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The adaptive beam-tracing algorithm

The most popular models to predict sound propagation in architectural spaces involve the tracing of rays, images, or beams. Most current beam-tracing methods use conical or triangular beams that may produce overlaps and holes in the predicted sound field. Hence a new method has been developed whereby the shape of reflected beams is governed by the shape of reflecting surfaces so as to produce a geometrically perfect description of the sound propagation for halls with occluding surfaces. The method also facilitates the calculation of diffuse sound propagation by managing the energy transfer from a specular model to a diffuse model. This adaptive beam-tracing method compares well with other methods in terms of speed and accuracy.

Journal Article↗

Cortical map reorganization enabled by nucleus basalis activity.

Little is known about the mechanisms that allow the cortex to selectively improve the neural representations of behaviorally important stimuli while ignoring irrelevant stimuli. Diffuse neuromodulatory systems may facilitate cortical plasticity by acting as teachers to mark important stimuli. This study demonstrates that episodic electrical stimulation of the nucleus basalis, paired with an auditory stimulus, results in a massive progressive reorganization of the primary auditory cortex in the adult rat. Receptive field sizes can be narrowed, broadened, or left unaltered depending on specific parameters of the acoustic stimulus paired with nucleus basalis activation. This differential plasticity parallels the receptive field remodeling that results from different types of behavioral training. This result suggests that input characteristics may be able to drive appropriate alterations of receptive fields independently of explicit knowledge of the task. These findings also suggest that the basal forebrain plays an active instructional role in representational plasticity.

Acetylcholine↗

Formation and transport of chylomicrons by enterocytes to the lymphatics.

Digestion of triglyceride in the intestine results in the production of 2-monoglyceride and fatty acid. Phosphatidylcholine is hydrolyzed in the lumen to form lysophosphatidylcholine before its absorption. These digestion products are absorbed by the enterocytes through simple diffusion. In contrast, cholesterol absorption seems specific and is energy dependent. After entry into the enterocytes, these lipid digestion products migrate to the endoplasmic reticulum. Both fatty acid-binding protein and sterol carrier protein may be involved in the intracellular transport of fatty acid and cholesterol, respectively. Through predominantly the monoglyceride pathway, monoglycerides and fatty acids are resynthesized to form triglyceride in the endoplasmic reticulum. The lipid droplets, coated with cholesterol, phospholipid, and apolipoproteins, are then further processed in the Golgi apparatus before being released by the enterocytes through exocytosis. As yet, little is known of the factors regulating the formation and release of these chylomicrons by the enterocytes. Although apolipoprotein B is a prerequisite for the formation of chylomicrons, the question of whether its supply is rate limiting for chylomicron formation remains to be demonstrated. Other factors that may play a role in chylomicron formation are luminal phospholipid supply, Ca2+, and microtubules. Chylomicrons and very low-density lipoproteins are probably produced by the enterocytes via different pathways. For example, Pluronic L-81, a hydrophobic surfactant, affects only chylomicron formation and has little effect on very low-density lipoprotein production. The movement of chylomicrons from the intercellular space through the basement membrane to the lamina propria is not fully understood. Once inside the lamina propria, the movement of chylomicrons is probably by diffusion and is greatly facilitated by interstitial hydration; thus the lymphogogic effect of fat absorption may serve an important function for the transfer of chylomicrons from the enterocytes to the lacteal.

Animals↗

Public-private partnerships in the siting of hazardous waste facilities: the importance of trust.

As the need for hazardous waste management facilities becomes critical, several strategies including public-private partnership have been suggested and adopted by states to diffuse public opposition, and facilitate the siting of these facilities. Public-private partnership involves state ownership of the facility, and its operation by a private company. Proponents of this strategy contend that state ownership of facility and therefore an assumption of long-term liability would reduce public concerns regarding site maintenance. Also, that state or public ownership of the land on which the facility is built would thwart any local land use regulation that may be enacted to derail the site selection process. This paper, through a case study provides an analysis of this strategy, and surmised that factors identified in past research-public trust in institutions of government, in the perceived competence and integrity of the developer, and in the waste treatment technology to pose minimal risks to public health and the environment, are necessary to enhance the efficacy of public-private partnership in the siting of hazardous waste management facilities.

Decision Making↗

The controversial nuclear matrix: a balanced point of view.

