Comparison of diaphragmatic fatigue in newborn and older rabbits

The ability to maintain occlusion pressure (i.e., fatigability) during activation of the diaphragm via phrenic nerve stimulation was compared in newborn (less than 14 days old) and older (greater than 30 days old) rabbits. The younger animals had lower maximum inspiratory pressures (MIP) and markedly greater falls in pressure during sustained diaphragmatic contractions at greater than 40% MIP than did the older animals. Histological analysis showed a paucity of high-oxidative type I fibers in the diaphragms of the young animals. We therefore conclude that the newborn rabbit diaphragm is extremely susceptible to fatigue and that this susceptibility correlates with the distribution of muscle fiber types.     

Ultrastructural and morphometric analysis of long-term peripheral nerve regeneration through silicone tubes

Brain Cell Biology - Light and electron microscopy were used to investigate long-term regeneration in peripheral nerves regenerating across a 10 mm gap through silicone tubes. Schwann cells and...     

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.     

In situ hybridization as a rapid means to assess meiotic pairing and detection of alien DNA transfers in interphase cells of wide crosses involving wheat and rye

Summary The objectives of this study were to determine if biotin-labelled total genomic DNA of rye (Secale cereale L.) could be used to (i) preferentially label rye meiotic chromosomes in triticale and (ii) detect translocation stocks at interphase and/or early prophase by in situ hybridization. Welsh triticale, a wheat-rye segmental amphiploid, and Kavkaz wheat, a wheat-rye translocation were used. The results indicated that labelled chromosomes of rye and unlabelled chromosomes of wheat could be observed throughout all meiotic stages in the triticale. For Kavkaz wheat, the presence of the translocated 1RS chromosome arm of rye was detected at the interphase or very early prophase stage. Rapid assessment of feasibility of gene transfers and detection of alien DNA in somatic cells at the interphase stage by in situ hybridization allows for rapid decision-making and saves time and expense in plant breeding programs.Plant Research Centre Contribution No. 1276