Targeted effect of ischemic preconditioning on the gas exchange threshold in healthy males and females.

Ischemic preconditioning (IPC) appears to improve exercise performance although there is uncertainty about the intensity dependence of this effect. The present study sought to clarify effects of IPC on physiological responses at and below peak oxygen uptake, including the gas exchange threshold (GET). Ten male and female participants completed five cycling ramp tests (10 W/min) to failure, with the final two tests preceded by either IPC (4 × 5 min 220 mmHg bilateral leg occlusions) or SHAM (20 mmHg), in a randomised crossover design. The rates of O2 uptake ( V ˙ O2), carbon dioxide output ( V ˙ CO2), and expired ventilation ( V ˙ E) were measured at rest and throughout exercise. Exercise data were fitted using several functions to identify GET, two ventilatory thresholds and peak V ˙ O2. IPC increased V ˙ O2 at GET by ~ 9% (IPC: 1.89 ± 0.51 L/min, SHAM: 1.73 ± 0.56 L/min; p = 0.055) and power output at GET by ~ 11% (IPC: 133 ± 36 W, SHAM: 120 ± 39 W; p = 0.022). In addition, peak power output increased by 2.4% following IPC (IPC: 217 ± 50 W, SHAM: 212 ± 51 W; p = 0.052), but there was no significant effect of IPC on peak V ˙ O2 (IPC: 2.87 ± 0.68 L/min, SHAM: 2.84 ± 0.73 L/min; p = 0.60) or the ventilatory thresholds. The present results suggest that IPC improves GET and peak power output but not peak V ˙ O2 during a maximal graded test.

Craniofacial Bones and Teeth in Spacefarers: Systematic Review and Meta-analysis.

INTRODUCTION: Estimating the risk of dental problems in long-duration space missions to the Moon and Mars is critical for avoiding dental emergencies in an environment that does not support proper treatment. Previous risk estimates were constructed based on the experience in short-duration space missions and isolated environments on Earth. However, previous estimates did not account for potential changes in dental structures due to space travel, even though bone loss is a known problem for long-duration spaceflights. The objective of this study was to systematically analyze the changes in hard tissues of the craniofacial complex during spaceflights. METHODS: Comprehensive search of Medline, Embase, Scopus, the NASA Technical Report Server, and other sources identified 1,585 potentially relevant studies. After screening, 32 articles that presented quantitative data for skull in humans (6/32) and for calvariae, mandible, and lower incisors in rats (20/32) and mice (6/32) were selected. RESULTS: Skull bone mineral density showed a significant increase in spacefaring humans. In spacefaring rodents, calvariae bone volume to tissue volume (BV/TV) demonstrated a trend toward increasing that did not reach statistical significance, while in mandibles, there was a significant decrease in BV/TV. Dentin thickness and incisor volume of rodent incisors were not significantly different between spaceflight and ground controls. DISCUSSION: Our study demonstrates significant knowledge gaps regarding many structures of the craniofacial complex such as the maxilla, molar, premolar, and canine teeth, as well as small sample sizes for the studies of mandible and incisors. Understanding the effects of microgravity on craniofacial structures is important for estimating risks during long-duration spaceflight and for formulating proper protocols to prevent dental emergencies. KNOWLEDGE TRANSFER STATEMENT: Avoiding dental emergencies in long-duration spaceflights is critical since this environment does not support proper treatment. Prior risk estimates did not account for changes in dental structures due to space travel. We reviewed and synthesized the literature for changes in craniofacial complex associated with spaceflight. The results of our study will help clinicians and scientists to better prepare to mitigate potential oral health issues in space travelers on long-duration missions.

HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells.

