Formation of H2 on graphene using Eley-Rideal and Langmuir-Hinshelwood processes.

A hydrogen atom can either physisorb or chemisorb onto a graphene surface. To describe the interaction of H with graphene, we trained the C-C, H-H, and C-H interactions of the ReaxFF CHO bond order potential to reproduce Density Functional Theory (DFT) generated values of graphene cohesive energy and lattice constant, H2 dissociation energy, H on graphene adsorption potentials, and H2 formation on graphene using the Eley-Rideal (ER) and Langmuir-Hinshelwood (LH) processes. The results, generated from the trained H-graphene potentials, are in close agreement with the corresponding results from DFT. The advantage of using optimized CH potentials is, for example, the inclusion of physisorption interactions and quantum mechanical features of chemical bonding in the functional forms of the potentials. The trained CH potentials are utilized to study the energetics of formation of an H2 molecule on graphene using the Eley-Rideal and Langmuir-Hinshelwood processes. Potential energy surfaces for the formation of H2 through ER are generated for the collinear and oblique approach of the second hydrogen atom. Energetics of the formation of H2 through LH is studied for a variety of cases such as when hydrogen atoms are chemisorbed or physisorbed and when hydrogen occupies ortho, meta, or para chemisorption sites. The likelihood of H2 formation through LH for various configurations is discussed. Furthermore, the tunneling probability of an atom through a continuous symmetric/asymmetric barrier is calculated and applied to an adsorbed hydrogen atom on graphene.

Parkinson's Disease Genetic Loci in Rapid Eye Movement Sleep Behavior Disorder.

Rapid eye movement (REM) sleep behavior disorder (RBD) is a prodromal condition for Parkinson's disease (PD) and other synucleinopathies, which often occurs many years before the onset of PD. We analyzed 261 RBD patients and 379 controls for nine PD-associated SNPs and examined their effects, first upon on RBD risk and second, on eventual progression to synucleinopathies in a prospective follow-up in a subset of patients. The SCARB2 rs6812193 (OR = 0.67, 95 % CI = 0.51-0.88, p = 0.004) and the MAPT rs12185268 (OR-0.43, 95 % CI-0.26-0.72, p = 0.001) were associated with RBD in different models. Kaplan-Meier survival analysis in a subset of RBD patients (n = 56), demonstrated that homozygous carriers of the USP25 rs2823357 SNP had progressed to synucleinopathies faster than others (log-rank p = 0.003, Breslow p = 0.005, Tarone-Ware p = 0.004). As a proof-of-concept study, these results suggest that RBD may be associated with at least a subset of PD-associated genes, and demonstrate that combining genetic and prodromal clinical data may help identifying individuals that are either more or less susceptible to develop synucleinopathies. More studies are necessary to replicate these results, and identify more genetic factors affecting progression from RBD to synucleinopathies.

Are oxybutynin and trospium efficacious in the treatment of detrusor overactivity in spinal cord injury patients?

OBJECTIVES: To evaluate the efficacy of anticholinergic agents in the treatment of neurogenic overactive bladder (NOAB) and neurogenic detrusor overactivity (NDO) in spinal cord injury (SCI) patients on clean intermittent catheterisation (CIC). METHODS: Chronic suprasacral SCI patients on CIC presenting with at least one urinary leakage a day were included. Urodynamics and voiding diaries were performed at baseline and 1 month follow-up. In case of NDO at baseline, an anticholinergic drug was prescribed. RESULTS: The 231 SCI patients presented with one to five urinary leakages per day (mean 2.1). Urodynamics showed NDO in all patients. A new anticholinergic treatment was started in all, either in monotherapy (134 patients) or in association with the existing anticholinergic drug (oxybutynin+trospium bitherapy, 97 patients). The mean maximum bladder capacity significantly increased from 225 to 441 ml, and the mean involuntary detrusor contractions (IDC) significantly decreased from 67 to 41 cm H2O. Only 75 SCI patients (32%) were fully continent. However, 25 out of these 75 patients showed persistent NDO, with amplitudes of IDC above 40 cm H2O in 12 patients. Incontinence was still found in 156 SCI patients (67%), with an average of 1,2 leakages a day. In 100 patients, amplitudes of IDC remained above 40 cm H2O. There was no statistical difference between patients on anticholinergic monotherapy or bitherapy at follow-up. CONCLUSION: Anticholinergic treatment is not always satisfactory in terms of control of NDO and rarely allows full continence. Urodynamic follow-up is mandatory in all patients, even in those showing clinical continence.

