Micropore Filtration Research Articles

Calcium translocation by Golgi and lateral-basal membrane vesicles from rat intestine: decrease in vitamin D-deficient rats.

Intestinal Ca2+ transport was studied in membrane vesicles isolated from microvillus, Golgi, and lateral-basal membrane preparations. Ca2+ uptake by these vesicles was measured by determination of 45Ca2+ associated with these membranes after collection by micropore filtration. Golgi membranes showed the highest initial rate and equilibration level of Ca2+ uptake. Approximately 90% of this Ca2+ uptake was into an osmotically responsive space, suggesting that what was measured was predominantly Ca2+ translocation. Vitamin D-deficient rats showed a markedly diminished rate of uptake and level of equilibration. These data indicate that a Ca2+-translocating process was associated with Golgi membranes to a greater extent than with surface membranes and that this process was markedly decreased in vitamin D-deficient rats. The results suggest that the Golgi apparatus participates in intestinal Ca2+ absorption.

Effects of Intersept micropore filtration of blood on microaggregates and other constituents.

Micropore filtration (Intersept) of whole, stored blood was examined in two studies. In Study A, 1 unit of 14-day-old blood flowed by gravity across the filter. In Study B, the filter was preloaded by passage of 2 units of blood, and the effects on a third, consisting of 21-day-old blood, flowing under 150 mm Hg pressure, were examined. Filtration did not significantly alter erythrocyte count, total hemoglobin, plasma hemoglobin, erythrocyte fragility, plasma sodium, potassium, albumin, or globulin in either study, although some platelets and leukocytes were removed. Microaggregates, assessed by Coulter counting, screen filtration pressure, total screen proteins, wet and dry weights of material retained, and scanning electron microscopy, were satisfactorily removed over the whole range of particle sizes. Comparison with the Bentley PFS-127, Fenwal 4C2417, Pall Ultipore, and Swank IL200 filters led to the conclusion that the Intersept is the most efficient filter available at the present time for removing microaggregates during massive blood transfusion.

Catecholamine Binding to Plasma Membrane Enriched Fractions of Heart and Skeletal Muscle

Binding of [3H]epinephrine to plasma membrane enriched fractions from guinea pig heart and rabbit skeletal muscle was investigated using the micropore filtration technique. [3H]Epinephrine and [3H]norepinephrine were found to be degraded rapidly in aqueous buffer at pH 7.6 and 37 degrees C. Deterioration of the compounds could be prevented by low concentrations of dithiothreitol. Binding of [3H]epinephrine to both membrane preparations was a slow process requiring 60 min to approach equilibrium in the case of cardiac membranes at 37 degrees C, and 20 min for skeletal muscle membranes at O degrees C. Binding was antagonized by the unlabeled beta-agonists, isopropylnorepinephrine, epinephrine, and norepinephrine but all were equipotent. A variety of catechol compounds were as effective antagonists of binding as the catecholamines. The beta-adrenergic antagonists propranolol, pronethalol, and dichloroisoproterenol were not effective in inhibiting binding to either membrane preparation. D-Norepinephrine and L-norepinephrine were equi-effective in antagonizing binding of [3H]norephinephrine to skeletal muscle membranes. It was concluded that binding of labeled catecholamine to isolated tissue membranes using the micropore filtration technique does not represent interaction with the specific beta-adrenergic receptor, but more likely reflects a less specific binding of compounds having one or more hydroxyl groups on a ring.

The Purification of Red Cells for Transfusion by Freeze‐Preservation and Washing. IV. The Use of Micropore Filtration To Reduce the Residual HL‐A Antigenicity of Previously Frozen, Washed Red Cells

Using a semi‐quantitative HL‐A antibody adsorption assay, a reduction in HL‐A antigenicity was observed in 15 of 22 units of previously frozen and washed red blood cells after passage through a micropore transfusion filter. Red blood cell viability was unaffected by this procedure. Micropore filtration was a simple adjunct to the freezing and washing of red blood cells that may further reduce histocompatibility antigen exposure in transfused recipients.

Particulate matter in bubble oxygenators

The degree of contamination was evaluated in seven blood oxygenators in common use. The Bentley Q 100 unit was found to have the highest degree of particulate contamination and the Travenol 6LF the lowest. Evidence is presented in favor of adopting a micropore filtration and washing technique of the bubble oxygenator immediately before open-heart surgery. Vast amounts of particulate matter were removed from the seven types of blood oxygenators studied. The incidence of postoperative neurologic deficits decreased after the incorporation of this technique into the preparation of the heart-lung machine.

The purification of red cells for transfusion by freeze preservation and washing. I. The mechanism of leukocyte removal from washed, freeze-preserved red cells.

Leukocytes were removed from washed, freeze‐preserved red blood cells primarily by the clumping together of damaged cells. This agglomeration was associated with membrane and nuclear changes which were usually manifested after the removal of glycerol from the red blood cells. Washing glycerolized, freeze‐preserved leukocytes with solutions containing deoxyribonuclease prevented the agglomeration. Polymorphonuclear granulocytes were preferentially damaged and removed. Of the residual leukocytes that were isolated by the dextran sedimentation method, 70 per cent were lymphocytes. The dye exclusion test showed that the majority of the agglomerated leukocytes were nonviable, while the majority of the leukocytes isolated by dextran sedimentation were viable. The agglomerated material ranged in size from macroscopic aggregates to microscopic particles with a minimum diameter of 40‐50μ. HL‐A antibody adsorption demonstrated that particulate antigenic material was present in the washed red blood cells which could he decreased by micropore filtration.

