A novel whole "Joint-in-Motion" device reveals a permissive effect of high glucose levels and mechanical stress on joint destruction.

OBJECTIVE: Osteoarthritis (OA) has recently been suggested to be associated with diabetes. However, this association often disappears when accounting for body mass index (BMI), suggesting that mechanical stress may be a confounding factor. We investigated the combined influence of glucose level and loading stress on OA progression using a novel whole joint-in-motion (JM) culture system. DESIGN: Whole mouse knee joints were placed in an enclosed chamber with culture media and actuated to recapitulate leg movement, with a dynamic stress regimen of 0.5 Hz, 8 h/day for 7 days. These joints were treated with varying levels of glucose and controlled for osmolarity and diffusion. Joint movement and joint space were examined by X-ray fluoroscopy and microCT. Cartilage matrix levels were quantified by blinded Mankin scoring and immunohistochemistry. RESULTS: Culturing in the JM device facilitated proper leg extension and flexion movements, and adequate mass transport for analyzing the effect of glucose on cartilage. Treatment with higher levels of glucose either via media supplementation or intra-articular injection caused a significant decrease in levels of glycosaminoglycan (GAG) and an increase in aggrecan neoepitope in articular cartilage, but only under dynamic stress. Additionally, collagen II level was slightly reduced by high glucose levels. CONCLUSIONS: High levels of glucose and dynamic stress have permissive effects on articular cartilage GAG loss and aggrecan degradation, implicating that mechanical stress confounds the association of diabetes with OA. The JM device supports novel investigation of mechanical stress on the integrity of an intact living mouse joint to provide insights into OA pathogenesis.

Acute promyelocyte leukemia arose from CALR 1 mutated post essential thrombocythemia- myelofibrosis with splanchnic vein thrombosis: A case report.

Major disease complications for patients with essential thrombocythemia (ET) include thrombosis and fibrotic or leukemic transformation. Calreticulin (CALR) mutation type 1 frequencies in ET are estimated between 7% and 11% and ET patients carrying CALR type 1 mutation are associated with lower risk of thrombosis but higher risk of myelofibrosis transformation compared to ET patients with JAK2 mutation. Leukemic transformation rates at 20 years are estimated at less than 5% for ET and risk factors for leukemic transformation are advanced age, thrombosis history, leukocytosis, and anemia. Amongst the subtypes of blast phase myeloproliferative neoplasms, acute promyelocytic leukemia is extremely rare. Herein, we present a case of a promyelocytic blast crisis of post-ET myelofibrosis with associated life-threatening splanchnic vein thrombosis. This case suggests that inflammation plays a key role in thrombotic events and fibrotic/leukemic transformation in ET patients, regardless the molecular landscape.

5-aminolevulinic acid photodynamic therapy for the treatment of high-grade gliomas.

INTRODUCTION: Photodynamic therapy (PDT) is a two-step treatment involving the administration of a photosensitive agent followed by its activation at a specific light wavelength for targeting of tumor cells. MATERIALS/METHODS: A comprehensive review of the literature was performed to analyze the indications for PDT, mechanisms of action, use of different photosensitizers, the immunomodulatory effects of PDT, and both preclinical and clinical studies for use in high-grade gliomas (HGGs). RESULTS: PDT has been approved by the United States Food and Drug Administration (FDA) for the treatment of premalignant and malignant diseases, such as actinic keratoses, Barrett's esophagus, esophageal cancers, and endobronchial non-small cell lung cancers, as well as for the treatment of choroidal neovascularization. In neuro-oncology, clinical trials are currently underway to demonstrate PDT efficacy against a number of malignancies that include HGGs and other brain tumors. Both photosensitizers and photosensitizing precursors have been used for PDT. 5-aminolevulinic acid (5-ALA), an intermediate in the heme synthesis pathway, is a photosensitizing precursor with FDA approval for PDT of actinic keratosis and as an intraoperative imaging agent for fluorescence-guided visualization of malignant tissue during glioma surgery. New trials are underway to utilize 5-ALA as a therapeutic agent for PDT of the intraoperative resection cavity and interstitial PDT for inoperable HGGs. CONCLUSION: PDT remains a promising therapeutic approach that requires further study in HGGs. Use of 5-ALA PDT permits selective tumor targeting due to the intracellular metabolism of 5-ALA. The immunomodulatory effects of PDT further strengthen its use for treatment of HGGs and requires a better understanding. The combination of PDT with adjuvant therapies for HGGs will need to be studied in randomized, controlled studies.