[Evaluation of the management of erectile and ejaculatory dysfunction in a series of 90 patients with spinal cord injuries]. / Évaluation du traitement des dysfonctions érectiles et éjaculatoires dans une série de 90 blessés médullaires.

OBJECTIVE: To evaluate the erectile dysfunction and ejaculatory on patients injured in conus medullaris (CMI) and the efficacy of treatment. METHODS: Ninety patients with injured conus medullaris underwent a retrospective and monocentric study. They have all an assessment of erectile dysfunction and ejaculatory by the International Index of Erectile Function (IIEF-15) and a clinic scenario. We evaluated the erection by a clinical scoring scale (Shrameck). Seventy-seven out of 90 (85.55%) had therapeutic tests to restore erection: prostaglandin (PGE1), phosphodiesterase type 5 inhibitors (PDE5) and Papaverine. Seventy-four out of 90 (82.22%) underwent a penile stimulation tests (VM) more or less associated with Midodrine(®) (alpha mimetic) to cause ejaculation with a systematic search of spermatozoa in urine. Data were entered and analyzed using Microsoft Office Excel. RESULTS: Ninety patients with a complete lesion of the sacral metameres (S2S3S4) were included. They were responding to PGE1, PDE5, respectively 81.63% and 30.76%. The association VM/Midodrine(®) improves ejaculation in 52.63% of cases. Orgasm is absent in all our CMI. CONCLUSION: In this series of BCM patients, we observed a good efficacy of PGE1 and PDE 5 on erection. We also observed positive results of vibromassage and alpha-agonists on ejaculation.

Therapeutic patient education for stroke survivors: Non-pharmacological management. A literature review.

OBJECTIVES: Focus on the different therapeutic patient education (TPE) programs for stroke survivors found in the literature. Verify their content and efficacy. METHOD: A literature review was conducted by searching for entries from 1966 to 2011 in the Medline and Cochrane Library databases. The references for the accepted articles were taken into consideration and the articles corresponding to the criteria inclusion but not present within the initial search were selected. The keywords used were "self care", "self management", "patient education" and "stroke". Given the multiplicity of symptoms that may be addressed in TPE programs, and following expert advice, the symptoms were grouped after expanding the bibliographic search using the following, additional keywords: "dysphagia"; "swallowing disorder"; "urinary incontinence"; "caregiver"; "fall prevention"; "falling"; "injury"; "shoulder pain"; "physical activity"; "exercise"; "aphasia" and "cognitive impairment". RESULTS: We found 30 article abstracts. In the end, we only accepted seven articles on general TPE programs that were well structured and detailed enough. The TPE programs found in the literature were often of questionable methodological quality. The multiplicity of symptoms led to very general TPE programs that covered all possible stroke after-effects. The purpose of these programs was to reduce stress and anxiety, to improve quality of life and to alleviate psychosocial after-effects. A change in caregiver and patient behavior was observed at times. We expanded the bibliographic search to include scientific arguments that could help implement TPE programs for more specific targets. CONCLUSION: TPE programs for stroke survivors could be improved by standardizing and assessing programs that focus on a specific problem caused by the various possible after-effects of strokes. In order to promote education for stroke survivors, specific training for health care professionals and appropriate funding are necessary.

Absence of deficits in social behaviors and ultrasonic vocalizations in later generations of mice lacking neuroligin4.

