Distribution of 4a-hydroxytetrahydropterin dehydratase in rat tissues. Comparison with the aromatic amino acid hydroxylases.

A 4a-carbinolamine intermediate is generated stoichiometrically during the tetrahydrobiopterin-dependent phenylalanine hydroxylation reaction catalyzed by phenylalanine hydroxylase. The dehydration of the carbinolamine is catalyzed by the enzyme, 4a-hydroxytetrahydropterin dehydratase. We have now examined the distribution of the dehydratase activity in various rat tissues by activity measurements and by immunoblot analysis to explore the possibility that the dehydratase may also play a role in tyrosine and tryptophan hydroxylation. The only two tissues that express relatively high dehydratase activity are liver and kidney, which are also the only two tissues that express phenylalanine hydroxylase activity. The dehydratase activity was generally very low in those tissues which contain high levels of tyrosine and tryptophan hydroxylase activity, except for the pineal gland. These results suggest that the dehydratase may not play an important role in the regulation of the synthesis of those neurotransmitters which are derived from the hydroxylated aromatic amino acids.     

Localization of anti-clathrin antibody in the sarcomere and sensitivity of myofibril structure to chloroquine suggest a role for clathrin in myofibril assembly

Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and alpha-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.     

The pattern of X-chromosome inactivation in the embryonic and extra-embryonic tissues of post-implantation digynic triploid LT/Sv strain mouse embryos

Spontaneously cycling LT/Sv strain female mice were mated to hemizygous Rb(X.2)2Ad males in order to facilitate the distinction of the paternal X chromosome, and the pregnant females were autopsied at about midday on the tenth day of gestation. Out of a total of 222 analysable embryos recovered, 165 (74.3%) were diploid and 57 (25.7%) were triploid. Of the triploids, 26 had an XXY and 31 an XXX sex chromosome constitution. Both embryonic and extra-embryonic tissue samples from the triploids were analysed cytogenetically by G-banding and by the Kanda technique to investigate their X-inactivation pattern. The yolk sac samples were separated enzymatically into their endodermally-derived and mesodermally-derived components, and these were similarly analysed, as were similar samples from a selection of control XmXp diploid embryos. In the case of the XmXmY digynic triploid embryos, a single darkly-staining Xm chromosome was observed in 485 (82.9%) out of 585, 304 (73.3%) out of 415, and 165 (44.7%) out of 369 metaphases from the embryonic, yolk sac mesodermally-derived and yolk sac endodermally-derived tissues, respectively. The absence of a darkly staining X-chromosome in the other metaphase spreads could either indicate that both X-chromosomes present were active, or that the Kanda technique had failed to differentially stain the inactive X-chromosome(s) present. In the case of the XmXmXp digynic triploid embryos, virtually all of the tissues analysed comprised two distinct cell lineages, namely those with two darkly-staining X-chromosomes, and those with a single darkly staining X-chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exfoliative colonic cytology. A simplified method of collection and initial results

Exfoliative colonic cytology for the diagnosis of colorectal cancer has been largely abandoned due to (1) the widespread use of colonoscopy, (2) the cumbersome methods of cell collection and (3) the occasional difficulty of interpreting the cytologic findings in the presence of inflammatory bowel disease or adenomas. This paper describes a newly formulated bowel preparation for routine colonoscopy, based on imbibing 2 L to 4 L of a balanced electrolyte solution, in which the recovered precolonoscopic effluent (using a convenient disposable collecting kit) yielded cells for cytologic evaluation from 70% of a group of 80 patients at high risk for large bowel neoplasia. Cytology demonstrated neoplastic cells in most cases of endoscopically proven cancer. These results suggest that colonic exfoliative cytology may be useful as a supplemental test to routine colonoscopy. This could be enhanced by further methodologic modifications to the collecting and cytologic methods; large long-term studies are needed to evaluate the potential usefulness of colonic exfoliative cytology.     

A rapid and specific method for the immunological identification of mycelial form cultures ofParacoccidioides brasiliensis

We developed two simple methods for extracting specific, cell-free, soluble antigens of the mold form ofParacoccidioides brasiliensis. Detection of these antigens by a microimmunodiffusion test permits the rapid and accurate identification of cultures ofP. brasiliensis. ThirtyP. brasiliensis isolates treated by these techniques produced specific exoantigens detectable by the immunodiffusion test. None of the other 78 fungal pathogens or saprophytes tested produced identical exoantigens. Personnel in any diagnostic laboratory who want a rapid and specific method for identifying or confirming suspected isolates ofP. brasiliensis can use the simple procedures described.     

Studies on acidification of media by Avena stem segments in the presence and absence of gibberellic Acid

The rate of acidification of media by Avena stem segments was studied with a titrimeter. GA₃ increased this rate by an average of 17% if supplied to the segments 90 min prior to measurement. GA₃ inhibited the rate by 15% if supplied 10 min prior to measurement. After 90 min incubation, stimulation of elongation had started; at 10 min, GA₃ had not yet started to stimulate elongation in the segments.     

The influence of various factors on fluid secretion by in vitro salivary glands of ixodid Ticks.

1. Salivary glands of the female ixodid tick, Dermacentor andersoni, secrete fluid in vitro when bathed in a slightly modified version of the mammalian tissue culture medium 'TC 199'. 2. Rate of salivation in vitro increases with progression of feeding, but there is no comparable increase in dry weight of the salivary glands during the early phase of engorgement. Engorged ticks secreted at only 25% the rate of 90-250 mg ticks, indicating that salivary gland degeneration has already begun in the very early post-engorgement stage. 3. A salivary gland stimulating factor can be detected in the nervous system but not in other tissues. 4. Male salivary glands secrete at only 1/20th the rate of female glands. Thus males probably do not use their salivary glands as osmoregulatory organs. 5. From the uniform lack of response to ACh and uniform response to DA in 7 ixodid tick species, it is suggested that the control of salivation is similar throughout the ixodid family.