Confocal laser scanning microscopy of rat follicle development.

This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pro, LysoTracker Red, hydroethidine, ethidium bromide, and 7-aminoactinomycin-d) were applied either to fresh tissue or to tissue that had been fixed with glutaraldehyde or paraformaldehyde. After fixation and staining, the tissue was dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. CLSM was then used with the appropriate laser excitation, dichroics, and bandpass filters to acquire images of oocytes contained in follicles. Analysis of the data revealed three principal findings. First, a rapid increase in oocyte size occurred in the preantral stages of follicle development. In the antral stage of follicle development, there was a rapid increase in follicle size without any substantial increase in oocyte size. Second, accompanying these changes in oocyte and follicle growth was a differential staining pattern in which the nucleus stained more than the cytoplasm in a young follicle, but stained less than the cytoplasm as the follicle enlarged into the late antral stage. Lastly, using CLSM, atretic follicles showed increased LysoTracker Red staining in the granulosa region of the antral follicle, suggestive of cell death.     

Aspects of cardiovascular reflexes in pathologic states

Cardiovascular reflexes that are mediated by receptors in the heart and blood vessels control a variety of important hemodynamic and humoral functions. The action of these receptors can be shown to be abnormal in several pathologic states. Left atrial receptors exhibit a depressed discharge sensitivity in dogs with chronic congestive heart failure caused by an aortocaval fistula. The reflex effects of atrial receptor stimulation are also depressed in heart failure. Left ventricular receptor stimulation has been implicated in the abnormal vascular responses to exercise in patients with aortic stenosis. The arterial baroreflex control of heart rate is abnormal in animals and humans with various forms of hypertension. Arterial baroreceptors from hypertensive animals show a resetting of their pressure-discharge curve to higher pressures. The arterial baroreflex is also depressed in chronic heart failure. This effect may result from an abnormality of the efferent limb of the reflex arc or from changes in the interaction between baroreceptors and cardiac receptors centrally. A final possibility may be abnormal arterial baroreceptor discharge characteristics in heart failure.     

Presynaptic calcium diffusion from various arrays of single channels. Implications for transmitter release and synaptic facilitation.

A one-dimensional model of presynaptic calcium diffusion away from the membrane, with cytoplasmic binding, extrusion by a surface pump, and influx during action potentials, can account for the rapid decay of phasic transmitter release and the slower decay of synaptic facilitation following one spike, as well as the very slow decline in total free calcium observed experimentally. However, simulations using this model, and alternative versions in which calcium uptake into organelles and saturable binding are included, fail to preserve phasic transmitter release to spikes in a long tetanus. A three-dimensional diffusion model was developed, in which calcium enters through discrete membrane channels and acts to release transmitter within 50 nm of entry points. Analytic solutions of the equations of this model, in which calcium channels were distributed in active zone patches based on ultrastructural observations, were successful in predicting synaptic facilitation, phasic release to tetanic spikes, and the accumulation of total free calcium. The effects of varying calcium buffering, pump rate, and channel number and distribution were explored. Versions appropriate to squid giant synapses and frog neuromuscular junctions were simulated. Limitations of key assumptions, particularly rapid nonsaturable binding, are discussed.     

Prevention by a platelet-derived factor (platelet factor 4) of induction of low dose tolerance to pneumococcal polysaccharides

It was previously shown that human or mouse serum, and platelet factor 4 (PF4) prepared from human platelet releasate, counteracts nonspecific immunosuppression induced in mice by injection of concanavalin A or syngeneic gamma-irradiated lymphoma cells. The present studies show that PF4 prepared from normal mouse or human serum by absorption to heparin-agarose and elution between 0.5 and 1.5 M NaCl is also active in this respect. The ability of PF4 to counteract antigen-specific suppression of the antibody response to pneumococcal polysaccharide (pps) was now studied. PF4 derived from human or mouse serum as well as recombinant PF4 interferes with induction of antigen-specific low dose tolerance when they are injected at the same time as a low dose (0.2 microgram) of type 14 pps 3 days before an optimal immunizing dose (25 micrograms). Furthermore, injection of platelet releasate at the time of an optimal primary immunizing dose of pps type 14 enhances the secondary response to killed bacteria injected 2 weeks later, but not the primary response itself. Both effects are interpreted as due to interference with antigen-specific suppressor cell induction during primary immunization. Injection of PF4 is much less effective in reversing low dose tolerance to an optimal immunizing dose (0.1 microgram) of type 3 pps induced by injection of 0.005 microgram of this antigen. Differences in the mechanism of tolerance induction for the two pps types that might be responsible for this are discussed.     

Purification of human DNA (cytosine-5-)-methyltransferase

We have developed a facile procedure for the purification of DNA methyltransferase activity from human placenta. The procedure avoids the isolation of nuclei and the dialysis and chromatography of large volumes. A purification of 38,000-fold from the whole cell extract has been achieved. The procedure employs ion exchange, affinity, and hydrophobic interaction chromatography coupled with preparative glycerol gradient centrifugation. A protein of 126,000 daltons was found to copurify with the activity and was the major band seen in the most highly purified material after SDS gel electrophoresis. This observation, coupled with an observed sedimentation coefficient of 6.3S, suggests that the enzyme is composed of a single polypeptide chain of this molecular weight. Hemimethylated DNA was found to be the preferred substrate for the enzyme at each stage in the purification. The ratio of the activity of the purified product on hemimethylated to that on unmethylated M13 duplex DNA was about 12 to 1. Thus, the purified activity has the properties postulated for a maintenance methyltransferase. The availability of highly purified human DNA methyltransferase should facilitate many studies on the structure, function, and expression of these activities in both normal and transformed cells.     

