Histochemical, immunofluorescence, and ultrastructural differences in fetal cartilage among three genetically distinct chondrodystrophic mice

The severe lethal chondrodystrophies in man result in a common clinical syndrome including shortening of the face, mandible, and limbs. Studies of three lethal chondrodystrophic mutants in mice, viz., chondrodysplasia (cho), cartilage matrix deficiency (cmd), and disproportionate micromelia (Dmm), which share this syndrome, were performed with the aim of identifying histochemical, immunofluorescence, or ultrastructural differences which might exist among these hereditary cartilage disorders. We examined limb cartilage epiphyses from day 18 normal and mutant fetuses and observed repeatable, mostly qualitative differences. All observations were made relative to the normal control. Histochemical staining of matrix proteoglycan was moderately decreased in cho and Dmm cartilage and markedly decreased in cmd when compared to the normal control. Staining of matrix collagen was irregular in distribution in cho, increased in cmd, and decreased in Dmm. Immunofluorescence of proteoglycan was increased in the matrix of cho and Dmm and decreased in cmd. Immunofluorescence of type II collagen was heterogeneous and moderately decreased in the matrix of cho, increased in cmd, and markedly decreased in Dmm. Immunofluorescence of link protein in cho was localized in the cellular-pericellular region as in the normal and appeared increased in the matrix of cmd and Dmm. Immunofluorescence of chondronectin was localized in the cellular-pericellular region and appeared normal in all three mutants. Major differences in cellular and matrix ultrastructure were observed among the mutants, including a decreased frequency of small-diameter collagen fibrils in cho and Dmm, increased density of collagen fibrils in cmd, and dilated RER in Dmm. These observations demonstrate that distinct structural and possibly molecular differences exist among the chondrodystrophies. In the case of cmd, the differences correlated with a previously reported molecular defect, viz., absence of core protein of cartilage specific proteoglycan in the cartilage of this mutant. It is anticipated that the methods used in the present study can be applied to humans in case classification and in identifying potential mouse-human correlates.     

Interrupter resistance elucidated by alveolar pressure measurement in open-chest normal dogs

The interrupter method for measuring respiratory system resistance involves rapidly interrupting flow at the mouth while measuring the pressure just distal to the point of interruption. The pressure signal observed invariably exhibits two distinct phases. The first phase is a very rapid jump, designated delta Pinit, which occurs immediately on interruption of flow. The second phase is designated delta Pdif and is a further pressure change in the same direction as delta Pinit but evolving over several seconds. The physiological interpretations of delta Pinit and delta Pdif have been somewhat unclear. Delta Pinit has been taken to equal the pressure drop across the pulmonary airways, possibly with a contribution from the tissues of the respiratory system. Delta Pdif can arise, in principle, from two sources: gas redistribution throughout the lung after interruption of flow and stress recovery within the tissues. To resolve these issues we performed interruption experiments on anesthetized paralyzed, tracheotomized, open-chest normal dogs during passive expiration while measuring alveolar pressures at three sites with alveolar capsules. We found that, in the absence of the chest wall, delta Pinit reflects only the resistance of the airways and that delta Pdif can be ascribed almost entirely to the stress recovery properties of lung tissues.     

Taxol-induced reorganization of the microtubule system in murine splenic lymphocytes inhibits response to allogeneic cells but not to concanavalin A

The exposure of mouse splenic lymphocytes to the microtubule assembly-promoting drug taxol (10 microM for 4 h) results in an extensive reorganization of the microtubule system to form one to a few large bundles of microtubules, which extend from the centrosome. Lymphocytes pretreated with taxol for 4 h, or cultured in the continued presence of taxol, respond normally to the mitogen concanavalin A up to, and including, the stage of DNA replication. In contrast, the induction of DNA synthesis during the alloactivation of lymphocytes is inhibited when taxol is present in the mixed leukocyte culture. If the stimulators are pretreated with this drug, the mixed leukocyte reaction occurs normally, but pretreatment of the responders inhibits the proliferative response markedly. Microscopic observations of nuclear morphologies in these populations and autoradiography indicate that taxol inhibition occurs early in alloactivation, prior to DNA replication. The responding ability of taxol-treated lymphocytes is not restored to control levels by the addition of interleukin 2, leading to the suggestion that interleukin 2 receptors do not emerge or function normally in these cells. We conclude that the capacity to respond to allogeneic cells, but not to a mitogen, is dependent on the presence of the normal submembranous organization of the microtubule system.     

