundance was decreased in Tx soleus and plantaris, and was not restored with the interventions. These results indicate that, following a relatively long postinjury interval, exercise and transplantation combined restore muscle size. Satellite cell fusion and restoration of myofiber nuclear number contributed to increased muscle size in the soleus, but not in plantaris, suggesting that cellular mechanisms regulating muscle size differ between muscles with different fiber type composition.

Aged human muscle demonstrates an altered gene expression profile consistent with an impaired response to exercise.

The gene expression profile of skeletal muscle from healthy older (62-75 years old) compared with younger (20-34 years old) men demonstrated elevated expression of genes typical of a stress or damage response, and decreased expression of a gene encoding a DNA repair/cell cycle checkpoint protein. Although the expression of these genes was relatively unaffected by a single bout of resistance exercise in older men, acute exercise altered gene expression in younger men such that post-exercise gene expression in younger men was similar to baseline gene expression in older men. The lack of response of muscle from older subjects to resistance exercise was also apparent in the expression of the inflammatory response gene IL-1beta, which did not differ between the age groups at baseline, but increased within 24 h of the exercise bout only in younger subjects. Other genes with potentially important roles in the adaptation of muscle to exercise, specifically in the processes of angiogenesis and cell proliferation, showed a similar response to exercise in older compared with younger subjects. Only one gene encoding the multifunctional, early growth response transcription factor EGR-1, showed an opposite pattern of expression in response to exercise, acutely decreasing in younger and increasing in older subjects. These results may provide a molecular basis for the inherent variability in the response of muscle from older as compared with younger individuals to resistance training.

Enterotrophic effect of insulin-like growth factor-I but not growth hormone and localized expression of insulin-like growth factor-I, insulin-like growth factor binding protein-3 and -5 mRNAs in jejunum of parenterally fed rats.

BACKGROUND: Administration of insulin-like growth factor (IGF)-I, but not growth hormone (GH), stimulates mucosal hyperplasia in surgically stressed rats with intestinal atrophy induced by hypocaloric total parenteral nutrition (TPN). Our aim was to characterize the basis for this disparity in enterotrophic action by assessing the relationships between stimulation of intestinal growth, nutritional adequacy, and localization of expression of IGF-I, insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mRNAs in jejunum. METHODS: Rats were maintained with TPN for 8 days and treated with IGF-I or GH and adequate nutrition for 5 days after recovery from surgery. Jejunal mass, morphology, and sucrase activity were assessed. Localization of expression of IGF-I, IGFBP-3, and IGFBP-5 mRNAs in jejunum was accomplished by in situ hybridization. RESULTS: Serum IGF-I and body weight gain were significantly increased by IGF-I or GH. Jejunal mucosal dry mass, morphology, and sucrase activity were improved with IGF-I but not GH. There were no differences in IGF-I mRNA. IGFBP-3 mRNA was localized in the lamina propria of the villi. IGF-I or GH stimulated IGFBP-3 expression. IGF-I strongly stimulated IGFBP-5 expression in the lamina propria and the muscularis and induced a twofold increase in IGFBP-5 mRNA based on RNase protection assay of intact jejunum total RNA. GH induced a modest increase in IGFBP-5 expression in the muscularis with no effect on intact jejunum total RNA. CONCLUSIONS: The GH resistance observed in the jejunal mucosa of TPN rats cannot be fully explained by inadequate nutrition. The expression of IGFBP-5 in the lamina propria suggests it may modulate the enterotrophic action of exogeneous IGF-I.

Radial hydraulic conductivity along developing onion roots.

