FBN1 isoform expression varies in a tissue and development-specific fashion

Mutations in FBN1 cause Marfan syndrome, a heritable disorder of connective tissue. FBN1 encodes the extracellular matrix protein, fibrillin. Our objective was to elucidate the extent that variation in RNA splicing contributes to FBN1 isoforms. To identify FBN1 splice variants, we scanned each of its 64 internal exons in a set of pooled human brain cDNA samples. FBN1 splicing is generally efficient as we identified only two variants. Neither variant has previously been reported in the literature and include (i) an isoform which contains a cryptic 105 basepair exon between exons 54 and 55 (54A-FBN1) and (ii) an isoform which contains a cryptic 62 basepair exon between exons 57 and 58 (57A-FBN1). We compared 57A-FBN1 and FBN1 expression in multiple human tissues, including adult skeletal muscle and brain, as well as fetal skeletal muscle, brain, liver, aorta, lung, skin, and heart. 57A-FBN1 represents 8–44% of FBN1 mRNA and varies in a tissue- and development-specific fashion. In adult brain, 57A-FBN1 represented 39 ± 3 (%, mean ± SD) of total FBN1 expression. In contrast, 57A-FBN1 represented 19 ± 2 (%, mean ± SD) of FBN1 expression in skeletal muscle. In fetal tissue, the 57A-FBN1 proportion was highest in brain (27%) and low elsewhere, e.g., skin, aorta and lung (9–13%). In summary, a significant proportion of FBN1 is expressed as 57A-FBN1 and this proportion varies in a tissue- and development-specific fashion. Since the 57A insertion creates a premature stop codon that mimics Marfan-associated mutations, the protein encoded by 57A-FBN1 is likely to not be functional. These results suggest that altered splicing may modulate disease severity, regulate FBN1 expression, and potentially represent a therapeutic target.     

Characterization of Exocytotic Events From Single PC12 Cells: Amperometric Studies in Native PC12h, DA-Loaded PC12h and Bovine Adrenal Chromaffin Cells

Exocytotic events from rat pheochromocytoma (PC12) cells were characterized by amperometric analysis. For single-cell amperometric recordings, PC12h cells cultured onto poly-L-lysine corted glass-base dish were incubated with 1 mM dopamine (DA) for 60 min. Amperometric recordings, with a carbon fiber microelectrode (5 μm diameter), of catecholamine release from the individual cells were conducted under an inverted microscope at 25 ^∘C. To characterize a single exocytotic event that is detected as a single spike current, the spike number, spike parameters (rise time, middle width and area) and spike shape were analyzed. Exposure of DA-loaded PC12h cells to 60 mM KCl (1000 hps) for 5 min and for 4 s evoked a train of events with the event number of 114± 19 (spikes/response for 5 min) and 12± 3 (spikes/response for 15 s), respectively. We observed distinctive kinetics in the events (rise time = 0.83± 0.19 ms, middle width = 2.89± 0.62 ms, area = 62± 7.6 fC and the spikes with a “foot” = 15.4± 2.7% of total spikes). The number and mean height of the events were 3- to 4-fold higher than that in DA-unloaded cells, and the values of rise time and middle width in DA-loaded PC12h cells were approx. 5- and 10-fold less than those observed in cultured adrenal chromaffin cells. The successful application of amperometry to monitor DA released from secretory vesicles in DA-loaded PC12h cell suggest that this technique is applicable to characterize exocytotic events in neurons.     

