Freeze-fracture localization of filipin-sterol complexes in plasma- and cyto-membranes of Pneumocystis carinii

The polyene antibiotic, filipin, was used as the probe for demonstrating sterols in the freeze-fractured plasma- and cytomembranes of Pneumocystis carinii. The distribution of filipin-sterol complexes was homogeneous on the plasma membrane throughout all developmental stages from trophozoite to cyst; however, the density of the complexes gradually decreased with the progress of development. In the trophozoite, the density of the complexes was 485 +/- 42/micron2 on the P face and 341 +/- 27/micron2 on the E face. It was 249 +/- 50 on the P face and 132 +/- 48 on the E face in the precyst and 138 +/- 24 and 59 +/- 20, respectively, in the cyst. The membranes of nucleus, mitochondria, and small round bodies showed more or fewer complexes while no complexes were found in the membranes of one endoplasmic reticulum. In nuclear and mitochondrial membranes, some small scattered clusters of complexes were observed. Two types of vacuoles were distinguished: one having many complexes in its membrane and the other having none at all.     

Freeze-Fracture Study of the Trophozoite and the Cyst of Entamoeba histolytica

ABSTRACT The fine structure of the trophozoite and cyst of Entamoeba histolytica from the stool of a patient was compared using the freeze-fracture method. The intramembranous particles (IMP's) were heterogeneously distributed on the plasma membrane of the trophozoite and their density was 1139 ± 105/μm² on the P face and 27 ± 9/μm² on the E face. Particle-rich depressions and linear particle arrays, reported by other investigators on cultured trophozoites, were also observed on the P face while on the E face such special particle arrangement was not recognized. Particle-free, small protrusions were frequently observed on the P face of the trophozoite membrane. The existence of these protrusions is a new finding. In the cyst, the IMP's were also distributed heterogeneously on both the P and E faces of the plasma membrane. The density of the IMP's, however, was much lower than in the trophozoite: 6 ± 2/μm² on the P face and averaging less than 1/μm² on the E face. In freeze-fracture images, the plasma membrane of the cyst showed a variety of configurations from smooth to uneven or ridged surfaces. These morphological alterations of the plasma membrane may be attributed to the aging of the cyst. The thick wall of the cyst had a filamentous tri- or tetra-lamellar structure. The cytoplasm of the cyst was similar in structure to that of the trophozoite and the diameter of the nuclear pores was equal in both trophozoites and cysts.     

Freeze-fracture study of the trophozoite and the cyst of Entamoeba histolytica

The fine structure of the trophozoite and cyst of Entamoeba histolytica from the stool of a patient was compared using the freeze-fracture method. The intramembranous particles (IMP's) were heterogeneously distributed on the plasma membrane of the trophozoite and their density was 1139 +/- 105/micron 2 on the P face. Particle-rich depressions and linear particle arrays, reported by other investigators on cultured trophozoites, were also observed on the P face while on the E face such special particle arrangement was not recognized. Particle-free, small protrusions were frequently observed on the P face of the trophozoite membrane. The existence of these protrusions is a new finding. In the cyst, the IMP's were also distributed heterogeneously on both the P and E faces of the plasma membrane. The density of the IMP's, however, was much lower than in the trophozoite: 6 +/- 2/micron 2 on the P face and averaging less than 1/micron 2 on the E face. In freeze-fracture images, the plasma membrane of the cyst showed a variety of configurations from smooth to uneven or ridged surfaces. These morphological alterations of the plasma membrane may be attributed to the aging of the cyst. The thick wall of the cyst had a filamentous tri- or tetra-lamellar structure. The cytoplasm of the cyst was similar in structure to that of the trophozoite and the diameter of the nuclear pores was equal in both trophozoites and cysts.     

