ARTHRITIS—The Use of Physical Therapy as an Aspect of Management

In some cases arthritis can be controlled even in the acute stage by physical therapy alone. In many cases, recovery depends on how thoroughly and intelligently such methods are applied.Heat, ultrasound and therapeutic baths relieve pain and spasm, permitting greater freedom in passive and active exercise. Exercise is necessary in maintaining and restoring function to arthritic limbs. Because the atrophy produced by rheumatoid arthritis is greater than that due to mere disuse for the same length of time, resistive exercise is especially valuable in building muscle, and this can be carried out even during active inflammation. Without exercise the muscles weaken and throw a greater burden on the already disabled joints.At the same time the patient must be relieved of undue stress and trauma by planned rest, splinting, bed posture and, later, crutches and other aids to ambulation. Efforts should be made to prevent contractures and deformities.Occupational therapy increases muscle strength, range of motion, work tolerance and mental status. A dynamic and carefully planned rehabilitation program hastens restoration of the patient''s independence and usefulness.     

Translation in vitro of RNA from the human malarial parasitePlasmodium falciparum

RNA has been isolated from the human malarial parasite, Plasmodium falciparum, and translated in vitro using two systems: rabbit-reticulocyte lysate and Xenopus laevis oocytes. Polypeptides are synthesized that are characteristic of the parasite isolate and the developmental stage from which the RNA was purified. Many of these polypeptides are precipitated by antibody from an immune monkey. Analysis of the primary structure of these polypeptides should give the information necessary to produce a vaccine against this parasite.     

Modification of a commercial cell sorter to support efficient and reliable preparation of ALDH-bright cells for clinical use

BACKGROUND: Cell populations manufactured by conventional commercial cell sorters have been safely infused into patients, but reliably sterilizing these instruments remains challenging. We are developing clinical protocols involving use of ALDH bright cells manufactured by cell sorting in patients. However, we encountered problems when we attempted to reliably sterilize the FACSAria cell sorter using standard methods. RESULTS: We have identified and modified potential sources of microbial contamination in several FACSAria systems. We added new filter systems to the sheath and sample air lines, to the wet cart fluid supply, and to the sample line. Sheath was provided from an external sterile, disposable bag through sterile disposable tubing sets. The plenum reservoirs were modified in several ways to allow efficient decontamination of internal surfaces. A new bubble filter assembly was added and one valve was eliminated from the sample pathway to improve flow cell sterilization. A new cleaning and sterilization protocol was developed and validated. All cell products manufactured using the modified instrument and validated cleaning protocol have met lot release criteria for prevention of microbial contamination and safe clinical use. DISCUSSION: The instrument modification and cleaning protocol described enable reliable manufacture of ALDH bright cell populations that are suitable for clinical trials. We have manufactured nineteen consecutive samples that meet all clinical release criteria in an on-going Phase 1 human trial.     

Gabapentin's minimal action on markers of rat brain arachidonic acid metabolism agrees with its inefficacy against bipolar disorder

In rats, FDA-approved mood stabilizers used for treating bipolar disorder (BD) selectively downregulate brain markers of the arachidonic acid (AA) cascade, which are upregulated in postmortem BD brain. Phase III clinical trials show that the anticonvulsant gabapentin (GBP) is ineffective in treating BD. We hypothesized that GBP would not alter the rat brain AA cascade. Chronic GBP (10mg/kg body weight, injected i.p. for 30 days) compared to saline vehicle did not significantly alter brain expression or activity of AA-selective cytosolic phospholipase A(2) (cPLA(2)) IVA or secretory (s)PLA(2) IIA, activity of cyclooxygenase-2, or prostaglandin E(2) or thromboxane B(2) concentrations. Plasma esterified and unesterified AA concentration was unaffected. These results, taken with evidence of an upregulated AA cascade in the BD brain and that approved mood stabilizers downregulate the rat brain AA cascade, support the hypothesis that effective anti-BD drugs act by targeting the brain AA cascade whereas ineffective drugs (such as GBP) do not target this pathway, and suggest that the rat model might be used for screening new anti-BD drugs.     