The nuclear matrix is defined as the residual framework after the removal of the nuclear envelope, chromatin, and soluble components by sequential extractions. According to several investigators the nuclear matrix provides the structural basis for intranuclear order. However, the existence itself and the nature of this structure is still uncertain. Although the techniques used for the visualization of the nuclear matrix have improved over the years, it is still unclear to what extent the isolated nuclear matrix corresponds to an in vivo existing structure. Therefore, considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the situation in living cells. Here, we summarize the experimental evidence in favor of, or against, the presence of a diffuse nucleoskeleton as a facilitating organizational nonchromatin structure of the nucleus.

Animals↗

Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors.

The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABAA receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABAA receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABAA receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABAA receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABAA receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABAA receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABAA receptors, facilitating their accumulation at inhibitory synapses.

Animals↗

Polypeptide delivery across the blood-brain barrier.

The blood-brain barrier (BBB) used to be considered impermeable to polypeptides. However, this view has evolved rapidly over the past two decades. Not only do polypeptides have the potential to serve as carriers for selective therapeutic agents, but they themselves may directly cross the BBB after delivery into the bloodstream to become potential treatments for a variety of CNS disorders, including neurodegeneration, autoimmune diseases, stroke, depression, and obesity. The interactions of polypeptides with the BBB can take many forms, such as simple diffusion, saturable transport, or facilitation of entry of another peptide or protein. In some instances, interactions in the blood compartment (outside the BBB) or within the endothelial cells (at the BBB level) can significantly impede the passage of polypeptides across the BBB. We shall review the different aspects of interactions between peptides/proteins and the BBB that affect their delivery as potential drugs in their natural form, and discuss recent advances in the cell biology of polypeptide transport across the BBB. Better understanding of the BBB will provide insight and direction for future research in the treatment of CNS disorders.

Animals↗

Derivatives of melphalan designed to enhance drug accumulation in cancer cells.

The objective of this study was to develop chemical strategies to improve the uptake and accumulation of melphalan (L-Mel and D-Mel), a cytotoxic agent, into cancer cells. Dipeptides synthesized from L- (or D-) Mel and L-glutamic acid (L-Glu) or L-valine (L-Val) and their methyl or ethyl esters (all compounds were trifluoroacetic acid salts) were evaluated for cytotoxicity and cellular uptake using Caco-2 cells, a human colon carcinoma cell line, and RT-2 cells, a rat brain glioma cell line. Treatment of Caco-2 cells with L-Mel or D-Mel (0.5 mg/ml equivalent of melphalan) for 48 h resulted in approximately 50% cell survival. Treatment of the Caco-2 cells with dipeptide derivatives of L-Mel (or D-Mel) (11c-d, 12c-d and 13) caused similar cytotoxicity effects (approximately 50-70% of cell survival). When the cytotoxicities of the esters of L-Mel, D-Mel and their dipeptide derivatives (11a-b, 12a-b and 14) in Caco-2 cells were determined, less than 10% cell survival was observed. Similar results were observed in RT-2 cells. When the cellular uptake properties of these compounds were determined in Caco-2 cell monolayers, L-Glu-L-Mel (12c), L-Glu-D-Mel (12d), and L-Mel-L-Glu (11c) generated slightly lower intracellular levels of L-Mel or D-Mel than when the cell monolayer was treated with the amino acids (L-Mel or D-Mel). In Caco-2 cells treated with 11c, 12c or 12d, low levels of the dipeptides were also detected. Caco-2 cell monolayers treated with D-Mel-L-Glu (11d) or D-Mel-L-Val (13) showed very low levels of the amino acids (L-Mel or D-Mel), but generally higher levels of the dipeptides. In contrast to the amino acids (L-Mel, D-Mel) or the dipeptide derivatives (11c-d, 12c-d and 13), the ester derivatives of the amino acids [L-Mel(OEt), D-Mel(OEt)] or the dipeptides (11a-b, 12a-b and 14) produced 5-20 times higher intracellular concentrations of potentially cytotoxic metabolites (e.g., L-Mel, D-Mel, Mel-containing dipeptides or Mel-containing dipeptide monoesters). L-Mel(OEt), D-Mel(OEt), L-Glu(OEt)-L-Mel(OEt) (12a), L-Glu(OEt)-D-Mel(OEt) (12b), and L-Mel-L-Glu(OEt)2 (11a) accumulated mainly as either L-Mel or D-Mel, and the percentages of L-Mel or D-Mel were 99%, 99%, 90%, 75% and 98% of the total intracellular concentration of potentially cytotoxic agents, respectively. D-Mel-L-Glu(OEt)2 (11b) accumulated as its monoester (> 95%) and D-Mel-L-Val(OMe) (14) accumulated as its dipeptide metabolite (> 98%). Inclusion of Gly-Pro, carnosine, L-Phe or L-Glu did not inhibit uptake of the dipeptide derivatives of L-Mel (or D-Mel) or their esters. These results suggest that the cellular uptake of the dipeptide derivatives of melphalan and their esters is probably via passive diffusion rather than being facilitated by an amino acid transporter or a di/tripeptide transporter. The higher intracellular levels of cytotoxic agents generated from the ester derivatives of the amino acids and the dipeptides are probably due to their higher lipophilicity and the overall neutral charge of the esters and subsequent intracellular formation of the more polar amino acids (L- or D-Mel) and/or Mel-containing dipeptides. Finally, these studies suggest that dipeptides of D-Mel [11b, 11d, 13] have inherent cytotoxicity properties.

