Calculation of effective diffusivities for biofilms and tissues.

In this study we describe a scheme for numerically calculating the effective diffusivity of cellular systems such as biofilms and tissues. This work extends previous studies in which we developed the macroscale representations of the transport equations for cellular systems based on the subcellular-scale transport and reaction processes. A finite-difference model is used to predict the effective diffusivity of a cellular system on the basis of the subcellular-scale geometry and transport parameters. The effective diffusivity is predicted for a complex three-dimensional structure that is based on laboratory observations of a biofilm, and these numerical predictions are compared with predictions from a simple analytical solution and with experimental data. Our results indicate that, under many practical circumstances, the simple analytical solution can be used to provide reasonable estimates of the effective diffusivity.     

Epidermal cells in activation of suppressor lymphocytes: further characterization

Intravenous administration of hapten-coupled, high-density (density greater than 1.077) epidermal cells (HD-EC) to mice results in the appearance of transferable splenic T suppressor (Ts) cells as assayed in adoptive transfer experiments. Depletion of I-A bearing cells from the HD-EC population before hapten coupling prevents these cells from inducing Ts cell formation, whereas depletion of Thy-1-bearing cells from the HD-EC cell preparation has no effect. When HD-EC are adhered to glass for 2 hr, the ability to induce Ts cell formation resides in the adherent population. Exposure of HD-EC to a dose of ultraviolet radiation (UVR) that largely abrogates the ability of hapten-coupled EC to immunize mice for a DTH response does not affect the ability of these cells to activate Ts cells. Treatment of mice with i.p. administration of 20 mg/kg of cyclophosphamide 2 days before EC harvesting abrogates the ability of HD-EC from these mice to induce Ts cell formation. HD-EC from B10.A(3R) (I-Jb) but not B10.A(5R) (I-Jk) mice induce Ts cell formation in B10.A(3R) mice, demonstrating that the ability to do so is restricted by the I-J locus. Transmission electron microscopy of adherent HD-EC populations demonstrated that two cell types were present. One type had the characteristics of keratinocytes; the other was monocyte-like and resembled Langerhans cells or indeterminate cells in many aspects. Immunoelectron microscopy revealed this second cell type to bear I-A/I-E antigen. These cells were T-200 positive and Mac-1 negative by immunoperoxidase staining. Extensive examination by light and electron microscopy failed to reveal any dermal components in the EC populations; however, a very small degree of dermal contamination cannot be excluded. Thus, EC that activate afferent-acting Ts cells are high-density, I-A+, Thy-1-, I-J restricted, glass adherent, and functionally UVR resistant and cyclophosphamide sensitive.     

Bombesin stimulation of inositol 1,4,5-trisphosphate generation and intracellular calcium release is amplified in a cell line overexpressing the N-ras proto-oncogene.

Human neutrophils and HL-60 leukaemic cells possess an NADPH oxidase which catalyses superoxide (O2-) formation and is activated by the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). In dibutyryl cyclic AMP-differentiated HL-60 cells, ATP and UTP in the presence of cytochalasin B activated O2- formation with EC50 values of 5 microM and efficacies amounting to 30% of that of fMet-Leu-Phe. The potency order of purine nucleotides in activating O2- generation was ATP = adenosine 5'-O-(3-thiotriphosphate) greater than ITP greater than dATP = ADP. Pyrimidine nucleotides activated NADPH oxidase in the potency order UTP greater than dUTP greater than CTP = TTP = UDP. Pertussis toxin completely prevented activation of NADPH oxidase by fMet-Leu-Phe and UTP, whereas the effect of ATP was only partially inhibited. ATP and UTP enhanced O2- generation induced by fMet-Leu-Phe by up to 8-fold, and primed the cells to respond to non-stimulatory concentrations of fMet-Leu-Phe. Activation of NADPH oxidase by UTP but not by ATP was inhibited by various activators of adenylate cyclase. In dimethyl sulphoxide-differentiated HL-60 cells and in human neutrophils, ATP and UTP per se did not activate NADPH oxidase, but they potentiated the effect of fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via purino- and novel pyrimidinoceptors respectively, which are coupled to guanine nucleotide-binding proteins leading to the activation of NADPH oxidase. As ATP and UTP are released from cells under physiological and pathological conditions, these nucleotides may play roles as intercellular signal molecules in the activation of O2- formation.     

Comparison of a new inorganic membrane filter (Anopore) with a track-etched polycarbonate membrane filter (Nuclepore) for direct counting of bacteria.

Bacterial counts obtained by using a new Anopore inorganic membrane filter were 21 to 33% higher than those obtained by using a Nuclepore polycarbonate membrane filter. In addition, the inorganic filter had higher flow rates, permitting lower vacuum pressures to be used, while the intrinsically flat, rigid surface resulted in easier focusing and sharp definition of bacteria across the whole field of view.     

