Hypoxia and fatigue-induced modification of function and proteins in intact and skinned murine diaphragm muscle

Fatigue studies of isolated, intact muscles typically utilize solutions saturated with O2. However, under in vivo fatiguing conditions, less oxygen is delivered to the muscles and they actually experience hypoxia. No studies to date have correlated the effects of acute hypoxia on the isometric contractile properties of intact muscles, skinned fibers isolated from the same muscles, and the cellular content of specific muscle proteins. Therefore, we have studied the effects of in vitro acute hypoxia on the fatigability of intact diaphragm muscle strips and on the isometric contractile properties of single Triton-skinned fibers isolated from control and hypoxic diaphragm muscles. We found that hypoxia and fatiguing stimulation per se affect the tetanic force of intact muscle strips without exhibiting any significant deleterious effects on the calcium-activated force of skinned muscle fibers dissected from the intact muscles. In contrast, fatiguing stimulation under hypoxic conditions decreased both the tetanic force of muscle strips and the calcium-activated force of skinned muscle fibers. Gel electrophoresis of muscles subjected to hypoxia and hypoxic-fatigue revealed that there is a significant reduction in three protein bands when compared to control muscles. Protein modification may be the underlying mechanism of muscle fatigue under physiologic conditions.     

Intranasal Tat Alters Gene Expression in the Mouse Brain

Intranasal (IN) delivery of HIV-1 Tat in aging mice was investigated as a possible model for HIV-1 infection in the brain. After IN administration, the distribution of [¹²⁵I]-labeled Tat in the brains of Swiss Webster mice was evaluated by autoradiography and gamma counting. [¹²⁵I]-labeled Tat was detected at the highest concentrations in the olfactory bulb, cervical nodes, and trigeminal nerve tract. In another experiment, APPSw transgenic mice were used to model chronic Tat exposure. The mice were treated intranasally with 6 μg Tat (n = 4) or vehicle (n = 4) three times per week for 4 weeks. Total RNA was isolated from the frontal cortex, and differential gene expression analysis was performed using gene microarrays. Gene ontology profiles indicated innate immunity, inflammatory and apoptotic responses. Five genes of interest in the Tat-treated mice that were significantly elevated in the microarrays were validated by RT-PCR. One gene, the Toll-like receptor 9 (Tlr9), has previously been shown to activate signaling cascades leading to innate immunity and enhanced HIV-1 gene expression. A second gene, Fas, plays a key role in neuroinflammation. Two cysteine-rich cytokines associated with chemotaxis were elevated: MCP-1 (Ccl2), which is chemotactic for monocytes, and Ccl17 (TARC), which is chemotactic for lymphocytes. Finally, the gene sestrin was significantly elevated and has been associated with oxidative stress, in particular amyloid beta-induced oxidative stress. This IN Tat model of neuroinflammation may be useful to study HIV-1-induced neurodegeneration. This paper was presented at an NIMH workshop “HIV Preclinical–Clinical Therapeutics Research Meeting”, May 5–16, 2006.     

Protective effects of Hibiscus tiliaceus L. methanolic extract to V79 cells against cytotoxicity and genotoxicity induced by hydrogen peroxide and tert-butyl-hydroperoxide

Plants of the genus Hibiscus thrives produce a diversity of molecules with bioactive properties. In a previous study of Hibiscus tiliaceus L. methanolic extract (HME) using bacteria and yeast, as test media, it has been shown that HME strongly inhibited the mutagenic action of H₂O₂ or tert-butyl-hydroperoxide (t-BHP). Here, our interest is to evaluate the genotoxicity and the antigenotoxic/antimutagenic properties of HME using oxidative challenge with H₂O₂ and t-BHP in V79 cells. We determined cytotoxicity using clonal survival assay; evaluated DNA damage using the comet assay and the micronucleus test in binucleated cells besides of the lipid peroxidation degree and the reduced glutathione content. We examined the ability of HME in quenching hydroxyl radical by means of a HPLC-based method utilizing the hypoxanthine/xanthine oxidase assay. At concentrations ranging from 0.001 to 0.1 mg/mL, HME was not cytotoxic, genotoxic or mutagenic. Treatment with non-cytotoxic concentrations of HME increased cell survival after H₂O₂ and t-BHP exposure and prevented DNA damage. The pre-treatment with HME also was able to decrease the mutagenic effect of these genotoxins, evaluated using the micronucleus test. HME prevented the increase in lipid peroxidation and decrease in GSH content in response to the oxidative challenge. Therefore, the ability in preventing against H₂O₂- and t-BHP-induced GSH depletion and lipid peroxidation was probably a major contribution to the cytoprotective effects. Moreover, HME acts as a hydroxyl radical scavenger. In summary, HME did not have a harmful or inhibitory effect on the growth of V79 cells and presented antioxidant activity, consequently, both antigenotoxic and antimutagenic effects against oxidative DNA damage.     

Identification of a Novel Receptor Tyrosine Kinase Expressed in Acute Myeloid Leukemic Blasts

Using the polymerase chain reaction with degenerate oligonucleotides derived from conserved motifs within the catalytic kinase domain of protein tyrosine kinases, and RNA extracted from embryonic stem cells, sequences that encode a segment of the kinase domain of several potentially novel receptor tyrosine kinases (RTKs) have been identified. One of these was selected for further study because in Northern analysis it hybridized to RNA from multipotential hematopoietic cell lines, but not from lines representative of lineage-committed cells. A cDNA for this receptor, designated developmental tyrosine kinase (DTK), was isolated and encodes a protein with structural similarities to AXL. Together these receptors form a new class of RTK. DTK is expressed in a number of human leukemic cell lines, and in the blasts of 6 of 11 patients with acute myeloid leukemia (AML) analyzed. The structure of DTK suggests that it may function as a cell adhesion molecule, and mediate cell-to-cell or cell-matrix interactions between hematopoietic cells and their respective microenvironments.     

