Biomedical subjects

K A Johnson

Publications and source records attributed to K A Johnson.

At least 55 records · Page 3Linked to original sources

PubMed2001

Effects of an orally administered mixture of chondroitin sulfate, glucosamine hydrochloride and manganese ascorbate on synovial fluid chondroitin sulfate 3B3 and 7D4 epitope in a canine cruciate ligament transection model of osteoarthritis.

OBJECTIVE: To evaluate effects of an orally administered mixture of chondroitin sulfate, glucosamine hydrochloride and manganese ascorbate (CS-G-M) on articular cartilage metabolism in dogs with cranial cruciate ligament (CCL) deficient and reconstructed knees, as reflected by concentrations of synovial fluid 3B3, 7D4 and total sulfated glycosaminoglycan (GAG). METHODS: Sixteen adult dogs that underwent unilateral CCL transection were randomized into four groups. Thereafter, group I (N=3) had a sham CCL reconstruction, group II (N=3) had CS-G-M and sham CCL reconstruction, group III (N=5) had CCL reconstruction, and group IV (N=5) had CS-G-M and CCL reconstruction. Synovial fluid collected at 0, 1, 3 and 5 months was examined by ELISA for 3B3 and 7D4 epitope, and by DMMB assay for total GAG. RESULTS: Synovial fluid from CCL transected knees of CS-G-M treated dogs contained significantly elevated concentrations of 3B3 (P=0.029), 7D4 (P=0.036) and 7D4/GAG (P=0.007) in comparison to controls, in a cross-sectional analysis at 3 months. Furthermore, 7D4 and 7D4/GAG concentrations remained significantly elevated (P=0.012) in CCL transected knees of CS-G-M treated dogs over the 5 month period. However, when epitope concentrations were expressed as a ratio of CCL-transected to contralateral non-operated knee, treatment effect of CS-G-M was no longer significant. Reconstruction of the CCL had no significant effect on synovial fluid epitope. CONCLUSIONS: Administration of CS-G-M was associated with altered concentrations of 3B3 and 7D4 epitope in synovial fluid, suggesting that these compounds may act to modulate articular cartilage matrix metabolism in vivo.

Administration, Oral↗

PubMed2001

Parental alcoholism as a risk factor for DSM-IV-defined alcohol abuse and dependence in American women: the protective benefits of dyadic cohesion in marital communication.

Important trends in research over the past decade indicate that women are as greatly affected by familial alcoholism as are men. Although it is increasingly recognized that the adverse drinking outcomes predicted for adult children of alcoholics (COAs) are not inevitable, and only a small percentage develop alcohol dependence or grow up to be alcoholic, relatively little knowledge exists regarding moderating factors that reduce their vulnerability. This study identifies a multiple mediator latent structural model of the intergenerational transmission of risk for DSM-IV-assessed alcohol abuse and dependence among women COAs in adulthood. The effects of both parental alcoholism and family environment are estimated at three time points spanning 10 years across 5-year intervals (1984, 1989, and 1994) using data from a subsample of 4,449 women in the National Longitudinal Survey of Youth (NLSY). Dyadic cohesion in marital communication (greater marital cohesion, harmony, and less verbal disagreement, discord, and conflict) is a proposed moderating factor that may operate in adulthood to lower the risk of female COAs developing alcohol abuse and dependence. Maximum likelihood standardized estimates of the effects of alcohol mediators measured over time indicate that direct parental effects for adverse outcomes decline when COAs are in their late 20s and early 30s. Indirect parental effects through environmental influences dramatically increase the risk of abuse and dependence among COAs at this time if they have one or more alcoholic siblings, especially an alcoholic sister. Dyadic cohesion and positive interpersonal communication patterns were found to moderate effectively the relationship that existed among parental alcoholism, environmental influences, and adverse alcohol consequences. COAs with satisfactory marital communication also evidenced higher levels of intimacy with their partners, perceived the division of housework to be fairer, shared more responsibilities and burdens of the household, and had less conflict over critical domestic issues than other women COAs. The protective benefits of a good marriage against the risks of alcoholism remained when applied to younger and older subjects, across diverse backgrounds, and after adjusting for other factors such as employment status.

