Regional assignment of two genes of the human branched-chain alpha-keto acid dehydrogenase complex: the E1 beta gene (BCKDHB) to chromosome 6p21-22 and the E2 gene (DBT) to chromosome 1p31

Maple syrup urine disease (MSUD) is caused by the deficiency of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The multienzyme complex is a macromolecule (Mr 4 X 10(6] consisting of at least six distinct subunits. In this study, the human E1 beta gene (BCKDHB) has been localized to human chromosome 6 by hybrid somatic cell analysis, and regionally assigned to chromosome bands 6p21-22 by in situ hybridization. The E2 gene (DBT), which was previously localized to chromosome 1, is regionally assigned to the chromosome band 1p31 also by in situ hybridization. Localization of the E1 beta gene to chromosome 6p21-22 assigns another major human disease locus to a region that contains several important genes, including the major histocompatability complex, tumor necrosis factor, and heat-shock protein HSP70. Mapping of the E1 beta and the E2 genes may provide information for the linkage analysis of MSUD families with mutations in these two loci.     

Proteolytic processing in a non-lysosomal compartment is required for transcytosis of protein-polylysine conjugates in cultured Madin-Darby canine kidney cells

The transcytosis of horseradish peroxidase, as well as its poly(L-lys) and poly(D-lys) thioether conjugates, was investigated in Strain I Madin-Darby canine kidney (MDCK) cell monolayers grown on 0.4 microns pore size polycarbonate membranes in Costar Transwells. The 3 types of HRP had almost identical rates of transport during the first 2 hr of incubation. However, a significant increase of basal-to-apical transport was detected beginning at 3 hr only in Transwells containing the poly(L-lys) conjugate. This increase was inhibited by colchicine (2 microM) and by the Bowman-Birk protease inhibitor (0.1 mg/ml), but not by NH4Cl (10 mM) or chloroquine (0.1 mM). The increase was abolished either by prior trypsinization of the conjugate or by incubation at 4 degrees C. Ultrafiltration studies indicated that the transcytosed poly(L-lys) conjugate was smaller in size than the original conjugate. These results indicate that the conjugate was processed during transcytosis in a non-lysosomal proteolytic compartment, where its poly(L-lys) moiety was selectively degraded, allowing active peroxidase to be released into the apical medium.     

Inhibition of the bovine branched-chain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates

A novel class of inhibitors for the branched-chain 2-oxo acid dehydrogenase (BCOAD) complex has been synthesized and studied. The sodium salts of arylidenepyruvates: e.g., furfurylidenepyruvate (compound I), 4-(3-thienyl)-2-oxo-3-butenoate (compound II), cinnamalpyruvate (compound III) and 4-(2-thienyl)-2-oxo-3-butenoate (compound IV) inhibit the overall and kinase reactions of the BCOAD complex from bovine liver. Inhibitions of the overall reaction occur at the decarboxylase (E1) step as determined by a spectrophotometric assay with 2,6-dichlorophenolindophenol as an electron acceptor. Inhibition of the E1 reaction by compound I (Ki = 0.5 microM) is competitive, whereas inhibitions by compounds II (Ki = 150 microM) and III (Ki = 500 microM) are non-competitive with respect to the substrate 2-oxoisovalerate. The Km value for 2-oxoisovalerate is 6.7 microM as measured by the E1 assay. Inhibition of the E1 step by compounds I, II and III are reversible at low inhibitor concentrations based on the Michaelis-Menten kinetics observed. By comparison, compound I does not significantly inhibit pyruvate and 2-oxoglutarate dehydrogenase complexes. The arylidenepyruvates (compounds I, II and IV) inhibit the BCOAD kinase reaction in a manner similar to the substrate 2-oxo acids. The inhibition of the kinase reaction by compound I is non-competitive with respect to ATP, with an apparent Ki value of 4.5 mM. The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.     

Gap junctions and impulse propagation in embryonic epithelium of Amphibia

Summary Epithelium of amphibian embryos (Cynops orientalis, Xenopus laevis) was found in preceding experiments to generate and conduct impulses during a limited stage (26–37) of development. In order to elucidate the structural basis of impulse propagation, epithelial cells of four stages were examined by the freeze-etching method: (I) before and (II) during acquisition of conductivity; (III) when propagation was fully established, and (IV) when it was no longer present. Only few gap junctions (GJ) of small size were found in groups I and IV. GJ in epithelia of group III were increased in number and size, and appeared morphologically coupled, i.e., with more loosely arranged connexons. The size of gap-junctional particles did not differ significantly between coupled and uncoupled stages. Zonulae occludentes seemed leaky in stage I, and tight in stages II–IV. Thus, the morphological characteristics of specialized junctions between non excitable cells correlated with the opening and closing of low resistance intercellular current pathways during embryonic development.Gap junctions in particular seem to form an essential link in the non-neural stimulus-response system, which may facilitate the mobility of the embryo during early phases of aquatic life before the reflex pathways have been established. Coupling and uncoupling of gap junctions may also play an important role in the regulation of cell differentiation and morphogenetic movement. The experimental model used in this study provides a useful tool for further investigations of structural correlates of gap junctional permeability under physiological conditions.     