Animals↗

NIH conference. Respiratory disease in the immunosuppressed patient.

Pulmonary complications, both infectious and noninfectious, are an important cause of morbidity in patients with various types of immunosuppression. The appropriate response to these clinical problems requires an understanding of pulmonary host defense and of the various types of systemic immunosuppression. Infectious and noninfectious pulmonary complications may vary according to the type of immunosuppression as well as to the degree and duration of immunosuppression. Appropriate clinical management also requires an understanding of the clinical problems commonly seen in specific groups of immunosuppressed patients and an understanding of the sensitivity, specificity, and potential complications associated with the available diagnostic approaches to those patients. Because respiratory disease in these patient groups may progress rapidly to respiratory failure, an expeditious evaluation based on the knowledge of likely causes of respiratory disease and prompt specific or empiric therapy are indicated. Specific sets of algorithms for the evaluation of both focal and diffuse pulmonary disease may facilitate such an evaluation. In addition, an aggressive approach to the prevention of pulmonary disease including immunization, prophylaxis, and immunomodulation (for example, colony stimulating factors) may be warranted in specific subgroups at risk.

Algorithms↗

Upflow anaerobic sludge blanket reactor--a review.

Biological treatment of wastewater basically reduces the pollutant concentration through microbial coagulation and removal of non-settleable organic colloidal solids. Organic matter is biologically stabilized so that no further oxygen demand is exerted by it. The biological treatment requires contact of the biomass with the substrate. Various advances and improvements in anaerobic reactors to achieve variations in contact time and method of contact have resulted in development of in suspended growth systems, attached growth or fixed film systems or combinations thereof. Although anaerobic systems for waste treatment have been used since late 19th century, they were considered to have limited treatment efficiencies and were too slow to serve the needs of a quickly expanding wastewater volume, especially in industrialized and densely populated areas. At present aerobic treatment is the most commonly used process to reduce the organic pollution level of both domestic and industrial wastewaters. Aerobic techniques, such as activated sludge process, trickling filters, oxidation ponds and aerated lagoons, with more or less intense mixing devices, have been successfully installed for domestic wastewater as well as industrial wastewater treatment. Anaerobic digestion systems have undergone modifications in the last two decades, mainly as a result of the energy crisis. Major developments have been made with regard to anaerobic metabolism, physiological interactions among different microbial species, effects of toxic compounds and biomass accumulation. Recent developments however, have demonstrated that anaerobic processes might be an economically attractive alternative for the treatment of different types of industrial wastewaters and in (semi-) tropical areas also for domestic wastewaters. The anaerobic degradation of complex, particulate organic matter has been described as a multistep process of series and parallel reactions. It involves the decomposition of organic and inorganic matter in the absence of molecular oxygen. Complex polymeric materials such as polysaccharides, proteins, and lipids (fat and grease) are first hydrolyzed to soluble products by extracellular enzymes, secreted by microorganisms, so as to facilitate their transport or diffusion across the cell membrane. These relatively simple, soluble compounds are fermented or anaerobically oxidized, further to short-chain fatty acids, alcohols, carbon dioxide, hydrogen, and ammonia. The short-chain fatty acids (other than acetate) are converted to acetate, hydrogen gas, and carbon dioxide. Methanogenesis finally occurs from the reduction of carbon dioxide and acetate by hydrogen. The initial stage of anaerobic degradation, i.e. acid fermentation is essentially a constant BOD stage because the organic molecules are only rearranged. The first stage does not stabilize the organics in the waste. However this step is essential for the initiation of second stage methane fermentation as it converts the organic material to a form, usable by the methane producing bacteria. The second reaction is initiated when anaerobic methane forming bacteria act upon the short chain organic acids produced in the 1st stage. Here these acids undergo methane fermentation with carbon dioxide acting as hydrogen acceptor and getting reduced to methane. The methane formed, being insoluble in water, escapes from the system and can be tapped and used as an energy source. The production and subsequent escape of methane causes the stabilization of the organic material. The methane-producing bacteria consist of several different groups. Each group has the ability to ferment only specific compounds. Therefore, the bacterial consortia in a methane producing system should include a number of different groups. When the rate of bacterial growth is considered, then the retention time of the solids becomes important parameter. The acid fermentation stage is faster as compared to the methane fermentation stage. This means that a sudden increase in the easily degradable organics will result in increased acid production with subsequent accumulation of acids. This inhibits the methanogenesis