Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Eccentric exercise induces transient insulin resistance in healthy individuals.

Euglycemic-hyperinsulinemic clamps were performed on six healthy untrained individuals to determine whether exercise that induces muscle damage also results in insulin resistance. Clamps were performed 48 h after bouts of predominantly 1) eccentric exercise [30 min, downhill running, -17% grade, 60 +/- 2% maximal O2 consumption (VO2max)], 2) concentric exercise (30 min, cycle ergometry, 60 +/- 2% VO2max), or 3) without prior exercise. During the clamps, euglycemia was maintained at 90 mg/dl while insulin was infused at 30 mU.m-2.min-1 for 120 min. Hepatic glucose output (HGO) was determined using [6,6-2H]glucose. Eccentric exercise caused marked muscle soreness and significantly elevated creatine kinase levels (273 +/- 73, 92 +/- 27, 87 +/- 25 IU/l for the eccentric, concentric, and control conditions, respectively) 48 h after exercise. Insulin-mediated glucose disposal rate was significantly impaired (P less than 0.05) during the clamp performed after eccentric exercise (3.47 +/- 0.51 mg.kg-1.min-1) compared with the clamps performed after concentric exercise (5.55 +/- 0.94 mg.kg-1.min-1) or control conditions (5.48 +/- 1.0 mg.kg-1.min-1). HGO was not significantly different among conditions (0.77 +/- 0.26, 0.65 +/- 0.27, and 0.66 +/- 0.64 mg.kg-1.min-1 for the eccentric, concentric, and control clamps, respectively). The insulin resistance observed after eccentric exercise could not be attributed to altered plasma cortisol, glucagon, or catecholamine concentrations. Likewise, no differences were observed in serum free fatty acids, glycerol, lactate, beta-hydroxybutyrate, or alanine. These results show that exercise that results in muscle damage, as reflected in muscle soreness and enzyme leakage, is followed by a period of insulin resistance.     

Effects of increased meal frequency on fat oxidation and perceived hunger

Objective:

Consuming smaller, more frequent meals is often advocated as a means of controlling body weight, but studies demonstrating a mechanistic effect of this practice on factors associated with body weight regulation are lacking. The purpose of this study was to compare the effect of consuming three (3M) vs. six meals (6M) per day on 24‐h fat oxidation and subjective ratings of hunger.

Design and Methods:

Lean (body mass index <25 kg/m²) subjects (7M, 8F) were studied in a whole‐room calorimeter on two occasions in a randomized cross‐over design. Subjects were provided isoenergetic, energy balanced diets with a 1‐ to 2‐week washout between conditions. Hunger, fullness, and “desire to eat” ratings were assessed throughout the day using visual analog scales and quantified as area under the curve (AUC).

Results:

There were no differences (P < 0.05) in 24‐h energy expenditure (8.7 ± 0.3 vs. 8.6 ± 0.3 mj d^?1), 24‐h respiratory quotient (0.85 ± 0.01 vs. 0.85 ± 0.01), or 24‐h fat oxidation (82 ± 6 vs. 80 ± 7 g day^‐1) between 3M and 6M, respectively. There was no difference in fullness 24‐h AUC, but hunger AUC (41850 ± 2255 vs. 36612 ± 2556 mm.24 h, P = 0.03) and “desire to eat” AUC (47061 ± 1791 vs. 41170 ± 2574 mm.24 h, P = 0.03) were greater during 6M than 3M.

Conclusion:

We conclude that increasing meal frequency from three to six per day has no significant effect on 24‐h fat oxidation, but may increase hunger and the desire to eat.

     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

HRT preserves increases in bone mineral density and reductions in body fat after a supervised exercise program

The aims of thisstudy were to confirm our previous finding that hormone-replacementtherapy (HRT) augments exercise-induced increases in bone mineraldensity (BMD) in older women and to determine whether HRT preserves theadaptations when exercise is reduced or discontinued. The studyincluded an 11-mo treatment phase and a 6-mo follow-up phase.Participants, aged 66 ± 3 yr, were assigned to control (Con;n = 10), exercise (Ex;n = 18), HRT(n = 10), and Ex+HRT(n = 16) groups. HRT was continuedduring the follow-up. After the treatment phase, changes in total body BMD were 0.5 ± 1.7, 1.5 ± 1.4, 1.2 ± 0.8, and 2.7 ± 1.2% in Con, Ex, HRT, and Ex+HRT, respectively. Ex+HRT was moreeffective than HRT in increasing BMD of the total body and tended(P = 0.08) to be more effective at thelumbar spine. Ex+HRT was more effective than Ex in increasing BMD ofthe total body, lumbar spine, and trochanter. Exercise-induced gains inBMD were preserved during the follow-up only in those individuals onHRT. HRT also attenuated fat accumulation, particularly in theabdominal region, after the exercise program. These findings suggestthat HRT is an important adjunct to exercise for the prevention notonly of osteoporosis but also of diseases related to abdominal obesity.

     

Pyrazole inhibitors of HMG-CoA reductase: an attempt to dramatically reduce synthetic complexity through minimal analog re-design

An extraordinarily potent and hepatoselective class of HMG-CoA reductase inhibitors containing a pyrazole core was recently reported; however, its development was hampered by a long and difficult synthetic route. We attempted to circumvent this obstacle by preparing closely related analogs wherein the key dihydroxyheptanoic acid sidechain was tethered to the pyrazole core via an oxygen linker ('oxypyrazoles'). This minor change reduced the total number of synthetic steps from 14 to 7. Although the resulting analogs maintained much of the in vitro and cell activity of the pyrazoles, inferior in vivo activity precluded further development. Caco-2 cell permeability data suggest that enhanced cellular efflux of the oxypyrazoles relative to the pyrazoles may be responsible for the poor in vivo activity.     