Selecting blood glucose monitoring systems.

A wide range of blood glucose monitoring systems is available for use in different applications. The systems have varying characteristics which should be taken into consideration during selection. Operator technique can influence the quality of the results.

Community reinforcement training for family and significant others of drug abusers: a unilateral intervention to increase treatment entry of drug users.

We randomly assigned 32 concerned family members and significant others (FSOs) of drug users (DUs) to a community reinforcement training intervention or a popular 12-step self-help group. We measured problems arising from the DU's behavior, social functioning of the DU and FSO, and mood of the FSO at baseline and 10 weeks later. We also monitored the FSOs' treatment attendance and treatment entry of the DUs. The treatment groups showed equal reductions from baseline to follow-up in problems and improvements in social functioning and mood of the FSO. However the community reinforcement intervention was significantly better at retaining FSOs in treatment and inducing treatment entry of the DUs.

Blood lancing systems for skin puncture.

Blood tests increasingly require only a few microlitres of blood. Lancing systems used for the collection of blood have the potential to transmit infection if used improperly. These systems vary in the amount of blood and pain produced.

Blood glucose measuring systems.

Glucose monitoring and lancing systems have proved useful in a range of patient testing applications in hospitals, general practice and for use by patients at home. Products are becoming easier to use and less technique dependent. It is important to be aware of their specific characteristics and to adhere to manufacturers' and local hospital laboratory guidelines.

High-speed screening of polymerase chain reaction products by capillary electrophoresis.

In an effort to develop capillary electrophoresis (CE) for high-throughput polymerase chain reaction (PCR) molecular diagnostics, a method was developed to rapidly screen small PCR products of similar molecular weights. The assay of interest required the separation of two PCR products (375 and 400 bp) in an assay of TGF-beta 1 knockout mice to determine the genotype of neonates. Using a commercially available CE instrument, the two PCR products were separated in 12 min with a replaceable gel buffer, a 20-cm effective length DB-17 capillary, and 185 V/cm field strength. With the coinjection of a 20-bp ladder, the sizes of the PCR products were determined from the electropherogram without using a calibration plot and curve-fitting program. Faster separation was obtained using the combination of a short effective length capillary and high field strength. The two PCR products were separated in 82 s with a 7-cm effective length capillary and 556 V/cm. A 60% buffer further reduced the separation time in about a minute. This high-speed separation, with minimum postrun data processing, is highly desirable for the high-throughput screening of PCR products using a single-capillary CE system.

Cloning of the gene encoding rat JAK2, a protein tyrosine kinase.

A complete cDNA clone encoding the rat JAK2 protein tyrosine kinase was isolated from an Nb2-SP (rat pre-T lymphoma cell line) cDNA library. The nucleotide (nt) and deduced amino acid (aa) sequences for this clone were determined and an open reading frame of 3399 bp, encoding a protein of a deduced mass of 130 kDa, was found. The coding regions of the rat and murine Jak2 clones share 93.4% nt identity and 97.1% aa identity. Northern analysis demonstrated that the 5-kb mRNA is highly abundant in brain and spleen, less abundant in skeletal muscle and testis, and detectable in kidney, heart, lung and liver. Translation of the rat Jak2 mRNA in rabbit reticulocytes results in a protein which is specifically immunoprecipitated by antibodies (Ab) recognizing JAK2, but not by Ab recognizing JAK1.

Spatial working memory score of humans in a large radial maze, similar to published score of rats, implies capacity close to the magical number 7 +/- 22.