CONTRIBUTION OF POLLEN MORPHOLOGY TO SYSTEMATICS OF COLLOMIA (POLEMONIACEAE)

Pollen morphology of 14 species of Collomia (Polemoniaceae) was examined by light microscopy, and by both scanning and transmission electron microscopy. Four distinct pollen types were observed which are based principally upon 1) shape, number and distribution of apertures, and 2) surface sculpturing: Type 1—zonocolporate with striate ridges; Type 2—zonocolporate with striato-reticulate ridges; Type 3—pantoporate with radiate ridges; Type 4—pantoporate with irregularly reticulate ridges. Evaluation of pollen morphology reveals considerable discrepancy with respect to presently accepted sectional classification. Collomia grandiflora of sect. Collomia has a pollen type similar to that of members of sect. Collomiastrum and is now interpreted as representing an independent evolutionary line derived from the latter section. Collomia diversifolia of sect. Courtoisia has a pollen morphology similar to that of sect. Collomia. whereas C. heterophylla of the same section possesses pollen unique within the genus. This last pollen type shows close similarity to the pollen of members of Polemonium, Gilia, Leptodactylon, and Ipomopsis. Pollen of C. tinctoria and C. tracyi of sect. Collomia are anomalous within Polemoniaceae. No significant difference in exine stratification was discernible among the four pollen types.     

Sensitivity of cultured rat hepatocytes to aflatoxin B1 and benzo(a)pyrene.

We have tested the sensitivity of a cloned rat hepatocyte line, RL-PR-C, to aflatoxin B1 and benzo(a)pyrene as a function of population-doubling level. The cells were much more sensitive to the cytotoxic action of these agents subsequent to 230 population doublings. This sensitivity corresponded to the enhanced inducibility of aryl hydrocarbon hydroxylase activity by 3-methylcholanthrene.     

Sensitivity of cultured rat hepatocytes to aflatoxin B1 and benzo(a)pyrene

Summary We have tested the sensitivity of a cloned rat hepatocyte line, RL-PR-C, to aflatoxin B₁ and benzo(a)pyrene as a function of population-doubling level. The cells were much more sensitive to the cytotoxic action of these agents subsequent to 230 population doublings. This sensitivity corresponded to the enhanced inducibility of arylhydrocarbon hydroxylase activity by 3-methylcholanthrene. Supported by Grants CA 21258 and CA 12056 from the National Cancer Institute.     

Study of a possible structural relationship between thymopoietin and the facteur thymique serique

Background peptide chemistry, and the known 49-amino acid sequence of thymopoietin and the known 9-amino acid sequence of the facteur thymique serique (FTS) allowed the concept that Arg⁴⁹ of thymopoietin might be linked to Gln¹ of FTS in a new 58-amino acid peptide in tissue. Cleavage between Arg⁴⁹ and Gln⁵⁰ adjacent to the unique Lys⁴⁸-Arg⁴⁹ moiety could liberate thymopoietin and the [H-Gln¹]-FTS which could cyclize to FTS by the known reaction. In support of, rather than negating, this concept, synthetic FTS and the new dodecapeptide consisting of Val-Lys-Arg linked to the N-terminal of [H-Gln¹]-FTS showed comparable immune stimulating activity, $\text{in vivo}$; both peptides appeared more active than synthetic thymopoietin II.     

The subcellular distribution and partial characterization of cholinesterase activities of canine platelets.

The multiple cholinesterase activities in canine platelets have been investigated. Platelets were homogenized by rapid decompression under nitrogen, glass tube/Teflon pestle, and glycerol lysis techniques. Rapid decompression under nitrogen technique was found to be the most efficient and gentle method for cell disruption. Homogenates were subfractionated using sodium diatrizoate density gradients. Marker enzyme assays and pulse labeling experiments with 5-hydroxyl[14C] tryptamine and [125I] thrombin on prepared subcellular fractions confirmed that the soluble, plasma membrane and the granule-1 fractions were all in reasonably pure form. Furthermore, labeling of the plasma membrane with [125I] thrombin is cited as the first successful attempt at attaining significantly bound marker for this structure. Cholinesterase activity distributions measured in these fractions indicated that about 30% of the activity was present in the plasma membrane, 50% in granule-1 and 5% in soluble fractions. Kinetic data of cholinesterase activities obtained from intact platelets, plasma membrane preparations and platelet release supernatants indicated that they are strikingly similar.     

Fungi isolated from asthmatic homes in the Taipei area.

Indoor fungi in Taipei were surveyed during 1969-1974 using Sabouraud's agar plates. No remarkable yearly variation of fungal incidence was observed. The fungal population was found to be different from home to home. The dominant members were Aspergillus, Penicillium, Mycelia sterila, Oospora, Rhodotorula, Hormodendrum and yeasts. The order of frequency of fungi found indoors was quite different from that of outdoor fungi. The fungal spores were more numerous during summer months, and this trend was more apparent for Aspergillus, Oospora, Rhodotorula, and Hormodendrum. The kind and the incidence of fungi found in asthmatic homes were not different from those detected in non-asthmatic homes, except that in asthmatic homes there were greater numbers of fungi.