Adolescent↗

PubMed2001

Concentrations of chondroitin sulfate epitopes 3B3 and 7D4 in synovial fluid after intra-articular and extracapsular reconstruction of the cranial cruciate ligament in dogs.

OBJECTIVE: To evaluate effects of intra-articular and extracapsular reconstruction of the cranial cruciate ligament (CCL) on metabolism of articular cartilage as reflected by concentrations of chondroitin sulfate epitopes 3B3 and 7D4 in synovial fluid. ANIMALS: 13 adult dogs. PROCEDURE: Each dog underwent unilateral CCL transection (CCLT). One month after CCLT, sham CCL reconstruction (3 dogs), intra-articular CCL reconstruction (5), or extracapsular CCL reconstruction (5) was performed. Synovial fluid was collected by direct arthrocentesis from CCLT and contralateral stifle joints immediately before (time 0) and 1, 3, and 5 months after CCLT. Fluid was examined for concentrations of 3B3 and 7D4 epitopes and total sulfated glycosaminoglycan (GAG) content. RESULTS: Concentrations of 3B3, 7D4, and GAG, 3B3:GAG, or 7D4:GAG in CCLT joints did not differ significantly among treatment groups nor in the ratios of these variables in CCLT joints to contralateral joints at 3 months. In a longitudinal analysis, concentrations of 3B3 and 7D4, 3B3:GAG, and 7D4:GAG in CCLT joints in all groups changed significantly with time, but we did not detect time X group interactions. CONCLUSIONS AND CLINICAL RELEVANCE: Transection of CCL resulted in significant perturbation in articular cartilage metabolism as reflected by alterations in concentrations of 3B3 and 7D4 in synovial fluid. These changes over time were not significantly influenced by method of CCL reconstruction. We did not find evidence that surgical stabilization of CCL-deficient joints by intra-articular or extracapsular techniques had any effect on preventing alterations in composition of synovial fluid that have been associated with secondary osteoarthritis.

Animals↗

PubMed2001

Site-specific adaptive remodeling of Greyhound metacarpal cortical bone subjected to asymmetrical cyclic loading.

OBJECTIVE: To quantify geometric, inertial, and histomorphometric properties at the mid-diaphyseal level of left and right metacarpal bones (MCB) of racing Greyhounds. SAMPLE POPULATION: MCB from 7 racing Greyhounds euthanatized for reasons unrelated to MCB abnormalities. PROCEDURES: Mid-diaphyseal transverse sections of left and right MCB were stained with H&E or microradiographed. Images of stained sections were digitized, and cross-sectional area, cortical area, and maximum and minimum area moments of inertia of each bone were determined. Histomorphometric data (osteonal density, osteonal birefringence, and endosteal new lamellar bone thickness) were collected in 4 quadrants (dorsal, palmar, lateral, medial). Values were compared between limbs and among bones and quadrants. RESULTS: Cross-sectional area, cortical area, and maximum and minimum moments of inertia of left MCB-IV and -V were significantly greater, compared with contralateral bones. Overall osteonal densities in the dorsal quadrants of left MCB were greater, compared with lateral and medial quadrants. Also, percentage of birefringent osteons was significantly greater in the dorsal quadrant of left MCB-III, -IV, and -V, compared with the palmar quadrant. Thickness of new endosteal lamellar bone was not significantly influenced by limb, bone, or quadrant. CONCLUSIONS AND CLINICAL RELEVANCE: Increased cortical thickness and geometric properties of left MCB-IV and -V of Greyhounds, together with altered turnover and orientation of osteons in the dorsal quadrants of left MCB, are site-specific adaptive responses associated with asymmetric cyclic loading as a result of racing on circular tracks. Site-specific adaptive remodeling may be important in the etiopathogenesis of fatigue fractures in racing Greyhounds.

Adaptation, Physiological↗

PubMed2000

Probing the stoichiometry and oxidation states of metal centers in iron-sulfur proteins using electrospray FTICR mass spectrometry.