Mutations in NLGN4X have been identified in individuals with autism spectrum disorders and other neurodevelopmental disorders. A previous study reported that adult male mice lacking neuroligin4 (Nlgn4) displayed social approach deficits in the three-chambered test, altered aggressive behaviors and reduced ultrasonic vocalizations. To replicate and extend these findings, independent comprehensive analyses of autism-relevant behavioral phenotypes were conducted in later generations of the same line of Nlgn4 mutant mice at the National Institute of Mental Health in Bethesda, MD, USA and at the Institut Pasteur in Paris, France. Adult social approach was normal in all three genotypes of Nlgn4 mice tested at both sites. Reciprocal social interactions in juveniles were similarly normal across genotypes. No genotype differences were detected in ultrasonic vocalizations in pups separated from the nest or in adults during reciprocal social interactions. Anxiety-like behaviors, self-grooming, rotarod and open field exploration did not differ across genotypes, and measures of developmental milestones and general health were normal. Our findings indicate an absence of autism-relevant behavioral phenotypes in subsequent generations of Nlgn4 mice tested at two locations. Testing environment and methods differed from the original study in some aspects, although the presence of normal sociability was seen in all genotypes when methods taken from Jamain et al. (2008) were used. The divergent results obtained from this study indicate that phenotypes may not be replicable across breeding generations, and highlight the significant roles of environmental, generational and/or procedural factors on behavioral phenotypes.

Metastatic paraplegia: care management characteristics within a rehabilitation center.

STUDY DESIGN: Retrospective study. OBJECTIVES: To determine the potential impact of rehabilitation care on associated symptoms and functional improvements of paraplegic patients with metastatic spinal cord compression. SETTING: CMN Propara, Montpellier (France). MEASURES: Demographics, Functional Independence Measure (FIM), Frankel Modified Score and Visual Analog Scale (VAS) for pain, intercurrent adverse medical events and neurological outcome, duration of stay, survival time, rehospitalization in a non-Spinal Cord Injury unit, number of contracts defining the patients rehabilitation goals, number of contracts defining the patients duration of stay within the rehabilitation center. RESULTS: We reviewed the charts of 26 patients. The initial neurological profile was paraplegia or paraparesis for 24 patients and quadriparesis for 2 patients. Regarding functional improvements: four patients demonstrated a poor functional evolution, five patients showed no functional improvements or very slight improvements and all the other patients showed an increase in their overall functional aptitudes. At the end of the stay, 14 patients were urinary independent. Our study reports 52 rehospitalizations in an another unit and 101 outpatient visits during their rehabilitation stay in a physical medicine and rehabilitation (PM&R) center. For the 14 patients who were deceased at the time of data collection, the median survival rate post-paraplegia was 12.7 months. A total of 12 of the 14 patients spent more than a third of their remaining survival time in a rehabilitation center. DISCUSSION: Compared to the patients' life expectancy, their stay in a rehabilitation center is too long and prevents them from spending time with family and loved ones. The occurrence rate of the associated symptoms is high because of both cancer-related disorders and neurological disorders caused by the spinal cord lesion. PM&R professionals are faced with patients affected by chronic pain and fatigue as well as frequent rehospitalizations, short stays and outpatient stays, in the primary oncology unit. This study focuses on the need to privilege the patients' comfort over their functional rehabilitation.

[Assessment of functional abilities, handicap and quality of life in patients with spinal cord injuries]. / Evaluation des aptitudes fonctionnelles, du handicap et de la qualité de vie chez le blessé médullaire.

OBJECTIVES: A literature review of the methods of evaluating function, handicap and quality of life in patients with spinal cord injuries. METHODS: The literature review was based on the available French and English articles published since 1990 in 3 databases: MEDLINE, Pascal and Embase. RESULTS: The literature is dominated by descriptions of tools for evaluating functional limitations in motor deficiencies. Such descriptions involve the validation of generic tools for patients with spinal cord injuries or of specific tools during the evaluation of a particular intervention such as surgery of the tetraplegic hand or adaptation of technical help. CONCLUSION: The tools to assess patients with spinal-cord injuries are sufficiently numerous and varied to allow us to evaluate physical, functional and psychosocial dimensions. Rigorous methodological validation is continuously at the base of those proposed tools and thus reinforces our choice to use them. Unfortunately, few evaluation tools for patients with spinal cord injuries have been published, translated into French and validated.

Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats ages 0-21 days: I. Two groups of proteinases cleave the core protein of aggrecan.