step. Acclimatization of the microorganisms to a substrate has been reported to take more than five weeks. Sufficiently acclimated bacteria have shown greater stability towards stress-inducing events such as hydraulic overloads, fluctuations in temperature, fluctuations in volatile acid and ammonia concentrations etc. Several environmental factors can affect anaerobic digestion, by altering the parameters such as specific growth rate, decay rate, gas production, substrate utilization, start-up and response to changes in input. It has long been recognized that an anaerobic process is in many ways ideal for wastewater treatment and has following merits: A high degree of waste stabilization A low production of excess A low nutrient requirements No oxygen requirement Production of methane gas Anaerobic microorganisms, especially methanogens have a slow growth rate. At lower HRTs, the possibility of washout of biomass is more prominent. This makes it difficult to maintain the effective number of useful microorganisms in the system. To maintain the population of anaerobes, large reactor volumes or higher HRTs are required. This may ultimately provide longer SRTs upto 20 days for high rate systems. Thus, provision of larger reactor volumes or higher HRTs ultimately lead to higher capital cost. Among notable disadvantages, it has low synthesis/reaction rate hence long start up periods and difficulty in recovery from upset conditions. Special attention is, therefore, warranted towards, controlling the factors that affect process adversely; important among them being environmental factors such as temperature, pH and concentration of toxic substances. The conventional anaerobic treatment process consists of a reactor containing waste and biological solids (bacteria) responsible for the digestion process. Concentrated waste (usually sewage sludge) can be added continuously or periodically (semi-batch operation), where it is mixed with the contents of the reactor. Theoretically, the conventional digester is operated as a once-through, completely mixed, reactor. In this particular mode of operation the hydraulic retention time (HRT) is equal to the solids retention time (SRT). Basically, the required process efficiency is related to the sludge retention time (SRT), and hence longer SRT provided, results in satisfactory population (by reproduction) for further waste stabilization. By reducing the hydraulic retention time (HRT) in the conventional mode reactor, the quantity of biological solids within the reactor is also decreased as the solids escape with the effluent. The limiting HRT is reached when the bacteria are removed from the reactor faster than they can grow. Methanogenic bacteria are slow growers and are considered the rate-limiting component in the anaerobic digestion process. The first anaerobic process developed, which separated the SRT from the HRT was the anaerobic contact process. In 1963, Young and McCarty (1968) began work, which eventually led to the development of the anaerobic upflow filter (AF) process. The anaerobic filter represented a significant advance in anaerobic waste treatment, since the filter can trap and maintain a high concentration of biological solids. By trapping these solids, long SRT's could be obtained at large waste flows, necessary to anaerobically treat low strength wastes at nominal temperatures economically. Another anaerobic process which relies on the development of biomass on the surfaces of a media is an expanded bed upflow reactor. The primary concept of the process consists of passing wastewater up through a bed of inert sand sized particles at sufficient velocities to fluidize and partially expand the sand bed. One of the more interesting new processes is the upflow anaerobic sludge blanket process (UASB), which was developed by Lettinga and his co-workers in Holland in the early 1970's. The key to the process was the discovery that anaerobic sludge inherently has superior flocculation and settling characteristics, provided the physical and chemical conditions for sludge flocculation are favorable. When these conditions are met, a high solids retention time (at high HRT loadings) can be achieved, with separation of the gas from the sludge solids. The UASB reactor is one of the reactor types with high loading capacity. It differs from other processes by the simplicity of its design. UASB process is a combination of physical & biological processes. The main feature of physical process is separation of solids and gases from the liquid and that of biological process is degradation of decomposable organic matter under anaerobic conditions. No separate settler with sludge return pump is required, as in the anaerobic contact process. There is no loss of reactor volume through filter or carrier material, as in the case with the anaerobic filter and fixed film reactor types, and there is no need for high rate effluent recirculation and concomitant pumping energy, as in the case with fluidized bed reactor. Anaerobic sludge inherently possesses good settling properties, provided the sludge is not exposed to heavy mechanical agitation. For this reason mechanical mixing is generally omitted in UASB-reactors. At high organic loading rates, the biogas production guarantees sufficient contact between substrate and biomass. Regarding the dynamic behaviour of the water phase UASB reactor approaches the completely mixed reactor. For achieving the required sufficient contact between sludge and wastewater, the UASB-system relies on the agitation brought about by the natural gas production and on an even feed inlet distribution at the bottom of the reactor. (ABSTRACT TRUNCATED)

