Construction of Partially Balanced Incomplete Block Designs with Nested Rows and Columns

A number of methods of construction of partially balanced incomplete block designs with nested rows and columns are developed and new balanced incomplete block designs with nested rows and columns are obtained as a by-product.     

Quantitative gap junction alterations in mammalian heart cells quickly frozen or chemically fixed after electrical uncoupling

Summary The gap junction morphology was quantified in freeze-fracture replicas prepared from rat auricles that had been either quickly frozen at 6 K or chemically fixed by glutaraldehyde, in a state of normal cell-to-cell conduction or in a state of electrical uncoupling. The general appearance of the gap junctions was similar after both preparative procedures. A quantitative analysis of three gap junctional dimensions provided the following measurements in the quickly frozen conducting auricles (mean±sd): (a) P-face particles' diameter 8.27±0.74 nm (n =5709), (b) P-face particles' center-to-center distance 10.78±2.12 nm (n=4800), and (c) E-face pits' distance 9.99±2.19 nm (n=1600). Corresponding values obtained from chemically fixed tissues were decreased by about 3% for the particle's diameter and about 5% for the particles' and pits' distances. Electrical uncoupling by the action of either 1 mM 2–4-dinitrophenol (DNP), or 3.5 mMn-Heptan-1-ol (heptanol), induced a decrease of the particle's diameter, which amounted to –0.69±0.01 nm (mean ±se) in the quickly frozen preparations and –0.71±0.01 nm in the chemically fixed ones. The particles' distance was decreased by –0.96±0.04 nm in the quickly frozen samples and by –0.90 ±0.03 nm in the chemically fixed ones and the E-face pits' distance was similarly reduced. All differences were statistically significant (P<0.001 for all dimensions). Electrical recoupling after the heptanol effect promoted a return of these gap junctional dimensions towards normal values, which was about 50% complete within 20 min. It is concluded that very similar morphological alterations of the gap junctional structure are induced in the mammalian heart by different treatments promoting electrical uncoupling and that these conformational changes appear independently of the preparative procedure. The suggestion that the observed decrease of the particles' diameter is genuinely related to the closing mechanism of the unit cell-to-cell channel set in thei centers is thus confirmed.     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Influence of single amino acids on the development of hamster one-cell embryos in vitro

One-cell hamster embryos placed in culture have always shown a complete block to development at the two-cell stage. In a preliminary study using a chemically defined culture medium containing 20 amino acids (HECM-1), many one-cell embryos were able to escape the "two-cell block" and develop to the four-cell stage. Use of a simpler formulation containing only the amino acids hypotaurine and glutamine revealed marked inhibitory and stimulatory effects of adding the other amino acids. In the first experiment, 19 amino acids were separately examined for effects on one-cell embryo development. Six amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited embryo development (reduced mean cell number; MCN), and three others (glycine, cystine, and lysine) stimulated development (increased MCN), compared with basic medium containing only glutamine and hypotaurine (low control). When the responses with the six inhibitory amino acids were totalled, only 3 of 185 (2%) one-cell embryos reached the six-or seven-cell stage compared to a total of 15 of 76 (20%) embryos that developed to these stages using the three stimulatory amino acids. When tested together in a second experiment, the six inhibitory amino acids significantly reduced the MCN, from 4.28 +/- 0.44 (low control) to 3.71 +/- 0.55. In this group, 17 of 117 (15%) of one-cell embryos reached more than four-cell and only 4 of 117 (3%) reached six- or 7-cell stages, compared with 39 of 117 (33%) and 12 of 117 (10%), respectively, for the basal medium group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimating denitrification rates in estuarine sediments: A comparison of stoichiometric and acetylene based methods