Milk intake,growth and energy consumption in pups of ice-breeding grey seals (Halichoerus grypus) from the Gulf of St. Lawrence,Canada

In this study we document growth, milk intake and energy consumption in nursing pups of icebreeding grey seals (Halichoerus grypus). Change in body composition of the pups, change in milk composition as lactation progresses, and mass transfer efficiency between nursing mothers and pups are also measured. Mass transfer efficiency between mother-pup pairs (n=8) was 42.5±8.4%. Pups were gaining a daily average of 2.0±0.7 kg (n=12), of which 75% was fat, 3% protein and 22% water. The total water influx was measured to be 43.23±8.07 ml·kg^-1·day^-1. Average CO₂ production was 0.85±0.20 ml·g^-1·h^-1, which corresponds to a field metabolic rate of 0.55±0.13 MJ·kg^-1·day^-1, or 4.5±0.9 times the predicted basal metabolic rate based on body size (Kleiber 1975). Water and fat content in the milk changed dramatically as lacation progressed. At day 2 of nursing, fat and water content were 39.5±1.9% and 47.3±1.5%, respectively, while the corresponding figures for day 15 were 59.6±3.6% fat and 28.4±2.6% water. Protein content of the milk remained relatively stable during the lactation period with a value of 11.0±0.8% at day 2 and 10.4±0.3% at day 15. Pups drank an average of 3.5±0.9 kg of milk daily, corresponding to a milk intake of 1.75 kg per kg body mass gained. The average daily energy intake of pups was 82.58±19.80 MJ, while the energy built up daily in the tissue averaged 61.72±22.22 MJ. Thus, pups assimilated 74.7% of the energy they received via milk into body tissue. The lactation energetics of ice-breeding grey seals is very similar to that of their land-breeding counterparts.Abbreviations bm body mass - BMR basal metabolic rate - FMR field metabolic rate - IU international unit - RQ respiration quotient - HTO tritiated water - HT¹⁸O doubly labeled water - TBW total body water - VHF very high frequency     

Stomatal movement in Zea mays: Shuttle of potassium and chloride between guard cells and subsidiary cells

Summary When stomates of Zea mays open K and Cl migrate from the subsidiary cells into the guard cells; when the stomates close both elements return to the subsidiary cells. Subsidiary cells function as reservoirs for K and Cl. Import of K and Cl into the guard cells and loss of both elements from the guard cells become observable 1 or 2 min after light is turned on or off, both when histochemical methods and the electron-probe microanalyzer are used for detection. Each stomatal complex of maize contains on the average 10±3×10^-13 gram equivalents (eq) of K and 4±1×10^-13 eq of Cl. Guard cells accumulate K in the light and CO₂-free air at an average rate of 10×10^-15 eq K per minute, and Cl at approximately half that rate.     

The effect of superficial pinealectomy on reproduction and brown fat in the adult white-footed mouse,Peromyscus leucopus

Summary A method for superficial pinealectomy of the adult white-footed mouse,Peromyscus leucopus, is presented. Histological examination of the brain of pinealectomized mice showed that the deep pineal gland was left intact. The survival rate of pinealectomized mice was 80%. Pinealectomized mice were exposed to a short day photoperiod (8L:16D) at 15°C for 7 weeks. After this time male mice maintained active gonads with a testicular index (TI, testis width×length/body weight) of 2.0±0.1. Testis weight was 202±35 mg, and the seminiferous tubules contained abundant spermatozoa (spermatogenic index [SI]=4.5±0.2). Sham operated animals had regressed testes. TI was 1.2±0.2, testis weight was 97±26 mg, and the SI was 2.7±0.7 (allP<0.05 relative to pinealectomized mice). Pinealectomized females were reproductively competent in that all of the mice had a perforate vagina, the reproductive tract weight (vagina, uterus, oviducts, and ovaries) was 111±15 mg, and the ovaries from each animal contained preovulatory follicles. Sham operated mice had an imperforate vagina, reproductive tract weights were 34±5 mg, and in only 1 out of 5 mice did the ovaries contain a preovulatory follicle (allP<0.05). The weight of the lipid-free interscapular brown fat was 28% less in pinealectomized mice relative to sham operated animals (P<0.01). These results support the role of the pineal gland as regulator of short day, cold induced reproductive regression and brown fat hypertrophy.     