Bacteria, Anaerobic↗

The real new economy.

During the soar-and-swoon days of the late 1990s, many people believed that information technology, and the Internet in particular, were "changing everything" in business. A fundamental change did happen in the 1990s, but it was less about technology than about competition. Under director Diana Farrell, the McKinsey Global Institute has conducted an extensive study of productivity and its connection to corporate IT spending and use during that period. The study revealed that information technology is important--but not central--to the fate of industries and individual companies. So if information technology was not the primary factor in the productivity surge, what was? The study points to competition and innovation. In those industries that saw increases in competitive intensity, managers were forced to innovate aggressively to protect their revenues and profits. Those innovations--in products, business practices, and technology--led to the gains in productivity. In fact, a critical dynamic of the new economy--the real new economy--is the virtuous cycle of competition, innovation, and productivity growth. Managers can innovate in many ways, but during the 1990s, information technology was a particularly powerful tool, for three reasons: First, IT enabled the development of attractive new products and efficient new business processes. Second, it facilitated the rapid industrywide diffusion of innovations. And third, it exhibited strong scale economies--its benefits multiplied rapidly as its use expanded. This article reveals surprising data on how various industries in the United States and Europe were affected by competition, innovation, and information technology in the 1990s and offers insights about how managers can get more from their IT investments.

Commerce↗

Rejection of trace organic compounds by high-pressure membranes.

High-pressure membranes, encompassing reverse osmosis (RO), nanofiltration (NF), and low-pressure RO, may provide an effective treatment barrier for trace organic compounds including disinfection by-products (DBPs), pesticides, solvents, endocrine disrupting compounds (EDCs) and pharmaceutically active compounds (PhACs). The objective is to develop a mechanistic understanding of the rejection of trace organic compounds by high-pressure membranes, based on an integrated framework of compound properties, membrane properties, and operational conditions. Eight trace organic compounds, four DBPs and four chlorinated (halogenated) solvents, are being emphasized during an initial study, based on considerations of compound properties, occurrence, and health effects (regulations). Four polyamide FilmTec membranes; three reverse osmosis/RO (BW-400, LE-440, XLE-440) and one nanofiltration/NF (NF-90); are being characterized according to pure water permeability (PWP), molecular weight cutoff (MWCO), hydrophobicity (contact angle), and surface charge (zeta potential). It is noteworthy that rejections of compounds of intermediate hydrophobicity by the candidate membranes were observed to be less than salt rejections reported for these membranes, suggesting that transport of these solutes through these membranes is facilitated by solute-membrane interactions. We are continuing with diffusion cell measurements to describe solute-membrane interactions by estimation of diffusion coefficients through membranes pores, either hindered or facilitated.

Diffusion↗

Drug dissolution into micellar solutions: development of a convective diffusion model and comparison to the film equilibrium model with application to surfactant-facilitated dissolution of carbamazepine.