Prior experiments in explants of human lymphoid tissue have demonstrated that human immunodeficiency virus type 1 (HIV-1) productively infects diverse cellular targets including T cells and tissue macrophages. We sought to determine the specific contribution of macrophages and T cells to the overall viral burden within lymphoid tissue. To block infection of macrophages selectively while preserving infection of T cells, we used viruses deficient for viral protein R (Vpr) that exhibit profound replication defects in nondividing cells in vitro. We inoculated tonsil histocultures with matched pairs of congenic viruses that differed only by the presence of a wild-type or truncated vpr gene. Although these viruses exhibited no reduction in the infection or depletion of T cells, the ability of the Vpr-deficient R5 virus to infect tissue macrophages was severely impaired compared with matched wild-type R5 virus. Interestingly, the Vpr-deficient R5 virus also exhibited a 50% reduction in overall virus replication compared with its wild-type counterpart despite the fact that macrophages represent a small fraction of the potential targets of HIV-1 infection in these tissues. Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1.

Specific protein synthesis in cellular differentiation. Production of eggshell proteins by silkmoth follicular cells.

Silkmoth follicles, arranged in a precise developmental sequence within the ovariole, yield pure and uniform populations of follicular epithelial cells highly differentiated for synthesis of the proteinaceous eggshell (chorion). These cells can be maintained and labeled efficiently in organ culture; their in vitro (and cell free) protein synthetic activity reflects their activity in vivo. During differentiation the cells undergo dramatic changes in protein synthesis. For 2 days the cells are devoted almost exclusively to production of distinctive chorion proteins of low molecular weight and of unusual amino acid composition. Each protein has its own characteristic developmental kinetics of synthesis. Each is synthesized as a separate polypeptide, apparently on monocistronic messenger RNA (mRNA), and thus reflects the expression of a distinct gene. The rapid changes in this tissue do not result from corresponding changes in translational efficiency. Thus, the peptide chain elongation rate is comparable for chorion and for proteins synthesized at earlier developmental stages (1.3-1.9 amino acids/sec); moreover, the spacing of ribosomes on chorion mRNA (30-37 codons per ribosome) is similar to that encountered in other eukaryotic systems.

Separation of transit of auxin from uptake: average velocity and reversible inhibition by anaerobic conditions.

An applied pulse of (14)C-indole-3-acetic acid moved down aerobic corn coleoptiles at about 15 millimeters per hour. When sections with a moving pulse were transferred to nitrogen, the rate fell below 2 millimeters per hour. This inhibition was completely reversible; sections returned to air moved the same amount of auxin as untreated aerobic controls.

Early signal transduction by the antigen receptor without commitment to T cell activation.

The T lymphocyte antigen-receptor complex mediates antigen-specific cell activation, at least in part, through the production of inositolphospholipid-derived second messengers. Little is known about how second messenger events, typically measured within minutes of ligand binding, eventually lead to distal biologic responses such as expression of lymphokine genes. Several monoclonal antibodies directed against the receptor complex were tested for their ability to elicit transmembrane signaling in the parental Jurkat line and in a somatic mutant (J.CaM1) with a deficient receptor function. One antibody elicited substantial early Ca2+ mobilization responses in both cells but was unable to promote expression of the interleukin-2 gene in J.CaM1. In J.CaM1 there was a diminished production of phosphatidylinositol second messengers, and the elevation in intracellular free Ca2+ was transient. Thus, short-term Ca2+ mobilization does not always indicate complete signal transmission and lead to a full cellular response.

Drug discrimination learning in lead-exposed rats.

Lead acetate (0.02 or 0.5 percent) was administered to dams throughout the lactation period with half of the litters continuing on lead after weaning. Drug thresholds for d-amphetamine were determined by using the drug-discrimination learning paradigm. All the offspring that had been exposed to lead were less sensitive to the stimulus properties of d-amphetamine irrespective of whether or not they had continued on lead after weaning.

Field focusing nuclear magnetic resonance (FONAR): visualization of a tumor in a live animal.

A nuclear magnetic resonance (NMR) image of a tumor in a live animal is reported. The field focusing NMR method or FONAR process that now achieves the tumor outline is described.

Multiple extracellular elements of CCR5 and HIV-1 entry: dissociation from response to chemokines.