Anatomical evidence for androdioecy in the clam shrimp Eulimnadia texana

Recent genetic evidence suggests that the clam shrimp Eulimnadia texana exhibits androdioecy, a rare mating system. In this system, individuals are either hermaphrodites or males, pure females not being found. Through the use of light microscopy, this study provides anatomical evidence that egg-bearing individuals are, indeed, hermaphrodites. All such individuals observed possess ovotestes with testicular tissue restricted to the posterior region of the gonad. Developing sperms in hermaphrodites are similar in appearance to those seen in male-only individuals. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microbial Community Metabolic Modeling: A Community Data‐Driven Network Reconstruction

Metabolic network modeling of microbial communities provides an in‐depth understanding of community‐wide metabolic and regulatory processes. Compared to single organism analyses, community metabolic network modeling is more complex because it needs to account for interspecies interactions. To date, most approaches focus on reconstruction of high‐quality individual networks so that, when combined, they can predict community behaviors as a result of interspecies interactions. However, this conventional method becomes ineffective for communities whose members are not well characterized and cannot be experimentally interrogated in isolation. Here, we tested a new approach that uses community‐level data as a critical input for the network reconstruction process. This method focuses on directly predicting interspecies metabolic interactions in a community, when axenic information is insufficient. We validated our method through the case study of a bacterial photoautotroph–heterotroph consortium that was used to provide data needed for a community‐level metabolic network reconstruction. Resulting simulations provided experimentally validated predictions of how a photoautotrophic cyanobacterium supports the growth of an obligate heterotrophic species by providing organic carbon and nitrogen sources. J. Cell. Physiol. 231: 2339–2345, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Light-promoted changes in apoplastic K+ activity in the Samanea saman pulvinus,monitored with liquid membrane microelectrodes

The movement of Samanea saman (Jacq.) Merrill leaflets is a consequence of the re-distribution of K⁺ and anions between motor cells on opposite sides of the pulvinus. We used a K⁺-sensitive microelectrode to study dynamic changes in K⁺ transport through motor-cell membranes during and immediately after change in illumination. Potassium-ion-sensitive and reference microelectrodes were inserted into extensor or flexor tissue of a whole pulvinus in white light (WL). A brief pulse of red light (RL) followed by darkness (D) (a) increased K⁺ activity in the extensor apoplast, indicating K⁺ release by the protoplast; and (b) decreased K⁺ activity in the flexor apoplast, indicating K⁺ uptake by the protoplast. White light after 35–40 min D reversed K⁺ activity in the extensor apoplast to approximately its original value. Blue light substituted partially for WL in this regard. Potassium-ion activity in the flexor apoplast reverted to approximately its original value after 2 h, with or without white illumination. Our data support the hypothesis that K⁺ efflux from extensor cells and K⁺ uptake by flexor cells following a WLRLD transition occurs by way of K⁺ channels.Abbreviations L light - WL white light - RL red light - BL blue light - D darkness     

Differentiation of HL-60 cells: cell volume and cell cycle changes

HL-60 promyelocytic leukemic cells can differentiate into more mature myeloid cells with the addition of dimethylsulfoxide, butyric acid or retinoic acid and can differentiate into macrophages with the addition of phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). After the addition of an inducer, the HL-60 cell volume shows a daily decrease while the cell number increases at a rate similar to the untreated control cells. Flow cytometry measurements show an increase in G1 cells and a decrease in S cells after day 1. Since the generation time is constant, the data suggest that the length of time spent in the different cell cycle stages has changed during differentiation. Within 3 hours after the addition of TPA to HL-60 cells, selective adhesion of G1 cells occurs. Smaller sized cells are recovered from the flask bottom and larger sized cells are recovered from the supernate. Flow cytometric analysis reveals a G1 and S block in cells obtained from both the supernatant and from the flask bottom. After 1 day of TPA incubation, there is preferential adhesion of G1 and G2 cells with the nonadherent cells being primarily in the S and G2 cell cycle stages and undergoing a cell cycle traverse.     

Evaluation of serum parameters relevant to vitamin D status in tamarins

Serum levels of 25(OH)D, alkaline phosphatase (AP), and parathormone (PTH) were evaluated to investigate the vitamin D requirement of Saguinus fuscicollis. Diets with various vitamin D content were fed 4 weeks and longer. The values of 25(OH)D (30-300 nmol/l), AP (less than 300 U/l), and PTH (less than or equal to 1,000 equl/l) considered as normal were obtained with 2,000 IU D3/kg diet, or 33 IU/animal/day, which we regard as the level required. Animals depleted of vitamin D for 215 days developed a secondary hyperparathyroidism.