The intrinsic dissolution rate of carbamazepine in solutions of sodium lauryl sulfate was measured to study the convective diffusion transport of drug-loaded micelles from a rotating disk. Alternative definitions for effective diffusivity and reaction factor are presented and compared with those commonly used for this type of transport problem. The conventional and alternative approaches are based on the same fundamental assumptions differing only in their interpretation of the diffusional boundary layer. For example, in this study it was observed that, above the cmc, a 2% w/v solution of sodium lauryl sulfate increased the dissolution rate approximately 6-fold and the solubility approximately 20-fold. This difference between the solubility and dissolution enhancement was attributed to the contribution to the total transport of both the enhanced solubility, a 20-fold increase, and the effective diffusivity of the drug-micelle complex, a 3-fold decrease, hence a net 6-fold increase in dissolution. The diffusivity of the drug-loaded micelle estimated from the dissolution data using the new definitions compared well with values determined by other methods (Dsm = 8.4 x 10(-7) cm2/s). On the basis of these results, the new definitions for the effective diffusivity and reaction factor offer a practical method for estimating micellar diffusion coefficients and predicting drug dissolution under the well-defined hydrodynamics of the rotating disk. It may also be possible to extend the application of these definitions to study the dissolution of water-insoluble drugs in other media, such as emulsions, to better understand drug dissolution under fed conditions in vivo.

Carbamazepine↗

Flow-enhanced epitaxial ordering of brush-like macromolecules on graphite.

Long-range orientational order in monolayers of brush-like macromolecules was achieved during spreading of a polymer melt on the surface of highly oriented pyrolytic graphite. The combination of wetting-induced flow and epitaxial adsorption of poly(n-butylacrylate) side chains on graphite led to the formation of large domains of uniaxially oriented rodlike molecules. The domain size varied from ca. 1 to 10 microm which is noticeably larger than the submicrometer-sized mosaic domains typically observed upon adsorption from solution. The increase in the degree of order is attributed to the flow-enhanced diffusion of the macromolecules within spreading monolayers which facilitates the epitaxial alignment of the large macromolecules. The diffusion coefficient was shown to increase linearly with the spreading rate. Even though the ordering occurred during flow, no correlation was observed between the molecular orientation and the flow direction. Thus, the role of the flow was not to induce the molecular orientation but to facilitate the intrinsic ordering process. This finding can inspire and lead to new strategies for constructing large scale ordered structures on surfaces.

Journal Article↗

Preferential accumulation of GABAA receptor gamma 2L, not gamma 2S, cytoplasmic loops at rat spinal cord inhibitory synapses.

Alternative splicing generates two variants of the GABAAR gamma2-subunit, gamma2S and gamma2L, which differ by insertion of the amino acid sequence LLRMFSFK into the large cytoplasmic loop between transmembrane domains 3 and 4. This additional sequence within the GABAAR gamma2L-subunit contains the potential protein kinase C (PKC) phosphorylation site serine 343 (Ser343). In the present study we intended to determine the capacity of these two splice variants to accumulate at inhibitory synaptic terminals and to colocalize with gephyrin, and to find out whether phosphorylation of Ser343 has any effect on GABAAR distribution. Green fluorescent protein (GFP)-tagged large cytoplasmic loops of GABAAR gamma2S and gamma2L (GFP::gamma2S/L) were used as surrogates for full-length receptors to study the function of the individual gamma2S and gamma2L peptides in transfected spinal cord neurones (SCNs) and COS-7 cells. It was found that GFP::gamma2L displayed a significantly higher capacity to accumulate at inhibitory synapses than GFP::gamma2S. GABAAR GFP::gamma2S accumulation at inhibitory postsynaptic sites was suppressed to the extent that GFP::gamma2S assumed a diffuse cytosolic distribution. PKC activation facilitated the postsynaptic clustering of GFP::gamma2L but not of GFP::gamma2S. This required the Ser343 residue, since substituting Ala343 for Ser343 produced a diffuse cytosolic localization pattern, like that of GFP::gamma2S. Furthermore, upon PKC activation Discosoma Red2-tagged GABAAR gamma2L (DsRed 2::gamma2L) colocalized with gephyrin in transfected COS-7 cells. These results support the idea that alternative splicing regulates the access of GABAARs to inhibitory postsynaptic sites in a Ser343 phosphorylation-regulated way.

Amino Acid Substitution↗

Gap junctions with varied permeability properties establish cell-type specific communication pathways in the rat seminiferous epithelium.