Although most studies have shown that water uptake varies along the length of a developing root, there is no consistent correlation of this pattern with root anatomy. In the present study, water movement into three zones of onion roots was measured by a series of mini-potometers. Uptake was least in the youngest zone (mean hydraulic conductivity, Lpr = 1.5 x 10(-7) +/- 0.34 x 10(-7) m MPa-1 s-1; +/- SE, n = 10 roots) in which the endodermis had developed only Casparian bands and the exodermis was immature. Uptake was significantly greater in the middle zone (Lpr = 2.4 x 10(-7) +/- 0.43 x 10(-7) m MPa-1 s-1; +/- SE, n = 10 roots) which had a mature exodermis with both Casparian bands and suberin lamellae, and continued at this level in the oldest zone in which the endodermis had also developed suberin lamellae (Lpr = 2.8 x 10(-7) +/- 0.30 x 10(-7) m MPa-1 s-1; +/- SE, n = 10 roots). Measurements of the hydraulic conductivities of individual cells (Lp) in the outer cortex using a cell pressure probe indicated that this parameter was uniform in all three zones tested (Lp = 1.3 x 10(-6) +/- 0.01 x 10(-6) m MPa-1 s-1; +/- SE, n = 60 cells). Lp of the youngest zone was lowered by mercuric chloride treatment, indicating the involvement of mercury-sensitive water channels (aquaporins). Water flow in the older two root zones measured by mini-potometers was also inhibited by mercuric chloride, despite the demonstrated impermeability of their exodermal layers to this substance. Thus, water channels in the epidermis and/or exodermis of the older regions were especially significant for water flow. The results of this and previous studies are discussed in terms of two models. The first, which describes maize root with an immature exodermis, is the 'uniform resistance model' where hydraulic resistances are evenly distributed across the root cylinder. The second, which describes the onion root with a mature exodermis, is the 'non-uniform resistance model' where resistances can be variable and are concentrated in a certain layer(s) on the radial path.

Cortical development in roots of the aquatic plant Pontederia cordata (Pontederiaceae).

Adventitious roots of marsh-grown Pontederia cordata were examined to determine cortical development and structure. The innermost layer of the ground meristem forms the endodermis and aerenchymatous cortex. The outermost layer of the early ground meristem undergoes a precise pattern of oblique and periclinal cell divisions to produce a single or double layer of prohypodermis with an anchor cell for each radial file of aerenchyma cells. At maturity, endodermal cell walls are modified only by narrow Casparian bands. The central regions of the ground meristem become proaerenchyma and exhibit asymmetric cell division and expansion. They produce an aerenchymatous zone with barrel-shaped large cells and irregularly shaped small cells traversing the aerenchyma horizontally along radii; some crystalliferous cells with raphides are present in the aerenchyma. The walls of the hypodermis are modified early by polyphenols. The outermost layer of the hypodermis later matures into an exodermis with Casparian bands that are impermeable to berberine, an apoplastic tracer dye. The nonexodermal layer(s) of the hypodermis has suberin-modified walls. Radial files of aerenchyma are usually connected by narrow protuberances near their midpoints, the aerenchyma lacunae having been produced by expansion of cells along walls lining intercellular spaces. We are terming this type of aerenchyma development, which is neither schizogenous nor lysigenous, "differential expansion."

Norgestomet- and oestradiol valerate-induced luteolysis is dependent upon the uterus.

Beef heifers were assigned to three groups: (1) untreated controls (n=4), (2) Syncro-Mate B(R) (SMB)-treated (n=5), and (3) hysterectomized and SMB-treated (n=4). SMB was administered 8 or 9 days after oestrus, approximately 30 days after hysterectomy. This study was conducted to determine if the uterus was necessary for SMB to induce luteolysis. SMB induced premature luteolysis as only 20% of the intact SMB-treated heifers had >/=0.75 ng/ml of progesterone 7 days after the time of SMB treatment, compared to all (100%) of the untreated heifers (p<0.05). By 9 days after the time of SMB treatment, 25% of the untreated heifers and none (0%) of the intact SMB-treated heifers had >/=0.75 ng/ml of progesterone; however, all (100%) of the hysterectomized SMB-treated heifers had >/=0.75 ng/ml of progesterone (p<0.05). Therefore, SMB-induced luteolysis required the involvement of the uterus. The luteolysin, prostaglandin F(2alpha), is probably the secretion from the uterus that mediates the SMB-induced luteolysis. SMB treatment, however, required 7-8 days to induce luteolysis.

Requirement for PCNA and RPA in interstrand crosslink-induced DNA synthesis.