The formation of a secondary ossification center in the cartilaginous epiphysis of long bones requires the excavation of canals and marrow space and, therefore, the resorption of cartilage. On the assumption that its resorption requires the lysis of the major cartilage component aggrecan, it was noted that the core protein may be cleaved in vitro by proteinases from two subfamilies: matrix metalloproteinases (MMPs) and aggrecanases. Such cleavage results in aggrecan being replaced by a fragment of itself referred to as a "G1-fragment." To find out if this cleavage occurs in the developing epiphysis of the rat tibia, the approach has been to localize the G1 fragments. For this purpose two neoepitope antisera were applied, one capable of recognizing the MMP-generated G1-fragment that bears the C-terminus ...FVDIPEN341 and the other capable of recognizing the aggrecanase-generated G1-fragment that carries the C-terminus ...NITEGE373. With the aid of these antisera, we report here that aggrecan cleavage is localized to newly developed sites of erosion. Thus, at 6 days of age, canals allowing the entry of capillaries are dug out from the surface of the epiphysis in a radial direction (stage I), whereas immunostaining indicative of aggrecan cleavage by MMPs appears at the blind end of each canal. The next day, the canal blind ends fuse to create a marrow space in the epiphysis (stage II), whereas immunostaining produced by MMPs occurs along the walls of this space. By 9 days, clusters of hypertrophic chondrocytes are scattered along the marrow space wall to initiate the formation of the secondary ossification center (stage III), where the resorption sites are unreactive to either antiserum. From the 9th to the 21st day, the center keeps on enlarging and, as the distal wall of the marrow space recedes, it is intensely immunostained with both antisera indicating that both MMPs and aggrecanases are involved in this resorption. We conclude, that both enzyme subfamilies contribute to the lysis of aggrecan. However, the results suggest that the respective subfamilies target different sites and even stages of development in the tissue, suggesting some diversity in the mode of aggrecan lysis during the excavation of a secondary ossification center.

Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats aged 0-21 days: II. Two proteinases, gelatinase B and collagenase-3, are implicated in the lysis of collagen fibrils.

In the transformation of the cartilaginous epiphysis into bone, the first indication of change in the surfaces destined for resorption is the cleavage of aggrecan core protein by unidentified matrix metalloproteinases (MMPs) (Lee et al., this issue). In cartilage areas undergoing resorption, the cleavage leaves as superficial, 6-microm-thick band of matrix, referred to as "pre-resorptive layer." This layer harbors G1-fragments of the aggrecan core protein within a framework of collagen-rich fibrils exhibiting various stages of degeneration. Investigation of this layer in every resorption area by gelatin histozymography and TIMP-2 histochemistry demonstrates the presence of an MMP whose histozymographic activity is inhibited by such a low dose of the inhibitor CT1746 as to identify it as gelatinase A or B. Attempts at blocking the histozymographic reactions with neutralizing antibodies capable of inhibiting either gelatinase A or B reveals that only those against gelatinase B do so. Immunostaining of sections with anti-gelatinase B IgG confirms the presence of gelatinase B in every pre-resorptive layer, that is, at the blind end of excavated canals (stage I; 6-day-old rats), at sites along the walls of the forming marrow space (stage II; 7days), at sites within the walls of this space as it becomes the ossification center (stage III; 9 days) and along the wall of the maturing center (stage IV; 10-21 days). We also report the presence of collagenase-3 in precisely the same sites, possibly as active enzyme, but this remains to be proven. Because the results reveal that collagenase-3 is present beside gelatinase B in every pre-resorptive layer and, because these sites exhibit various signs of degradation including fibrillar debris, reduction in fibril number, or overt loss, we propose that gelatinase B and collagenase-3 mediate the lysis of this pre-resorptive layer-most likely through a cooperative attack leading to the disintegration of the collagen fibril framework.

Transcutaneous electric nerve stimulation reduces neglect-related postural instability after stroke.