Dye coupling experiments were performed to determine whether the gap junctions connecting Sertoli cells with other Sertoli cells and different germ cell stages in rats showed functional variations. Chop loading of adult rat seminiferous tubules was conducted using fluorescent dextran controls and a variety of low-molecular-weight tracers (lucifer yellow, biotin-X-cadaverine, biotin cadaverine, and neurobiotin) to evaluate dye coupling in situ, and scrape loading was used to study dye coupling in Sertoli-germ cell cocultures established using prepuberal rats. Sertoli-Sertoli coupling is relatively short range and nonselective in situ, whereas coupling between Sertoli cells and chains of spermatogonia is strongly selective for the positively charged biotin tracers relative to negatively charged lucifer yellow. Coupling between Sertoli cells and spermatogonia was also asymmetric; lucifer yellow in germ cells never diffused into Sertoli cells, and biotinylated tracers only weakly diffused from spermatogonia to Sertoli cells. Asymmetric coupling would facilitate the concentration in germ cells of molecules diffusing through junctions from Sertoli cells. Dye coupling between Sertoli cells and adluminal germ cells was too weak to detect by fluorescence microscopy, suggesting that the junctional communication between these cells may be functionally different from that between Sertoli and basal germ cells. The results show that there are multiple routes of gap junction communication in rat seminiferous tubules that differ in permeability properties and show alternative gating states. Functional diversity of gap junctions may permit regulated communication among the many interacting Sertoli cells and germ cell stages in the seminiferous epithelium.

Animals↗

Direct membrane method for the study of interface-controlled transport of cholesterol in aqueous media.

Studies were designed to demonstrate the use of a silicone rubber membrane diffusion cell in the mechanistic study of cholesterol mass transfer in aqueous media. The method is shown to be simple, precise, and well suited for delineating conditions which facilitate cholesterol transport. Traditional membrane diffusion resistance was determined with cholesterol solubilized in the nonionic surfactant, polyoxyethylene(10)-nonylphenol ether. The use of a charged surfactant additive, either sodium oleate or benzyldimethyltetradecylammonium chloride, reduced cholesterol membrane flux in a manner consistent with a transport barrier residing in the membrane and micelle interfacial regions. Quantitative determination of total transport resistance was good (CV of greater than 95%) for cases more than 99% interface controlled. Interfacial resistance imparted by the charged surfactant additive was essentially abolished by strong electrolyte (sodium chloride). Electrolyte was utilized in either the upstream or the downstream aqueous compartment to enhance cholesterol transport by a mechanism which is consistent with a marked increase in the frequency of micelle collision with the corresponding membrane surface. When the downstream interfacial component of total transport resistance was "short circuited" by electrolyte in sequential transport runs using the same membrane, a "dumping" of cholesterol by the membrane compartment was observed. Limited studies with a second nonionic surfactant, polyoxyethylene(15)-tridecyl ether, suggest that the structure of separate micelle components may also be related to cholesterol mass transfer which occurs via a micelle collision in the interfacial region.

Cholesterol↗

Diffusion of tricarboxylic acid cycle enzymes in the mitochondrial matrix in vivo. Evidence for restricted mobility of a multienzyme complex.

It has been proposed that enzymes in many metabolic pathways, including the tricarboxylic acid cycle in the mitochondrial matrix, are physically associated to facilitate substrate channeling and overcome diffusive barriers. We have used fluorescence recovery after photobleaching to measure the diffusional mobilities of chimeras consisting of green fluorescent protein (GFP) fused to the C terminus of four tricarboxylic acid cycle enzymes: malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinyl-CoA synthetase. The GFP-enzyme chimeras were localized selectively in the mitochondrial matrix in transfected Chinese hamster ovary (CHO) and COS7 cells. Laser photobleaching using a 0.7-microm diameter spot demonstrated restricted diffusion of the GFP-enzyme chimeras. Interestingly, all four chimeras had similar diffusional characteristics, approximately 45% of each chimera was mobile and had a diffusion coefficient of 4 x 10(-8) cm(2)/s. In contrast, unconjugated GFP in the mitochondrial matrix (targeted using COX8 leader sequence) diffused freely (nearly 100% mobility) with a greater diffusion coefficient of 20 x 10(-8) cm(2)/s. The mobility of the GFP-enzyme chimeras was insensitive to substrate source, ATP depletion, or inhibition of the adenine nucleotide translocase. These results indicate similar mobility characteristics of unrelated tricarboxylic acid cycle enzymes having different sizes and physical properties, providing biophysical evidence for a diffusible multienzyme complex in the mitochondrial matrix.

Animals↗