Crop production in the USA is frequently limited by a lack of pollinators.

Most of the world's crops depend on pollinators, so declines in both managed and wild bees raise concerns about food security. However, the degree to which insect pollination is actually limiting current crop production is poorly understood, as is the role of wild species (as opposed to managed honeybees) in pollinating crops, particularly in intensive production areas. We established a nationwide study to assess the extent of pollinator limitation in seven crops at 131 locations situated across major crop-producing areas of the USA. We found that five out of seven crops showed evidence of pollinator limitation. Wild bees and honeybees provided comparable amounts of pollination for most crops, even in agriculturally intensive regions. We estimated the nationwide annual production value of wild pollinators to the seven crops we studied at over $1.5 billion; the value of wild bee pollination of all pollinator-dependent crops would be much greater. Our findings show that pollinator declines could translate directly into decreased yields or production for most of the crops studied, and that wild species contribute substantially to pollination of most study crops in major crop-producing regions.

Myonuclei acquired by overload exercise precede hypertrophy and are not lost on detraining.

Effects of previous strength training can be long-lived, even after prolonged subsequent inactivity, and retraining is facilitated by a previous training episode. Traditionally, such "muscle memory" has been attributed to neural factors in the absence of any identified local memory mechanism in the muscle tissue. We have used in vivo imaging techniques to study live myonuclei belonging to distinct muscle fibers and observe that new myonuclei are added before any major increase in size during overload. The old and newly acquired nuclei are retained during severe atrophy caused by subsequent denervation lasting for a considerable period of the animal's lifespan. The myonuclei seem to be protected from the high apoptotic activity found in inactive muscle tissue. A hypertrophy episode leading to a lasting elevated number of myonuclei retarded disuse atrophy, and the nuclei could serve as a cell biological substrate for such memory. Because the ability to create myonuclei is impaired in the elderly, individuals may benefit from strength training at an early age, and because anabolic steroids facilitate more myonuclei, nuclear permanency may also have implications for exclusion periods after a doping offense.

Reduced arterial elasticity after anabolic-androgenic steroid use in young adult males and mice.

High-doses of anabolic-androgenic steroids (AAS) is efficient for building muscle mass, but pose a risk of cardiovascular side effects. Little is known of the effect of AAS on vasculature, but previous findings suggest unfavorable alterations in vessel walls and vasoreactivity. Here, long-term effect of AAS on vascular function and morphology were examined in male weightlifters, and in a mimicking animal model. Arterial elasticity and morphology were tested with ultrasound, pulse wave velocity (PWV) and carotid intima media thickness (cIMT) in 56 current male AAS users, and 67 non-exposed weightlifting controls (WLC). Female mice were treated with testosterone for 14 days and echocardiography were applied to evaluate vascular function and morphology. Male AAS users had higher PWV (p = 0.044), reduced carotid artery compliance (p = 0.0005), and increased cIMT (p = 0.041) compared to WLC. Similar functional changes were found in the ascending aorta of mice after 7- (p = 0.043) and 14 days (p = 0.001) of testosterone treatment. This animal model can be used to map molecular mechanisms responsible for complications related to AAS misuse. Considering the age-independent stiffening of major arteries and the predictive power of an increase in PWV and cIMT, the long-term users of AAS are at increased risk of severe cardiovascular events.

Is it possible to predict the origin of epithelial cells? - A comparison of secondary transfer of skin epithelial cells versus vaginal mucous membrane cells by direct contact.

In alleged sexual assault and rape cases, the focus has often been to collect samples from the victim's body, for detection of body fluids or skin cells from the offender. But in many cases intimate body samples from the perpetrator(s) can also be informative. However, in cases where the female victim claims vaginal penetration, the defendant may display an alternative explanation to the DNA findings, i.e. that the victim's skin cells has been secondarily transferred to his penis. We hypothesized that female DNA will be detected in a significantly greater amount on swabs from penis after intercourse than after secondary transfer by skin contact. Fourteen male-female couples were recruited to test the above hypothesis, by collecting penile swabs from 3 specified anatomical locations: Glans, shaft, and the coronal sulcus, after two different situations: Vaginal intercourse and secondary transfer of epithelial cells by skin contact. The results show that penile swabs following intercourse produce significantly higher DNA concentrations than after secondary transfer by skin contact. Our results, indicates which of the anatomical regions is best suited for sampling. The DNA profiling results show a preponderance of female profiles over male profiles following intercourse compared to secondary skin contact. Based on these data, it is possible to make a statistical model to distinguish between samples taken after intercourse and samples taken after secondary transfer by skin contact based on the amount of female DNA and mixture proportion (Mx) between female and male DNA in samples collected from penis swabs.