The human beta-chemokine receptor CCR5 is an important cofactor for entry of human immunodeficiency virus-type 1 (HIV-1). The murine form of CCR5, despite its 82 percent identity to the human form, was not functional as an HIV-1 coreceptor. HIV-1 entry function could be reconstituted by fusion of various individual elements derived from the extracellular region of human CCR5 onto murine CCR5. Analysis of chimeras containing elements from human CCR5 and human CCR2B suggested that a complex structure rather than single contact sites is responsible for facilitation of viral entry. Further, certain chimeras lacking the domains necessary to signal in response to their natural chemokine ligands retained vigorous HIV-1 coreceptor activity.

Janus kinases in interleukin-2-mediated signaling: JAK1 and JAK3 are differentially regulated by tyrosine phosphorylation.

BACKGROUND: Cytokines mediate a variety of effector cell functions, including cellular proliferation, differentiation, and modulation of the immune response. Many cytokines activate receptor-associated Janus kinases (JAKs) that promote tyrosine phosphorylation of signal transducers and activators of transcription (STAT) factors. Although JAK activation has been correlated with phosphorylation, the role of this tyrosine phosphorylation in the regulation of JAK1 and JAK3 remains unclear. Furthermore, the relative roles of JAK1 and JAK3 in the activation of STAT5 by interleukin-2 (IL-2) remain poorly understood. RESULTS: We targeted two conserved tyrosine residues within the activation loop of the JAK1 and JAK3 kinase domains for substitution with phenylalanines. In an overexpression system, the catalytic function of JAK1 strictly required the presence of the first of these tyrosines, Y1033. In contrast, JAK3 retained catalytic activity when either or both of these activation-loop tyrosines were mutated. Analysis of JAK1/3 chimeras demonstrated that JAK activity was also controlled by intramolecular interactions involving the amino-terminal domain of the JAK as well as by the inherent signaling properties of the kinase domain. Finally, we have reconstituted IL-2-dependent STAT5 induction in a cell line that lacks detectable expression of JAK1 and JAK3. Catalytically active versions of both JAK1 and JAK3 must be present for effective induction of STAT5. CONCLUSIONS: JAK1 and JAK3 are differentially regulated by specific tyrosines within their respective activation loops. Additionally, the amino-terminal domain of JAK3 appears to contain regulatory sequences that modify the function of the kinase domain. Finally, both JAK1 and JAK3 must retain catalytic function for IL-2-induced STAT5 activation.

A trans-receptor mechanism for infection of CD4-negative cells by human immunodeficiency virus type 1.

Chemokine receptors, particularly CCR5 and CXCR4, act as essential coreceptors in concert with CD4 for cellular entry by human immunodeficiency virus type 1 (HIV-1; reviewed in [1]). But infection of CD4(-) cells has also been encountered in various tissues in vivo, including astrocytes, neurons and microvascular endothelial cells of the brain [2] [3] [4] [5] [6], epithelial cells [5] [7], CD4(-) lymphocytes and thymocytes [8] [9], and cardiomyocytes [10]. Here, we present evidence for the infection of CD4(-) cell lines bearing coreceptors by well-known HIV-1 strains when co-cultured with CD4(+) cells. This process requires contact between the coreceptor-bearing and CD4(+) cells and supports the full viral replication cycle within the coreceptor-bearing target cell. Furthermore, CD4 provided in trans facilitates infection of primary human cells, such as brain-derived astrocytes. Although the pathobiological significance of infection of CD4(-) cells in vivo remains to be elucidated, this trans-receptor mechanism may facilitate generation of hidden reservoirs of latent virus that confound antiviral therapies and that contribute to specific AIDS-associated clinical syndromes.

Parallel evolution of CCR5-null phenotypes in humans and in a natural host of simian immunodeficiency virus.