OBJECTIVE: To test the existence of a neglect-related component of postural imbalance in some stroke patients to determine whether neglect patients (1) show worse postural control compared with nonneglect patients and healthy subjects and (2) have latent postural capacities that could be unmasked by an appropriate somatosensory manipulation. DESIGN: Intervention study with and without transcutaneous electric nerve stimulation (TENS). SETTING: Rehabilitation center research laboratory. PARTICIPANTS: Twenty-two stroke patients (mean age, 58.3 +/- 2.5yr; average days since stroke, 83.2d) and 14 age-matched healthy subjects. Stroke patients were subdivided into 3 groups: 6 with spatial neglect and 16 without (8 with left lesion, 8 with right lesion). INTERVENTIONS: All participants were subjected to a dynamic balance task, performed while sitting for 8 seconds on a laterally rocking platform. Seated on this mobile support, they were asked to maintain actively an erect posture, sitting as still as possible. In patients, TENS was applied on the contralesional side of the neck during the postural task. An effective stimulation (intensity corresponding to the threshold of perception, TENS+) was compared with a placebo stimulation (.01 x threshold of perception, TENS-). MAIN OUTCOME MEASURES: Postural performance in each trial was monitored by using 2 criteria: the number of aborted trials caused by loss of balance, and the angular dispersion of the support oscillations in roll. The latter criterion, which increased with body instability, was defined as 2 standard deviations of the angular distribution. RESULTS: Patients showing neglect displayed pronounced postural instability compared with other patients and controls. Although dramatic postural instability in the neglect patients was spectacularly and systematically reduced with TENS, no effect was observed in patients without neglect. CONCLUSION: This is among the first studies to provide clinical evidence supporting the "postural body scheme" concept.

The polymodal sensory cortex is crucial for controlling lateral postural stability: evidence from stroke patients.

In modern literature, internal models are considered as a general neural process for resolving sensory ambiguities, synthesising information from disparate sensory modalities, and combining efferent and afferent information. The polymodal sensory cortex, especially the temporoparietal junction (TPJ), is thought to be a nodal point of the network underlying these properties. According to this view, a pronounced disruption of the TPJ functioning should dramatically impair body balance. Surprisingly, little attention has been paid to this possible relationship, which was the subject of investigation in this study. Twenty-two brain-damaged patients and 14 healthy subjects were subjected to a self-regulated lateral balance task, performed while sitting for 8 s on a rocking platform. Their lateral body balance was analysed both with and without vision (darkness). Support displacements in the frontal plane were recorded by means of an accelerometer. Two criteria were taken into account to evaluate body stability in each trial: the number of aborted trials due to balance loss and the angular dispersion of the supporting surface. Lesions involving the temporoparietal junction were found to markedly increase body instability, both with and without vision. Therefore, the temporoparietal junction plays a pivotal role in lateral body stabilisation, irrespective of the sensory condition in which the task is performed. This suggests that body stability is controlled throughout internal model(s).

Active gelatinase B is identified by histozymography in the cartilage resorption sites of developing long bones.

In order to determine which proteinases mediate the resorption of endochondral cartilage in the course of long bone development, a novel assay called "histozymography" has been developed. In this assay, frozen sections of tibial head from 21-day-old rats are placed for 4 hr at room temperature on light-exposed photographic emulsion (composed of silver grains embedded in gelatin). We report a localized but complete digestion of emulsion gelatin facing two tissue sites which are, therefore, presumed to contain an active proteinase. One of the sites is localized at the growth plate surface forming the epiphysis/metaphysis interface. The other consists of small patches located within the epiphysis at the edge of the marrow space. Both sites are engaged in the resorption of endochondral cartilage. In both sites, inhibitor tests have established that the involved proteinase is a gelatinase. Furthermore, the use of neutralizing antibodies against gelatinase A or B have demonstrated that only those that are specific for the latter block the reaction. That gelatinase B is present in the two sites has been confirmed by light microscopic immunohistochemistry. Finally, when immunoelectron microscopy is used for fine localization of the cartilage structures that form the epiphysis/metaphysis interface, the enzyme is detected within the 0.5-microm thick edge of the cartilage, and outside the cartilage, it is present in debris composed of type II collagen-rich fibrils in various states of digestion. It is concluded that gelatinase B attacks the edge of an endochondral cartilage and helps to solubilize the type II-collagen-rich fibrillar framework, which is then released as debris for further digestion. This final step opens the way to invasion by capillaries, thereby making possible the replacement of cartilage by bone. Dev Dyn 1999;215:190-205.

The eleven stages of the cell cycle, with emphasis on the changes in chromosomes and nucleoli during interphase and mitosis.