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry is used to determine the stoichiometry and oxidation states of the metal centers in several iron-sulfur proteins. Samples are introduced into the ESI source under nondenaturing conditions in order to observe intact metal-containing protein ions. The stoichiometry and oxidation state of the metal or metal-sulfur cluster in the protein ion can be derived from the mass spectrum. Mononuclear metal-containing proteins and [4Fe-4S] centers are very stable and yield the molecular ion with little or no fragmentation. Proteins that contain [2Fe-2S] clusters are less stable and yield loss of one or two sulfur atoms from the molecular species, although the molecular ion is more abundant than the fragment peaks. [3Fe-4S]-containing proteins are the least stable of the species investigated, yielding abundant peaks corresponding to the loss of one to four sulfur atoms in addition to a peak representing the molecular ion. Isotope labeling experiments show that the sulfur loss originates from the [3Fe-4S] center. Negative ion mode mass spectra were obtained and found to produce much more stable [3Fe-4S]-containing ions than obtained in positive ion mode. ESI analysis of the same proteins under denaturing conditions yields mass spectra of the apo form of the proteins. Disulfide bonds are observed in the apoprotein mass spectra that are not present in the holoprotein. These result from oxidative coupling of the cysteinyl sulfur atoms that are responsible for binding the metal center. In addition, inorganic sulfide is found to incorporate itself into the apoprotein by forming sulfur bridges between cysteine residues.

Iron-Sulfur Proteins↗

PubMed2000

Response to genetic counseling and testing for the APC I1307K mutation.

The APC I1307K gene mutation is associated with increased colorectal cancer (CRC) risk in Ashkenazi Jews. Factors predicting acceptance of this and other hereditary colon cancer mutation tests in a clinical setting are unknown. We analyzed sex, age, family history, personal history, and gene test results of patients at increased risk for cancer who sought cancer risk counseling at the Johns Hopkins (JH) CRC Risk Assessment Clinic (n = 91), and those submitting samples to the JH Pathology Molecular Diagnostic Laboratory (n = 256) for APC I1307K testing. Of patients seen at the JH Clinic, 77/91 (84.6%) elected APC I1307K testing after pretest counseling (acceptors). There were no statistically significant differences in demographic characteristics between acceptors and decliners. In comparison, only 8 of 57 (14.0%) patients offered HNPCC testing proceeded with testing (P < 0.001). Of 256 individuals tested at the JH laboratory, most were male (61.3%) and most had a personal or family history of colorectal cancer or polyps. Test positivity correlated with increasing risk of colorectal cancer. Acceptance of testing for the APC I1307K mutation is high, with more men than women pursuing counseling and testing. The reported association between the APC I1307K mutation and colon cancer risk was supported by a correlation in these data between personal or family history of CRC or polyps and a gene mutation.

Adult↗

PubMed2000

Human mitochondrial DNA polymerase holoenzyme: reconstitution and characterization.

We have reconstituted the holoenzyme of the human mitochondrial DNA polymerase from cloned and overexpressed catalytic and accessory subunits. We have examined the polymerization activity of the catalytic subunit alone and of the holoenzyme to establish the function of the accessory subunit in this two subunit enzyme. The accessory subunit associates with the catalytic subunit with a dissociation constant of 35 +/- 16 nM as measured by the concentration dependence of its effect in stimulating maximal DNA binding and polymerization. At saturating concentrations, the accessory subunit contributes to every kinetic parameter examined to facilitate tighter binding of DNA and nucleotide and faster replication. The accessory protein makes the DNA binding 3.5-fold tighter (K(d) of 9.9 +/- 2.1 nM compared to 39 +/- 10 nM for the catalytic subunit alone) without significantly affecting the DNA dissociation rate (0.02 +/- 0.001 compared to 0.03 +/- 0.001 s(-)(1)). The ground-state nucleotide binding is improved from 4.7 +/- 2.0 to 0.78 +/- 0.065 microM, and the maximum DNA polymerization rate is increased from 8.7 +/- 1.1 to 45 +/- 1 s(-)(1) by the addition of the accessory protein. This leads to an increase in processivity from an estimated 290 +/- 46 to 2250 +/- 162. Although the accessory protein has been described as a "processivity factor" because of its effect on the ratio of rate constants defining processivity, this terminology falls short of adequately describing the profound effects of the small subunit on nucleotide-binding and incorporation catalyzed by the large subunit. By using the complete holoenzyme, we can now proceed with a comprehensive analysis of the structural and mechanistic determinants of enzyme specificity that govern toxicity of nucleoside analogues used in the treatment of viral infections such as AIDS.

