Systolic pressure gradients between the wall of the left ventricle, the left ventricular chamber, and the aorta during positive inotropic states: implications for left ventricular efficiency

To study systolic pressure gradients developed between the left ventricular wall, its chamber, and the aortic root, in one group of dogs left ventricle ventral wall intramyocardial pressure, left ventricular outflow tract pressure, and aorta pressure were compared with aortic flow as well as left ventricular dimension changes during control conditions as well as during positive intropic states induced by isoproterenol, stellate ganglion stimulation, and noradrenaline. In another group of dogs systolic pressures in the ventral wall of the left ventricle, the main portion of the left ventricular chamber, and the aorta were compared with aortic flow during similar interventions, before and after the administration of phentolamine. Pressure gradients between the wall of the left ventricle and the outflow tract of the left ventricle were minimal during control states, but during the three positive inotropic states were increased significantly. In contrast, pressure gradients between the outflow tract of the left ventricle and the aortic root were insignificant during positive inotropic states; those between the wall and main portion of the chamber were only significantly different during left stellate ganglion stimulation. The data derived from these experiments indicate that useful peak power output of the left ventricle (systolic aortic pressure X flow) is unchanged following isoproterenol infusion, but is increased by stellate ganglion stimulation and noradrenaline. The useful peak power output index (an index of left ventricular efficiency derived by dividing useful peak power output by peak intramyocardial pressure) was reduced more by isoproterenol than the other two interventions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Establishment and characterization of murine macrophage hybrids

Proteose peptone-induced peritoneal macrophages from CBA/J (H-2k) mice have been fused to a hypoxanthine phosphoribosyltransferase-negative variant of the P388D1 (H-2d) murine macrophage cell line. Six hybrid clones were isolated following HAT selection and further characterized. Five of the six clones express class I antigens of both parental haplotypes by microelisa and by flow cytometric analysis. Class II antigen expression of both haplotypes was apparent following a 72-hr incubation of the hybrids with concanavalin A-stimulated rat spleen cell supernatant. However, I-Ad was expressed in all hybrids to a greater extent than I-Ak. Three clones with the highest level of I-Ak expression, E5, C2, and C4, were capable of antigen presentation to the I-Ak-restricted T-cell line, D10.G4.1. LPS induction of the hybrids resulted in a 2- to 15-fold increase in the amount of IL-1 produced relative to the P388D1 parent. Finally, in distinction to P388D1, all hybrids demonstrated increased Fc-mediated erythrophagocytosis of chromium-labeled antibody-coated erythrocytes. These murine macrophage hybrids appear stable and should serve as useful models in understanding the regulation of macrophage function.     

Characteristics of cellular immune responses to collagen type I or collagen type II

We have examined the murine cell-mediated immune (CMI) response to collagens type I (CI) and type II (CII) as measured by in vivo delayed-type hypersensitivity responses. We have verified the histopathology and kinetics of the cell-mediated immune responses. Predominant cell-mediated responses were obtained 7, 10, or 14 days following immunization. A presumed antibody-mediated reaction was observed at later times (e.g., greater than 21 days following immunization). The CMI responses to the collagens show a strain-dependent relationship. For CI, the CMI response profile shows H-2b greater than or equal to H-2k = H-2q much greater than H-2d. For bovine CII, the response profile is H-2d greater than H-2b = H-2k = H-2q; the chick CII response profile is H-2q = H-2k greater than H-2b = H-2d, and in limited testing, only the H-2q strain could generate murine CII-specific cell-mediated immune responses. The CII-specific CMI response is cross-reactive with CII from several species of animals, but not with CI. Further, the collagen-specific CMI response can be elicited with certain cyanogen-bromide fragments of bovine CII. Finally, our study also demonstrates that there is a non-H-2-linked locus(i) involved in the development of CII-induced arthritis.     

Properties of in vitro recombinant derivatives of pJV1, a multi-copy plasmid from Streptomyces phaeochromogenes

The 10.8 kb plasmid pJV1, isolated from Streptomyces phaeochromogenes, has a high copy number (about 150) and a broad host range among Streptomyces spp. Several pJV1 derivatives carrying the thiostrepton resistance gene (tsr) of S. azureus were made. One derivative, pWOR191, was shown to promote its own transfer and to mobilize chromosomal markers in S. lividans. Another derivative, pWOR109, was non-transmissible. Deletion in vitro of a segment of pWOR109 gave pWOR120 (5.6 kb), which has single BamHI and Bg/II sites shown to be capable of accepting 'foreign' DNA such as a previously cloned S. antibioticus DNA fragment encoding tyrosinase, giving vectors (pWOR125, pWOR126) with properties resembling the well-established multicopy vector pIJ702. Shuttle vectors capable of functioning in both S. lividans and Escherichia coli were also constructed. The region of pJV1 essential for replication and maintenance was localized to a 2.5 kb segment. Stable maintenance of pWOR109 and pWOR120 was observed in the presence of derivatives of pIJ101, the progenitor of pIJ702.     

Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous and heterozygous fibroblasts

The esterification of cholesterol derived from human low density lipoprotein (LDL) or fetal bovine serum (FBS) was deficient in cultured fibroblasts from subjects with heterozygous and homozygous type C Niemann-Pick (NPC) disease. Failure to significantly esterify LDL-derived cholesterol resulted in abnormal accumulation of predominantly unesterified cholesterol in homozygous NPC fibroblasts. Compared with normal and homozygous fibroblasts, heterozygous NPC fibroblasts synthesized intermediate levels of cholesteryl ester during the initial 6 h of incubation with LDL. The rate of cholesterol esterification in heterozygous cells was normal when measured over a 24-h period of incubation with LDL. In addition to demonstrating a defect in cholesterol esterification, homozygous NPC fibroblasts accumulated more total cholesterol when incubated with LDL or FBS than normal fibroblasts accumulated. When heterozygous NPC fibroblasts were incubated with LDL or FBS, cellular accumulation of cholesterol reached levels that were high-normal or intermediary between levels observed in normal and homozygous NPC fibroblasts. The partial expression of these metabolic errors in the heterozygous genotype relevantly links these errors to the primary mutation of this disorder.     

Comparison of material properties in fascicle-bone units from human patellar tendon and knee ligaments

The fascicle material properties in bone-fascicle-bone units were determined for the anterior and posterior cruciate ligaments (ACL, PCL), the lateral collateral ligament (LCL) and the patellar tendon (PT) from three young human donor knees. Groups of fascicles from each tissue were isolated with intact bone ends and failed at a high strain rate in a saline bath at 37 degrees C. In each knee tested the load related material properties (linear modulus, maximum stress and energy density to maximum stress) for the patellar tendon were significantly larger than corresponding values for the cruciate and collateral ligaments. Bundles from different ligaments in the same knee were similar to each other in their mechanical behavior. In addition, no significant differences were present in the maximum strains recorded for any of the four tissue types examined. The results presented have implications in studies of ligament injury. They are also important in the design and use of synthetic and biological ligament replacements and in tissue and whole knee modeling.     

Microheterogeneity of Micro tubule-Associated τ Proteins Is Due to Differences in Phosphorylation

We have studied the heterogeneity of the microtubule-associated tau proteins using tau-specific antibodies and two-dimensional electrophoresis. Both monoclonal and polyclonal antibodies to tau proteins recognize five bands in cow brain microtubule proteins run on sodium dodecyl sulfate (SDS)-polyacrylamide gels, with apparent molecular weights between 56,000 and 66,000. Immunoblots of cow brain microtubules separated on two-dimensional gels, using nonequilibrium pH gradient electrophoresis in the first dimension and SDS-gel electrophoresis in the second, reveal that greater than 30 isoforms of tau exist. The tau proteins vary in pI from 6.5 to 8.5, with the higher-molecular-weight forms being more acidic. The microheterogeneity of tau is not induced by cycling of microtubules, because two-dimensional immunoblots of tau from total brain are almost identical to those of tau from cycled tubules. Adult rat brain tau, which appears as three doublet bands on SDS gels, also exhibits considerable isoelectric heterogeneity, as does tau from 7-day-old rats, which appears as only one band on SDS gels. After dephosphorylation of cow brain tau with alkaline phosphatase, the highest-molecular-weight band disappears on SDS gels. On two-dimensional gels, the number of tau variants decreases by more than half after dephosphorylation, and the more basic species increase greatly in intensity. Preliminary experiments with tau labeled in vivo with 32PO4 also indicate that the more acidic tau proteins are the more highly phosphorylated forms. Thus, isoelectric heterogeneity of tau proteins exists at all ages and is due, at least in part to differences in the state of phosphorylation of tau isoforms.     

Nonhistone chromosomal protein HMG 1 interactions with DNA. Fluorescence and thermal denaturation studies

The interaction of high mobility group protein 1 (HMG 1) isolated from chicken erythrocytes with DNA has been characterized using the intrinsic tryptophan fluorescence of the protein as a probe. It was found that the fluorescence is quenched approximately 30% upon binding to either single- or double-stranded DNA. Fluorescent titrations indicate that the physical site size for HMG 1 binding on native DNA is approximately 14 base pairs (or 14 bases for binding to single-stranded DNA). Binding to single-stranded poly(dA) is only slightly dependent on ionic strength, although the affinity for double-stranded DNA is strongly ionic strength-dependent and has an optimum at approximately 100-120 mM Na+. Above this range, binding to native DNA is virtually all electrostatic in nature. Although the affinity of HMG 1 for single-stranded DNA is higher than that for double-stranded DNA at the extremes of the ionic range studied, no clear evidence for a helix-destabilizing activity was obtained. At low ionic strength, the protein actually stabilized DNA against thermal denaturation, while at high ionic strength, HMG 1 appears to undergo denaturation below the Tm of the DNA. Studies of the environment of the tryptophan fluorophores using collisional quenchers iodide, cesium, and acrylamide suggest that the predominant fluorophore is relatively exposed but constrained in a rigid, positively charged environment.