Determination of dimethylamine in biological samples by high-performance liquid chromatography

A reversed-phase HPLC method for the quantification of dimethylamine in serum and urine is presented. Dimethylamine (DMA) is converted into a stable fluorescent product by precolumn derivatization with fluorenylmethylchloroformate. The DMA derivative is resolved from derivatives of other amines and amino acids by gradient elution with a total run-time of 15 min. The lower limit of determination in biological samples is 0.1 μmol/1. Recoveries from spiked serum samples were 99–107%. Within- and between-run precision were better than 6%. Concentrations of DMA in serum from normal human subjects (n=8) and from continuous ambulatory peritoneal dialysis patients (n=15) were 3.3±1.5 and 29.2±12.1 μmol/1, respectively.     

Presence of activated lymphocytes in the urine of patients with superficial bladder cancer after intravesical immunotherapy with bacillus Calmette-Guérin

Summary To study the mode of action of intravesical bacillus Calmette-Guérin (BCG) immunotherapy in the prevention and cure of superficial bladder cancer, flow-cytofluorometric analysis of the cellular immunological reaction in the urine of patients was performed. Fresh urinederived leucocytes were obtained from eight patients before (t ₀) and 24 h (t ₂₄) and 48 h (t ₄₈) after repeated intravesical BCG instillations (at least 5 instillations). For two patients urine-derived leucocytes were investigated at the first BCG instillation. The number of leucocytes in the urine was markedly increased 24 h after repeated BCG instillations, indicating a local cellular immunological reaction induced by BCG. The mean number of cells per milliliter of urine at that time was 2.9×10⁶±3.6×10⁶ (n = 8). These leucocytes consisted mainly of granulocytes (75±11%,n = 8). In addition monocytes/macrophages (4±2%,n = 8) and T lymphocytes were present (1±1%,n = 5). The relative increase of monocytes/macrophages in the urine after BCG application tended to be higher compared to the other leucocyte subtypes. As T lymphocytes may play an important role in the BCG-mediated antitumour activity, subsets of lymphocytes were further characterized att ₀,t ₂₄, andt ₄₈ after repeated BCG instillations. The lymphocyte population consisted mainly of T cells (86% CD3⁺,t ₀). Most of the T cells were CD4⁺ (helper/inducer) and were significantly decreased at 48 h (62±9% att ₀ vs 49±6% att ₄₈). Lymphocytes partly expressed HLA-DR antigens (44%,t ₀). The percentage of lymphocytes with interleukin-2 (IL-2) receptors (CD25⁺) was significantly increased at 24 h and 48 h, compared to pre-instillation values (19±11% and 10±4% vs 3±3% respectively). Natural killer cells (CD 16⁺ and/or CD56⁺) and B cells (CD 19⁺) were less numerous (10% and 19% att ₀ respectively). After the first BCG instillation the increase in the number of leucocytes in urine seemed to be less compared to the numbers after repeated BCG instillations. Lymphocytes could not be detected in the urine collected before or after the first BCG instillation. In conclusion, we demonstrated the presence of considerable numbers of leucocytes in the urine 24 h after repeated BCG instillations, i.e. shortly after immunological activation. The antigen expression of the lymphocytes suggested that they may represent the lymphocytes in the bladder wall. Expression of HLA-DR and IL-2 receptors on lymphocytes indicated activation of T cells by the intravesical BCG treatment. These leucocytes may be useful for functional studies, which are essential to elucidate the actual effector mechanism(s) in the mode of action of BCG against superficial bladder cancer in man.     

Rate of activation and deactivation of K∶Cl cotransport by changes in cell volume in hemoglobin SS,CC and AA red cells