The C-C chemokine receptor CCR5 in humans and rhesus macaques (Macaca mulatta) serves as the primary coreceptor for cellular entry by macrophagetropic strains of human immunodeficiency virus type 1 (HIV-1) and all reported strains of simian immunodeficiency virus (SIV) [1-6]. Humans homozygous for a 32 bp deletion allele of CCR5, resulting in a null phenotype, are highly resistant to infection by HIV-1 [7-9], prompting development of therapies and vaccines targeting CCR5. We now report a novel deletion allele of CCR5, with an allele frequency of 0.04, in sooty mangabey monkeys (Cercocebus torquatus atys), a natural host of SIV (SIVsmm) [10]. The mutant protein was not expressed at the cell surface and accordingly did not function as a viral coreceptor. Primary activated lymphocytes from mangabeys heterozygous for the deletion allele expressed significantly less CCR5 on the cell surface. Moreover, SIV seroprevalence and viremia were comparable among CCR5 heterozygotes and wild-type animals. Parallel evolution of CCR5-null alleles in humans and sooty mangabeys suggests that similar negative selection pressures have acted against CCR5, as would occur during epidemics of infectious agents that require CCR5 for pathogenesis. Sooty mangabeys bred to homozygosity for the deletion allele will be useful for experimental studies on the context-dependent role of CCR5 in host defense and microbial pathogenesis.

Shared gamma(c) subunit within the human interleukin-7 receptor complex. A molecular basis for the pathogenesis of X-linked severe combined immunodeficiency.

Genetic evidence suggests that mutations in the gamma(c) receptor subunit cause X-linked severe combined immunodeficiency (X-SCID). The gamma(c) subunit can be employed in receptor complexes for IL-2, -4, -7, -9, and -15, and the multiple signaling defects that would result from a defective gamma(c) chain in these receptors are proposed to cause the severe phenotype of X-SCID patients. Interestingly, gene disruption of either IL-7 or the IL-7 receptor (IL-7R) alpha subunit in mice leads to immunological defects that are similar to human X-SCID. These observations suggest the functional importance of gamma(c) in the IL-7R complex. In the present study, structure/function analyses of the IL-7R complex using a chimeric receptor system demonstrated that gamma(c) is indeed critical for IL-7R function. Nonetheless, only a limited portion of the cytoplasmic domain of gamma(c) is necessary for IL-7R signal transduction. Furthermore, replacement of the gamma(c) cytoplasmic domain by a severely truncated erythropoeitin receptor does not affect measured IL-7R signaling events. These findings support a model in which gamma(c) serves primarily to activate signal transduction by the IL-7R complex, while IL-7R alpha determines specific signaling events through its association with cytoplasmic signaling molecules. Finally, these studies are consistent with the hypothesis that the molecular pathogenesis of X-SCID is due primarily to gamma(c)-mediated defects in the IL-7/IL-7R system.

Abnormal polyamine metabolism in hereditary muscular dystrophies: effect of human growth hormone.

Previous studies showed hyperre-sponsiveness to human growth hormone (hGH) in men with myotonic or limb girdle dystrophies (MMD or LGD). Because polyamines may mediate some actions of hGH, we have now investigated polyamine metabolism in these and other dystrophies. Under metabolic balance study conditions, serum and urine levels of putrescine (Pu), spermidine (Sd), spermine (Sm), and cadaverine (Cd) were measured in six normal men (36-44 yr), four men with MMD (38-44 yr), and three men with LGD (30-36 yr), before and during treatment with 0.532 U/kg body wt ((3/4)/d) of hGH. Daily balances of N, P, and K were also monitored. In the normal subjects, hGH did not influence elemental balances or serum and urine polyamines. In MMD, hGH caused significant retention of N, P, and K (P < 0.005). Basal levels of Sm and Cd were significantly elevated above normal (P < 0.005), and Pu, Sm, and Cd increased two- to fourfold above basal during hGH treatment (P < 0.005). In LGD, hGH also caused retention of N, P, and K. Basal levels of nearly all the polyamines (not serum Pu) were significantly above normal in serum and urine (P < 0.05). During hGH treatment, all four polyamines rose significantly above basal (P < 0.005). Serum and urine polyamine levels in five boys with Duchenne muscular dystrophy, age 8-13, did not differ from those in five age-matched normal boys. Skeletal muscle polyamines were measured in five men (31-40 yr) without muscle disease and in three men with LGD (30-38 yr). Average concentrations of Pu, Sd, Sm, and Cd were 46, 306, 548, and 61 nmol/g wet wt in LGD and 1, 121, 245, and 14 in the normal subjects, respectively (P < 0.05 in each instance). Polyamines were determined in skeletal muscle, liver, kidney, and brain of male mice with hereditary muscular dystrophy and in age- and sex-matched normal controls. Pu, Sd, Sm, and Cd levels were two to three times higher than normal in muscle, but did not differ in liver, kidney, and brain. Similar findings were made in male hamsters with hereditary dystrophy and in their controls. The abnormality in hamster muscle polyamines appeared between 1 and 6 wk of age and persisted or intensified until 30 wk. These data reveal abnormalities of polyamine metabolism in men with MMD, in men with LGD, and in mice or hamsters with hereditary muscular dystrophy. The polyamine disorder could be related to dystrophic patients' hyperresponsiveness to hGH.