Impaired communication between nurses and patients' family members

This study aims at presenting the analysis of an interaction between a nurse and a patient's family member in which impaired communication was observed. The interpersonal-relationship theoretical framework was used. The patient was young, 20 years old, bore a dead fetus and presented various complications. The interaction took place with her aunt (stepmother) and as to the structure, it was diagnostic, therapeutic and made it possible to establish a proposal of continuous assistance. As to content, it was possible to find the points of support given by family members and identify new facts so that the nursing team could improve the assistance given to the patient.     

Waiver of consent in studies of acute brain injury

A multicenter trial of hypothermia in patients with acute brain injury, designed to accrue 140 patients per year and randomizing in less than 6 h from injury, enrolled 392 patients. The design was to achieve 33 degrees C within 8 h after injury. For the first 9 months of the trial, the only consent mechanism permitted by federal regulations was prospective, informed consent. In the subsequent 33 months, after a change in federal regulations, waiver of consent could be used when family could not be located. Waiver of consent was used in 62% of patients enrolled. In the first 9 months of the trial, accrual was 65 patients. In the subsequent 3 years, an average yearly accrual was 127 patients. In the first 9 months, time from injury to randomization was 4.5 +/- 1.2 h; time to achievement of target temperature was 11.7 +/- 2.6 h. In years when waiver of consent was permitted, randomization time was 4.1 +/- 1.1 h, and time to target temperature was 7.9 +/- 2.7 h. For all years of the study, waiver of consent was used for 53% of minorities, 47% of unskilled workers, 33% of nonminorities, and 29% of skilled or professional workers. Minorities were underrepresented by 30% in the first 9 months of the study. We conclude that it is impracticable and unjust to perform studies of acute brain injury without use of waiver of consent when the treatment window is less than 6 h.     

Phenotype markers and function of neutrophils in children with hemolytic uremic syndrome

The hemolytic uremic syndrome (HUS) is the most-common cause of acute renal failure in children. Several researchers have reported the presence of neutrophil (PMN) activating cytokines, such as interleukin-8 and tumor necrosis factor-α, in the sera of HUS patients. Moreover, PMN-derived products, such as elastase, were increased. These observations have lead to the hypothesis that activated PMN could act as mediators of endothelial damage. The objective of this investigation was to directly evaluate the activation status of peripheral PMN from children with HUS. For this purpose, 12 children with typical HUS were bled during the acute period, before dialysis and/or transfusion, and 8 of them were also bled after 1 month follow-up. Additionally, blood samples from healthy control children admitted for routine surgical procedures, chronic uremic children, and neutrophilic children with acute infections not related to HUS were collected and processed in an identical manner. The function and membrane activation markers of PMN from these groups were evaluated.We found that during the acute period of HUS, PMN had reduced expression of FcγRIII (CD16) and CD11b, were degranulated, and exhibited an impaired antibody-dependent cellular cytotoxicity. These parameters returned to normal after clinical recuperation. We conclude that PMN activation in HUS patients is a very early and transient event, and upon hospitalization before dialysis PMN show a phenotype and functional pattern of partial deactivation. Received: 25 June 2001 / Revised: 16 November 2001 / Accepted: 20 November 2001     

A portable microkeratome-based anterior corneal surface harvesting device

PURPOSE: To determine the reproducibility of anterior sclerokeratectomy using a portable nonelectric microkeratome-based device capable of harvesting the entire anterior corneal surface for lamellar transplantation. METHODS: A modified gas turbine-driven microkeratome (LSK One, Moria/Microtech, Doylestown, PA) with a redesigned head large enough to incorporate the whole human anterior corneal surface in a pass and was coupled to a manual vacuum pump. This instrument was tested on 25 fresh porcine globes divided into 2 groups (170-microm and 200-microm head). To assess cut reproducibility the physical dimensions (diameter and thickness) of the obtained lenticules were measured. RESULTS: The obtained lenticules were fairly circular (horizontal versus vertical diameters, p >0.2), with average diameters of 12.85 +/- 0.52 mm and 13.25 +/- 1.15 mm for the 170 and 200-microm heads, respectively. The average central lenticule thickness was 176.92 +/- 34.68 microm and 166.00 +/- 53.74 microm for the 170 and 200-microm heads, respectively. CONCLUSION: This new system presents an economical and portable alternative to electric-powered systems. In addition to being used by surgeons in the operating room, eye bank technicians in the field could theoretically use this system; including in developing countries where cost, availability of electricity, and portability are issues.     

Differential activation of protein kinase C isoforms by euxanthone,revealed by an in vivo yeast phenotypic assay

The protein kinase C (PKC) modulatory effects of euxanthone, isolated from the wood of Cratoxylum maingayi, on isoforms alpha, betaI, delta, eta and zeta were characterised using an alternative in vivo yeast phenotypic assay. The present study shows that euxanthone can activate isoforms alpha, betaI, delta, eta and zeta, being more effective on PKC-betaI, -delta, -eta and -zeta than the established PKC activators used (the phorbol ester PMA and arachidonic acid for PKC-zeta). Furthermore, euxanthone presents differences on its potency towards individual PKC isoforms, showing a remarkable selectivity for PKC-zeta. These results can help to clarify the molecular basis of the euxanthone-mediated effects.