Red blood cells (RBC) of subjects homozygous for hemoglobin A (AA), C (CC) and S (SS) exhibit different cell volumes which might be related to differences in cell volume regulation. We have investigated how rapidly K:Cl cotransport is activated and deactivated to regulate the cell volume in these cells. We measured the time course of net K⁺ efflux after step changes in cell volume and determined two delay times: one for activation by cell swelling and a second for deactivation by cell shrinkage. Cell swelling induced by 220 mOsm media activated K⁺ efflux to high values (10–20 mmol/ liter cell x hr) in CC and SS; normal AA had a threefold lower activity. The delay time for activation was very short in blood with a high percentage of reticulocytes (retics): (SS, 10% retics, 1.7±0.3 min delay, n=8; AA, 10% retics, 4±1.5 min, n=3; CC, 11.6% retics, 4±0.3, n=3) and long in cells with a smaller percentage of reticulocytes: (AA, 1.5% retics, 10±1.4 min, n=8; CC whole blood 6% retics, 10±2.0 min, n=10, P<0.02 vs. SS). The delay times for deactivation by cell shrinking were very short in SS (3.6±0.4 min, n=8, P<0.02) and AA cells with high retics (2.7±1 min, n=3) and normal retics (2.8±1 min, n=3), but 8–15-fold longer in CC cells (29±2.8 min, n=9).Density fractionation of CC cells (n=3) resulted in coenrichment of the top fraction in reticulocytes and in swelling-activated cotransport (fourfold) with short delay time for activation (4±0.3 min) and long delay for deactivation (14±4 min). The delay time for activation, but not for deactivation, increased markedly with increasing cell density. These findings indicate that all CC cells do not promptly shut off cotransport with cell shrinkage and high rates of cellular K⁺ loss persist after return to isotonic conditions.In summary, (i) K:Cl cotransport is not only very active in young cells but it is also very rapidly activated and deactivated in young AA and SS cells by changes in cell volume. (ii) Delay times for cotransport activation markedly increased with RBC age and in mature cells with low cotransport rates, long delay times for activation were observed. (iii) The long delay time for deactivation exhibited even by young CC cells induces a persistent loss of K⁺ after cell shrinkage which may contribute in vivo to the uniformly low cell volume, low K⁺ and water content of CC cells.This research was supported by National Institutes of Health grants Shannon Award HL-35664, HL-42120, Sickle Cell Center grant HL-38655, and a Grant-in-Aid of the New York Branch of the American Heart Association. The technical help of Sandra M. Suzuka, M.S. is gratefully acknowledged.     

Ionic and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon, Acipenser oxyrinchusde sotoi

The salinity tolerance, and hydromineral regulation capabilities of three size groups (small 110–170 g; medium 230–290 g, large 460–700 g; n=48 for each group) of 13-month-old juvenile Gulf of Mexico sturgeon were investigated. Fish (n=6 for each salinity) were transferred directly from freshwater (FW) to a series of experimental salinity treatments (0, 5, 10, 15, 20, 25, 30, and 35 parts per thousand (ppt)). Fish were also acclimated in brackish water (20 ppt) for 2 weeks and transferred to a salinity of 34 ppt. In this condition juvenile Gulf of Mexico sturgeon adapted to saltwater (SW) and maintained their hydromineral balance. FW adapted sturgeon (n=6) had an average blood hemotocrit of 28.2±0.8%, plasma osmolality of 260.7±1.6 mOsm kg⁻¹ H₂O, and plasma ion concentrations of 135.7±1.2 mM l⁻¹ Na⁺, 106.9±1.9 mEq l⁻¹ Cl⁻, and 2.9±0.1 mM l⁻¹ K⁺. In SW adapted sturgeon (n=8) blood parameters averaged 26.9±0.7% for hematocrit, 294.2±2.3 mOsm kg⁻¹ H₂O for osmolality, 152.0±1.7 mM l⁻¹ Na⁺, 149.2±1.4 mEq l⁻¹ for Cl⁻, and 3.1±0.1 mM l⁻¹ K⁺. The method of transfer (abrupt or slow acclimation) directly affected fish survival and the time they took to achieve ionic and osmotic regulation. This SW adaptation appears to be related to body size, the larger the fish the easier the adaptation process. A threshold size of about 170 g was apparent for the fish to adapt to saltwater after 2 weeks of acclimation. Chloride cells were present in both FW and SW adapted sturgeon with SW and brackish water fish having chloride cells significantly (P<0.05) more numerous (561±53 and 598±45 cells mm⁻²) and larger in size (41.0±3.85 and 34.2±4.49 μm²) than FW adapted sturgeon (10±1.0 cells mm⁻² and 22±2.53 μm²). Few chloride cells were observed in the opercular membrane, however, none were found in the pseudobranch and spiracle.     