The C-Terminal Domain of CENP-C Displays Multiple and Critical Functions for Mammalian Centromere Formation

CENP-C is a fundamental component of functional centromeres. The elucidation of its structure-function relationship with centromeric DNA and other kinetochore proteins is critical to the understanding of centromere assembly. CENP-C carries two regions, the central and the C-terminal domains, both of which are important for the ability of CENP-C to associate with the centromeric DNA. However, while the central region is largely divergent in CENP-C homologues, the C-terminal moiety contains two regions that are highly conserved from yeast to humans, named Mif2p homology domains (blocks II and III). The activity of these two domains in human CENP-C is not well defined. In this study we performed a functional dissection of C-terminal CENP-C region analyzing the role of single Mif2p homology domains through in vivo and in vitro assays. By immunofluorescence and Chromatin immunoprecipitation assay (ChIP) we were able to elucidate the ability of the Mif2p homology domain II to target centromere and contact alpha satellite DNA. We also investigate the interactions with other conserved inner kinetochore proteins by means of coimmunoprecipitation and bimolecular fluorescence complementation on cell nuclei. We found that the C-terminal region of CENP-C (Mif2p homology domain III) displays multiple activities ranging from the ability to form higher order structures like homo-dimers and homo-oligomers, to mediate interaction with CENP-A and histone H3. Overall, our findings support a model in which the Mif2p homology domains of CENP-C, by virtue of their ability to establish multiple contacts with DNA and centromere proteins, play a critical role in the structuring of kinethocore chromatin.     

The IsdG‐family of haem oxygenases degrades haem to a novel chromophore

Enzymatic haem catabolism by haem oxygenases is conserved from bacteria to humans and proceeds through a common mechanism leading to the formation of iron, carbon monoxide and biliverdin. The first members of a novel class of haem oxygenases were recently identified in Staphylococcus aureus (IsdG and IsdI) and were termed the IsdG‐family of haem oxygenases. Enzymes of the IsdG‐family form tertiary structures distinct from those of the canonical haem oxygenase family, suggesting that IsdG‐family members degrade haem via a unique reaction mechanism. Herein we report that the IsdG‐family of haem oxygenases degrade haem to the oxo‐bilirubin chromophore staphylobilin. We also present the crystal structure of haem‐bound IsdI in which haem ruffling and constrained binding of oxygen is consistent with cleavage of the porphyrin ring at the β‐ or δ‐meso carbons. Combined, these data establish that the IsdG‐family of haem oxygenases degrades haem to a novel chromophore distinct from biliverdin.     

Classification of DNA sequences based on thermal melting profiles

A new classification scheme based on the melting profile of DNA sequences simulated thermal melting profiles. This method was applied in the classification of (a) several species of mammalian - β globin and (b) α-chain class II MHC genes. Comparison of the thermal melting profile with the molecular phylogenetic trees constructed using the sequences shows that the melting temperature based approach is able to reproduce most of the major features of the sequence based evolutionary tree. Melting profile method takes into account the inherent structure and dynamics of the DNA molecule, does not require sequence alignment prior to tree construction, and provides a means to verify the results experimentally. Therefore our results show that melting profile based classification of DNA sequences could be a useful tool for sequence analysis.     

Distribution of the scaffolding proteins PSD-95, PSD-93, and SAP97 in isolated PSDs

We compared the distribution of three scaffolding proteins, all belonging to a family of membrane-associated guanylate kinases, thought to have key roles in the organization of the postsynaptic density (PSD). Isolated PSDs readily adhered to treated glass coverslips where they were labeled with immunogold and rotary shadowed for analysis by EM. The distribution of proteins within individual PSDs were measured by counting and mapping individual immunogold particles. PSD-95, as previously described, is distributed evenly throughout the PSD. We find here that PSD-93 has a nearly identical distribution suggesting that PSD-95 and PSD-93 could perform similar roles. SAP97, in contrast, is concentrated near edges of cleft sides of the PSDs, and in small clumps on their cytoplasmic sides. The homogenous distribution of PSD-95 and PSD-93 throughout the PSD is consistent with their being part of a backbone that stabilizes their various binding partners within the PSD. The distribution of SAP97 confirms that this protein is actually an integral component of the PSD, and suggests that it may have a role in inserting or stabilizing its main binding partner, Glu-R1, at the edge of the PSD.