Freeze-fracture studies on Pneumocystis carinii. I. Structural alteration of the pellicle during the development from trophozoite to cyst

Pneumocystis carinii has generally been distinguished in three developmental stages, namely, trophozoite, precyst and cyst. The fine structure of the pellicle--the plasma membrane and the outer layer existing outside this plasma membrane--of each stage was studied by freeze-fracture technique. By this technique, P. carinii was cleaved through the cytoplasm or through the hydrophobic region of the plasma membrane, and the cross-fractured face of the outer layer was revealed on the replicas. The outer layer, which is electron-dense in the thin section, consisted of numerous fine granules about 15 nm in diameter in freeze-fracture images, whereas the electron-lucent middle layer which appeared in the precyst and cyst was less granular. Measurement of the intramembranous particles (IMP) also was carried out. The number of IMP per square micrometer of the plasma membrane of the trophozoite was 1,512 +/- 125 on the P face and 417 +/- 44 on the E face. In the precyst, the IMP density decreased, and 1,037 +/- 56 on the P face and 262 +/- 22 on the E face. In the cyst, it further decreased, nd 875 +/- 59 and 150 +/- 20 respectively. It is generally assumed that the density of IMP is related to the physiological activity of the cell membrane, so that the present results obtained in P. carinii suggest that the trophozoite is the most active stage, and that metabolic activity of the pellicle gradually decreases with the progress of development to the precyst then to the cyst.     

Endothelial cells of the meningeal capillaries in the rainbow trout: A freeze-fracture study

Meningeal capillaries are unfenestrated. They are made up of endothelial cells that have a pinocytotic caveolae density of 41 ± 11/μm² and 89 ± 21/μm² on the abluminal and luminal sides respectively. The total density of intramembrane particles is not significantly different between the luminal and the abluminal membranes; however, the coefficients of partition are significantly different (P< 0.001). One or two strands of tight junctions occur between adjacent cells but no gap junctions nor desmosomes exist. The density of nuclear pores is less than 3.2/μm². An abundance of intermediate filaments and free vesicles, some of which are seriated, characterize the cytoplasm. The functional significance of these findings is discussed.     

Tritrichomonas foetus: Fine Structure of Freeze-Fractured Membranes1

ABSTRACT. Freeze-fracture techniques reveal differences in fine structure between the anterior three flagella of Tritrichomonas foetus and its recurrent flagellum. The anterior flagella have rosettes of 9–12 intramembranous particles on both the P and E faces. The recurrent flagellum lacks rosettes but has ribbon-like arrays of particles along the length of the flagellum, which may be involved in the flagellum's attachment to the cell body. This flagellum is attached to the membrane of the cell body along a distinct groove that contains few discernible particles. Some large intramembranous particles are visible on the P face of the cell body membrane at the point where the flagellum emerges from the cell body. The randomly distributed particles on the P and E faces of the plasma membrane have a particle density of 919/μm² and 468/μm² respectively, and there are areas on both faces that are devoid of particles. Freeze-fracture techniques also reveal numerous fenestrations in the membrane of the Golgi complex and about 24 pores per μm² in the nuclear. membrane.     

Ordered membrane particles in rhabdomeric microvilli of the housefly (Musca domestica L.)

Rhabdomeric microvilli of the housefly were freeze-fractured (FF) and thin sectioned (TS) for ultrastructural examination. Ordered files of closely packed membrane particles (82 Å wide, 250 Å long) were seen (FF) on the microvillar membrane (usually E face). The long axis of each particle was canted about 45° to that of the microvillus. Occasionally particles in this array appeared on the P face. It is hypothesized that ordered particles may represent either a photopigment precursor stock, a second photolabile pigment, or the newly discovered sensitizing, UV-absorbing, photostable visual pigment. In the underlying membrane leaflet (P face) were found spherical (85 Å diameter) unoriented particles in a concentration of about 6,000/μm². The size, shape and density of these structures are compatible with those of rhodopsin particles. These particles also covered the basal area of each microvillus. The findings from TS material were difficult to correlate with those from FF replicas. At high magnification the former showed that the plasma membrane of the transected microvillus is composed of spherical, hollow subunits (averaging 43 Å diameter), sometimes fused to form double, 86 Å units. These substructures were closely packed and continuous around the microvillus. This beaded plasma membrane, in rare cases, was doubled around the microvillus. In other instances the plasma membranes were continuous between neighboring microvilli. The physiological implications of these ultrastructural features are discussed.     

