A sensitive assay for detecting low-affinity interactions at the cell surface reveals no additional ligands for the adhesion pair rat CD2 and CD48

The ligand for the T cell antigen CD2 is CD48 in rodents, but CD58 in humans. The extracelluar parts of these three antigens are structurally related in that all contain two immunoglobulin superfamily (IgSF) domains. There have been reports of alternative ligands for CD2 in the human, but not so far in rodents. We describe the analysis of ligands for rat CD2 and CD48 using fluorescent beads capable of displaying a high ligand density and detecting low-affinity interactions like that of CD2 with CD48 (Kd = 60 ? 90 μM). Monovalent chimeric proteins containing the two IgSF domains of rat CD48 or CD2 and domains 3 and 4 of rat CD4 (CD4d3+4) were anchored to fluorescent covaspheres^TM via a CD4 monoclonal antibody (mAb) with the CD48 or CD2 domains available for ligand binding. Multivalent CD48-CD4d3 + 4 covaspheres^TM gave strong specific binding to rat CD2 expressed on the surface of transfected Jurkat cells. CD48-CD4d3+4 was compared with CD48-IgG and CD48-IgM as tools for detecting binding at the cell surface. Soluble divalent CD48-IgG and decavalent CD48-IgM bound to soluble CD2 with a K_off of around 10^?3 s^?1 as determined using a BIAcore^TM biosensor. However, binding to cells by CD48-IgG and CD48-IgM was only detectable when they were immobilized on covaspheres^TM and represented no increase in sensitivity over CD48-CD4 covaspheres^TM when tested for binding to cells expressing high and low levels of CD2. CD48-CD4d3 + 4 covaspheres^TM only bound to rat cells expressing CD2. In the reverse orientation, binding of CD2-CD4d3 + 4 covaspheres^TM was dependent on expression of CD48. Pre-incubation of cells with CD2 or CD48 mAb abolished all binding of CD48-CD4d3 + 4 or CD2-CD4d3 + 4, respectively. The data provide no evidence for an alternative ligand for rat CD2 or CD48.     

CD39 activity correlates with stage and inhibits platelet reactivity in chronic lymphocytic leukemia

Background  

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of mature appearing lymphocytes and is rarely complicated by thrombosis. One possible explanation for the paucity of thrombotic events in these patients may be the presence of the ecto-nucleotidase CD39/NTDPase-1 on the surface of the malignant cells in CLL. CD39 is the major promoter of platelet inhibition in vivo via its metabolism of ADP to AMP. We hypothesize that if CD39 is observed on CLL cells, then patients with CLL may be relatively protected against platelet aggregation and recruitment and that CD39 may have other effects on CLL, including modulation of the disease, via its metabolism of ATP.     

Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs

 Glial cell line-derived neurotrophic factor (GDNF) has recently been shown to signal by binding to GDNF receptor-alpha (GDNFR-α), after which the GDNF-GDNFR-α associates with and activates the tyrosine kinase receptor Ret. We have localized Ret messenger RNA (mRNA) in the developing and adult rodent and compared with to the expression of GDNF and GDNFR-α mRNA. Ret mRNA is strongly expressed in dopamine neurons and α-motorneurons as well as in thalamus, ruber and occlumotor nuclei, the habenular complex, septum, cerebellum, and brain stem nuclei. Ret mRNA was also found in several sensory systems, in ganglia, and in nonneuronal tissues such as teeth and vibrissae. Very strong Ret mRNA signals are present in kidney and the gastrointestinal tract, where Ret and GDNF mRNA expression patterns are precisely complementary. The presence of Ret protein was confirmed in adult dopamine neurons using immunohistochemistry. GDNFR-α mRNA was strongly expressed in the developing and adult dopamine neurons. It was also found in neurons in deep layers of cortex cerebri, in hippocampus, septum, the dentate gyrus, tectum, and the developing spinal cord. In the kidney and the gastrointestinal tract, GDNFR-α mRNA and Ret mRNA distribution overlapped. Dorsal root ganglia, cranial ganglia, and developing peripheral nerves were also positive. GDNFR-α was additionally found in sensory areas and in developing teeth. Sensory areas included inner ear, eye, olfactory epithelium, and the vomeronasal organ, as well as developing tongue papillae. The temporospatial pattern of expression of GDNFR-α mRNA did not always match that of Ret mRNA. For instance, GDNFR-α mRNA was also found in the developing ventral striatum, including the olfactory tubercle, and in hippocampus. These areas seemed devoid of Ret mRNA, suggesting that GDNFR-α might also have functions unrelated to Ret. Received: 2 January 1997 / Accepted: 26 February 1997     