Transferrin binding by mammalian cortical cells

Predominantly neuronal (neuronal) or non-neuronal (glial) cerebral cortical cell cultures were employed to study the kinetics and changes with maturation of¹²⁵I-diferric-transferrin uptake. The diferric-transferrin association curve of neuronal cultures at 37°C was nonphasic and indicated equilibrium at 90 minutes. Dissociation was completed by 70 minutes. Diferric-transferrin specific uptake (80% of total) in neuronal cells (evaluated at days 6, 9, 13, 16, and 23 in culture) increased with maturation. Scatchard transformation of the data revealed increasingB _max from day 6 to day 16 in culture (1626 to 2740 fmoles/mg protein). However, theK _uptake was statistically unchanged over time and equaled 48.7±13.9 nM (mean ±SD). In contrast, association studies of glial cultures documented equilibrium by 45 minutes and dissociation by 40 minutes. The concentration curves for differric-transferrin uptake in glial cells, evaluated at days 11, 15, and 18 in culture, revealed virtually identical uptake at the three ages studied, but the percent specific uptake (58%) was less than for neurons (88%). Scatchard transformation of the data revealed no statistical alteration ofB _max orK _uptake from days 11 to 18 in culture.B _max ranged from 595 to 751 fmol/mg protein; overallK _uptake was 48.3±13.2 nM (mean±SD).     

Effects of angiotensin-II receptor blocker candesartan cilexetil in rats with dilated cardiomyopathy

We examined effects of an angiotensin-II receptor blockers, candesartan cilexetil, in rats with dilated cardiomyopathy after autoimmune myocarditis. Candesartan cilexetil showed angiotensin-II blocking action in a dose-dependent manner in rats with dilated cardiomyopathy. Twenty-eight days after immunization, surviving Lewis rats were divided into four groups and given candesartan cilexetil at 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg per day (Group-C0.05, n = 15, Group-C0.5, n = 15 and Group-C5, n = 15, respectively) or vehicle alone (Group-V, n = 15). After oral administration for 1 month, the left ventricular end-diastolic pressure and heart weight/body weight ratio were lower in Group-C0.05 (13.3± 1.1 mmHg and 3.7± 0.2 g/kg, respectively), in Group-C0.5 (8.0± 0.9 mmHg and 3.3± 0.1 g/kg, respectively) and in Group-C5 (5.5± 1 mmHg and 3.1± 0.1 g/kg, respectively) than in Group-V (13.5± 1.0 mmHg and 3.8± 0.2 g/kg, respectively). The area of myocardial fibrosis was also lower in Group-C0.05 (25± 3%), in Group-C0.5 (20± 3%), and in Group-C5 (12± 1%) than in Group-V (32± 4%). Furthermore, expressions of transforming growth factor-1 and collagen-III mRNA were suppressed in Group-C0.05 (349± 23% and 395± 22%, respectively), Group-C0.5 (292± 81% and 364± 42%, respectively) and in Group-C5 (204± 63% and 259± 33%, respectively) compared with those in Group-V (367± 26% and 437± 18%, respectively). These results suggest that candesartan cilexetil can improve the function of inefficient heart. (Mol Cell Biochem 269: 137–142, 2005)     

Purification ofPseudomonas fluorescens subsp.cellulosa endoglucanases produced inEscherichia coli

Both plasmid pPFC4, which contains 10.6 kb, and a derivative of pPFC4—viz., pPFC4-4.6—which contains 4.6 kb ofPseudomonas fluorescens subsp.cellulosa DNA, direct the synthesis of six distinct endoglucanases inEscherichia coli. Two of these enzymes were purified to homogeneity in a single step by means of anion exchange HPLC. One enzyme has a molecular weight of 30.0 ± 1.0 kDa, an isoelectric point of 7.5, and a specific activity of 3470 U of activity/mg of protein, whereas the other has a molecular weight of 38.5 ± 1.0 kDa, an isoelectric point of 6.7, and a specific activity of 18,050 U of activity/mg of protein. On the basis of the amino acid composition, the 38.5 kDa enzyme appears to be a modified version of the 30.0 kDa enzyme. Thus, the multiplicity of endoglucanases produced inE. coli/pPFC4-4.6 cells may be owing to the posttranslational modification of a smaller number of primary translation products.     