To compare the working memory (WM) capacity of humans to rats, we tested humans with a 17-arm radial maze and, in a follow up experiment, with a 13-arm radial maze. Both mazes were 15.2 meters in diameter, painted on a grassy field. In one version of the 13-arm experiment, we required a concurrent nonsense vocalization to impede subjects' use of language to remember locations. Subjects were instructed to choose arms of the radial maze unsystematically--as rats generally appear to do--and to visit the end of each arm only once. In additional procedures, we tested working memory capacity in a verbal task that is more analogous to the radial maze than is the typical ordered recall test. Subjects were asked to try to recite a sequence of 17 numbers (i.e., 18 through 34) or letters (A through Q) in unsystematic order, with no repeats. In another experiment subjects recited 13 numbers (14-26) or letters (A-M). In all tests, subjects were allowed only as many responses as there were distinct items (17 or 13, respectively). Average correct-response (nonrepeat) scores were 14.4 for the 17-arm maze and 14.1 for both of the verbal 17-item tests; these scores are close to the reported score for rats in a 17-arm radial maze. Average scores were between 10.8 and 11.4 in all of the 13-item maze and recitation tasks.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence and genome organization of biologically active proviruses of the bovine immunodeficiency-like virus.

The complete nucleotide sequences and translations of major open reading frames (ORF) of two distinct, infectious, proviral molecular clones (106 and 127) of the bovine immunodeficiency-like virus (BIV), obtained from a single virus isolation, were determined and compared. The genomes of BIV 127 and 106 are 8482 and 8391 nucleotides (nt), respectively, in the form predicted for the viral RNA. The structural organization of the genomes of BIV 127 and 106 are identical to one another and most similar to that of the lentivirus subfamily of retroviruses. In addition to gag, pol, and env genes, the BIV genome contains five short ORFs between and overlapping pol and env in the "central region," a hallmark of the lentiviruses which is believed to play an important role in their pathogenesis. Three of the short ORFs in the central region of BIV have been identified by location and structural similarity to the nonstructural/regulatory genes (vif, tat, and rev) of other lentiviruses; we also discovered two unique ORFs, termed W and Y, which may serve as exons for novel genes. BIV does not have the nef gene found in primate lentivirus genomes. The proviral LTR of BIV 127 is 589 nt, contains regulatory signals for initiation, enhancement, and termination of viral transcription, and has sequences related to the Sp1 and NF-kappa B binding sites. A major deletion (87 nt) in the env gene and 2 minor deletions (2 nt each) in the R regions of the LTRs account for the smaller size of clone 106. Numerous point mutations were also present; some caused coding substitutions that were most prevalent in the env encoding ORF. These data suggest that, within a single virus isolate, BIV displays extensive genomic variation. These infectious clones of BIV represent well-defined tools with which to analyze the function of the various ORFs and to dissect the molecular mechanisms of replication and pathogenesis.

Development of the bovine immunodeficiency-like virus as a model of lentivirus disease.

The bovine immunodeficiency-like virus (BIV) is morphologically, serologically, and genetically related to the lentivirus subfamily of retroviruses which includes human and simian immunodeficiency viruses and other lentiviruses causally associated with debilitating diseases of domestic animals. There are many parallels in the biology and pathologic characteristics of BIV infections with those of HIV that make its development as a model of HIV-like infection and disease potentially attractive. In order to obtain a better understanding of the molecular basis of BIV-induced disease, two biologically active proviruses of BIV were molecularly cloned and sequenced. The BIV genome is 9.0 kilobases in the form of the proviral DNA. It contains the obligate retroviral structural genes, gag, pol, and env. In addition, in the BIV central region, between and overlapping pol and env, there are five potential coding regions for non-structural/regulatory genes; three are analogous to vif, tat, and rev in HIV and two, called W and Y, are unique to BIV. There is no coding region analogous to nef in BIV. Sequence comparisons of two functional proviruses obtained from the DNA of cells carrying an infection from a single virus isolation indicate that the genome of BIV is highly variable within a single biological isolate. Moreover, the greatest number of substitutions occur in the env gene. The results suggest the presence of multiple genotypes which may be of significance in defining the disease potential of a BIV isolate. These clones will be useful in dissecting the replicative cycle and mechanisms of pathogenesis of BIV in various animal models.

Characterization of a deletion mutant of bacteriophage phi 29.

A spontaneous deletion mutant of bacteriophage phi 29 (phi 29 delta 1) has been characterized. This mutant has a 1112-bp deletion, which covers almost the entire sequence of genes 14 and 15, including an early promoter (B2). While lysis is very delayed, the phage DNA synthesis and internal phage development appear to be normal in the cells infected with this deletion mutant. These results indicate that the early functions are intact in phi 29 delta 1. Our results also suggest that genes 14 and 15 are dispensable for bacteriophage phi 29 growth and that the B2 promoter may also be dispensable for early functions in phi 29.