Amino Acid Sequence↗

PubMed2000

Chronic toxicity and oncogenicity studies of ingested 1, 3-dichloropropene in rats and mice.

Fischer 344 rats and B6C3F1 mice were administered 1, 3-dichloropropene (1,3-D) via their diets for up to 2 years, at dose levels of 0, 2.5, 12.5, or 25 mg 1,3-D/kg body wt/day for rats and 0, 2.5, 25, or 50 mg 1,3-D/kg body wt/day for mice. The test material was stabilized in the feed by microencapsulation in a starch/sucrose matrix (80/20%). Rats given 12.5 or 25 mg/kg/day, and mice given 25 or 50 mg/kg/day, had decreased body weights and body weight gains. There were no effects on survival or clinical pathology parameters for rats or mice. Histopathologic effects attributed to treatment in rats consisted of basal cell hyperplasia of the nonglandular mucosa of the stomach in males and females given 12.5 or 25 mg/kg/day for 12 and 24 months and an increased number of hepatocellular adenomas in males given 12.5 or 25 mg/kg/day and females given 25 mg/kg/day for 24 months. The increase in hepatocellular adenomas was statistically identified by pairwise comparison only in males given 25 mg/kg/day. An increased incidence of eosinophilic foci of altered cells in the liver was also noted in all treated groups of rats at 24 months. The latter observation, however, was considered of equivocal toxicological significance because of the common spontaneous occurrence of liver foci in aged Fischer 344 rats. The only histologic change attributed to treatment in mice was decreased size of hepatocytes in males given 50 mg/kg/day for 12 months. The decreased size of hepatocytes was consistent with decreased cytoplasmic glycogen content and corresponded to decreased liver weights. This effect was not present at 24 months. There was no oncogenic response observed in mice. The low-dose level of 2.5 mg/kg/day was interpreted as the no-observed-adverse-effect level (NOAEL) for systemic chronic toxicity of 1,3-D in the Fischer 344 rat. The no-observed-effect level (NOEL) for chronic systemic toxicity was 2.5 mg/kg/day in the B6C3F1 mouse.

Adenoma↗

PubMed2000

Bimanual co-ordination in Huntington's disease.

The ability of Huntington's disease patients to co-ordinate their two hands with and without external cueing was investigated. Twelve Huntington's disease patients and sex- and age-matched controls performed a bimanual cranking task at two speeds (0.5 Hz, 1.5 Hz) and phase relationships (in-phase, anti-phase), with and without an external metronome cue. Data were sampled at 200 Hz, and raw displacement data for each hand, mean and standard deviation measures of the relative positions of the two hands and their velocities were then calculated. All participants could perform the in-phase movement, at both speeds; however. the Huntington's disease patients were more variable and less accurate than the control participants, particularly at the fast speed. While controls could perform the anti-phase movement, in which rotation of the cranks differed by 180 degrees at both speeds, Huntington's disease patients were unable to do so at either speed, reverting to the in-phase movement at the slow speed. An external metronome cue did not improve the performance of the Huntington's disease patients, which differentiated this group from patients suffering from Parkinson's disease. The Huntington's disease patients' inability to perform the anti-phase movement may be due to damage to the basal ganglia and its output regions.

Adult↗

PubMed2000

An axonemal dynein at the Hybrid Sterility 6 locus: implications for t haplotype-specific male sterility and the evolution of species barriers.