Serum and urine polyamines in normal and in short children.

The serum and urine polyamines putrescine, spermidine, and spermine were measured in 112 normal subjects from 0 to 70 yr of age, and in three groups of short children from 7 to 20 yr: 21 growth hormone (GH) deficient patients, 20 normal variant short stature children, and 9 girls with 45, X Turner's syndrome. Urine polyamines were expressed as micromoles per gram of creatinine or per kilogram body weight, and serum polyamines were expressed as nanomoles per milliliter. In normals, the three polyamines were highest in urine and serum at birth. The mean levels declined progressively with age, the rate of change decreasing with age. The mean for the normal subjects, and its 95% confidence and prediction intervals, were estimated from birth to age 70 for each serum and urine polyamine. In GH-deficient children, serum and urine values were significantly lower (P < 0.05) than the age-specific normal values (with the exception of serum spermidine and spermine), averaging 25-55% below normal. This abnormality was corrected during 1 wk of treatment with human GH. In Turner's syndrome, serum and urine values were significantly reduced (P < 0.05), averaging 35-80% below age-specific normals. GH treatment had no corrective effect. In 6 of 20 normal variant short stature children, polyamine levels were significantly (P < 0.01) subnormal, averaging 50-80% below age-specific normals in both serum and urine. Treatment with GH had no corrective effect. These data show that levels of polyamines in serum and urine are correlated with linear growth primarily during the first decade of life. Subnormal polyamine levels are generally associated with growth retardation.

JAK/STAT signaling by cytokine receptors.

The JAK/STAT pathway is recognized as one of the major mechanisms by which cytokine receptors transduce intracellular signals. This system is regulated at multiple levels, including JAK activation, nuclear trafficking of STAT factors, and negative feedback loops. Gene deletion studies have implicated selected STAT factors as predominant mediators for a limited number of lymphokines. This signaling pathway influences normal cell survival and growth mechanisms and may contribute to oncogenic transformation.

5' Nucleotide sequences influence serum-modulated expression of a human dihydrofolate reductase minigene.

Human dihydrofolate reductase (DHFR) gene sequences were isolated from DHFR gene-amplified breast cancer cell line MCF-7. These genomic sequences plus human DHFR cDNA sequences were used to construct a DHFR minigene. Calcium phosphate-mediated transfer of minigene DNA into DHFR gene-deleted Chinese hamster ovary cells converted these cells to a DHFR+ phenotype at a frequency of 0.12%. Minigene-transfected cells contained 20 to 30 minigene copies per cell and had DHFR enzyme levels similar to those of wild-type MCF-7 human cells (1.4 pmol/mg of protein). In contrast to gene-amplified MCF-7 cells, which contained multiple DHFR mRNA species (1.1, 1.6, 3.8, and 5.3 kilobases), only a single 3.8-kilobase DHFR mRNA was found in minigene-transfected cells. Previous studies on normal cells demonstrated modulation of DHFR levels by a variety of conditions which altered cell growth. When cell growth was induced in minigene-transfected cells by release from serum deprivation and DHFR levels were assayed at the time of maximum DNA synthesis, these levels were increased 2.4 to 3.7-fold. In contrast, the DHFR levels in cells transfected with a construct made from DHFR cDNA and viral promoter, intron, and termination sequences were unchanged. Minigene deletions were made and analyzed to determine the DHFR gene sequences responsible for regulation. Deletion of sequences upstream from 322 base pairs 5' to the start of transcription or 90 base pairs downstream from the termination of translation (which removed most of the 3' nontranslated region of the gene) did not alter the responsiveness of minigene-transfected cells to serum deprivation. However, when sequences between 322 and 113 base pairs 5' to the start of transcription were deleted, serum-dependent expression in minigene-transfected cells was affected.