Neural regulation of muscle acetylcholine receptor epsilon- and alpha-subunit gene promoters in transgenic mice.

The effects of denervation were investigated in mice with transgenes containing promoter elements from the muscle acetylcholine receptor epsilon- and alpha-subunit genes. The promoter sequences were coupled to a nuclear localization signal-beta-galactosidase fusion gene (nlacZ) as a reporter. While many postsynaptic specializations form in the embryo, expression of the epsilon subunit is induced during the first two postnatal weeks. When muscles were denervated at birth, before the onset of epsilon expression, epsilon nlacZ still appeared at the former synaptic sites on schedule. This result suggests that the nerve leaves a localized "trace" in the muscle that can continue to regulate transcription. An additional finding was that epsilon nlacZ expression was much stronger in denervated than in intact muscles. This suggests that the epsilon promoter is similar to the other subunits in containing elements that are activated on cessation of neural activity. However, even after denervation, epsilon nlacZ expression was always confined to the synaptic region whereas alpha nlacZ expression increased in nuclei along the entire length of the fiber. This suggests that while the epsilon gene is similar in its activity dependence to other subunit genes, it is unique in that local nerve-derived signals are essential for its expression. Consequently, inactivity enhances epsilon expression only in synaptic nuclei where such signals are present, but enhances expression throughout the muscle fiber. Truncations and an internal deletion of the epsilon promoter indicate that cis-elements essential for the response to synaptic signals are contained within 280 bp of the transcription start site. In contrast to these results in young animals, denervation in older animals leads to an unexpected reduction in nlacZ activity. However, mRNA measurements indicated that transgene expression was increased in these animals. This discordance between nlacZ mRNA and enzyme activity, demonstrates a previously unknown limitation of nlacZ as a reporter gene in transgenic animals.

Myogenin induces a shift of enzyme activity from glycolytic to oxidative metabolism in muscles of transgenic mice.

Physical training regulates muscle metabolic and contractile properties by altering gene expression. Electrical activity evoked in muscle fiber membrane during physical activity is crucial for such regulation, but the subsequent intracellular pathway is virtually unmapped. Here we investigate the ability of myogenin, a muscle-specific transcription factor strongly regulated by electrical activity, to alter muscle phenotype. Myogenin was overexpressed in transgenic mice using regulatory elements that confer strong expression confined to differentiated post-mitotic fast muscle fibers. In fast muscles from such mice, the activity levels of oxidative mitochondrial enzymes were elevated two- to threefold, whereas levels of glycolytic enzymes were reduced to levels 0.3-0.6 times those found in wild-type mice. Histochemical analysis shows widespread increases in mitochondrial components and glycogen accumulation. The changes in enzyme content were accompanied by a reduction in fiber size, such that many fibers acquired a size typical of oxidative fibers. No change in fiber type-specific myosin heavy chain isoform expression was observed. Changes in metabolic properties without changes in myosins are observed after moderate endurance training in mammals, including humans. Our data suggest that myogenin regulated by electrical activity may mediate effects of physical training on metabolic capacity in muscle.

Specific labelling of myonuclei by an antibody against pericentriolar material 1 on skeletal muscle tissue sections.

