Reassociation of complementary strand-specific adenovirus type 2 DNA with viral DNA sequences of transformed cells.

Complementary strand-specific adenovirus DNA, either full length or from restriction enzyme cleavage fragments, was used to estimate the fractional representation and abundance of viral sequences in two adenovirus type 2 (Ad2)-transformed rat cell lines, A2F19 and A2T2C4. The reassociation method introduced is based on the linear relationship, after exhaustive hybridization, between the inverted fraction of hybrid DNA and the molar ratio of probe to cellular DNA in the reaction mixture. The amount of viral DNA in A2F19 cells represents 12 to 14% of the viral genome at a level of around seven copies per diploid cell equivalent. For the cell line A2T2C4, the pattern of integrated viral DNA sequences is more complex. With full-length Ad2 DNA strands as a probe, about 56% of the probe was represented in cellular DNA. When each of the four BamHI fragment strands of Ad2 DNA was used as a probe, the fraction of the viral DNA present also amounted to around 56% with one to five copies from different regions of the viral genome. The results demonstrate the advantage of using strand-specific viral DNA as a probe in reassociation analysis with denatured cell DNA. The method should be useful in any system in which complementary strand separation of viral DNA sequences can be achieved.     

Formation of primordial yolk granules in the avian oocyte

Summary Electron and light microscopical investigations of early oocytes (between 1.0 mm and 5.0 mm in diameter) from the ovary of 28–30 week-old chickens, suggested the formation of primordial yolk granules from cytoplasmic vesicles. These vesicles formed an aggregation which was observed to be surrounded by membranes, giving the aggregate a multivesicular body-like appearance. At a later stage the vesicles inside the membrane disintegrated and the multivesicular bodies acquired the appearance of primordial yolk granules. The contribution of other structures to the formation of yolk granules is discussed.For constructive criticism I am very grateful to Dr. Hadar Emanuelsson, Institute of Zoophysiology, Lund. The excellent technical assistance of Miss Inger Antonsson and Mrs. Annagreta Petersen is gratefully acknowledgedThis work was supported by Kungliga Fysiografiska Sällskapet, Lund     

The immunohistochemical demonstration of chymase and tryptase in human intestinal mast cells

Summary An immunohistochemical double-labelling technique for the simultaneous identification of mast cells containing tryptase alone (MC_T) or chymase together with tryptase (MC_TC) was evaluated quantitatively using two monoclonal antibodies, mAb 1222A (antitryptase) and mAb 1254B (antichymase). Saturation conditions were established for the binding of the antibodies to the mast cell enzymes by counting labelled mast cells in consecutive sections of normal human intestine incubated with serial dilutions of the antibodies. When, under such conditions, the antitryptase was applied after saturation with mAb 1254B, the reproducibility of the double-labelling procedure was excellent. MC_T were located preferentially in the intestinal mucosa but, in contrast to what has previously been reported, they were not the predominant type of mast cell at this site. The percentage of MC_T of the total number of immunopositive mast cells varied considerably in the colonic mucosa (7–67%, average 30%), while this was not the case in the small intestinal mucosa (5–26%, average 10%). Mast cell chymase, unlike tryptase, was not recognized by the antichymase antibody after aldehyde fixation and a higher apparent fraction of MC_T therefore occurred after double labelling. These findings suggest that the proteinase composition of human mast cells, unlike that of murine mast cells, should not be taken as evidence of phenotypic heterogeneity. Taken together with previous observations, they suggest instead that the lack of chymase may be related to functional activity or stage of maturation of the mast cells.     

Coxsackievirus B3 entry into the host cell interferes with G-protein-mediated transmembrane signalling

In the present work we used various cell lines in order to study the possible effect of coxsackievirus B3 (CVB3) entry on the adenylyl cyclase transmembrane signalling system. A significant decrease (by about 10–20%) was found in forskolin-augmented as well as in AlF ₄ ^– - and GTPS-sensitive adenylyl cyclase activity in plasma membranes isolated from HeLa, HEp-2, Vero and green monkey kidney cells shortly (up to 60 min) preincubated with CVB3 (5 PFU/cell). Moreover, the ability of G-proteins derived from plasma membranes of infected cells to reconstitute AC activity in the cyc^– mutant of S49 cells was also reduced. Content of G-protein subunits, however, remained unchanged after CVB3 attachment. Functional alterations in the G-protein-mediated adenylyl cyclase signalling system were accompanied by a marked decrease (by about 20–40%) of intracellular cAMP levels in virus-affected cells. These findings demonstrate clearly that CVB3 may affect functioning of the G-protein regulated adenylyl cyclase transmembrane signalling system in virus-sensitive cells as early as during the first period of its contact with the cellular plasma membrane.     

Mast cells in normal and sectioned peripheral nerve

Summary An investigation is reported on the properties and quantitative distribution of mast cells in normal and sectioned peripheral nerve. A considerable number of mast cells has been found in the epineurial connective tissue in normal rats, as well as scattered mast cells in the endoneurium. After nerve section there was an about five-fold increase in the number of endoneurial mast cells throughout the distal part of the sciatic nerve.The mast cell granules in normal and sectioned nerve showed the same histochemical properties as mast cell granules in other tissues, i.e. strong toluidine blue metachromasia resistant to alcohol dehydration, and persistence of dye binding and metachromasia at pH below 1. Furthermore, the metachromasia is unaffected by extraction with chloroform and methanol prior to staining. The metachromatic component of the mast cell granules can be differentiated by these properties from other metachromatic structures in normal and sectioned nerve. The significance of the findings is discussed, in particular the possible relation of endoneurial mast cells to the degradation of myelin. Acknowledgements. The authors are indebted to Miss Kristina Müntzing for skilful technical assistance.     

NATURAL HYBRIDS BETWEEN ORYZOPSIS AND STIPA. III. ORYZOPSIS HYMENOIDES × STIPA PINETORUM

Johnson, B. Lennart. (U. California, Los Angeles.) Natural hybrids between Oryzopsis and Stipa. III. Oryzopsis hymenoides × Stipa pinetorum. Amer. Jour. Bot. 50(3): 228–234. Illus. 1963.—On the basis of morphological characters, the spontaneous hybrid Oryzopsis hymenoides × Stipa pinetorum is included in 0. bloomeri which consists of a number of sterile, natural hybrids between O. hymenoides (2n = 48) and various American species of Stipa (2n = 32-82). While intermediate between its parents in many attributes, the pinetorum hybrid is different from the other hybrids included in O. bloomeri with respect to lemma-hair length and other characters which are diagnostic for S. pinetorum (2n = 32). The hybrid has 2n = 40 chromosomes, but some plants of the hybrid and some of O. hymenoides had small supernumerary, somatic chromosomes. The parents formed only bivalents at meiosis. The hybrid showed some affinity among 14 chromosomes per sporocyte. This affinity is nearly as great as that shown by other Stiporyzopsis hybrids with 56 or 58 chromosomes, and is consistent with the earlier suggestion that bivalent formation in polyploid species of Stipa may be gene-controlled.