Importance of some factors on the dimorphism of Candida albicans

The passage between the yeast and mycelial forms of Candida albicans B 311-10 was studied by using the minimal syntehtic medium of Shepherd et al. [19] modified without biotin and with low glucose concentrations. It was observed that biotin, aminoacids and particularly pH are not important factors in the dimorphism of C. albicans. The only factor of notable importance in the passage of yeast form to mycelial form in C. albicans was glucose concentration.     

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.     

Selective lung leukosequestration after complement activation

This study tests whether activated complement leads to a selective entrapment of polymorphonuclear leukocytes (PMN's) in the lungs. Awake sheep were infused for 5 min with zymosan-activated plasma (ZAP, 2.5 mg/ml) at a rate of 5 ml/min into the superior vena cava (IV, n = 4) or intra-arterially into the aortic arch or femoral artery (IA, n = 8). At the end of IV infusion, leukocyte counts fell from 8,862 to 1,631/mm3 (P less than 0.01). PMN counts across the lungs decreased by 74%. There were increases in plasma thromboxane (Tx) B2 from 114 to 2,733 pg/ml (P less than 0.01), mean pulmonary arterial pressure from 12 to 42 mmHg (P less than 0.01), and physiological shunt from 13 to 25% (P less than 0.05). Within 1 h lymph TxB2 levels had risen from 301 to 4,916 pg/ml (P less than 0.01), lung lymph flow (QL) rose from 3.7 to 11.1 ml/30 min (P less than 0.05), lymph-to-plasma protein ratio (L/P) remained unchanged at 0.63, and lymph protein clearance increased from 2.3 to 7.5 ml/30 min (P less than 0.05). Leukosequestration, quantitated by capillary PMN counting and by assaying the granulocyte marker myeloperoxidase, occurred relative to sham animals (P less than 0.05) in the lung and spleen but not in other organs. Intra-arterial ZAP infusion led to changes that were similar in magnitude and timing to the IV group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Volume density of syncytial sprouts and its regional variation in the human mature placenta

The authors studied by means of sterological methods the volume density of syncytial sprouts in different cotyledonary regions of the human term placenta. The syncytial sprouts account for 4.92% of the placenta volume. Meanwhile, there were significant differences according to the site from which the sample was obtained. The lowest value (2.72%) corresponds to the central-parabasal zone of the cotyledon, close to the entry of the maternal blood into the intervillous space, whereas the highest values correspond to the "venous" regions. The results of the present study, as well as those reported by others, suggest that the lateral-subchorial zone would be the growing region of the cotyledon.     

Long-term cryopreservation of dog granulocytes

Granulocytes isolated by counterflow centrifugation elutriation (CCE) from leukapheresed dog blood, frozen in liquid nitrogen at ?196 °C, were studied. The effects of long-term cryopreservation on cell recovery and in vitro function were detertmined. In seven separate experiments, an average of 1.7 × 10⁹ granulocytes were obtained. The white cell differential count was 91% granulocytes and 9% mononuclear cells. There was less than 5% red cells presrent and no platelets. Granulocytes were placed in Hemoflex bags and mixed slowly with equal volumes of sterile ice-cold hyperosmolar cryoprotectant buffer to make a final composition of 5% dimethylsulfoxide (DMS), 6% hydroxyethyl starch (HES), and 4% bovine serum albumin (BSA), pH 7.1. Total volumes of 40 ml were frozen at a cooling rate of 4 °C per minute and stored for periods of 1, 34, 60, 90, and 132 weeks in liquid nitrogen at ?196 °C. Thawing was done at a rate of 190 ° per minute to 10 °C. The recovery of cells was 95%, 105%, 100%, 100%, and 88% respectively. Ethidium bromide exclusion, indicative of viable nuclei, was 91%, 81%, 94%, 89%, and 80% respectively. Virtually all thawed cells ingested opsonized Fluolite particles, but the number ingested was approximately one-half that of prefreeze values. Thawed cells also demonstrated superoxide anion synthesis at rates approximating those in unfrozen granulocytes. These results indicate that dog granulocytes obtained by leukapheresis may be preserved in liquid nitrogen at ?196 °C with high cellular recovery and at least 50% phagocytic function.     

Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.     

Neutrophil adherence receptors (CD 18) in ischemia. Dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B4

Neutrophils and eicosanoid chemoattractants are centrally involved with ischemia-reperfusion (I/R) injury. The CD 18 complex of adhesive glycoproteins, readily up-regulated by chemoattractants in vitro, is required for polymorphonuclear leukocyte (PMN) adherence to endothelium. This study tests whether CD 18 is up-regulated by ischemia in vivo and its role in mediating PMN diapedesis. Anesthetized rabbits underwent 3 h of bilateral hindlimb tourniquet ischemia (n = 16). Ten min after tourniquet release, levels of plasma leukotriene (LT)B4 increased to 390 +/- 62 pg/ml (mean +/- SE), higher than 134 +/- 26 pg/ml in control rabbits (n = 13, p less than 0.01). Aliquots of plasma were added to whole blood from normal rabbits (n = 6) for flow cytometric analysis of neutrophils with the CD 18 mAb R 15.7. Addition of I/R plasma failed to demonstrate an increase in surface expression of CD 18. Similarly, no CD 18 up-regulation was observed in vivo upon reperfusion in ischemic animals pretreated with mAb R 15.7 (n = 3). However, I/R plasma when introduced into plastic chambers taped atop dermabrasion sites in normal rabbits (n = 12) resulted in diapedesis, measured by the accumulation after 3 h of 1130 +/- 125 PMN/mm3 in the chambers relative to 120 +/- 31 PMN/mm3 with control plasma (p less than 0.01). Diapedesis in response to I/R plasma was abolished by pretreatment with mAb R 15.7 (less than 5 PMN/mm3, n = 6), was reduced by U 75,302, an LTB4 receptor antagonist (253 +/- 101 PMN/mm3, n = 6) (both p less than 0.01) and was not protein synthesis dependent. These results demonstrate that PMN diapedesis in response to I/R plasma is exclusively dependent upon the CD 18 glycoprotein complex by an LTB4-dependent mechanism, despite the fact that CD 18 is not up-regulated on circulating PMN in ischemia. These data indirectly indicate the functional importance of conformational changes of CD 18 in determining PMN adhesion.