Localization of actin in mammalian spermatozoa: a comparison of eight species

The distribution of monomeric and polymeric actin in spermatozoa from the bull, boar, rabbit, human, rat, mouse, golden hamster, and guinea pig has been examined by using a monoclonal antiactin antibody and NBD-phallacidin. Actin was present in sperm from each species. When the monoclonal antibody was used, there was a species-specific distribution and intensity of fluorescence, but no generalized pattern. Specific fluorescence was noted in the neck and principal piece of human sperm; in the postacrosomal region, neck, and midpiece of bull and boar sperm; in the postacrosomal region, neck, and principal/equatorial segment border of rabbit sperm; in the neck region of hamster sperm; and in the neck, midpiece, and principal piece of rat, mouse, and guinea pig sperm. Sperm from all eight species displayed no specific fluorescence with NBD-phallacidin, indicating that actin was present in a nonfilamentous form. SDS extracts of sperm were analyzed by SDS-PAGE and Western blotting; in sperm from each species, a 42-kD protein with specific affinity for the monoclonal antibody was present.     

The slide centrifuge gram stain as a urine screening method.

A slide centrifuge Gram stain procedure was performed to screen for bacteriuria 4161 urine specimens submitted in urine preservative tubes for routine culture. For slide centrifuge Gram staining, each urine sample was mixed well. Thereafter, 0.2 mL of each sample was placed, using a pipette, into a slide centrifuge chamber and centrifuged at 2,000 rpm for 5 minutes. The slides were heat fixed, Gram stained, and read by laboratory personnel who scanned 12 consecutive oil-immersion fields using a set pattern. The presence of the same organism in six or more fields was defined as a positive urine screen. Urine samples were cultured using a 0.001-mL loop and a comparison of culture growth with slide centrifuge screening was made. When growth of 100,000 or more colony-forming units per milliliter (CFU/mL) was the reference for comparison, the screen had a sensitivity rate of 98%, a specificity rate of 90%, a negative predictive value of 99%, and a positive predictive value of 65%. When a lower colony count of 10,000 or more CFU/mL was the reference for comparison, the screen had a sensitivity rate of 88%, a specificity rate of 95%, a negative predictive value of 96%, and a positive predictive value of 84%. The slide centrifuge Gram stain is a very sensitive screening method to detect bacteriuria in an adult male population.     

Morphological evidence of reduced bone resorption in the osteosclerotic (oc) mouse

Osteopetrosis, a metabolic bone disease characterized by a generalized sclerosis of the skeleton, is inherited as an autosomal recessive in a number of mammalian species. The pathogenesis of congenital osteopetrosis is mediated by a reduction in bone resorption as a result of decreased osteoclast function. This hypothesis is based on both functional and structural evidence of reduced bone resorption in all mutations examined to date. The present study examined the histology of cartilage and bone, the ultrastructure of osteoclasts, and the morphology of mineralized bone surfaces in a lethal osteopetrotic mutation, the osteosclerotic (oc) mouse. Histologically, epiphyseal cartilage growth plates, especially the hypertrophic zone, are markedly thickened in oc mice and metaphyses contain excessive osteoid, features characteristic of rickets. Transmission electron microscopy revealed that less than one-quarter of osteoclasts in oc mice demonstrated evidence of ruffled border formation compared with three-quarters of the osteoclasts in normal littermates. In mutants, ruffled borders were less elaborate and cytoplasmic processes penetrated into bone surfaces, suggesting that bone may be removed by mechanical rather than by enzymatic means. There was little morphological evidence of cartilage degradation and broad laminae limitantes persisted in mutants. Mineralized surfaces that undergo resorption in normal mice showed no evidence of bone resorption by scanning EM in mutants. The presence of a rachitic condition, the observations of reduced bone resorption, and the possible contribution of undermineralized matrices to decreased bone resorption are charcteristics of the osteosclerotic mutation which suggest that it is a unqiue csteopetrotic mutant in which to study both the development and regulation of skeletal metabolism.     

Osteopetrosis in the toothless (tl) rat: Presence of osteoclasts but failure to respond to parathyroid extract or to be cured by infusion of spleen or bone marrow cells from normal littermates

Osteoclasts have been observed for the first time in toothless (tl ) rats, a mutation which inherits osteopetrosis as an autosomal recessive. The ability of tl rats to raise the serum calcium concentration after injection of parathyroid extract was severely limited when compared with normal littermates. In addition, osteopetrosis in tl rats is not cured by radiation and infusion of normal spleen or bone marrow cells from normal littermates, a method known to cure osteopetrosis in mutants of this and other species. This indirect evidence for a reduction in bone resorption as a cause of osteopetrosis in this mutation and the failure of transplanted cells to cure the disease are discussed in relation to the development and function of osteoclasts.