EXAMINATION OF FOOD AND WATER FOR CHOLERA VIBRIOS

A procedure to detect cholera vibrios in food and water, consisting of the examination of large samples, using pre-enrichment, concentration by micropore filtration, and enrichment in alkaline peptone broth is described. This method was used to examine ten types of foodstuffs exported from countries invaded by cholera. No cholera vibrios were found in these 435 samples, whereas 37 of 964 specimens collected in households of cholera patients harbored these organisms. It appeared that the risk of transmission of cholera by uncooked cereals, legumes, acidic produces, properly canned food, and dried fruits and cereal products is negligible. A search for salmonellae and shigellae was carried out simultaneously. One of the 10 exported smoked fish samples yielded Salmonella typhimurium. Of the 964 samples collected in homes, 10 harbored salmonellae, and 11 shigellae. Food from cholera-infested areas also should be examined for these organisms.

Inhibition of fertilization in rabbits during treatment with progesterone.

Adult female rabbits (Dutch strain) were artificially inseminated with 0.5 ml of diluted semen (10 X 106 to 40 X 106 spermatozoa) and ovulated with 100 to 200 IU of HCG. Corn oilor progesterone in corn oilwas injecteddailysubcutaneously for various periods, ranging from 0 to 6 days before insemination and continuing untilsacrifice48 hr after insemination. Fertilizationof ova was inhibited when 1 mg progesterone was injected daily for 2 or 6 days before insemination but not when the treatment began on the day of insemination or 4 days before insemination. An increasein the transport rate of ova occurred only when treatment was initiated 2 days before insemination. The fertilizationinhibitory response to progesterone was dose dependent between doses of 0.05 and 4 mg/day and the ED50 was 0.4 mg. Although none of the levelsemployed was 100% effective,only 2% of the ova recovered from the group given 4 mg/day were fertilized. A sperm index with values ranging from 0 to 3 was employed to indicate the total number of sperm counted in the mucin layer, zona pellucida, or perivitelline space of ova. Values in all three areas were low in ova recovered from animals given 0.5 mg progesterone/day and were reduced to 0 or near zero with all higher doses. When semen was deposited into the uterus of animals given 1 mg progesterone/day for 6 days, nearly all ova were fertilized. In animals given 1 mg progesterone/day, an increase in the semen concentration from 10 to 100% failed to significantly increase the percentage of ova fertilized, but successive injections of 50 U.S.P. units of oxytocin 1 hr before insemination and 1 and 4 hr afterwards increased the percentage of ova fertilized from 0 to 45% and increased the sperm index values approximately 50% in each of the layers of the ovum. Spermatozoa were recovered from the cervix and serial segments of the uterus and oviducts at 4, 8, 16, and 24 hr following insemination; they were counted using a micropore filtration technique. The numbers of sperm in all segments of the uterus and oviducts of the progesterone-treated animals were consistently less than in corresponding segments of control organs; they were absent from the anterior 2/3 of the oviduct until 16 hr after insemination at which time only a few in the middle oviducal segment were found. Progesterone inhibited fertilization of ova, we conclude, primarily by interference with sperm transport mechanisms in the uterus and/or oviduct.

Estimation of spermatozoa numbers using a micropore filtration technique.

The estimation of sperm concentration of semen samples has been accomplished by a variety of different methods. The equipment used in these techniques has ranged from the simple haemocytometer chamber to more highly sophisticated electronic means such as measuring light transmission (Willet & Buckner, 1951) and counting particle size (Glover & Phipps, 1962). Although electronic means of counting spermatozoa offer obvious advantages in situations where large numbers of routine counts are involved, estimates made with a haemocytometer have been the method of choice for most researchers because the technique is relatively accurate, utilizes simple inexpensive equipment and is rapid. In addition, the haemocytometer, unlike electronic methods, has been used successfully in conjunction with centrifugation to study the movement of spermatozoa through the female reproductive tract (Braden, 1953). This method, however, was not entirely satisfactory in our laboratory when it was applied to reproductive studies involving pharmacological means of impeding sperm migration because the total number of spermatozoa contained in oviducal washings from treated animals was frequently reduced to only a few spermatozoa or none at all. In an attempt to develop a more suitable method to use under these conditions, micropore filtration techniques that had been developed for the cytological study of cells in body fluids and the washings of various organs, were modified to count spermatozoa. In the sequence of experiments to be described, semen samples were used to establish the feasibility of the method, which was applied to the recovery of spermatozoa from the female reproductive tract. Rabbit semen was collected with the aid of an artificial vagina and ejaculates from several males were pooled to provide the samples for sperm counts. Samples were diluted with 0-9% NaCl solution and 0-1 ml of diluted semen was filtered. The filtration unit consisted of a 5-ml hypodermic syringe attached to a Swinny filter holder containing a 13-mm type HA filter with pore size of 0-45 + 0-2 µ. The filter was moistened with physiological saline (0-9% NaCl solution in distilled water) before use. Organ washings were obtained from adult oestrous rabbits (Dutch belted strain) that were inseminated artificially with 0-5 ml of semen diluted with physiological saline to a concentration of 20xl06 spermatozoa/ml. Animals were killed 16 hr after insemination. Physiological saline (2 ml) was forced through one end of each uterine horn or