Poor sperm motility characterized by a distinct aberration in flagellar waveform known as "curlicue" is a hallmark of t haplotype (t) homozygous male sterility. Previous studies have localized "curlicue" and a flagellar developmental defect, "whipless", to the Hybrid Sterility 6 locus (Hst6), between the markers Pim1 and Crya1. More recent heterospecific breeding experiments between Mus spretus (Spretus) and Mus musculus domesticus (Domesticus) have mapped the primary source(s) of both "curlicue" and "whipless" to a small sub-locus of Hst6, Curlicue a (Ccua). Here we report the complete physical isolation of the Ccua locus and the identification of a candidate gene for expression of both "whipless" and "curlicue" at its proximal end, an axonemal dynein heavy chain gene, Dnahc8, formerly mapped by interspecific backcross analysis near Pim1. Dnahc8 mRNA expression commences in the Domesticus wild-type testis just prior to flagellar assembly and is testis-specific in the adult male. However, expression of Dnahc8 is not readily evident in the testis of either Spretus or "whipless" animals (Domesticus males homozygous for the Spretus allele of Dnahc8). Our results argue that Dnahc8 is fundamental to flagellar organization and function in Domesticus, but not Spretus, and suggest that Dnahc8 is integral to both Hst6- and t-specific male infertility.

Animals↗

PubMed2000

A java-based application for differential diagnosis of hematopoietic neoplasms using immunophenotyping by flow cytometry.

We describe the implementation of a Java-based application for differential diagnosis of hematopoietic neoplasms using immunophenotyping by flow cytometry. The current version of this Java applet includes the knowledge-base for 33 hematopoietic neoplasms and 43 diagnostic immunophenotyping markers. Java, a new object-oriented computing language, helps facilitate development of this applet, a platform-independent module that can be implemented on the World Wide Web. As the Web rapidly becomes more accessible to users around the world, Web-based software may eventually form the core of decision-support systems in clinical settings. Java-based applications, such as the one described in this paper, are expected to contribute significantly in this area.

Antigens, CD↗

PubMed2000

Hidden variables: unstable Abeta chain genes encoding antigen recognition structures in tumor survivors.

Novel single exon genes Abeta4-7 comprising the Abeta6 gene family have been cloned from mouse mutants surviving transplantable metastatic tumors. Their protein coding sequences are similar to H2-Ab cDNA which encodes antigen-binding molecules of antigen presenting cells (APC); their promoters and/or signal sequences are unrelated to Ab sequences but found in other eukaryotic genes. Abeta4(b) protein was demonstrated on macrophages and B cells that are APC. The Abeta6(w302) appears to be an ancient gene ancestral to major histocompatibility complex (MHC) class II beta genes. However, unlike the MHC class II, the Abeta4-7 genes are not involved in skin graft rejection. Despite inbreeding, the Abeta6(w302) locus remains unfixed in several strains of mice. The number of Abeta genes and their alleles varied between individual mice; they do not map into the H2 region but appear to be scattered over the genome. The Abeta6 gene family is molecularly unstable in Abeta6(w302)-positive (but not in Abeta6(w302)-negative) mice which are somatic mosaics for these genes. Biological features of Abeta4-7 genes make them remarkably different from the classical MHC gene system. All available evidence strongly suggests that these genes control susceptibility/resistance to the spread of metastatic tumors.

Animals↗

PubMed2000

Findings from case studies of state and local immunization programs.