Cloning and purification of a unique lysozyme produced by Bacillus phage phi 29.

A DNA fragment of the bacteriophage phi 29 chromosome, encoding the entire sequence of phi 29 gene 15, has been cloned into the Escherichia coli expression vector pPLc245 under the control of the phage lambda major leftward promoter, PL. Upon heat induction, a protein with an apparent molecular mass of 26 kDa was overproduced. The molecular mass of this protein corresponds to the 28 kDa predicted for the product of gene 15 from its nucleotide sequence. The overproduced protein has been purified to near homogeneity and confirmed to be the product of gene 15 by amino acid sequence analysis of its N terminus. The purified product of gene 15 has a lysozyme activity similar to other phage-type lysozymes: products of phage T4 gene e and of phage P22 gene 19. However, to our knowledge phi 29 lysozyme is structurally unique among the phage-type lysozymes.

Nucleotide sequence of Bacillus phage phi 29 genes 14 and 15: homology of gene 15 with other phage lysozymes.

The nucleotide sequence of Bacillus phage phi 29 genes 14 (g14) and 15 (g15) have been determined and shown to encode proteins with molecular weights of 15,014 and 28,022, respectively. The g14 open reading frame (ORF) was confirmed by sequencing a sus14(1241) mutant. Gene product 15 (gp15) has considerable homology with Salmonella phage P22 lysozyme and lesser homology with Escherichia coli phage T4 lysozyme. Putative translation signals are identified. In addition, the role of a previously described promoter, B2, is discussed.

Significance of the hydrogen ion concentration in synovial fluid in rheumatoid arthritis.

The hydrogen ion (H+) concentration and pCO2 were measured in the synovial fluid (SF) from the knee joints of 130 patients with arthritis by an acid-base analyser (ABL2 Acid-Base Laboratory), using a simple technique which prevented contact with air. H+ concentration was significantly higher in SF from 60 RA patients (mean 64.4 n mol/l; range 38-142 n mol/l) compared with patients with OA (mean 44 n mol/l; range 29-56 n mol/l), and 40 with other arthritides (mean 52 n mol/l). The H+ concentration in the SF showed a significant association with other variables of local inflammation-platelet, total leucocyte and polymorph counts, 5-nucleotidase, acid phosphatase and IgA levels in the SF and the clinical knee score, but not with the volume of the effusion. A similar relationship between these variables of inflammatory activity and SF pCO2 was also established. A higher SF H+ concentration was also found in systemically active disease, but no difference in SF pH between seropositive and seronegative patients. Whilst the pH of SF approximated to that of the blood in OA, it was significantly lower in the SF in RA. SF pH is a useful marker of local inflammatory activity, and its measurement is simple, reliable and rapid. It is relevant because changes in pH influence many of the processes involved in inflammation and the pH difference between SF and blood influences the transfer of drugs into the joint.

The complete sequence of the Bacillus phage phi 29 right early region.

We have sequenced the rightmost 2216 bp of the Bacillus phage phi 29 genome. This region encompasses the right early region and completes the sequence of the phi 29 early functions. The sequence of gene 17, an early gene implicated in the replication process, is presented. From these results we predict that gene 17 encodes a 19.1-kDal protein. Further analysis of the sequence revealed five previously undetected potential genes, encoding 12.6-, 12.4-, 15.2-, 6.2- and 4.6-kDal proteins. The biological efficacies of some of these putative genes were demonstrated using an Escherichia coli in vitro transcription-translation system. We also examined the transcriptional and translational signals present on this region of the genome.

The complete sequence of Bacillus phage phi 29 gene 16: a protein required for the genome encapsidation reaction.

We have sequenced the region of the Bacillus phage phi 29 genome that encodes gene 16, the gene product of which catalyzes the in vivo and in vitro genome-encapsidation reaction. The identity of the coding frame was confirmed by sequencing a sus mutant, sus16(300). It is concluded that gene 16 encodes a 39-kDal protein and is comprised of 331 amino acids. Only 30 bp separates gene 16 from the last open reading frame of the right early region. Analysis of potential secondary structures in this region suggests that the same sequences may be involved in the termination of both the late and early transcripts.