Since we had subdivided the cell cycle into 11 stages--four for mitosis and seven for the interphase--and since we had experience in detecting DNA in the electron microscope (EN) by the osmium-amine procedure of Cogliati and Gauthier (Compt. Rend. Acad. Sci., 1973;276:3041-3044), we combined the two approaches for the analysis of DNA-containing structures at all stages of the cell cycle. Thin Epon sections of formaldehyde-fixed mouse duodenum were stained by osmium-amine for electron microscopic examination of the stages in the 12.3-hr long cell cycle of mouse duodenal crypt columnar cells. In addition, semi-thin Lowicryl sections of mouse duodenal crypts and cultured rat kidney cells were stained with the DNA-specific Hoechst 33258 dye and examined in the fluorescence microscope. The DNA detected by osmium-amine is in the form of nucleofilaments, seen at high magnification as long rows of 11 nm-wide rings (consisting of stained DNA encircling unstained histones). At all stages of the cycle as well as in nondividing cells, nucleofilaments are of three types: 'free,' 'attached' to chromatin accumulations, and 'compacted' in all chromatin accumulations, the form of dense spirals within. At stage I of the cycle, besides free and attached nucleofilaments, compacted ones are observed in the three heterochromatin forms (peripheral, nucleolus-associated, clumped). Soon after the S phase begins, chromatin 'aggregates' appear, which are small at stage II, mid-sized at stage III, and large at stage IV. Chromatin 'bulges' also appear at stage III and enlarge at stage IV, while heterochromatins disappear. At stage V, aggregates and bulges accrete into 'chromomeres,' a process responsible for the apparent chromosome condensation observed at prophase. The chromomeres gradually line up in rows and, at stage VIa (prometaphase), approach one another within each row and coalesce to build up the metaphase chromosomes which are fully formed at stage VIb (metaphase). Daughter chromosomes arising at stage VII (anaphase) are eventually packed into a chromosomal mass at each pole of the cell. During stage VIII (telophase), the chromosomal mass is split into large chunks. In the course of the G1 phase, the chunks thin out to give rise to irregular 'bands' at stage IX, the bands are then cleaved into central and peripheral fragments at stage X, and finally the central fragments are replaced by free nucleofilaments and clumps at stage XI, while the peripheral fragments are replaced by peripheral heterochromatin. The "nucleoli" at stages I-III are associated with stained heterochromatin but otherwise appear as unstained lucent areas, except for weakly stained patches composed of histone-free DNA filaments. During stage IV, nucleoli lose patches and associated heterochromatin, while weakly lucent, pale vesicles appear within nucleoli and in the nucleoplasm. By the end of substage VIa, nucleoli generally disappear, while pale vesicles persist around the chromosomes appearing at substage VIb. At stages VIII and IX, the vesicles seem to become strongly lucent and, at stages IX and X, they associate and fuse to yield homogeneous lucent areas, the 'prenucleolar bodies,' which include histone-free DNA patches. During stage XI, groups of these bodies associate to give rise to nucleoli. In conclusion, the cell cycle DNA changes can be classified into 4 broad periods (Fig. 6): 1) Stage I is a 2-hr long interphase "pause," during which the stained DNA shows no signs of either chromosome condensation or decondensation, while the overall nuclear pattern is similar to that in nondividing cell nuclei. Nucleoli are fully developed. 2) From stage II to VIa, the "chromosome condensation" period extends over about 7 hr, during which the events are interpreted as follows. Throughout the S phase (stages II-IV), newly-synthesized segments of nucleofilaments approach one another, adhere and thus build aggregates and later bulges on nuclear matrix sites. (ABSTRACT TRUNCATED)

Biased postural vertical in humans with hemispheric cerebral lesions.

This study was aimed at demonstrating the existence of a biased postural vertical in humans with a recent cerebral lesion. The postural vertical of patients and controls was analysed comparatively using a self-regulated balancing task, performed in sitting posture. Patients displayed a quite constant (19/22) contralesional tilt of the postural vertical (mean -2.6 degrees), varying with the severity of their spatial neglect and hemianaethesia. Eight of them showed a pathological contralesional bias (mean -5.5 degrees) as compared to normals. This result indicates an asymmetric process of somatic graviceptive information due to some cerebral lesions. When patients were subjected to a transcutaneous electrical stimulation applied onto the contralesional side of the neck, body verticality was especially improved in those who showed a pathological bias in the postural vertical. This effect could thus be due to a reduced distortion in the egocentric co-ordinate system for spatial information processing.