Quantitative prediction of drug toxicity in humans from toxicology in small and large animals.

The mouse, dog, and monkey toxicity data on 30 drugs was retrospectively analyzed in comparison with the actual clinical dose schedules used in man. Animal dose schedules were converted to the human schedule and comparisons were made of the human dose versus the large animal toxic dose low, toxic dose high, and lethal dose, the lethal doses for 10% and 90% of normal mice, and the optimal dose in tumor-bearing mice. If the starting dose in Phase 1 clinical trials had been selected by calculating one-third of the toxic dose low (in mg/sq m) in the most sensitive large animal species, 5 of the 30 drugs would have produced significant toxicity in the first patient. The lethal doses for 10 and 90% of normal mice and the optimal dose in L1210-bearing mice were found to offer good quantitative prediction of human toxicity. Determination of a safe and practical starting dose for Phase 1 studies should take into account not only dog and monkey data but also toxicology data in normal and tumor-bearing mice.

Enhanced adenovirus transgene expression in malignant cells treated with the histone deacetylase inhibitor FR901228.

The presence of coxsackie and adenovirus receptor (CAR) and alpha(v) integrin on cell surfaces is required for efficient adenovirus infection. Treatment of cells with the histone deacetylase inhibitor FR901228 (depsipeptide) increased CAR and alpha(v) integrin RNA levels in six cancer cell lines. Sodium butyrate and trichostatin A, other histone deacetylase inhibitors, caused similar increases. Cells treated with FR901228 prior to infection had a 4-10-fold increase in transgene expression from a beta-galactosidase-expressing adenoviral vector. These studies suggest that FR901228 increases the efficiency of adenoviral transgene expression and may be useful in cancer gene therapy.

Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human alpha class glutathione S-transferase gene.

Studies have suggested that the alpha class glutathione S-transferase (GST) may protect cells from the chemotherapeutic drugs chlorambucil and melphalan. In order to further define the function of human alpha class GST, a complementary DNA which encodes it was ligated into an expression vector under the direction of the human metallothionein-IIA promoter and stably transfected into human MCF-7 breast cancer cells in conjunction with the G418-selectable plasmid pSV2neo. Clonal cell lines were identified which expressed increased levels of GST enzyme activity (2.2- to 5.6-fold). The transfected cell lines also had increased peroxidase activity using cumene hydroperoxide as the substrate (1.9- to 3.8-fold) which is consistent with the intrinsic peroxidase activity of alpha class GSTs. Southern blot analysis indicated that genomic DNA from these cells contained a fragment indistinguishable from the transfected alpha class GST complementary DNA (850 base pairs); Northern blot analysis of total cellular RNA indicated that these cells contained appropriately sized alpha class GST RNA (980 nucleotides); and Western blot analysis indicated that, while MCF-7 cells contained no detectable alpha class GST protein, the transfected cells contained markedly elevated levels of alpha class GST but no detectable mu or pi class GST. These alpha class GST transfected cells had increased resistance to ethacrynic acid (2.1- to 3.0-fold). However, the transfected cells failed to show any increased resistance measured at the drug dosage which inhibited 50% of the colony formation to the chemotherapeutic drugs chlorambucil, melphalan, Adriamycin, or cisplatin under conditions of either continuous or 1-h drug exposure. Neither was there any change in sensitivity to the cytotoxins benzo(a)pyrene, benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti), or 1-chloro-2,4-dinitrobenzene. These studies indicate that expression of this human alpha class GST by itself in MCF-7 human breast cancer cells does not confer resistance to the chemotherapeutic drugs tested under the conditions used in these studies.