Crystalline inclusions in the pituitary gonadotrophs of estrogen-primed, spayed rhesus monkeys (Macaca mulatta)

17-β-Estradiol or cholesterol was subcutaneously implanted in bilaterally oophorectomized rhesus monkeys for 37 days, after which the pituitary glands were analyzed ultrastructurally for evidence of granule disposal. Plasma estrogen returned to a physiologic level of 97.6 ± 2.8 pg/ml in the estrogen-treated animals but not in the cholesterol-treated controls. The hypertrophied gonadotrophs in the controls contained numerous large vesicles and a few secretory granules. The gonadotrophs in the experimental animals showed a reduction in cell size, a diminution of the dilated endoplasmic reticulum, and an accumulation of secretory granules. Moreover, crystalline inclusions composed of parallel and orthogonally arranged fibers were present in some gonadotrophs, most frequently in animals with lower levels of circulating estrogen. These inclusions are thought to be involved in the process of intracellular secretion in monkeys.     

Isotope Label-Aided Mass Spectrometry Reveals the Influence of Environmental Factors on Metabolism in Single Eggs of Fruit Fly

In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar (¹³C₆-glucose) for 12 h – either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate – possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism.     

Comparison of the electrophysiological effect of amiodarone, lidocaine and quinidine on rat ventricular cells.

The effects of 20 microM each of amiodarone, lidocaine and quinidine on action potential and membrane currents were studied in rat ventricular cells. At a stimulation frequency of 0.1 Hz, quinidine prolonged the action potential duration (APD50) from 120 +/- 26 to 660 +/- 8 msec and increased the time to peak (Tp) amplitude from 7 +/- 1 msec to 32 +/- 6 msec. Lidocaine shortened APD50 from 123 +/- 15 to 83 +/- 6 msec without altering Tp. Amiodarone changed neither APD50 nor Tp. Voltage clamp study revealed that quinidine inhibited sodium inward current (INa) even when this current was elicited by depolarizing pulses at 0.1 Hz from a holding potential of -90 mV. For amiodarone and lidocaine, the inhibition was observed when INa was elicited from a holding potential of -70 mV. A frequency-dependent inhibition of INa by amiodarone and lidocaine was observed at frequencies higher than 1 Hz. Quinidine showed this inhibition even at 1 Hz. In correlation with the stronger frequency dependent inhibition of INa, a greater delay of the recovery and increase of the non-recovery fraction of INa was induced by quinidine. For lidocaine and amiodarone, only the recovery time constant was delayed. In cells treated with sea anemone toxin (ATX, 0.2 microM), APD50 was prolonged to 4-5 sec in 5 min. Quinidine, but not amiodarone, completely reversed the effect of ATX. Quinidine showed use-dependent inhibition of INa in these ATX-treated cells. Amiodarone, however, did not show this inhibition. It is likely that amiodarone suppresses INa by delaying the recovery of INa instead of blocking the open-state Na(+)-channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of epidermal growth factor on the sheet formation of the human keratinocyte in cell culture

Epidermal growth factor is an important element in maintaining keratinocyte proliferation and maturation. To evaluate its effect on keratinocyte growth in vitro, human foreskins were cultured. The epidermal keratinocyte growth in culture was separated into two stages by a conditional medium: the proliferation stage, in which the cells were maintained in a monolayer; and the differentiation stage, in which the cells grew to stratification and keratinization. The keratinocytes were cultured in various concentrations of epidermal growth factor, and their morphology and growth behavior were closely observed. Our results demonstrated that cultured keratinocytes grew in a confluent layer under the influence of epidermal growth factor. In contrast, in a culture without epidermal growth factor, the proliferation rate of cultured keratinocytes slowed down and eventually the cells stopped growing. During serum stimulation, with or without additional exogenous epidermal growth factor, the cultured keratinocytes grew continuously to the normal terminal differentiation. Under this two-stage culture model, the cultured keratinocytes could grow into an intact sheet of graftable epidermis.     

A unique feeding strategy of the extinct marine mammal Kolponomos: convergence on sabretooths and sea otters

Mammalian molluscivores feed mainly by shell-crushing or suction-feeding. The extinct marine arctoid, Kolponomos, has been interpreted as an otter-like shell-crusher based on similar dentitions. However, neither the masticatory biomechanics of the shell-crushing adaptation nor the way Kolponomos may have captured hard-shelled prey have been tested. Based on mandibular symphyseal morphology shared by Kolponomos and sabre-toothed carnivores, we hypothesize a sabretooth-like mechanism for Kolponomos prey-capture, whereby the mandible functioned as an anchor. Torque generated from jaw closure and head flexion was used to dislodge prey by prying, with prey then crushed using cheek teeth. We test this hypothesized feeding sequence using phylogenetically informed biomechanical simulations and shape analyses, and find a strongly supported, shared high mandibular stiffness in simulated prey-capture bites and mandibular shape in Kolponomos and the sabre-toothed cat Smilodon. These two distantly related taxa converged on using mandibles to anchor cranial torqueing forces when prying substrate-bound prey in the former and sabre-driving forces during prey-killing in the latter. Simulated prey-crushing bites indicate that Kolponomos and sea otters exhibit alternative structural stiffness-bite efficiency combinations in mandibular biomechanical adaptation for shell-crushing. This unique feeding system of Kolponomos exemplifies a mosaic of form-function convergence relative to other Carnivora.     

Microtubule-Associated Type II Protein Kinase A Is Important for Neurite Elongation

Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process.