Cholinesterases and peanut agglutinin binding related to cell proliferation and axonal growth in embryonic chick limbs

Embryonic cholinesterases are assigned important functions during morphogenesis. Here we describe the expression of butyrylcholinesterase and acetylcholinesterase, and the binding of peanut agglutinin, and relate the results to mitotic activity in chick wing and leg buds from embryonic day 4 to embryonic day 9. During early stages, butyrylcholinesterase is elevated in cells under the apical ectodermal ridge and around invading motoraxons, while acetylcholinesterase is found in the chondrogenic core, on motoraxons and along the ectoderm. Peanut agglutinin binds to the apical ectodermal ridge and most prominently to the chondrogenic core. Measurements of thymidine incorporation and enzyme activities were consistent with our histological findings. Butyrylcholinesterase is concentrated near proliferative zones and periods, while acetylcholinesterase is associated with low proliferative activity. At late stages of limb development, acetylcholinesterase is concentrated in muscles and nonexistent within bones, while butyrylcholinesterase shows an inverse pattern. Thus, as in other systems, in limb formation butyrylcholinesterase is a transmitotic marker preceding differentiation, acetylcholinesterase is found on navigating axons, while peanut agglutinin appears in non-invaded regions. These data suggest roles for cholinesterases as positive regulators and peanut-agglutinin-binding proteins as negative regulators of neural differentiation.     

Spatial and temporal pattern of purkinje cell degeneration in shaker mutant rats with hereditary cerebellar ataxia

Temporal-spatial patterns of surviving Purkinje cells were studied quantitatively in a rat mutant (shaker) with differential hereditary cerebellar ataxia and Purkinje cell degeneration. Shaker rat mutants are characterized behaviorally as mild if they are ataxic or as strong if they have ataxia and tremor. Purkinje cells degenerate in both mild and strong shaker mutants, but the temporal and spatial patterns of cell death are strikingly different. In mild shaker mutants, Purkinje cell death is temporally restricted, with 31-46% of the Purkinje cells in lobules I-IX dying by 3 months of age. Very few Purkinje cells degenerate after this age. Purkinje cell death is spatially random. In lobules I-IX, every second, third, or fourth Purkinje cell degenerates. Purkinje cells in lobule X do not degenerate. In strong shaker mutants, Purkinje cell degeneration is temporally protracted and spatially restricted. By 3 months of age, most Purkinje cells in lobules I-VIa, -b, and -d, and -d have degenerated. Numerous Purkinje cells in the paravermis of lobules VIIb-VIII have also degenerated. Surviving Purkinje cells in the vermis and lateral hemisphere of lobules VIIb-VIII are aligned in parasagittally oriented stripes or transversely oriented bands. Purkinje cells continue to degenerate in localized areas of the posterior lobe such that, by 18 months of age, surviving Purkinje cells are limited primarily to lobules VIc, VIIa, IXd, and X. Quantitative analysis indicates that none of the Purkinje cells in these lobules degenerate.     

In vivo gene transfer: focus on the kidney

Renal gene transfer techniques are being developed as a novelexperimental approach to understand the pathogenesis of renaldisease and to potentially develop new therapeutic tools. Wereview the currently available technology to introduce foreigngenetic material into renal tissue, i.e., retroviral, adenoviral,and liposomal transfer systems with their respective advantagesand caveats. Today, the transfer efficiency of these methodsappears to be sufficiently high to study the effects of transducedgenes on renal function and morphology in rat kidney. This willallow (i) the elucidation of the function of genes on the courseof renal disease in experimental animal models and (ii) themodulation of local expression of endogenous genes which presumptivelycontribute to renal pathology in these models. One strategyto accomplish this aim is the use of recombinant DNA technologyto design antisense DNA constructs or oligonucleotides, whichinterfere with the renal expression of target genes. We willalso discuss some of the shortcomings of the currently usedtechniques with respect to potential therapeutic use of genetransfer systems and gene modulation.     