Cultivation of Chlorella pyrenoidosa in soybean processing wastewater

Chlorella pyrenoidosa was cultivated in soybean processing wastewater (SPW) in batch and fed-batch cultures without a supply of additional nutrients. The alga was able to remove 77.8 ± 5.7%, 88.8 ± 1.0%, 89.1 ± 0.6% and 70.3 ± 11.4% of soluble chemical oxygen demand (SCOD_Cr), total nitrogen (TN), NH₄⁺-N and total phosphate (TP), respectively, after 120 h in fed-batch culture. C. pyrenoidosa attained an average biomass productivity of 0.64 g L⁻¹ d⁻¹, an average lipid content of 37.00 ± 9.34%, and a high lipid productivity of 0.40 g L⁻¹ d⁻¹. Therefore, cultivation of C. pyrenoidosa in SPW could yield cleaner water and useful biomass.     

Outward-rectifying chloride channels in cultured adult and fetal human nasal epithelial cells

Summary The patch-clamp technique was used to characterize ion channels in the apical membranes of cultured human nasal epithelial cells, dissociated from fetal nasal mucosa and from adult nasal polyps. Outward-rectifying chloride channels were found in 4.3% of the cell-attached patches from fetal cells (n=258) and in 3.1% of the patches from adult cells (n=320). After exeision the number of patches containing active chloride channels increased threefold to 13% of the patches from the fetal cells and 10% from adult cells. The single-channel conductance at 0 mV in symmetrical 150mm NaCl solutions was 24.3 ±0.9 pS (n=28) and 26.0 ± 1.2 pS (n=30), respectively, in adult and fetal cells and showed outward rectification in the potential range from –80 to +80 mV. In fetal cells as well as in adult cells the channels were anion selective, and were almost impermeable for larger anions and monovalent cations. In cell-free patches the channels were Ca²⁺ independent. In most of the channels the open probability was voltage independent and high (±0.86); in 20% of the channels, however, the open probability increased with depolarization. In conclusion, fetal nasal epithelial cells contain chloride channels in their apical membranes with singlechannel properties and regulatory mechanisms similar to those found in cells from adults.     

Heritability of Reproductive Traits in the Medicinal Leech Hirudo medicinalis L.

The phenotype variability and inheritance of reproductive traits were investigated in the medicinal leech. Distribution parameters were determined for the following traits: batch size (X¯ = 4.3 ± 0.2, = 1.7, CV = 40%, As = 0.23 ± 0.25, Ex = 0.19 ± 0.51), number of juveniles in a cocoon ( X¯= 10.9 ± 0.3, = 4.6, CV = 42%, As = 0.31 ± 0.15, Ex = 0.23 ± 0.30), and juvenile weight ( X¯= 32.0 ± 0.3, = 14.9, CV = 47%, As = 1.38 ± 0.05, Ex = 3.32 ± 0.11). A nonlinear negative correlation between the number of juveniles in a cocoon and their weight was found (correlation ratio R= 0.86). It was shown that the environmental variance dominated over the genotypic one in the structure of phenotypic variance of the traits studied. The genetic variability is determined mainly by additive gene interactions and, to a small extent, intralocus dominance. The narrow-sense heritability, h ², for batch size was 0.35–0.40; for the number of juveniles in a cocoon, 0.35; for juvenile weight, 0.42.     