Expression of Hox-4 genes in the chick wing links pattern formation to the epithelial-mesenchymal interactions that mediate growth.

The relationship between the expression of Hox-4 genes in the mesenchyme and the apical ectodermal ridge was investigated in both normal chick wing buds and wing buds treated with retinoic acid. Two conclusions emerge. One is that the activation of Hox-4 domains and the elaboration of Hox-4 gene expression patterns involve cooperation with a signal from the apical ridge. The second is that the domains of expression of 5'-located members of the complex correlate with the maintenance of the thickened ridge which is required for subsequent bud outgrowth.     

Roles of PRP8 protein in the assembly of splicing complexes.

Three different approaches have been used to investigate the roles of the yeast U5 snRNP protein PRP8 in spliceosome assembly: genetic depletion of PRP8 protein in vivo, heat inactivation of temperature-sensitive prp8 protein in protoplasts and inhibition of PRP8 function with antibodies in vitro. In each case, U5 and U4/U6 snRNPs failed to assemble into the forming spliceosomes. In addition, extract prepared from PRP8-depleted cells and extract containing inactivated PRP8 protein had substantially reduced amounts of U4/U6.U5 triple snRNP complexes. Thus, functional PRP8 protein is required for the stable formation of U4/U6.U5 complexes without which spliceosomes fail to form. As spliceosome formation was also blocked by anti-PRP8 antibodies that apparently do not disrupt triple snRNPs, PRP8 protein may play a separate role in the assembly of triple snRNPs into spliceosomes. As a consequence of PRP8 depletion the levels of the U4, U5 and U6 snRNAs declined dramatically. We discuss this in the context of the known genetic interactions between PRP8 and putative RNA helicase (DEAD box protein) genes and propose that PRP8 protein plays a role in regulating dynamic RNA-RNA interactions in spliceosome assembly, possibly ensuring the correct directionality of these events.     

Biological responses to overload training in endurance sports

Five subjects undertook 10 days of twice daily interval training sessions on a treadmill followed by 5 days of active recovery. On days 1, 6, 11, and 16 the subjects were required to undertake a test of submaximal and maximal work capacity on a treadmill combined with a performance test consisting of a run to exhaustion with the treadmill set at 18 km.h-1 and 1% gradient. Also on these days a pre-exercise blood sample was collected and analysed for a range of haematological, biochemical and immunological parameters. The subjects experienced a significant fall in performance on day 11 which had returned to pretraining levels on day 16. Serum ferritin concentrations were depressed significantly from pretraining concentrations at the conclusion of the recovery period while the expression of lymphocyte activation antigens (CD25+ and HLA-DR+) was increased both after the training phase and the recovery phase. The number of CD56+ cells in the peripheral circulation was depressed at the conclusion of the recovery period. Several parameters previously reported to change in association with overload training failing to reflect the decrease in performance experienced by subjects in this study, suggesting that overtraining may best be diagnosed through a multifactorial approach to the recognition of symptoms. The most important factor to consider may be a decrease in the level of performance following a regeneration period. The magnitude of this decreased performance necessary for the diagnosis of overtraining and the nature of an "appropriate" regeneration period are, however, difficult to define and may vary depending upon the training background of the subjects and the nature of the preceding training. It may or may not be associated with biochemical, haematological, physiological and immunological indicators. Individual cases may present a different range of symptoms and diagnosis of overtraining should not be excluded based on the failure of blood parameters to demonstrate variation. However, blood parameters may be useful to identify possible aetiology in each separate case report of over-training. An outstanding factor to emerge from this study was the difficulty associated with an objective diagnosis of overtraining and this is a possible reason why there have been new accounts of overtraining research in the literature.     

