New Method for Visualization of Vascular Networks in Nonperfused Fixed Tissues

A new method of visualizing the angioarchitecture of tissues has been developed that uses blood components in nonperfused materials. Tissue blocks are fixed in 4% paraformaldehyde and cut with a vibratome into 50-60 μm sections. Endogenous peroxidase in red blood cells is then reduced in the presence of hydrogen peroxide with the resultant oxidation of the chromogen 3,3'-diaminobenzidine (DAB). This generates a dark, highly insoluble reaction product throughout the vascular system. The visualization of vascular components can be further enhanced by exposing the sections to peroxidase-conjugated IgG to increase the background staining of the blood plasma. The technique minimizes preparation artifact and permits the application of morphometric analytical methods, thus allowing parameters such as the volume density of the vascular bed to be quantified.     

Further segregation analysis of the fragile X syndrome with special reference to transmitting males

Summary A new series of 96 pedigrees with the fra(X) syndrome was analysed using complex segregation analysis with pointers, defining affection as any degree of mental impairment. These families were found to exhibit the same segregation pattern as the first series of 110 pedigrees (Sherman et al. 1984). The best estimate for penetrance of mental impairment in males was 79% and in females was 35% for the combined data. Again, there was little evidence for sporadic cases among affected males.Many more intellectually normal transmitting males have been observed since the existence of such males and the concomitant need to investigate the paternal side of pedigrees was recognized. On further investigation of all 206 pedigrees from the old and new data sets, the sibships of nonexpressing males appeared to be different from those of expressing males. Our analysis, using mental impairment as the phenotype, suggested that obligate carrier mothers and daughters of intellectually normal transmitting males are rarely, if ever, mentally impaired and that the sibs of transmitting males are much less likely to be retarded than the sibs of mentally impaired males. Though mothers and daughters of transmitting males are similar in phenotype, the expression of the gene in their offspring appears to be different: the penetrance of mental impairment is higher in offspring of intellectually normal daughters of transmitting males than in offspring of intellectually normal mothers of transmitting males. The implications of these observations for genetic counseling and for genetic models of the fra(X) syndrome are discussed.     

Time-budgeting and foraging strategy of the stoplight parrotfish Sparisoma viride Bonnaterre,in Jamaica

Quantitative data on the ways in which the different phases of the stoplight parrotfish (Sparisomaviride Bonnaterre) distribute their time among various activities in different habitats are presented. Individuals spent from 84–97% of their diurnal time swimming, feeding, and hovering. Additionally, large adults spent a significant amount of time sheltering among crevices. Phase-related differences in these activities are statistically significant, as are differences in duration and rates of change of the activities. Large individuals spent more time swimming, while small individuals spent more time hovering. In addition, large individuals performed longer bouts of activity and switched activities less frequently than small individuals. Adult males and females spent approximately equal proportions of time in each of the activity states. Stochastic analyses of behavioural sequences show second order Markov chain dependencies, suggesting that preceding activity states affect subsequent behaviour. Possible relationships between behavioural sequencing and the species foraging strategy are discussed, and it is suggested that the sequence of behavioural activities can provide an estimation of the distribution of food resources in the environment.     

Dolichol and Dolichyl Phosphate Levels in Brain Tissue from English Setters with Ceroid Lipofuscinosis

Human ceroid lipofuscinosis (CL) is an inherited disease marked by cerebromacular degeneration and early death. We have utilized the canine model to investigate the possible role of dolichol and dolichyl phosphate in the developmental pathology of CL. We found that while brain levels of dolichol increase with age in both affected and unaffected dogs, the amount in the diseased animal was similar to that in controls. Brain levels of dolichyl phosphate ranged from 20 to 35 micrograms/g in control dogs at all ages examined, but increased substantially during development in the affected dogs, a value of 113 +/- 24 micrograms/g (mean +/- SD) being obtained in the end-stage animals. In addition to the results obtained in the canine model, dolichyl phosphate levels in human brain tissues from a 5-year-old with late infantile CL and from a 19-year-old with juvenile CL were found to be 153 and 382 micrograms/g, respectively, compared with a control that assayed 26 micrograms/g. The preliminary findings with human tissues provide further evidence for an association of elevated brain dolichyl phosphate levels with CL. Whether the increase is primary or secondary remains to be determined.     

Effects of kainic acid on synaptic transmission in skate electroreceptors (ampullae of Lorenzini)

The effects of kainic acid on synaptic transmission in electroreceptors were investigated in the skate using techniques of uninterrupted superfusion of the synaptic area with a solution containing this substance and then recording the spike activity of single nerve fibers of the ampullae of Lorenzini. Kainic acid at threshold concentrations of the order of 10^–9 M effectively changed spontaneous and evoked activity of the receptors. Level of background activity served as an indication of the effects taking place. During blockage of synaptic transmission produced by magnesium ions the addition of kainic acid to the magnesium-saturated solution restored both spontaneous and evoked activity. It was deduced that the action of kainic acid on skate electroreceptors is of a primarily presynaptic nature.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 147–153, March–April, 1986.     

HLA-JY328: Mapping studies and expression of a polymorphic HLA class I gene

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk^– cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from,L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed —a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.     

Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

Inhibition of microbial growth by interference with siderophore biosynthesis. Oxidation of primary amino groups in aerobactin synthesis by Escherichia coli

Abstract The first step of aerobactin biosynthesis, oxidation of an aliphatic primary amino group to an N -hydroxy-amino compound seems to be involved in the biosynthesis of most of the hydroxamatetype siderophores which are widely distributed among bacteria and fungi. Therefore, the first step of aerobactin biosynthesis, oxidation of lysine to N ⁶-hydroxylysine was studied as a model reaction using a strain of Escherichia coli that contains the first gene aerA of aerobactin synthesis on a multi-copy plasmid and which is lacking the gene for the subsequent step in the pathway. In addition, culture conditions are described which lead to the secretion of N ⁶-hydroxylysine into the medium in amounts that can easily be quantitatively determined by a simple, reliable chemical assay. This assay can be used for screening inhibitors of the oxidation of α-amino groups, which should interfere with the biosynthesis of siderophore hydroxamates and thus should create bacteriostatic conditions.