Quantitation of protein adducts as a marker of genotoxic exposure: immunologic detection of benzo(a)pyrene -- globin adducts in mice

Immunologic methods have been developed for the determinationof benzo(a)pyrene (BP)-protein adducts and validated in animalstreated with (³H)BP. A previously developed antibody, 8E11,which recongnizes 7ß, 8-dihydroxy-9, 10-epoxy-7, 8,9, 10tetrahydrobenzo(a)pyrene (BPDE-I)-modified DNA or proteinas well as BPDE-I- tetraols, was used. The sensitivity of theassay was increased by enzymatic digestion of the modified proteinwith insoluble protease into peptides and amino acids beforeanalysis. In a competitive enzymelinked immunosorbent assay(ELISA) with digested BPDE-I-modified bovine serum albumin,50% inhibition occured at 400 fmol of adduct compared to 1450fmol for the nondigested albumin. Analysis of globin (Gb) isolatedfrom animals treated in vivo with 0.3–3 mg (³H)BP indicatedthat the ELISA could detect 90–100% of the adducts determinedby radioactivity. Levels of adducts in lung and liver DNA andserum albumin were correlated with the levels of Gb adducts.Of the total radioactivity associated with hemoglobin, only10% was from Gb while {small tilde}80% was from the heme fractionand the remainder from free BP metabolites. Significant cross-reactivityof antibody 8E11 was found with several BP-diols and phenols,suggesting that the immunoassay will not only be specific forBPDE-I adducts but will also detect adducts of other BP metabolitesas well as other aromatic hydrocarbon diol epoxides. An immunoaffinitycolumn of antibody 8E11 coupled to Sepharose 4B was used toisolate modified peptides from the digested Gb. About 65% ofthe applied radioactivity was retained on the column. Between1 and 2 mg of non-modified digested Gb could be added to thesample without interfering with binding of adducts. Proteindigestion and immunoaffinity chromatography should be usefulfor the measurement of protein adducts in biomonitoring studies.     

Complete bilateral internal ophthalmoplegia as sole clinical sign of botulism: confirmation of diagnosis by single fibre electromyography

Summary A case of complete bilateral internal ophthalmoplegia as the sole clinical sign of botulism is reported. Diagnosis was immediately confirmed by single-fibre electromyography (SFEMG), which revealed abnormally high blocking (14.3%), contrasting with moderately increased jitter (mean consecutive difference in the extensor digitorum communis muscle, 43.9 s). After giving equine botulinum antitoxin and simultaneous forced emptying of the bowels, ocular symptoms completely disappeared within 2 days. Six days, 5 weeks and 6 months after the first SFEMG study, the jitter was still abnormal, even becoming more so with time. Blocking, however, was only rarely observed in the follow-up studies. It is concluded that SFEMG may serve as a useful and sensitive method for the rapid diagnosis of botulinum intoxication, even in cases where no clinical signs of general muscular weakness are apparent.This paper was presented in part at the Congress of the International Medical Society of Motor Disturbances (ISMD), Rome, Italy, 2–4 June 1988     

Infection of rats with Taenia taeniformis metacestodes increases hepatic CYP450, induces the activity of CYP1A1, CYP2B1 and COH isoforms and increases the genotoxicity of the procarcinogens benzo[a]pyrene,cyclophosphamide and aflatoxin B(1)

Infection of rat liver by Taenia taeniformis metacestodes produced an increase in total CYP450 content and induced activity of the CYP1A1, CYP2B1 and COH isoforms. Variations in activity and p450 total content were found with increasing time of infection. During increased activity of p450 isoforms, rats were challenged with carcinogens metabolized by the mentioned isozymes and an increased amount of genotoxic damage was found when benzo[a] pyrene, cyclophosphamide and aflatoxin B(1) were used. No change was seen in CYP2E1 activity. These results support previous findings regarding an increased susceptibility to genotoxic damage of infected organisms.     

The influence of surface roughness of titanium on beta1- and beta3-integrin adhesion and the organization of fibronectin in human osteoblastic cells

Mechanisms of cell adhesion and extracellular matrix formation are primary processes in the interaction with the material surface of an implant which are controlled by integrin receptors. The aim of our study was to find out whether beta1- and beta3-integrins of osteoblastic cells sense the surface topography of titanium, and if structural alterations of integrin adhesions were involved in the organization of fibronectin. Pure titanium surfaces were modified by polishing (P), machining (NT), blasting with glass spheres (GB), and blasting with corundum particles (CB) resulting in increasing roughness. Confocal microscopic investigations revealed fibrillar adhesions of beta1- and alpha5-integrins on P, NT, and GB, but on CB with its sharp edges these integrin subunits did not form fibrillar adhesions. beta3 generally appeared in focal adhesions. We observed aligned fibrillar structures of fibronectin on NT not only on the basal site but interestingly, also on the apical cell surface. In contrast, on CB, fibronectin appeared apically clustered. We suggest that this alignment of fibronectin fibrils depends on the directed actin cytoskeleton and in particular, on the capability of the beta1-integrins to form fibrillar adhesions, which is affected by the surface roughness of titanium.