Diabetic ulcers: The effect of thermal biofeedback-mediated relaxation training on healing

The present study was a preliminary investigation into the effect of thermal biofeedback-mediated relaxation training on healing of diabetic ulcers. Subjects were three males who had nonhealing diabetic ulcers of the toe, ankle, and leg, respectively. Treatment consisted of thermal biofeedback training at the ulcer site, handwarming training (in two of the three subjects), and home relaxation practice. Two subjects showed significant healing. Thermal biofeedback-mediated relaxation training may be a useful adjunct to traditional treatments for diabetic foot ulcers.The authors wish to acknowledge the assistance of Drs. Naomi Alazraki, Kathryn Morton, Louis Larsen, and Mr. Barry Burkinshaw of the Salt Lake VA Medical Center, without whom this study could not have been performed. The first author would also like to thank Dr. Edward Taub for his invaluable advice and support.     

Correlation of the appearance of morphinan alkaloids and laticifer cells in germinating Papaver bracteatum seedlings

The course of alkaloid accumulation and laticifer cell appearance was compared in germinating P. bracteatum seedlings. Seedlings of various ages (0–14 days old) were analyzed for their dopamine, thebaine, morphinan alkaloid immunoreactivity, and benzophenanthridine alkaloid levels. Simultaneous electron microscopic studies revealed that seedlings were devoid of laticifer initials until day 3, where-upon their numbers increased with time. The appearance of appreciable amounts of thebaine only occurred after day 4 of germination. Conversely, dopamine was rapidly formed at the onset of germination and reached millimolar concentrations well before laticifer cells were detected. Benzophenanthridine alkaloid levels remained fairly constant over the period analyzed. These results support the theory that the presence of laticifer cells is necessary for the accumulation of morphinan but neither benzophenanthridine alkaloids nor their mutual precursor, dopamine.Abbreviations RIA radioimmunoassay     

Age-Dependent Changes in the Subcellular Distribution of Rat Brain μ-Opioid Receptors and GTP Binding Regulatory Proteins

The relative subcellular distributions of mu-opioid receptors and guanine nucleotide binding regulatory proteins (G proteins) in 1-day-old (P1) and adult rat forebrain were compared. Light membranes (LMs) were resolved from heavy membranes (HM) by sucrose density gradient centrifugation. Marker enzyme analyses indicated that LMs contained most of the endoplasmic reticulum and Golgi complexes, whereas HMs were enriched in plasma membranes. Binding distribution and properties of mu-opioid sites were assessed using [3H] [D-Ala2,Me-Phe4,Gly-ol5]enkephalin. P1 LMs possessed 43% of the total mu-opioid binding detected compared to 16% in the adult. Although NaCl inhibited mu binding in LMs to a greater extent than in HMs, age-dependent differences were not observed. P1 LM mu binding possessed greater sensitivity to 5'-guanylylimidodiphosphate than their adult counterpart. Moreover, P1 LMs contained more Go alpha protein than P1 HMs or adult LMs, as demonstrated by immunoblotting with antisera against Go alpha after one- or two-dimensional gel electrophoresis. These results suggest that P1 LMs contain a greater proportion of newly synthesized intracellular mu sites than adult LMs.     

Agrobacterium-mediated transformation of almond leaf pieces

A protocol for Agrobacterium-mediated transformation of leaf pieces from two almond cultivars has been developed. This protocol gave a transformation percentage of 48.6 for the cultivar MN51 and 27.9 for the cultivar Supernova. The Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector plasmid pBinGUSint has been used. A critical step for optimal transformation was the pre-culture period of leaf pieces prior selection on medium containing kanamycin. Both cultivars showed an almost doubled transformation percentage when the selection was started at day 6 compared to day 0. The presence of neomycin phosphotransferase II and ß-glucuronidase genes carried by pBinGUSint was confirmed in transformed expiants both by Southern blotting analysis and enzyme activity assays.Abbreviations CaMV cauliflower mosaic virus - GUS ß-glucuronidase - NPTII neomycin phosphotransferase II - -NAA -naphthaleneacetic acid - BAP benzylaminopurine - IAA indole-3-acetic acid - Kin kinetin - kn kanamycin - cx cefotaxime - cb carbenicillin - Tris trishydroxymethylaminomethane - PVPP insoluble polyvinylpyrrolidone-40 - X-gluc 5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecil sulfate - SSC sodium chloride (3 M) sodium citrate (0.3 M)     

EMG scanning norms: Caveat emptor

Questions are raised as to the appropriateness of applying static normative data to EMG muscle scanning and whether currently available normative data meet standards prescribed by the principles of measurement. Potential sources of systematic measurement error emanating from hardware differences, the practitioner, and the standardization sample are also discussed. An alternative to the fixed normative approach is presented. Specifically it is suggested that raw EMG data values be scaled using a nonlinear data transformation which would result in a derived standard scan score scale. This would also allow for intermuscular and interindividual comparisons, which are not appropriate with the currently used fixed normative approach. Further, the alternative approach adheres to the principle that EMG data must be interpreted relatively and not absolutely. It is argued that this alternative would eliminate systematic error problems with regard to hardware differentials and preclude the necessity of, and the problems endemic to, norms development. Finally there is an explanation on how one could develop a muscle scanning profile similar to that in current use by many personality inventories. The profiles could then be used as normative.     

Human Glioblastoma Multiforme: p53 Reactivation by a Novel MDM2 Inhibitor

Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients.     

Developmental Aspects of Detoxifying Enzymes in Fish (Salmo Iridaeus)

The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism.

A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development.

In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney.

No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.     

Cysteine 10 is critical for the activity of Ochrobactrum anthropi glutathione transferase and its mutation to alanine causes the preferential binding of glutathione to the H-site

The role of the evolutionarily conserved residue Cys10 in Ochrobactrum anthropi glutathione transferase (OaGST) has been examined by replacing it with an alanine. A double mutant C10A/S11A was also prepared. The effect of the replacements on the coniugating and thiotransferase activities, and on the thermal and chemical stability of the enzyme was analyzed. Our data support the view that in OaGST, in contrast with other beta class GSTs that display significant differences in the glutathione-binding site, Cys10 is a key residue for glutathione coniugating activity. Furthermore, analysis of the OaGST-Cys10Ala structure, crystallized in the presence of glutathione, reveals that this mutation causes a switch between the high-affinity G-site and a low-affinity H-site where hydrophobic cosubstrates bind and where we observe the presence of an unexpected glutathione.