T4-induced cardiac hypertrophy and the perfused and total microvasculature of the heart

The purpose of the present investigation was to determine the effects of thyroxine (T4), which induces myocardial hypertrophy, on the number per square millimetre and volume per cubic millimetre of both the total and perfused portions of the arteriolar and capillary beds of the heart. Studies were conducted in the subendocardial and subepicardial regions of the left ventricle of anesthetized open-chest rabbits. Fluorescein isothiocyanate-dextran (i.v.) or radioactive microspheres (intra-atrial) were injected to label the perfused microvessels or to determine coronary flow in three groups of rabbits: controls, and rabbits given 0.5 mg/kg T4 for 3 days and for 16 days. Fluorescent photography was used to identify the perfused microvessels. An alkaline phosphatase stain was employed to locate the total microvascular bed. There were 2369 +/- 638 (SD) capillaries/mm2 and 4 +/- 3 arterioles/mm2 in control hearts. These decreased significantly to 1380 +/- 199/mm2 and 1 +/- 1/mm2, respectively, after 16 days of T4. In controls, 60 +/- 5% of the capillaries and 59 +/- 21% of the arterioles were perfused. This increased significantly to 90 +/- 5 and 86 +/- 18%, respectively, by 16 days of T4 treatment. Similar changes, although smaller, were observed after 3 days of T4. Coronary blood flow increased to 1.7 times control after 3 days and 2.9 times after 16 days of T4. No significant subepicardial versus subendocardial differences were observed in any condition or measurement. Thus, the physiological response to the increased work and increase in anatomic minimum diffusion distance is to increase flow and the proportion of the capillary bed perfused to at least maintain physiological diffusion distances.     

Anesthetic and supportive management during experimental pulsatile flow perfusion studies in calves

The purpose of this study was to determine the factors influencing successful experimental cardiopulmonary bypass studies using pulsatile flow perfusion and the medications and methodology necessary to produce successful bypass in calves. In six calves showing no cardiopulmonary pathology prior to bypass procedures, successful anesthesia and surgical intervention was accomplished. Animals were maintained on 5 hours of pulsatile flow bypass perfusion. Successful recovery from the procedures was accomplished. In two calves with pre-existing pulmonary pathology, anesthetic and surgical intervention was accomplished with the utilization of extensive anesthetic management and cardiac supportive medications until the animals could be initiated into 5 hours of pulsatile flow bypass perfusion, in spite of major pulmonary dysfunction. In these two animals, attempts to resuscitate upon termination of pulsatile flow perfusion were unsuccessful due to pre-existing excessive lesions in the lungs. This study shows a contrast between complete success of a pulsatile flow system in normal subjects versus the ultimate failure in experimental animals with pre-existing pulmonary pathology. The inability of experimental calves with a diseased lung to resume spontaneous cardiopulmonary function after the challenges of thoracic intervention indicates the unsuitability of animals with marked pre-existing pulmonary disease status for use in cardiopulmonary bypass studies.     

Relationship between two major immunoreactive forms of arginase in Neurospora crassa.

Two major immunoreactive proteins of Mr 41,700 and 36,100 have been detected in crude mycelial extracts with polyclonal antibodies raised against arginase purified from Neurospora crassa. The latter corresponded to the protein used to obtain the antibodies. Both polypeptides were either missing or present in very low amounts in mutant strains having little or no detectable arginase activity. The relative proportion of the two species was altered in strains containing the nitrogen catabolite regulatory mutation nit-2. Peptide mapping indicated that the two species are very closely related, but several of the peptides which appeared to be identical by staining reacted differently with the antibodies. Both species were produced by in vitro translation of poly(A)+ mRNA, although the larger species was produced to a much smaller extent than was expected from its abundance in vivo. The results suggest the existence of multiple forms of arginase in N. crassa which differ in their response to nitrogen catabolite regulation.     

Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block

Batrachotoxin-modified, voltage-dependent sodium channels from canine forebrain were incorporated into planar lipid bilayers. Single-channel conductances were studied for [Na+] ranging between 0.02 and 3.5 M. Typically, the single-channel currents exhibited a simple two-state behavior, with transitions between closed and fully open states. Two other conductance states were observed: a subconductance state, usually seen at [NaCl] greater than or equal to 0.5 M, and a flickery state, usually seen at [NaCl] less than or equal to 0.5 M. The flickery state became more frequent as [NaCl] was decreased below 0.5 M. The K+/Na+ permeability ratio was approximately 0.16 in 0.5 and 2.5 M salt, independent of the Na+ mole fraction, which indicates that there are no interactions among permeant ions in the channels. Impermeant and permeant blocking ions (tetraethylammonium, Ca++, Zn++, and K+) have different effects when added to the extracellular and intracellular solutions, which indicates that the channel is asymmetrical and has at least two cation-binding sites. The conductance vs. [Na+] relation saturated at high concentrations, but could not be described by a Langmuir isotherm, as the conductance at low [NaCl] is higher than predicted from the data at [NaCl] greater than or equal to 1.0 M. At low [NaCl] (less than or equal to 0.1 M), increasing the ionic strength by additions of impermeant monovalent and divalent cations reduced the conductance, as if the magnitude of negative electrostatic potentials at the channel entrances were reduced. The conductances were comparable for channels in bilayers that carry a net negative charge and bilayers that carry no net charge. Together, these results lead to the conclusion that negative charges on the channel protein near the channel entrances increase the conductance, while lipid surface charges are less important.     

A single bone marrow-derived stromal cell type supports the in vitro growth of early lymphoid and myeloid cells

A clonal cell line (ALC) derived from murine bone marrow stroma is capable of supporting the continuous, in vitro growth of early lymphoid and myeloid cell populations. The growth-promoting effects of ALC are in part mediated through M-CSF and a pre-B cell growth factor, both of which accumulate in ALC-culture supernatant. To analyze the lymphoid growth factor produced by ALC cells, we derived a pre-B cell indicator line that is dependent on ALC-growth-conditioned medium. Using a combination of biological and biochemical analyses, we have established that the pre-B cell growth factor produced by ALC cells is distinct from IL-1, IL-2, IL-3, and IL-4 (BSF-1), suggesting that the early stages of B-cell development are regulated by a unique stroma-derived growth factor.     

Normal yeast tRNA(CAGGln) can suppress amber codons and is encoded by an essential gene

We have isolated a gene that can encode yeast tRNA(CAGGln). When present on a multicopy plasmid, this gene suppresses the phenotype of a number of amber mutants, but has no effect on the ocher mutants tested. We therefore conclude that the anticodon CUG in tRNA(CAGGln) can decode the amber codon UAG by G-U mispairing, possibly by wobble base-pairing in the first codon position. This represents the second example we have observed in this laboratory of nonsense suppression in yeast by natural tRNA(Gln), involving G-U mispairing in the first codon position. Replacing the genomic copy of the cloned gene with a disrupted tRNA gene results in recessive lethality in heterozygous diploids and is lethal to haploid cells. This lethality can be rescued by transformation of cells with a single copy plasmid containing the tRNA(CAGGln) gene. Thus, the gene encoding tRNA(CAGGln) is apparently essential for viability in yeast, suggesting that it is normally present as a single copy gene.     

Inhibitory effect of toluene on tumor promotion in mouse skin

In the two-stage mouse model for skin tumorigenesis with phorbol-12-myristate-13-acetate (PMA) as promoter, topical application of 40 microliters of toluene 2X/week at the initiation/promotion site (the back) reduced the average number of tumors/mouse (ANT/M) to approximately one-fourth that of controls. Control procedure involved initiation of C3H mice with benzo[a]pyrene (BaP) and CD-1 mice with 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with from 1 to 5 micrograms PMA in 40 microliters acetone 2X/week. Forty microliters of toluene 2X/week per se was a weak promoter (6-13% of control ANT/M), and produced mild skin irritation at the application site but behavior and body weights were normal. The toluene inhibition of tumorigenesis was not a direct chemical action on PMA since similar effects occurred whether toluene was the vehicle for PMA or whether it was applied up to 1 day before PMA (i.e., prepromotion). Prepromotion with acetone had no effect on tumorigenesis, substantiating its use as control vehicle and suggesting that the toluene inhibition was a specific tissue reaction. The inhibitory effect appeared to be on PMA promotion rather than on initiation since toluene and acetone produced similar numbers of tumors when used as the vehicle for BaP or DMBA in two-stage or BaP in single-stage trials. The inhibition was not permanent since tumorigenesis returned to control rates 2-3 weeks after prepromotion with toluene ceased but promotion with PMA in acetone continued. Toluene may be unique among reported promotion inhibitors in that it is a widely used commercial chemical which sometimes serves as a vehicle in cancer-screening trials. Since its metabolism is reasonably well defined, it may be of value in exploring further the process of tumor promotion.