TIP CELL GROWTH AND THE FREQUENCY AND DISTRIBUTION OF CELLULOSE MICROFIBRIL-SYNTHESIZING COMPLEXES IN THE PLASMA MEMBRANE OF APICAL SHOOT CELLS OF THE RED ALGA PORPHYRA YEZOENSIS1

The supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril-synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm^?2, whereas that of the exoplasmic fracture face was 325 μm^?2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm^?2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm² of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.     

Strong light-induced reorganization of pigment-protein complexes of thylakoid membranes in rye (spectroscopic study)

The supramolecular reorganization of LHCII complexes within the thylakoid membrane in Secale cereale leaves under low and high light condition was examined. Rye seedlings were germinated hydroponically in a climate chamber with a 16 h daylight photoperiod, photosynthetic photon flux density (PPFD) of 150 μmol m⁻² s⁻¹ and 24/16 °C day/night temperature. The influence of pre-illumination of the plants with high light intensity on the PSII antenna complexes was studied by comparison of the structure and function of the LHCII complexes and organization of thylakoid membranes isolated from 10-day-old plants illuminated with low (150 μmol m⁻² s⁻¹) or high (1200 μmol m⁻² s⁻¹) light intensity. Aggregated and trimeric with monomeric forms of LHCII complexes were separated from the whole thylakoid membranes using non-denaturing electrophoresis. Analyses of fluorescence emission spectra of these different LHCII forms showed that the monomer was the most effective aggregating antenna form. Moreover, photoprotection connected with LHCII aggregation was more effective upon LHCII monomers in comparison to trimer aggregation. Light stress induced specific organization of neighboring LHCII complexes, causing an increase in fluorescence yield of the long-wavelength bands (centered at 701 and 734 nm). The changes in the organization of the thylakoid membrane under light stress, observed by analysis of absorbance spectra obtained by Fourier transform infrared spectroscopy, also indicated light-induced LHCII aggregation.     

Characterization of the Na+/H+ Exchanger in the Luminal Membrane of the Distal Nephron

In the rabbit as well as the rat, a Na⁺/H⁺ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined the ethylpropylamiloride (EIPA)-sensitive ²²Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 μm EIPA in the membrane suspension decreased the 15 sec Na⁺ uptake to 75.70 ± 4.70% and 50.30 ± 2.23% of the control values in vesicles from proximal and distal tubules, respectively. The effect of EIPA on 35 mm Na⁺ uptake was concentration dependent, with a IC₅₀ of 700 μm and 75 μm for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine and clonidine up to a concentration of 2 mm, the 35 mm Na⁺ uptake by the distal membrane was strongly inhibited by cimetidine (IC₅₀ 700 μm) and modestly inhibited by clonidine (IC₅₀ 1.6 mm). The incubation of proximal tubule suspensions with 1 mm (Bu₂) cAMP decreased the 15-sec EIPA-sensitive Na⁺ uptake by the brush border membranes to 24.1 ± 2.38% of the control values. Unexpectedly, the same treatment of distal tubules enhanced this uptake by 46.5 ± 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 ± 3.04% and 79.7 ± 3.21% of the control values in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs from that of the proximal tubule and probably corresponds to isoform 1. Received: 6 March 1998/Revised: 6 July 1998     

Determination of protein mobility in mitochondrial membranes of living cells

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F₁F₀ ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D = 0.7 μm²/s and 0.6 μm²/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F₁F₀ ATPase: 0.4 μm²/s and 0.0005 μm²/s; COX: 0.3 μm²/s and 0.007 μm²/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.     