AIM: Skeletal muscle is a heterogeneous tissue containing several different cell types, and only about 40%-50% of the cell nuclei within the tissue belong to myofibres. Existing technology, attempting to distinguish myonuclei from other nuclei at the light microscopy level, has led to controversies in our understanding of the basic cell biology of muscle plasticity. This study aims at demonstrating that an antibody against the protein pericentriolar material 1 (PCM1) can be used to reliably identify myonuclei on histological cross sections from humans, mice and rats. METHODS: Cryosections were labelled with a polyclonal antibody against PCM1. The specificity of the labelling for myonuclei was verified using 3D reconstructions of confocal z-stacks triple-labelled for DNA, dystrophin and PCM1, and by co-localization with nuclear mCherry driven by the muscle-specific Alpha-Actin-1 promoter after viral transduction. RESULTS: The PCM1 antibody specifically labelled all myonuclei, and myonuclei only, in cryosections of muscles from rats, mice and men. Nuclei in other cell types including satellite cells were not labelled. Both normal muscles and hypertrophic muscles after synergist ablation were investigated. CONCLUSION: Pericentriolar material 1 can be used as a specific histological marker for myonuclei in skeletal muscle tissue without relying on counterstaining of other structures or cumbersome and subjective analysis of nuclear positioning.

Overexpression of myogenin in muscles of transgenic mice: interaction with Id-1, negative crossregulation of myogenic factors, and induction of extrasynaptic acetylcholine receptor expression.

To investigate the role of myogenin in regulating acetylcholine receptor expression in adult muscle, this muscle-specific basic helix-loop-helix transcription factor was overexpressed in transgenic mice by using regulatory elements conferring strong expression confined to differentiated postmitotic muscle fibers. Many of the transgenic mice died during the first postnatal week, but those that survived into adulthood displayed normal muscle histology, gross morphology, and motor behavior. The mRNA levels of all five acetylcholine receptor subunits (alpha, beta, gamma, delta, and epsilon) were, however, elevated. Also, the level of receptor protein was increased and high levels of receptors were present throughout the extrasynaptic surface membrane of the muscle fibers. Thus, elevated levels of myogenin are apparently sufficient to induce acetylcholine supersensitivity in normally innervated muscle of adult mice. The high neonatal mortality rate of the mice overexpressing myogenin hindered the propagation of a stable line. In an attempt to increase survival, myogenin overexpressers were mated with a line of transgenic mice overexpressing Id-1, a negative regulator that interacts with the basic helix-loop-helix family of transcription factors. The Id-1 transgene apparently worked as a second site suppressor and abolished the high rate of neonatal mortality. This effect indicates that Id-1 and myogenin interact directly or indirectly in these animals. Further study indicated that myogenin overexpression had no effect on the level of endogenous myogenin mRNA, while the levels of myoD and MRF4 mRNAs were reduced. Overexpression of the negative regulator Id-1 increased the mRNA levels of all the myogenic factors. These findings are consistent with a hypothesis suggesting that myogenic factors are influenced by mechanisms that maintain cellular homeostasis.

Distribution of myonuclei and microtubules in live muscle fibers of young, middle-aged, and old mice.

We have recently published a new technique for visualizing nuclei in living muscle fibers of intact animals, based on microinjection of labeled DNA into single myofibers, excluding satellite cells (Bruusgaard JC, Liestol K, Ekmark M, Kollstad K, and Gundersen K. J Physiol 551: 467-478, 2003). In the present study, we use this technique to study fiber segments of soleus and extensor digitorum longus (EDL) muscles from mice aged 2, 14, and 23 mo. As the animals maturing from 2 to 14 mo, they displayed an increase in size and number of nuclei. Soleus showed little change in nuclear domain size, whereas this increased by 88% in the EDL. For 14-mo-old animals, no significant correlation between fiber size and nuclear number was observed (R2=0.18, P=0.51) despite a fourfold variation in cytoplasmic volume. This suggests that size and nuclear number is uncoupled in middle-aged mice. When animals aged from 14 to 23 mo, EDL IIb, but not soleus, fibers atrophied by 41%. Both EDL and soleus displayed a reduction in number of nuclei: 20 and 16%, respectively. A positive correlation between number of nuclei and size was observed at 2 mo, and this reappeared in old mice. The atrophy in IIb fibers at old age was accompanied by a disturbance in the orderly positioning of nuclei that is so prominent in glycolytic fibers at younger age. In old animals, changes in nuclear shape and in the peri- and internuclear microtubule network were also observed. Thus changes in myonuclear number and distribution, perhaps related to alterations in the microtubular network, may underlie some of the adverse consequences of aging on skeletal muscle size and function.