BACKGROUND: As part of its examination of federal support for immunization services during the past decade, the Institute of Medicine (IOM) Committee on Immunization Finance Policies and Practices (IFPP) commissioned eight case studies of the states of Alabama, Maine, Michigan, New Jersey, North Carolina, Texas, and Washington; and a two-county study of Los Angeles and San Diego in California. Specifically, the IOM Committee and these studies reviewed the use of Section 317 grants by the states. Section 317 is a discretionary grant program that supports vaccine purchase and other immunization-related program activities. These studies afforded the Committee an in-depth look at local policy choices, the performance of immunization programs, and federal and state spending for immunization during the past decade. METHODS: The case-study reports were developed through interviews with state and local health department officials, including immunization program directors, Medicaid agency staff, budget analysts, and Centers for Disease Control and Prevention public health advisors to the jurisdiction. Other sources included state and federal administrative records and secondary sources on background factors and state-level trends. The case studies were supplemented by site visits to Detroit, Houston, Los Angeles, Newark, and San Diego. OBSERVATIONS: The nature of immunization "infrastructure" supported by the Section 317 program is shifting from primarily service delivery to a broader set of roles that puts the public effort at the head of a broad immunization partnership among public health, health financing, and other entities in both the public and private sectors. The rate and intensity of transition vary across the case-study areas. In the emerging pattern, service delivery increasingly takes place in the private sector and is related to managed care. "Infrastructure" is moving beyond supporting a core state staff and local health department service delivery to include such activities as immunization registries, quality improvement, and coordination with programs outside public health agencies. At the same time, the recent decline in federal Section 317 support is forcing difficult choices between old and new activities at the state and local levels. CONCLUSIONS: Immunization programs function as an organic component of the local health care financing and delivery systems of which they are a part. Immunization efforts are organized and conducted within distinctive state and local fiscal, economic, and health care contexts. Section 317 Financial Assistance grants, while playing a vital role in supporting immunization "infrastructure," have been too unstable and unpredictable to elicit the strategic planning, programming, and own-source spending that would be optimal for state and local programs. The predominant immunization function of state and local public health agencies is becoming assurance of age-appropriate immunization throughout the lifespan. To be successful in this emerging role, the health agencies must be supported with appropriate staffing, interagency collaboration, and clearly articulated authority.

Adult↗

PubMed2000

Federal immunization policy and funding: a history of responding to crises.

BACKGROUND: This article outlines the history of federal immunization policy and funding, with a focus on discretionary federal funding under Section 317 of the Public Health Service Act, paying particular attention to the role of Congress in shaping the program in the past 2 decades. This review of funding trends and initiatives indicates that when both a presidential administration and key congressional actors viewed immunization as a priority and made sufficient funds available to support the public health delivery system and its infrastructure, coverage levels would continue to rise and disease levels continue to decline. From the beginning, immunization financing was explicitly structured as a federal-state-private-sector partnership. Section 317 program's statute has not changed much in 35 years, despite significant changes to the health care delivery system, other federal immunization activities, and rates of immunization coverage. Although the creation and implementation of the Vaccines for Children (VFC) program in the mid-1990s resulted in some congressional deliberations over immunization policies, no explicit restructuring of the 317 program occurred as a result. The Section 317 program retains its traditional authority and mission to address urgent needs, sustain public delivery systems, and provide funds for purchase of vaccines. The question remains whether the resources to sustain progress in immunization can be secured during times with no crisis, to ensure constant "readiness" in immunization (as in defense), or whether another epidemic must occur before the federal government is willing to commit optimal resources.

Centers for Disease Control and Prevention, U.S.↗

PubMed2000

Asymmetric adaptive modeling of central tarsal bones in racing greyhounds.

Fatigue fracture of the cuboidal bones of the foot, especially the navicular tarsal bone, is common in athletes and dancers. The racing greyhound is a naturally occurring animal model of this injury because both microcracking and complete fracture occur in the right central (navicular) tarsal bone (CTB). The right limb is on the outside when racing in a counter-clockwise direction on circular tracks, and is subjected to asymmetric cyclic compressive loading. We wished to study in more detail adaptive modeling in the right CTB in racing greyhounds. We hypothesized that cyclic asymmetric loading of a cuboidal bone induced by racing on a circular track would induce site-specific bone adaptation. We also hypothesized that such an adaptive response would be attenuated in greyhounds that were retired from racing and no longer subjected to cyclic asymmetric loading. Central tarsal bones from racing greyhounds (racing group, n = 6) and retired greyhounds being used for breeding (nonracing group, n = 4) were examined using quantitative computed tomography (CT). Bone mineral density (BMD) was determined in a 3-mm diameter region-of-interest (ROI) in six contiguous 1-mm-thick sagittal CT slices of each CTB. Bones were subsequently examined histomorphometrically and percentage bone area (B.Ar./T.Ar., %) was determined in 10 ROI from dorsal to plantar in a transverse plane, mid-way between the proximal and distal articular surfaces. The BMD of the right CTB was greater than the left in all greyhounds (p < 0. 001). In comparing ipsilateral limbs between groups, BMD of the racing group was greater than the nonracing group for each side (p < 0.005). In sagittal plane histologic sections, bone in the dorsal region of the right CTB had undergone adaptive modeling, through thickening and compaction of trabeculae. B.Ar./T.Ar., % in the right CTB of the racing group was greater than in the contralateral CTB (p < 0.001), and the ipsilateral CTB of the nonracing group (p < 0.001). In the nonracing group, B.Ar./T.Ar., % in the right CTB was not significantly different from left CTB (p > 0.8; power = 80% at Delta = 48%). It was concluded that greyhounds racing on circular tracks develop site-specific bone adaptation with compaction of trabecular bone and increase in BMD in the right CTB in particular, the most common site for fatigue fracture. Our data also suggested that partial reversal of this adaptive process occurred in retired, nonracing greyhounds, after cessation of asymmetric cyclic loading at racing speed. Racing greyhounds provide a model in which to study fatigue fracture and adaptation of cuboidal foot bones subjected to cyclic loading.