Tritrichomonas foetus: Localization of filipin-sterol complexes in cell membranes

The polyene antibiotic, filipin, was used as a probe for the detection of sterols in the freeze-fractured plasma membrane and the flagellar membranes of the pathogenic protozoa, Tritrichomonas foetus. A homogeneous distribution of filipin-sterol complexes was seen throughout the plasma membrane, and the membrane of the three anterior and the one recurrent flagella. No or very few filipin-sterol complexes were observed in some specialized regions such as the base of the flagella (necklace), the portion of the recurrent flagellum, and that part of the cell body to which the flagellum was attached. The density of filipin-sterol complexes varied from one cell to the other. In some cells, about 205 complexes/μm² were seen. A larger number of filipin-sterol complexes were observed on both faces of the membrane of cytoplasmic structures, probably corresponding to vacuoles. No complexes were seen in the nuclear membrane and in the membrane of the endoplasmic reticulum. Very few or no complexes were observed in the membrane of the hydrogenosomes. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.     

Involvement of the alpha-subunit N-terminus in the mechanism of the Na+,K+-ATPase

Previous studies have shown that cytoplasmic K⁺ release and the associated E2 → E1 conformational change of the Na⁺,K⁺-ATPase is a major rate-determining step of the enzyme's ion pumping cycle and hence a prime site of acute regulatory intervention. From the ionic strength dependence of the enzyme's distribution between the E2 and E1 states, it has also been found that E2 is stabilized by an electrostatic attraction. Any disruption of this electrostatic attraction would, thus, have profound effects on the rate of ion pumping. The aim of this paper is to identify the location of this interaction. Using enhanced-sampling molecular dynamics simulations with a predicted N-terminal structure added to the X-ray crystal structure of the Na⁺,K⁺-ATPase, a previously postulated salt bridge between Lys32 and Glu233 (rat sequence numbering) of the enzyme's α-subunit can be excluded. The residues never approach closely enough to form a salt bridge. In contrast, strong interactions with anionic lipid head groups were seen. To investigate the possibility of a protein-lipid interaction experimentally, the surface charge density of Na⁺,K⁺-ATPase-containing membrane fragments was estimated from zeta potential measurements to be 0.019 (± 0.001) C m⁻². This is in good agreement with the charge density previously determined to be responsible for stabilization of the E2 state of 0.023 (± 0.009) C m⁻² and the membrane charge density estimated here from published electron-microscopic images of 0.018C m⁻². The results are, therefore, consistent with an interaction of the Na⁺,K⁺-ATPase α-subunit N-terminus with negatively-charged lipid head groups of the neighbouring cytoplasmic membrane surface as the origin of the electrostatic interaction stabilising the E2 state.     

Alteration of thylakoid membrane structure in Brassica rapa ssp. oleifera during ageing in high and low light

Abstract Alterations in the composition and structure of thylakoids were studied in Brassica rapa ssp. oleifera grown under high and low irradiance (800 μmol m^?2 s^?1 and 80 μmol m^?2 s^?1). During ageing, both high and low light induced a decrease in total protein particle density and in the relative amount of 80–90 Å cytochrome b6/f and 90–100 Å ATP-synthetase. The density of PSII complexes in stacked (EFs) and unstacked (EFu) thylakoids also decreased. In high light, a shift was noted towards smaller PSII complexes in the EFs face with decreasing attached antenna complex CP29, but the relative amount of the antenna chlorophyll a-protein complexes of photosystem II (CPa) remained stable. In contrast, the proportion of peripheral LHCH on the PFs face and the density of PFs particles increased together with an increase in grana size. In low light, a shift occurred towards larger PSII complexes on the EFs face, along with a decrease in the proportion of CPa complexes and the PFs particle density (peripheral LHCH), though a marked increase was observed in the proportion of chlorophyll a/b-protein complexes in SDS-PAGE. The amount of photosystem I in green gel remained fairly stable, although the density of PFu particles (including PSI) increased in low and slightly diminished in high light. The results indicate that the organization of thylakoid components depends strongly on the light conditions and stage of development.     