DNA injection into single cells of intact mice.

We describe a technique wherein single muscle fibers of intact mice are made transgenic by intracellular injection of DNA expression vectors for the reporter genes lacZ and green fluorescent protein (GFP). Application of in vivo imaging techniques allowed identification of single cells during the injections, and of the same single cells when the muscle was reexposed days or weeks later. DNA concentration by itself had little effect on fiber survival or expression efficacy, but it seemed crucial to exceed a threshold of about 10(6) injected plasmid molecules in order to obtain expression. On the other hand, experiments with coinjection of two different plasmids suggested that relatively few individual molecules were eventually transcribed in expressing cells. Plasmid DNA remained localized to the injection site, and expression was confined to nuclei in the vicinity. Expression was stable, as reporter was detected 2-61 days after injection.

Cholesterol-lowering effect of colestipol hydrochloride given twice daily in hypercholesterolemic patients.

In a randomized design study in 66 hypercholesterolemic patients, dosages of 10 g of colestipol HCl twice daily lowered serum cholesterol an average of 19% more than placebo therapy. These results are comparable to those in other studies in which the same total daily dose was given in three or four doses. The most common side effect was constipation, reported by 6 patients on colestipol HCl and 3 patients on placebo. No untoward systemic reactions or abnormal laboratory data were seen except for a slight rise in serum alkaline phosphatase during colestipol HCl therapy. The drug was well accepted by most patients.

Transgenic embryo yield is increased by a simple, inexpensive micropipet treatment.

A method for the treatment of micropipet tips for injection of DNA into pronuclei of mouse embryos is described. The method requires only standard laboratory equipment, a magnetic stirrer and an inexpensive commercially available grinding powder. The method involves little extra time and no special skills. After introduction of the method, the time required for injections was reduced by about 30%, and the yield of surviving embryos was increased by 28%.

Aldose reductase inhibitors in clinical practice. Preliminary studies on diabetic neuropathy and retinopathy.

Extensive animal data now exist to indicate potential benefit of sorbinil in the treatment of the major complications of diabetes mellitus. A clinical programme has been constructed to explore this therapeutic potential and encouraging evidence of drug effect has already been observed in patients with neuropathy and retinopathy. Two small preliminary studies in patients with painful neuropathy have shown that clinically significant reduction of pain was more frequently achieved with sorbinil than with placebo. A 6-month study of patients with retinopathy, using vitreous fluorophotometry as the criterion of retinal damage, showed significant (p = 0.03) benefit for the sorbinil group compared with the placebo group. Drug evaluation in these areas is complex and difficult but it is anticipated that the accumulation of additional data will further substantiate the efficacy suggested by these early findings. The only clinically important adverse effect of sorbinil is the hypersensitivity reaction. This usually occurs during the initial weeks of therapy and is similar to that seen with phenytoin. The long term use of sorbinil is without significant adverse effects.

Clinical experience with sorbinil--an aldose reductase inhibitor.

A considerable volume of animal pharmacologic data support the view that increased flux through the polyol pathway provides a unifying hypothesis for the major complications of diabetes. An extensive clinical program has been established to verify the extrapolation of the animal pharmacologic findings to man. Clinical data accumulated to date confirm the biochemical and electrophysiologic effects, and encouraging evidence of a drug effect in diabetic neuropathy and retinopathy has already been observed. In the large, controlled safety data base already available, the long-term clinical use of sorbinil is devoid of significant adverse effects in terms of both subjective side effects and laboratory parameters. The only clinically important adverse reaction reported to date has been a hypersensitivity reaction in the early weeks of therapy, which is similar to that seen with other hydantoins.

Psychosocial functioning in a prepubertal and early adolescent bipolar disorder phenotype.