Animals↗

PubMed2000

MJ0109 is an enzyme that is both an inositol monophosphatase and the 'missing' archaeal fructose-1,6-bisphosphatase.

In sequenced genomes, protein coding regions with unassigned function constitute between 10 and 50% of all open reading frames. Often key enzymes cannot be identified using sequence homology searches. For example, despite the fact that methanogens have an apparently functional gluconeogenesis pathway, standard tools have been unable to identify a fructose-1,6-bisphosphatase (FBPase) gene in the sequenced Methanoccocus jannaschii genome. Using a combination of functional and structural tools, we have shown that the protein product of the M. jannaschii gene MJ0109, which had been tentatively annotated as an inositol monophosphatase (IMPase), has both IMPase and FBPase activities. Moreover, several gene products annotated as IMPases from different thermophilic organisms also possess FBPase activity. Thus, we have found the FBPase that was 'missing' in thermophiles and shown that it also functions as an IMPase.

Amino Acid Sequence↗

PubMed2000

Synovial fluid chondroitin sulphate epitopes 3B3 and 7D4, and glycosaminoglycan in human knee osteoarthritis after exercise.

OBJECTIVE: Walking exercise alleviates some symptoms, such as pain, in patients with mild to moderate knee osteoarthritis (OA). However, a major concern is that weightbearing exercise on osteoarthritic joints may exacerbate articular cartilage degradation. Loading of proteoglycan depleted articular cartilage in vitro increased expression of the chondroitin sulphate epitope 3B3, suggesting that loading may influence metabolism of osteoarthritic cartilage. This study aimed at evaluating the effects of walking exercise on articular cartilage metabolism in patients with knee OA, as reflected by changes in concentrations of synovial fluid markers. METHODS: Thirty elderly patients with knee OA (Kellgren-Lawrence grades II to IV) were randomly allocated to control (n = 15) and 12 week exercise (n = 15) groups. Synovial fluid obtained from 21 of the patients at time zero and after 12 weeks was examined by enzyme linked immunosorbent assay (ELISA) for the chondroitin sulphate epitopes 3B3 and 7D4, and by a dye binding assay with 1, 9-dimethylmethylene blue for total sulphated glycosaminoglycan (GAG) concentrations. The 3B3/GAG and 7D4/GAG ratios were calculated. RESULTS: No significant changes in concentrations of 3B3, 7D4, GAG, 3B3/GAG, or 7D4/GAG between time zero and 12 weeks were found in either group. However, there were significant declines in 3B3 (p=0. 001), GAG (p=0.007), and the 3B3/GAG ratio (p=0.049) with aging. CONCLUSION: Twelve weeks of walking exercise had no demonstrable adverse effects on articular cartilage metabolism, as reflected by the concentrations of synovial fluid GAG or the chondroitin sulphate epitopes 3B3 and 7D4.

Aged↗

PubMed2000

Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1.

Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent (45)Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP-/- mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP(i)), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PP(i). Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PP(i) and inhibited (45)Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated (45)Ca precipitation. Furthermore, the PP(i) content of MV fractions was greater in cultured TNAP-/- than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP-/- mice. Thus TNAP attenuates PC-1/NTPPPH-induced PP(i) generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.

3T3 Cells↗