2-Methylisoborneol production characteristics of <Emphasis Type="Italic">Pseudanabaena</Emphasis> sp. FACHB 1277 isolated from Xionghe Reservoir,China

The cyanobacterium Pseudanabaena sp. FACHB 1277, a 2-methylisoborneol (2-MIB) producer isolated from Xionghe Reservoir, was identified by molecular biological methods based on the 16S rDNA sequence. Pseudanabaena sp. FACHB 1277 is a planktonic freshwater species with relatively high 2-MIB per cell density value (7.76?×?10^?6 ng cell^?1) and specific growth rate (0.25?±?0.01 d^?1). The effects of temperature and light intensity on 2-MIB production of Pseudanabaena sp. FACHB 1277 were investigated. Of the six temperatures tested, 10, 15, 20, 25, 30, and 35 °C, the maximum total 2-MIB per cell density and minimum cell density were observed at 10 °C, while the total 2-MIB and dissolved 2-MIB (including extracellular and dissolved intracellular 2-MIB) increased with increasing temperature. Among the six tested light intensities (10, 25, 40, 55, 70, and 85 μmol photons m^?2 s^?1), the minimum total 2-MIB per cell density and maximum cell density were observed at 25 μmol photons m^?2 s^?1. The total 2-MIB and extracellular 2-MIB increased with light intensity increasing from 10 to 40 μmol photons m^?2 s^?1, while no significant increase was observed when the light intensity was higher than 40 μmol photons m^?2 s^?1. The maximum intracellular 2-MIB (including dissolved and bound) occurred at 25 μmol photons m^?2 s^?1. The present study indicates that increasing temperature could favor the conversion of bound intracellular to dissolved 2-MIB, while increasing light intensity stimulates the release of dissolved intracellular 2-MIB into the environment.     

Ecosystem respiration derived from 222Rn measurements on Rishiri Island, Japan

We evaluated the nighttime CO₂ flux (ecosystem respiration) on Rishiri Island, located at the northern tip of Hokkaido, Japan, from 2009 to 2011, by using the relationship between atmospheric ²²²Rn and CO₂ concentrations. The annual mean CO₂ flux was 1.8 μmol m^?2 s^?1, with a maximum monthly mean in July (4.6 ± 2.6 μmol m^?2 s^?1) and a broad minimum from December to March (0.33 ± 0.29 μmol m^?2 s^?1). The annual mean was comparable to fluxes at the JapanFlux sites in northern Japan. During the season of snow cover (mid-December to early April), the CO₂ flux was low (0.45 ± 0.43 μmol m^?2 s^?1). Total annual respiration was estimated at 679 ± 174 g cm^?2, about 8 % of which occurred during the season of snow cover.     

The effects of synthetic estrogens on the metabolic clearance and production rates of estrone and estradiol

The metabolic clearance rates (MCR) of estrone (1) and estradiol were determined by pulse injections and constant infusions of ³H-estrone and ³H-estradiol in seven women taking mestranol-containing compounds and in seven women taking ethinyl estradiol-containing compounds. These results were compared with the results previously obtained in our laboratory (2, 3, 4, 5) in comparable women not taking these compounds. In the women taking mestranol the mean (± SE) MCR for estradiol, 750 ± 600 1/day/m², was similar to our normal mean value, 790 ± 30 1/day/m². However, the mean MCR for estrone was less 1,010 ± 60 1/day/m² than that in normals 1,230 ± 30 1/day/m². In the women taking ethinyl estradiol the mean MCR for estradiol, 1,070 ± 60 1/day/m² was significantly (P < 0.01) greater than the normal value. The mean MCR for estrone, 1,180 ± 80 1/day/m² was not different from the normal.Using an immunoassay to measure the concentrations of estradlol and. estrone in plasma, the mean level of estradlol in mestranol users was 40 ± 7 Pg/ml and in ethinyl estradlol users 69 ± 4 pg/ml.The mean calculated production rate for estradlol in the mestranol users was 47 ± 8 μg/day and in the ethinyl estradlol users was 120 ± 22 μg/day. The mean calculated, production rates for estrone were 71 ± 12 and 93 ± 12 μg/day in the respective groups.Thus while mestranol appears to have little effect on endogenous estrogen metabolism, the use of ethinyl estradiol appears to increase the MCR of estradlol, but not of estrone. The MCR of estradlol returns to the normal range when ethinyl estradlol is stopped.     

