Reactivities of Actin as a Contractile Protein

The molecular basis for the mechanism of contraction in striated muscle, with primary emphasis on the interaction between the thick and thin filaments and the role of the thin (actin) filaments, is the theme presented. Recent information relating to actin-myosin interaction points up the fact that definitive statements cannot be made regarding the molecular interaction(s) that lead to contraction. Nevertheless, the properties of actin indicate that (a) actin in the monomeric state has properties differing markedly from actin in the polymer (filament) state; (b) these property differences may be significant in the contractile process, for they include changes in the reactivity of the bound nucleotide and actin-myosin complex formation; (c) the bound nucleotide seems to be required in the contraction process. For these, and other, reasons discussed, the tentative hypothesis is advanced that the contraction reaction involves local changes in the actin filament providing local monomer or monomer-like actin units in the reaction with myosin.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Conservatism of sites of tRNA loci among the linkage groups of severalDrosophila species

Summary The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization.¹²⁵I-labeled tRNA probes fromDrosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from fourDrosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, andDrosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA ₂ ^Arg and tRNA ₂ ^Lys hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution ofDrosophila species.     

Effects of a New Calcium Channel Blocker, KB-2796, on Protein Synthesis of the CA1 Pyramidal Cell and Delayed Neuronal Death Following Transient Forebrain Ischemia

The effects of a new calcium channel blocker, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)-piperazine dihydrochloride (KB-2796), on delayed neuronal death (DND) in the hippocampus were examined in gerbils in comparison with those of pentobarbital and flunarizine. The neuronal density in the hippocampal CA1 subfield was counted on the seventh day of recirculation following 5 min of bilateral carotid occlusion, and protein biosynthesis in the brain was also determined at 1, 2, 4, 24, and 72 h following occlusion. The drugs were intraperitoneally administered after recirculation. KB-2796 (10 mg/kg) significantly prevented DND in the CA1 subfield. Pentobarbital (40 mg/kg), but not flunarizine (3 and 10 mg/kg), inhibited DND. Protein synthetic activity in the CA1 subfield was reduced by ischemia and the reduction was not restored even at 72 h after recirculation. KB-2796 did not ameliorate the reduction of protein synthesis in the CA1 subfield by 24 h after recirculation, but in one of three animals restoration of protein synthesis was observed at 72 h of recirculation. Pentobarbital also restored the reduced protein synthesis in two of three animals at 72 h. These results suggest that calcium influx into neurons participates in the pathogenesis of DND, and also that KB-2796 might prevent both morphological and functional cell damage in CA1 neurons induced by transient ischemia.     

Regionally selective alterations in enzymatic activities and metabolic fluxes during thiamin deficiency

To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.¹⁴CO₂ production from¹⁴C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO₂ production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC 2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3. - PDHC pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3 - TK transketolase (EC 2.2.1.1) - TPP thiamin pyrophosphate     

The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity

We have identified a minimum functional domain of human thrombomodulin for anticoagulant activity using deletion analysis. Four mutants were constructed by site-directed deletion mutagenesis to delete one or more epidermal growth factor (EGF)-like structures from the domain of human thrombomodulin containing six repeated EGF-like structures. These deletion mutants were expressed transiently in COS-1 cells, and their protein C-activating cofactor activities in the culture medium were examined. One mutant protein, E456, which contains the fourth, fifth, and sixth EGF-like structures expresses apparent cofactor activity. However, neither E456-N24 (24 NH2-terminal-residue deletion) nor E456-C16 (16 COOH-terminal-residue deletion) have cofactor activity. E456 was partially purified and its anticoagulant effects on plasma clotting time and platelet aggregation examined. E456 expressed almost the same anticoagulant activities as D123 which contains six consecutive EGF-like structures of thrombomodulin. It was concluded that E456 is the minimum functional domain for both protein C-activating cofactor activity and anticoagulant activity.     

The immune response and the eye. III. Anterior chamber-associated immune deviation can be adoptively transferred by serum

After the anterior chamber (AC) injection of trinitrophenol-coupled (TNP) spleen cells, it is observed that systemic delayed-type hypersensitivity responses to TNP are inhibited by Ag-specific suppressor T cells. We recently reported that suppression is initiated by viable TNP-coupled T cells within the inoculum and upon further analysis we found that these cells have the surface phenotype of CD4+ Ts inducer cells. We report here that treatment of these TNP-T cells with cycloheximide or cytochalasin-B before to AC injection abolishes suppression, whereas treatment with 2000 rad radiation does not. This indicates that protein synthesis and secretion are required to initiate suppression but proliferation is not. Further, we demonstrate the adoptive transfer of suppression by serum of AC inoculated animals. Detection of the component in serum in adoptive transfer assays, however, requires removal of the spleen before AC injection. We establish that the material in serum is a Ts cell product (T suppressor-inducer factor) based on three criteria: it is Ag specific, genetically restricted, and reactive with a mAb that specifically identifies these molecules. These results suggest that the signal leaving the eye to induce suppression of delayed-type hypersensitivity is T cell derived and that molecules mediating immune regulation for this organ are made within the eye and transported via the serum to the spleen.