Immunolocalization of the cleavage of the aggrecan core protein at the Asn341-Phe342 bond, as an indicator of the location of the metalloproteinases active in the lysis of the rat growth plate.

In view of the extensive lysis of hyaline cartilage known to take place during endochondral bone formation, the current study was designed to test the hypothesis that metalloproteinases are the agents that mediate this lysis. Since these enzymes have been shown in vitro to cleave the core protein of the major proteoglycan of cartilage, aggrecan, at the Asn341-Phe342 bond, an immunohistochemical method has been developed to find out whether or not there are sites in the growth plate of the rat tibia where cleavage of this bond takes place. The cleavage of aggrecan by metalloproteinases is followed by the retention of the fragment known as G1, for it includes the G1 domain. Since the G1 fragment terminates in the amino acid residues ...FVDIPEN, we prepared an antiserum against FVDIPEN, confirmed its specificity, then applied it to the growth plate of 21-day-old rat tibia in the hope of localizing the G1 fragments. The antiserum specificity was shown by its recognition of the ...FVDIPEN sequence at the C-terminus of peptides and of G1 fragments produced by aggrecan cleavage. When the antiserum was applied to Western blots of guanidinium chloride extracts prepared from epiphyseal growth plate, it recognized two species (56 and 52 kDa), which differed only in the degree of glycosylation. These fragments were comparable in size to the G1 fragments generated by the action of recombinant metalloproteinase in vitro, thus confirming antiserum specificity for these fragments. Applying the antiserum to cryosections of 21-day-old rat tibiae revealed immunostaining at two intensities within the growth plate matrix: a strong staining was observed in a 1-5 microm-wide layer designated "peripheral" matrix, which borders the epiphyseal and metaphyseal marrow spaces as well as the perichondrium, while a weak staining was found in the rest of the plate, designated "central" matrix. The abundance of G1 fragments terminating in ...FVDIPEN in the peripheral matrix indicates that this is where the growth plate is lysed to achieve longitudinal and latitudinal bone growth. The site where metalloproteinases exert their main lytic activity is a thin layer of matrix separating central from peripheral matrix.

Changes in the rate of RNA synthesis during the cell cycle.

BACKGROUND: Although the rate of RNA synthesis is known to drop at mitosis, the recent identification of 11 stages in the cell cycle (El-Alfy et al., 1994) makes it possible to measure the rate of this synthesis at each one of the stages and thus find out how it varies throughout the cell cycle. METHODS: Mice were injected intravenously with the RNA precursor, 3H-uridine; the duodenum was fixed 5-15 minutes later for embedment in Epon, and the duodenal crypts were cut in semithin serial sections for study of the rapidly dividing crypt columnar cells. Using Feulgen-stained sections, each cell nucleus was assigned to one of the 11 stages described in the cell cycle, and the same nucleus was identified in the next serial section that had been processed for radioautography, so that the overlying silver grains were enumerated. The count was taken as an index of the rate of RNA synthesis by this nucleus. RESULTS: Starting from stage I of the cell cycle (the period defined by the presence of a minimal amount of chromatin during which the S phase begins) and up to stage IV (when the S phase ends and the G2 phase begins), all or nearly all nuclei are synthesizing RNA with the rate peaking at stage III. During stages V to VIII (the period comprising the mitotic steps), the percentage of RNA-synthesizing nuclei decreases to over half at stage V (prophase), -10% at stages VIa (prometaphase) and VIb (metaphase) and none at stages VII (anaphase) and VIII (telophase). During stages IX-XI (which correspond to the G1 phase), the percentage rises sharply at stage XI to reach up to 100% at stages X and XI. Finally, on the average, 35% of nuclear silver grains are over the nucleolus (presumably representing ribosomal RNA precursors), whereas 65% are over the nucleoplasm (presumably representing mainly heterogeneous RNA precursors). CONCLUSIONS: Cells synthesize RNA during the interphase, but at a variable rate with a peak in S. The synthesis proceeds in a majority of the cells at prophase, but only in a few of them at prometaphase and metaphase, and in none at anaphase and telophase.