Five new species of Trichostomatida (ciliated protozoa) from the colon of wild African rhinoceroses

Five new species of Charonina Strand, 1928 were revealed, in addition to 41 species of 20 other apparent genera, in a survey of ciliated intestinal protozoan endocommensals of both black and white wild African rhinoceroses. Charonina species infected the ventral and dorsal region of the ascending colon, where the average total protozoan populations (× 10³/ml digesta fluid) were 100 and 80, respectively, in the white and 270 and 260, respectively, in the black rhinoceroses. Charonina species constituted up to 50% in the ventral and 25% in the dorsal populations. Measurements in micrometres and specific characteristics of the five species are: C. odontophora n. sp. length 70±5.7, width 32±7.2, dorso-ventral thickness 7±1.1, slender ovate-lanceolate-shaped body with frontal lobe and prong-like protrusion in oral-opening; C. tortuosa n. sp. length 87±9.3, width 42±5.3, dorso-ventral thickness 15±2.7, body-shape ovate-lanceolate without frontal lobe, oral-opening with longitudinal ridge, cytopharyngeal canal with sharp bend after emerging from oral-opening; C. dicerotis n. sp. length 67±8.6, width 37±4.0, dorso-ventral thickness 12±2.2, body-shape ovate-lanceolate without frontal lobe, oral-opening without ridge, cytopharyngeal canal curved without sharp bend; C. tenuis n. sp. length 56±10.0, width 16±4.1 at anterior end and 10±2.7 at posterior end, dorso-ventral thickness 7±1.2 at anterior end and 5±1.1 at posterior end, body-shape cone-like with longitudinal striations and frontal lobe; C. tetragona n. sp. length 58±4.7, width 26±3.2, dorso-ventral thickness 12±1.8, body-shape rectangular with frontal lobe and caudal flaps. The length, width and dorso-ventral thickness of the five species are on average in the approximate ratio of 6:3:1, thus showing them to be dorso-ventrally compressed. The body conformation of C. tetragona n. sp. closely resembles that of Didesmis quadrata Fiorentini, 1980, but is distinguished from the latter by the absence of a concrement vacuole and the presence of an elongate cytopharyngeal canal.     

Alteration in lymphocyte phenotype associated with administration of adjuvant levamisole and 5-fluorouracil

Levamisole (LMS) and 5-fluorouracil (5FU) administered adjuvantly are effective in reducing the relapse rate following surgical resection of Duke's stage C colon carcinoma. It has been postulated that LMS acts to stimulate the immune system and that this is one mechanism through which this drug exerts its antitumor effects. In this study, peripheral blood mononuclear cells (PBMC) were analyzed in nine patients with surgically resected colon carcinoma prior to initiation of adjuvant LMS/5FU and at several subsequent times while patients were on therapy. Changes in lymphocyte phenotype and soluble interleukin-2 receptor (sIL-2R) between pre-study samples and samples obtained during adjuvant LMS/5FU were evaluated. Significant increases were seen in the proportion of PBMC expressing natural killer (NK) antigen CD56 (14.7±2.4% versus 18.1±2.6%;P<0.05) and surface IL-2R (CD25; 0% versus 0.42±0.15%;P<0.05), in sIL-2R (314±86 U/ml versus 736±173 U/ml;P<0.05), and in the CD4CD8 ratio (2.34±0.93 versus 3.47±1.23;P<0.01). A significant decrease in the proportion of CD8⁺ PBMC (24.7±3.8% versus 18.8±2.6%;P<0.01) and total CD8⁺ PBMC (537±118 versus 324±37;P<0.01) was seen. The increase in CD56⁺ cells correlated with sIL2R levels (r=0.46;P<0.05). No changes were noted for CD3, CD4, CD5, CD14, CD16, CD19, CDw49a, or TCR. The greatest increase in CD56⁺ cells and the smallest reduction in CD8⁺ cells were seen in the subgroup of patients who remained disease-free following adjuvant chemotherapy. This study suggests that adjuvant LMS/5FU has significant stimulatory effects on the immune system, which correlate with patient outcome and may account at least in part for its clinical efficacy.Grant support: supported by a grant from the Elsa U. Pardee Foundation (R.F.H.) and the Centers for Excellence in Cancer Research and Arthritis & Rheumatology, LSU Medical Center, Shreveport, La., USA     

Changes in the Functioning of Ca2+-ATPases of Rat Exocrine Cells in Experimental Diabetes Mellitus