Glomerular bypass shunts and distribution of glomeruli in the kidney of the lesser spotted dogfish,Scyliorhinus caniculus

Summary Scanning electron microscopy of corroded resin casts of the renal vasculature of Scyliorhinus caniculus has revealed a novel vascular pathway arising from the afferent arteriole and bypassing the glomerulus. This glomerulus bypass shunt occurred in 36% of the glomerular casts examined. The shunt ran to join a peritubular network of capillaries and thereby offers the potential to vary the degree of glomerular perfusion and control the proportion of active glomeruli. In 29% of glomeruli two efferent arterioles drained the capillary knot. Glomeruli were located close to the dorsal margin of the posterior mass of the kidney, and towards the lateral edge of the anterior lobes of the kidney of female dogfish. In male dogfish, glomeruli were evenly distributed through the posterior mass of kidney, while in female dogfish 89% of glomeruli occurred in the posterior mass and 11% of glomeruli were located within the small anterior lobes.     

Ultrastructural identification and distribution of the adhesion molecules ICAM-1 and LFA-1 in the vascular and extravascular compartments of the human palatine tonsil

Summary Immunohistological analysis of sections prepared from human palatine tonsils revealed marked differences in the distribution of the adhesion molecule, leucocyte function antigen-1 (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1). Light microscopy showed that LFA-1 was restricted to the leucocytes, particularly the lymphocytes. In contrast, staining of ICAM-1 was predominantly confined to the vascular endothelium with the greatest expression seen on the morphologically distinct high endothelial venules in the parafollicular areas; these are the sites that appear to support lymphocyte migration. Electron microscopy revealed that ICAM-1 was present on the luminal and lateral surfaces of the high endothelium and absent from the abluminal surface supported by basal lamina. The ICAM-1 was also absent from those surfaces of the endothelium that were in close contact with intravascular lymphocytes. Other cells stained by the anti-ICM-1 antibody included dendritic cells, plasma cells and epithelial cells in the reticulated crypt epithelium and in the upper strata of the non-keratinised stratified squamous epithelium. The high expression of LFA-1 was most prominent on lymphocytes, low on antigen-presenting cells and activated lymphoid cells, and not detectable on plasma cells, epithelial and endothelial cells. We propose that LFA-1/ICAM-1 binding participates in mediating the transendothelial migration of lymphocytes across the high endothelial venules of palatine tonsil.     

The molecular structure of a 4'-epiadriamycin complex with d(TGATCA) at 1.7A resolution: comparison with the structure of 4'-epiadriamycin d(TGTACA) and d(CGATCG) complexes.

The structure of the complex between d(TGATCA) and the anthracycline 4'-epiadriamycin has been determined by crystallographic methods. The crystals are tetragonal, space group P4(1)2(1)2 with unit cell dimensions of a = 28.01, c = 52.95A. The asymmetric unit consists of one strand of hexanucleotide, one molecule of 4'-epiadriamycin and 34 waters. The R-factor is 20.2% for 1694 reflections with F greater than or equal to 2 sigma F to 1.7A. Two asymmetric units associate to generate a duplex complexed with two drug molecules at the d(TpG) steps of the duplex. The chromophore intercalates between these base pairs with the anthracycline amino-sugar positioned in the minor groove. The double helix is a distorted B-DNA type structure. Our structure determination of d(TGATCA) complexed to 4'-epiadriamycin allows for comparison with the previously reported structures of 4'-epiadriamycin bound to d(TGTACA) and to d(CGATCG). The three complexes are similar in gross features and the intercalation geometry is the same irrespective of whether a d(CpG) or d(TpG) sequence is involved. However, the orientation of the amino-sugar displays a dependence on the sequence adjacent to the intercalation site. The flexibility of this amino-sugar may help explain why this class of antibiotics displays a relative insensitivity to base sequence when they bind to DNA.     

Synthesis of oligodeoxyribonucleotides containing degenerate bases and their use as primers in the polymerase chain reaction.

Heptadecaoligodeoxyribonucleotides containing one or more of the bases, 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P), 2-amino-6-methoxyaminopurine (K), and hypoxanthine (I) and combinations of P with K and I have been synthesised on a DNA synthesiser. The stability of duplexes containing these basemodified oligomers with P/A, P/G, K/C and K/T; P/A, P/G, I/C, I/T and I/A, I/G, I/C, I/T base pairs were compared by measuring their melting transition (Tm) values. Oligomers containing both P and K and P and I were more stable than those with I alone or with mismatches. These oligomers together with one with a P base at the 3'-end were used as primers in polymerase chain reaction (PCR) experiments. They were all effective primers except one with I alone and a triple mismatch. Thus the use of the degenerate bases P and K in primer design is established.