Proliferating nuclear cell antigen (PCNA) and replication protein A (RPA) have proven to be essential elements in many aspects of DNA metabolism including replication, repair and recombination. We have developed an in vitro assay in which the presence of an interstrand crosslink stimulates the incorporation of radiolabeled nucleotides into both damaged and undamaged plasmid DNAs. Using this assay we have investigated the roles of PCNA and RPA in crosslink-induced DNA synthesis. p21, a potent inhibitor of PCNA, was found to strongly inhibit crosslink-induced incorporation. Addition of exogenous PCNA partially restored the resynthesis activity. Likewise, neutralization of RPA by monoclonal antibodies also inhibited incorporation, but the effect was somewhat more pronounced on the undamaged plasmid than the damaged plasmid. Addition of excess RPA also partially reversed antibody inhibition. These results indicate that both PCNA and RPA are required for efficient in vitro DNA resynthesis induced by interstrand crosslinks.

Cycling exercise and fetal spinal cord transplantation act synergistically on atrophied muscle following chronic spinal cord injury in rats.

The potential of two interventions, alone or in combination, to restore chronic spinal cord transection-induced changes in skeletal muscles of adult Sprague-Dawley rats was studied. Hind limb skeletal muscles were examined in the following groups of animals: rats with a complete spinal cord transection (Tx) for 8 weeks; Tx with a 4-week delay before initiation of a 4-week motor-assisted cycling exercise (Ex) program; Tx with a 4-week delay before transplantation (Tp) of fetal spinal cord tissue into the lesion cavity; Tx with a 4-week delay before Tp and Ex; and uninjured control animals. Muscle mass, muscle to body mass ratios, and mean myofiber cross-sectional areas were significantly reduced 8 weeks after transection. Whereas transplantation of fetal spinal cord tissue did not reverse this atrophy and exercise alone had only a modest effect in restoring lost muscle mass, the combination of exercise and transplantation significantly increased muscle mass, muscle to body mass ratios, and mean myofiber cross-sectional areas in both soleus and plantaris muscles. Spinal cord injury (SCI) also caused changes in myosin heavy chain (MyHC) expression toward faster isoforms in both soleus and plantaris and increased soleus myofiber succinate dehydrogenase (SDH) activity. Combined exercise and transplantation led to a change in the expression of the fastest MyHC isoform in soleus but had no effect in the plantaris. Exercise alone and in combination with transplantation reduced SDH activity to control levels in the soleus. These results suggest a synergistic action of exercise and transplantation of fetal spinal cord tissue on skeletal muscle properties following SCI, even after an extended post-injury period before intervention.

Activated satellite cells fail to restore myonuclear number in spinal cord transected and exercised rats.

In this study, possible mechanisms underlying soleus muscle atrophy after spinal cord transection and attenuation of atrophy with cycling exercise were studied. Adult female Sprague-Dawley rats were divided into three groups; in two groups the spinal cord was transected by a lesion at T10. One group was transected and killed 10 days later, and another group was transected and exercised for 5 days starting 5 days after transection. The third group served as an uninjured control. All animals received a continuous-release 5'-bromo-2'-deoxyuridine pellet 10 days before they were killed. Transection alone and transection with exercise lead to activation of satellite cells, but only the exercise group showed a trend toward an increase in the number of proliferating satellite cells. In all cases the number of activated satellite cells was significantly higher than the number that divided. Although the number of cells undergoing proliferation increased with exercise, no increase in fusion of satellite cells into muscle fibers was apparent. Spinal cord transection resulted in a 25% decrease in myonuclear number, and exercise was not associated with a restoration of myonuclear number. The number of apoptotic nuclei was increased after transection, and exercise attenuated this increase. However, the decrease in apoptotic nuclei with exercise did not significantly affect myonuclear number. We conclude that apoptotic nuclear loss likely contributes to loss of nuclei during muscle atrophy associated with spinal cord transection and that exercise can maintain muscle mass, at least in the short term, without restoration of myonuclear number.

Interstrand cross-links induce DNA synthesis in damaged and undamaged plasmids in mammalian cell extracts.

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.