Evidence for placental compensation in cattle

Prenatal development is known to be extremely sensitive to maternal and environmental challenges. In this study, we hypothesize that body growth and lactation during gestation in cattle reduce nutrient availability for the pregnant uterus, with consequences for placental development. Fetal membranes of 16 growing heifers and 27 fully grown cows of the Belgian Blue (BB) breed were compared to determine the effect of body growth on placental development. Furthermore, the fetal membranes of 49 lactating Holstein Friesian (HF) cows and 27 HF heifers were compared to study the impact of dam lactation compared to dam body growth. After parturition, calf birth weight and body measurements of dam and calf were recorded, as well as weight of total fetal membranes, cotyledons and intercotyledonary membranes. All cotyledons were individually measured to calculate both the surface of each individual cotyledon and the total cotyledonary surface per placenta. Total cotyledonary surface was unaffected by breed or the breed×parity interaction. Besides a 0.3 kg lower cotyledonary weight (P=0.007), heifer placentas had a smaller total cotyledonary surface compared with placentas of cows (0.48±0.017 v. 0.54±0.014 m², respectively, P<0.001). Within the BB breed, fetal membranes of heifers had a 1.5 kg lower total weight and 1.0 kg lower intercotyledonary membrane weight (P<0.005) compared with cows. A cotyledon number of only 91±5.4 was found in multiparous BB dams, while growing BB heifers had a higher cotyledon number (126±6.7, P<0.001), but a greater proportion of smaller cotyledons (<40 cm²). Within the HF breed, no parity effect on intercotyledonary membrane weight, cotyledon number and individual cotyledonary surface was found. Placental efficiency (calf weight/total cotyledonary surface) was similar in HF and BB heifers but significantly higher in multiparous BB compared with multiparous HF dams (106.0±20.45 v. 74.3±12.27 kg/m², respectively, P<0.001). Furthermore, a seasonal effect on placental development was found, with winter and spring placentas having smaller cotyledons than summer and fall placentas (P<0.001). Main findings of the present study are that lactation and maternal growth during gestation entail a comparable nutrient diverting constraint, which might alter placental development. However, results suggest that the placenta is able to manage this situation through two potential compensation mechanisms. In early pregnancy the placenta might cope by establishing a higher number of cotyledons, while in late gestation a compensatory expansion of the cotyledonary surface is suggested to meet the nutrient demand of the fetus.     

Giardia lamblia: Evaluation of roller bottle cultivation

A modified “outside-in” roller bottle with a high ratio of surface area to volume was used to cultivate Giardia lamblia. Yields were high, more so when bottles were rotated at 6 rph (9.3 ± 4.0 × 10⁸ trophozoites/bottle) than at 12 rph (4.2 ± 1.9 × 10⁸ trophozoites/bottle). The method was more efficient than stationary tube culture with respect to utilization of culture medium; trophozoite concentration after roller bottle culture (1.7 ± 0.8 × 10⁶ trophozoites/ml) was significantly higher (by a factor of 2.8) than concentrations obtained from stationary tube culture (0.6 ± 0.4 × 10⁶ trophozoites/ml, P < 0.002). Increased yields from roller bottle culture were not accounted for by a reduction in mean trophozoite generation time (roller culture, 10.7 ± 1.2 hr; stationary tube culture, 10.3 ± 0.6 hr) but may be related to prolongation of the period of log phase growth or increased trophozoite survival. Trophozoite yields expressed per unit surface area were significantly higher from roller bottle culture (7.2 ± 3.1 × 10⁵ trophozoites/cm²) than from stationary tubes (1.9 ± 1.0 × 10⁵ trophozoites/cm², P < 0.002). Attempts to cultivate G. lamblia in spin culture using polystyrene beads (Biosilon) as a microcarrier were unsuccessful, trophozoite growth being inhibited rather than promoted. Roller bottle culture of G. lamblia, however, is efficient, economical, and less laborious than stationary tube culture, particularly when more than 10⁸ trophozoites are required.     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997