Explanatory Models and Mental Health Treatment: Is Vodou an Obstacle to Psychiatric Treatment in Rural Haiti?

Vodou as an explanatory framework for illness has been considered an impediment to biomedical psychiatric treatment in rural Haiti by some scholars and Haitian professionals. According to this perspective, attribution of mental illness to supernatural possession drives individuals to seek care from houngan-s (Vodou priests) and other folk practitioners, rather than physicians, psychologists, or psychiatrists. This study investigates whether explanatory models of mental illness invoking supernatural causation result in care-seeking from folk practitioners and resistance to biomedical treatment. The study comprised 31 semi-structured interviews with community leaders, traditional healers, religious leaders, and biomedical providers, 10 focus group discussions with community members, community health workers, health promoters, community leaders, and church members; and four in-depth case studies of individuals exhibiting mental illness symptoms conducted in Haiti's Central Plateau. Respondents invoked multiple explanatory models for mental illness and expressed willingness to receive treatment from both traditional and biomedical practitioners. Folk practitioners expressed a desire to collaborate with biomedical providers and often referred patients to hospitals. At the same time, respondents perceived the biomedical system as largely ineffective for treating mental health problems. Explanatory models rooted in Vodou ethnopsychology were not primary barriers to pursuing psychiatric treatment. Rather, structural factors including scarcity of treatment resources and lack of psychiatric training among health practitioners created the greatest impediments to biomedical care for mental health concerns in rural Haiti.     

Novel and selective spiroindoline-based inhibitors of Sky kinase

We report the discovery of a novel series of spiroindoline-based inhibitors of Sky kinase that bind in the ATP-binding site and exhibit high levels of kinome selectivity through filling the Ala571-subpocket. These inhibitors exhibit moderate oral bioavailability in the rat due to low absorption across the gut wall.     

Importance of factors determining the effective lifetime of a mass, long-lasting, insecticidal net distribution: a sensitivity analysis

Background

Long-lasting insecticidal nets (LLINs) reduce malaria transmission by protecting individuals from infectious bites, and by reducing mosquito survival. In recent years, millions of LLINs have been distributed across sub-Saharan Africa (SSA). Over time, LLINs decay physically and chemically and are destroyed, making repeated interventions necessary to prevent a resurgence of malaria. Because its effects on transmission are important (more so than the effects of individual protection), estimates of the lifetime of mass distribution rounds should be based on the effective length of epidemiological protection.

Methods

Simulation models, parameterised using available field data, were used to analyse how the distribution's effective lifetime depends on the transmission setting and on LLIN characteristics. Factors considered were the pre-intervention transmission level, initial coverage, net attrition, and both physical and chemical decay. An ensemble of 14 stochastic individual-based model variants for malaria in humans was used, combined with a deterministic model for malaria in mosquitoes.

Results

The effective lifetime was most sensitive to the pre-intervention transmission level, with a lifetime of almost 10 years at an entomological inoculation rate of two infectious bites per adult per annum (ibpapa), but of little more than 2 years at 256 ibpapa. The LLIN attrition rate and the insecticide decay rate were the next most important parameters. The lifetime was surprisingly insensitive to physical decay parameters, but this could change as physical integrity gains importance with the emergence and spread of pyrethroid resistance.

Conclusions

The strong dependency of the effective lifetime on the pre-intervention transmission level indicated that the required distribution frequency may vary more with the local entomological situation than with LLIN quality or the characteristics of the distribution system. This highlights the need for malaria monitoring both before and during intervention programmes, particularly since there are likely to be strong variations between years and over short distances. The majority of SSA's population falls into exposure categories where the lifetime is relatively long, but because exposure estimates are highly uncertain, it is necessary to consider subsequent interventions before the end of the expected effective lifetime based on an imprecise transmission measure.     

Mechanical contributors to sex differences in idiopathic knee osteoarthritis

The occurrence of knee osteoarthritis (OA) increases with age and is more common in women compared with men, especially after the age of 50 years. Recent work suggests that contact stress in the knee cartilage is a significant predictor of the risk for developing knee OA. Significant gaps in knowledge remain, however, as to how changes in musculoskeletal traits disturb the normal mechanical environment of the knee and contribute to sex differences in the initiation and progression of idiopathic knee OA. To illustrate this knowledge deficit, we summarize what is known about the influence of limb alignment, muscle function, and obesity on sex differences in knee OA. Observational data suggest that limb alignment can predict the development of radiographic signs of knee OA, potentially due to increased stresses and strains within the joint. However, these data do not indicate how limb alignment could contribute to sex differences in either the development or worsening of knee OA. Similarly, the strength of the knee extensor muscles is compromised in women who develop radiographic and symptomatic signs of knee OA, but the extent to which the decline in muscle function precedes the development of the disease is uncertain. Even less is known about how changes in muscle function might contribute to the worsening of knee OA. Conversely, obesity is a stronger predictor of developing knee OA symptoms in women than in men. The influence of obesity on developing knee OA symptoms is not associated with deviation in limb alignment, but BMI predicts the worsening of the symptoms only in individuals with neutral and valgus (knock-kneed) knees. It is more likely, however, that obesity modulates OA through a combination of systemic effects, particularly an increase in inflammatory cytokines, and mechanical factors within the joint. The absence of strong associations of these surrogate measures of the mechanical environment in the knee joint with sex differences in the development and progression of knee OA suggests that a more multifactorial and integrative approach in the study of this disease is needed. We identify gaps in knowledge related to mechanical influences on the sex differences in knee OA.