Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

Buffalo Herd Structure and its Repercussions for Condition of Individual African Buffalo Cows

The African buffalo Syncerus caffer was studied in Lake Manyara National Park, Tanzania. Emphasis was placed on the study of (individual) buffalo cows, which live in mixed herds. Buffalo herds are discrete social units and females were never observed in another herd than their own. The herd showed a structure with respect to the distribution of sex-age classes. Individual cows generally kept the same location within the herd. The location in the herd appeared to be coupled to food intake and was strongly related to physical condition. The best location (highest intake and best condition) was between the front and the centre of the herd, the worst location was the rear of the herd (when moving or grazing). Females with calves appeared to have the highest position in the hierarchy as determined from the rate of displacement over food; adult bulls did not interact with cows. Few births were observed during the late dry season and it appears that there is a calving peak at the end of the long rains. Conception rate increased when cows increased in condition and dropped when cows lost condition. Cows showed a strong seasonality in condition but bulls on average hardly changed in condition, except for a loss in condition during the inferred conception peak. Buffalo herds in Manyara showed a fusion-fission pattern independent of season but strongly influenced by the size of the herd: large herds split more often than smaller ones. In large herds, buffalo grazed closer together than in small herds and it appeared likely that competition was more severe in large herds. Animals in the rear of a large herd lost condition faster during the dry season than animals in the best location in the herd, and especially cows in the rear split off most frequently from the herd to graze in a smaller fragment. From the literature on cattle, it is inferred that the reproductive success of cows in the rear of the herd will be lower than of cows in the best location, and this differential is confirmed by the behaviour of adult bulls. It is as yet unclear what the advantage is for adult cows in the rear of a large herd to stay in that herd but the sharing of information with more successful individuals seems a good candidate.     

Mapping the diabetes polygene Idd3 on mouse Chromosome 3 by use of novel congenic strains

Development of novel congenic mouse strains has allowed us to better define the location of the diabetogenic locus, Idd3, on Chromosome (Chr) 3. Congenic strains were identified by use of published and newly developed microsatellite markers, their genomes fingerprinted by a rapid, fluorescence-based approach, and their susceptibility to type 1 diabetes evaluated. The maximum interval containing Idd3 is now approximately 4 cM.     

Kinetic parameters of lactate dehydrogenases of some rumen bacterial species,the anaerobic ciliate Isotricha prostoma and mixed rumen microorganisms

A number of kinetic parameters of the lactate dehydrogenases of three rumen bacterial species (Peptostreptococcus productus, Propionibacterium acnes and Actinomyces viscosus), the rumen ciliate Isotricha prostoma and mixed rumen microorganisms (MRM) with respect to NADH, pyruvate, fructose-1,6-diphosphate (FDP) as well as the effects of several nucleotide phosphates were studied.Partially purified LDH of Peptostr. productus had the same kinetic parameters as in crude cell free extracts. Values for K_m, determined by Michaelis-Menten kinetics with pyruvate as the substrate, were in the same range for all lactate dehydrogenases. After feeding a cow, changes in the apparent K_m and V_max values for NADH of the total LDH activity in MRM were followed.It is suggested that of the factors studied the ratio NADH/NAD(H) and ATP are the most important regulatory factors for the lactate dehydrogenases of mixed rumen microorganisms.The investigation were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO)     

Inhibition of inward rectifying tonoplast channels by a vacuolar factor: Physiological and kinetic implications

Summary Regulation of ion-channel activity must take place in order to regulate ion transport. In case of tonoplast ion channels, this is possible on both the cytoplasmic and the vacuolar side. Isolated vacuoles of youngVigna unguiculata seedlings show no or hardly any channel activity at tonoplast potentials >80 mV, in the vacuole-attached configuration. When the configuration is changed to an excised patch or whole vacuole, a fast (excised patch) or slow (whole vacuole) increase of inward rectifying channel activity is seen. This increase is accompanied by a shift in the voltage-dependent gating to less hyperpolarized potentials. In the whole vacuole configuration the level of inward current increases and also the activation kinetics changes. Induction of channel activity takes up to 20 min depending on the age of the plants used and the diameter of the vacuole. On the basis of the estimated diffusion velocities, it is hypothesized that a compound with a mol wt of 20,000 to 200,000 is present in vacuoles of young seedlings, which shifts the population of channels to a less voltage-sensitive state.Ecotrans publication no. 27.     

Plasmalemma patch clamp experiments in plant root cells: procedure for fast isolation of protoplasts with minimal exposure to cell wall degrading enzymes

Summary A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato (Lycopersicon esculentum) andPlantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.Abbreviations BSA bovine serum albumin - BTP bis-tris propane - CAP cell-attached patch - OOP outside out patch - PEG polyethylene glycol - WC whole cell     

Benthic-pelagic flux rates on mussel beds: tunnel and tidal flume methodology compared

Material flux rates in an intertidal mussel bed were measured synchronously over two tidal cycles in June 1989 with Benthic Ecosystem Tunnels and a double lane flume. The tunnels enclosed the near bottom water, whereas the flume canalized the total water column. One tunnel was set up in a mussel bed and another one in an adjacent sand bottom as a control. The flume enclosed a mussel lane and a sand lane. In the tunnel and in the flume the mussel bed revealed ammonium and phosphate discharge. At the same time, phytoplankton, dominated byPhyaeocystis globosa, was taken up intensively. These flux rates showed the same tendency but they were higher in the flume than in the tunnel. Different tendencies and flux rates for oxygen and particulate organic matter (POC, PN) were found in flume and tunnel. These differences demonstrate the importance of water column processes regarding the material exchange of a mussel bed. Tunnels enclose smaller bodies of water and are therefore expected to detect even small effects of the benthos on the passing water. In flumes, benthic influence may be diluted over the entire water column but conditions are more natural. The use of flumes is restricted to shallow waters while tunnels have the potential to be used at any depth.     

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi.

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.     

Membrane potentials of vallisneria leaf cells and their relation to photosynthesis

A study has been made of the effects of the inhibitors carbonylcyanide m-chlorophenylhydrazone (CCCP), 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), and of anoxia on the light-sensitive membrane potential of Vallisneria leaf cells. The present results are compared with the known effects of these inhibitors on ion transport and photosynthesis (Prins 1974 Ph.D thesis). The membrane potential is composed of a diffusion potential plus an electrogenic component. The electrogenic potential is about −13 millivolts in the dark and −80 millivolts in the light. The inhibitory effect of DCMU and CCCP on the electrogenic mechanisms strongly depends on the light intensity used, the inhibition being less at a higher light intensity. This is of significance in view of the often conflicting results obtained with these inhibitors. With ion transport in Vallisneria the electrogenic pump derives its energy from phosphorylation; however, the process which causes the initial light-induced hyperpolarization and the process that keeps the membrane potential at a steady hyperpolarized state in the light have different energy requirements. The action of photosystem I alone is sufficient to induce the initial hyperpolarization. For continuous operation in the light the activity of photosystem II also is needed.