Fertilization rates in superovulating cows after deposition of semen on the infundibulum, near the uterotubal junction or after insemination with high numbers of sperm

Three experiments were conducted with 105 superovulating Holstein dairy cows in attempts to improve the fertilization rate. Cows were superovulated with follicle-stimulating hormone (FSH) and time of estrus was regulated with prostaglandin F(2)alpha (PGF(2)alpha). Semen was deposited on each infundibulum through a laparoscope inserted through the flank (Experiment 1) or near the uterotubal junctions through flexible tubing passed through the cervix and uterine horns (Experiment 2). In the third experiment, high numbers of sperm in fresh semen were deposited in the uterus. Cows were necropsied and ova were recovered and examined about 3.5 d after the beginning of estrus. Deposition of 0.5 ml of frozen-thawed semen on each infundibulum (Experiment 1) reduced both ovum recovery and fertilization. In ten cows inseminated on the infundibulum, ova representing 43% of ovulation points were recovered and 9% of these recovered ova were fertilized. In ten control cows, ova representing 80% of ovulation points were recovered and 62% of them were fertilized. In a 2 x 2 experiment with 36 superovulating cows (Experiment 2), 1 ml of diluted fresh or frozen semen was deposited either near the uterotubal junction or in the uterine body. The overall fertilization rate was 61%, with no significant effect of site of semen deposition or type of semen used. In Experiment 3, 2 or 3 ml of neat semen (average of 4.4 billion sperm) was deposited in the uterus of 12 cows; 183 of 197 intact ova (93%) were fertilized. In 56 control cows inseminated with 0.5 to 1.5 ml of frozen diluted semen (average of 70 million sperm), 502 of 947 intact ova were fertilized (53%, P<0.001). Insemination with high numbers of fresh sperm overcame problems of sperm loss or sperm transport and improved the fertilization rate.     

Neuropathological changes in scrapie and Alzheimer's disease are associated with increased expression of apolipoprotein E and cathepsin D in astrocytes.

With the rationale that the neuropathological similarities between scrapie and Alzheimer's disease reflect convergent pathological mechanisms involving altered gene expression, we set out to identify molecular events involved in both processes, using scrapie as a model to study the time course of these changes. We differentially screened a cDNA library constructed from scrapie-infected mice to identify mRNAs that increase or decrease during disease and discovered in this way two mRNAs that are increased in scrapie and Alzheimer's disease. These mRNAs were subsequently shown by sequence analysis to encode apolipoprotein E and cathepsin D (EC 3.4.23.5). Using in situ hybridization and immunocytochemistry to define the cellular and anatomic pathology of altered gene expression, we found that in both diseases the increase in apolipoprotein E and cathepsin D mRNAs and proteins occurred in activated astrocytes. In scrapie, the increase in gene expression occurred soon after the amyloid-forming abnormal isoform of the prion protein has been shown to accumulate in astrocytes. In Alzheimer's disease, the increased expression of cathepsin D also occurred in association with beta-amyloid. These studies reveal some of the molecular antecedents of neuropathological changes in scrapie and Alzheimer's disease and accord new prominence to the role of astrocytes in neurodegenerative conditions.     

Information transfer between T cell subsets is directed by I-J+ antigen nonspecific molecules

T cell antigen-specific suppressor factors (TsF) consist of two distinct polypeptide chains: one that binds antigen (ABM) and one that bears I-J region markers (I-J+ chain). We studied the functional role of these two molecules in delivering the biologic message of suppression to its appropriate target cell. Two different biologically active TsF were used in these studies: TsiF, a T suppressor-inducer factor consisting of an ABM secreted by Ly-1 T cells (Ti-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Ji), which initiates the suppressor circuit by inducing an Ly-1,2 T cell; and TseF, a T suppressor-effector factor consisting of an ABM secreted by Ly-2 T cells (Te-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Je), which delivers the biologic message of suppression to the T helper (TH) cell. In both TsF, the ABM and I-J+ chain are noncovalently associated and can be easily separated. Both molecules must be present, however, for biologic activity of the TsF to be manifest. We studied the role of each chain in delivering these biologically active messages by constructing "hybrid" factors made from mixing the ABM from TsiF with I-J+ chains from either TsiF or TseF and determined which of these chains could reconstitute functional TsiF activity. Likewise, we mixed the AMB from TseF with I-J+ chains of TsiF or TseF to determine which I-J+ chain could reconstitute TseF activity. We found that I-J+ chain from TsiF (I-Ji) can reconstitute ABM from TsiF to form a functional TsiF capable of inducing suppression but cannot reconstitute ABM from TseF to form a functional TsiF capable of suppressing the activity of TH cells. Likewise, the addition of I-J+ chain from TseF to ABM from TseF can reconstitute its ability to suppress TH responses, but I-J+ chain from TsiF plus ABM from TseF has no effect on these TH cell responses. We did find, however, that this hybrid TsF composed of the ABM from TseF and the I-J+ chain from TsiF is capable of suppressing the Ly-1,2 Ttrans cell, the cell normally induced by the ABM + I-J+ suppressor inducer complex from T suppressor-inducer cells (TsiF).(ABSTRACT TRUNCATED AT 400 WORDS)