OBJECTIVE: To compare psychosocial functioning (PF) in a prepubertal and early adolescent bipolar disorder phenotype (PEA-BP) sample to two comparison groups, i.e., attention-deficit/hyperactivity disorder (ADHD) and community controls (CC). METHOD: There were 93 PEA-BP (with or without comorbid ADHD), 81 ADHD, and 94 CC subjects who were participants in an ongoing study, the Phenomenology and Course of Pediatric Bipolar Disorders. Cases in the PEA-BP and ADHD groups were outpatients obtained by consecutive new case ascertainment, and CC subjects were from a survey conducted by the Research Triangle Institute. To fit the study phenotype, PEA-BP subjects needed to have current DSM-IV mania or hypomania with elation and/or grandiosity as one criterion. Assessments for PF were by experienced research nurses who were blind to group status. Mothers and children were separately interviewed with the Psychosocial Schedule for School Age Children-Revised. RESULTS: Compared with both ADHD and CC subjects, PEA-BP cases had significantly greater impairment on items that assessed maternal-child warmth, maternal-child and paternal-child tension, and peer relationships. CONCLUSIONS: Clinicians need to consider PF deficits when planning interventions. In the PEA-BP group, there was a 43% rate of hypersexuality with a <1% rate of sexual abuse, supporting hypersexuality as a manifestation of child mania.

Substance P in subdivisions of the sciatic nerve, and in red and white skeletal muscles.

Substance P (SP) has been determined by radioimmunoassay in the sciatic nerve and its fibular, tibial and sural branches; and in a red, slow-twitch and a white, fast-twitch muscle supplied by the sciatic nerve. The mixed sciatic nerve contained 25 ng SP per g tissue wet wt., while the sural branch, which is supplying mainly skin, and thus is rich in sensory fibres, had a significantly higher content (49 ng/g). The mixed fibular and tibial branches contained approximately the same amount as the sciatic nerve proper. SP was also found in the skeletal muscles. The red m. soleus had a significantly higher content (0.61 ng/g) than the white m. extensor digitorum longus (0.22 ng/g). A dorsal root lesion which leads to degeneration of sensory fibres, reduced the SP level more than 90% both in nerves and muscles. Ventral root section, which leads to degeneration of somato-motor fibres and terminals, had, on the other hand, no effect. We conclude that sensory neurons are the only source of importance for SP in the sciatic nerve and in skeletal muscles. Based on the above findings, the possibility that SP may function as a mediator of an axon reflex in skeletal muscle is discussed.

The Response of Denervated Muscle to Long-Term Electrical Stimulation.

Adapted from: Lømo T, Westgaard RH, Hennig R, Gundersen K. The response of denervated muscle to long-term electrical stimulation, In: Carraro U, Angelini C, eds. Proceedings of the First Abano Terme Meeting on Rehabilitation, 1985 August 28-30, Abano Terme, Padova, Italy, An International Symposium, Satellite Meeting of the XIII World Congress of Neurology, Hamburg 1985. Cleup Padova 1985. pp 81-90.

No change in myonuclear number during muscle unloading and reloading.

Muscle fibers are the cells in the body with the largest volume, and they have multiple nuclei serving different domains of cytoplasm. A large body of previous literature has suggested that atrophy induced by hindlimb suspension leads to a loss of "excessive" myonuclei by apoptosis. We demonstrate here that atrophy induced by hindlimb suspension does not lead to loss of myonuclei despite a strong increase in apoptotic activity of other types of nuclei within the muscle tissue. Thus hindlimb suspension turns out to be similar to other atrophy models such as denervation, nerve impulse block, and antagonist ablation. We discuss how the different outcome of various studies can be attributed to difficulties in separating myonuclei from other nuclei, and to systematic differences in passive properties between normal and unloaded muscles. During reload, after hindlimb suspension, a radial regrowth is observed, which has been believed to be accompanied by recruitment of new myonuclei from satellite cells. The lack of nuclear loss during unloading, however, puts these findings into question. We observed that reload led to an increase in cross sectional area of 59%, and fiber size was completely restored to the presuspension levels. Despite this notable growth there was no increase in the number of myonuclei. Thus radial regrowth seems to differ from de novo hypertrophy in that nuclei are only added during the latter. We speculate that the number of myonuclei might reflect the largest size the muscle fibers have had in its previous history.