It is obvious that disruption of functions of the nervous system in diabetes mellitus is to a great extent related to the changes of synthesis or exocytosis of neurotransmitters. Since the mechanisms underlying exocytosis are similar in cells of different types, it may be assumed that studying these mechanisms in secretory cells will allow experimenters to obtain information on ways to control this process in neurons. Based on the supposition that changes in the activity of Ca2+-controlling systems in exocrine cells play an important role in functional disorders in the salivary glands in diabetes mellitus, we demonstrated, using the fura-2/AM dye, that the intracellular calcium concentration ([Ca²⁺] _i ) in secretory cells of the above glands in rats with streptozotocin-induced diabetes mellitus (being in the resting state) is significantly increased (on average, by 65%). In our study, we showed that Ca²⁺-ATPases play an important role in the control of calcium homeostasis in secretory cells of salivary glands in diabetes mellitus. In particular, we demonstrated that the kinetic parameters of microsomal Ca²⁺-ATPases decreased: V ₀, by 50 ± 7, and P _max, by 52 ± 6%, on average. In diabetes mellitus, V _max of Ca²⁺-ATPases also dropped significantly, by 47 ± 8 and 79 ± 9%, on average, for PMCA and SERCA, respectively. The decrease in K _ATP was 71 ± 11% for SERCA and that in K _Ca was 92 ± 3% for PMCA. We concluded that the activity of Ca²⁺-ATPases of secretory cells in diabetes mellitus is suppressed because of a decrease in the turnover and/or in the specific number of active molecules of the enzyme.     

Hepatic Sinusoidal Endothelium Heterogeneity with Respect to the Recognition of Apoptotic Cells

The recognition and removal of human apoptotic peripheral lymphocytes in selected populations of periportal and perivenous endothelial cells was studied inin situandin vitroexperiments. Apoptotic peripheral blood lymphocytes once injected into the liver circulation were retained by the sinusoids showing a large heterogeneity of distribution: apoptotic cells are found in the periportal tract double the amount found in the perivenous region. Apoptotic PBL adhesion was lowered to a sixth of the control after preinjection with a sugar mixture (Mannose,N-acetylgalactosamine,N-acetylglucosamine,d-galactose), as suggested by the expression of modified surface glycoconjugates on the plasma membrane of apoptotic cells. A bimodal profile of the distribution of the hepatic sinusoidal cell population, regarding the number of galactose and mannose receptors and the porosity index, was found. Two endothelial cell subsets were present: low porosity cells (average index 14 ± 6%; periportal tract) with a high number of carbohydrate binding sites, and high porosity cells (average index 26 ± 7%; perivenous tract), with a low number of carbohydrate binding sites.     

Electrophysiological properties of onion guard cells

Intracellular electrical recordings in onion (Allium cepa L.) guard cells show that they maintain a membrane potential difference (MPD), inside negative. The MPD of intact cells averaged -72±29 mV (n=45); MPD of cells partially digested with a cellulolytic enzyme, -39±7 mV (n=65). Evidence indicates that the guard cells have two electrically distinct compartments, presumably delimited by the plasmalemma and tonoplast. Epidermal cells in partially digested preparations also showed MPD that could be either positive (+15±7 mV; n=23) or negative (-15 ±8 mV; n=13). Guard cells exposed to light-dark cycles hyperpolarized in the light and depolarized in the dark. The largest observed voltage changes reached 52 mV during hyperpolarizations and 60 mV during depolarizations. The light responses saturated with roughly exponential kinetics, with the depolarizations exhibiting a slower second phase that might be related to the contracting movements of the guard cells. Initial rates of the responses averaged about 14 mV min^-1 in the dark and about 8 mV min^-1 in the light. The results can be interpreted as electrical correlates of fluctuations in intracellular potassium concentration, as light-induced changes in membrane permeability, or as the photoactivation of an electrogenic proton pump. The last possibility seems to be the simplest interpretation of the data that also provides us with a mechanism driving the ion fluxes associated with stomatal function.