We compared denitrification rates obtained using an adaptation of the acetylene block technique to rates estimated from benthic flux nutrient stoichiometry in the subtidal sediments of Tomales Bay, California (USA). By amending whole cores with acetylene and saturating nitrate concentrations, we obtained potential denitrification rates, which ranged between 4 and 30 mmol N m^–2 d^–1. We determined the apparent Michaelis constant (K_app) and the maximum potential rate (V_mp) of the denitrifying community and used these constants in a rectangular hyperbola to estimatein situ denitrification rates. Both the K_app and V_mp of the denitrifying community exhibited significant variation over both depth in the sediment column and time of sampling.Estimates ofin situ denitrification obtained using our kinetic-fix adaptation of the acetylene block ranged between 1.8 (March) and 9 (Sept.) mmol N m^–1 d^–1. Denitrification rates obtained using benthic flux stoichiometry ranged between 0.7 and 4.1 mmol N m^–2 d^–1. Average denitrification rates obtained using the kinetic-fix acetylene block approach exceeded those obtained from net benthic flux stoichiometry; however, these differences were not significant. We conclude that our kinetic-fix adaptation of the acetylene block technique provides realistic estimates of denitrification in sediments, even when pore water nitrate concentrations are low and nitrification and denitrification are closely coupled.     

Nutritional environment of soil and roots in and around mycelial blocks of an ectomycorrhizal fungusTricholoma bakamatsutake in an evergreen Fagaceae forest

In the evergreen Fagaceae forests of Japan, an ectomycorrhizal fungusTricholoma bakamatsutake forms shiros or developing mycelial blocks. To determine the physiological characteristics of the mycelial blocks, organic acids of the soil and major nutrient elements of the soil and roots were compared at three types of sites: presently colonized mycelial blocks, previously colonized sites behind the blocks, and uncolonized sites in front of the blocks. The upper part of the mycelial blocks showed the following features compared with the uncolonized site: lower pH (5.1), higher concentrations of oxalic and gluconic acids, lower content of total nitrogen, a similar amount of total carbon, reduced total and available phosphorus, higher content of total calcium and lower content of exchangeable calcium. These findings suggested that the activity of the fungus led to soil acidification by the organic acids, an increase in C/N ratio, depletion of phosphorus and accumulation of calcium.     

Denitrification measurements in aquatic sediments: A comparison of three methods

Measurements of denitrification using the acetylene inhibition,¹⁵N isotope tracer, and N₂ flux methods were carried out concurrently using sediment cores from Vilhelmsborg sø, Denmark, in an attempt to clarify some of the limitations of each technique. Three experimental treatments of overlying water were used: control, nitrate enriched, and ammonia enriched water. The N₂ flux and¹⁵N tracer experiments showed high rates of coupled nitrification/denitrification in the sediments. The acetylene inhibition method did not capture any coupled nitrification/denitrification. This could be explained by acetylene inhibition of nitrification. A combined¹⁵N tracer/acetylene inhibition experiment demonstrated that acetylene inhibition of N₂O reduction was incomplete and the method, therefore, only measured approximately 50% of the denitrification due to nitrate from the overlying water. Similar rates of denitrification due to nitrate in the overlying water were measured by the N₂ flux method and the acetylene inhibition method, after correcting for the 50% efficiency of acetylene inhibition. Rates of denitrification due to nitrate from the overlying water measured by the¹⁵N tracer method, however, were only approximately 35% or less of those measured by the acetylene inhibition or N₂ flux methods.     

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann     

Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation

The ability of parthenogenetically activated mouse eggs to establish a plasma membrane (PM) block to sperm penetration was studied. Zona-free eggs preloaded with Hoechst 33342 were activated by exposure to ethanol or OAG (1-oleoyl-2-acetyl-sn-glycerol) and inseminated after different periods. Eggs challenged with sperm at 30- or 60-min postactivation displayed a fertilization frequency significantly lower than that of control eggs. Conversely, when insemination was carried out at 120-min postactivation, the proportion of fertilized eggs was equivalent to that observed in the control group. Moreover, we report that when the eggs were induced to resume meiosis without any notable loss of CGs (egg exposure to OAG at 100 μM external Ca²⁺ or to heat shock), a normal ability to be penetrated was recorded at 30-min postactivation. Similar behaviour was exhibited by eggs that underwent a CG exocytosis close to that triggered by sperm in absence of nuclear activation (microinjection of inositol 1,4,5-trisphosphate into the egg at 1 μM cytosolic concentration). Present data support the conclusion that parthenogenetically activated mouse eggs are capable of a transitory PM block response that requires both CG exocytosis and meiosis resumption to occur. © 1994 Wiley-Liss, Inc.