Distinct developmental patterns of short-term and long-term functioning lymphoid and myeloid precursors defined by competitive limiting dilution analysis in vivo

Functional abilities of individual marrow precursor cells were defined by competitive limiting dilution without enrichment, tissue culture, or induced marking, manipulations that might affect cell functions. We directly measured long-term repopulating abilities in limiting doses (0.25-1.0 x 10(5)) of genetically marked congenic marrow cells. These were mixed with a standard dose of 4 or 5 x 10(5) competitor marrow cells, which contained a predictable distribution of precursor cells and allowed quantitative assays. Percentages of donor type T and B lymphocytes, granulocytes, platelets, and erythrocytes were measured in recipient blood. Applying the maximum likelihood statistic, concentrations (per 10(5)) of precursors repopulating at least one lineage were: 4.7 and 6.0 after 6 wk, 1.6 and 2.7 after 14 to 15 wk, and 1.2 and 1.9 after 30 to 32 wk; concentrations repopulating at least three lineages were 2.3 and 3.4 after 6 wk, 0.9 and 1.7 after 14 to 15 wk, and 0.9 and 1.3 after 32 wk. Almost all precursors functioning after 14 wk repopulated all lineages. At 6 wk, similar levels of donor cells were produced in recipients of both short- and long-term precursors. However, after 14 to 32 wk, contributions by short-term precursors (about two-thirds of the precursors) dropped to zero, while contributions by long-term precursors (about one-quarter of the precursors) expanded severalfold. The latter permanently repopulated all lineages after 30 to 32 wk, functioning as the most primitive stem cells (PSC) in the immune and myeloid systems. Nearly all the variance in long-term repopulated recipients was explained using the Poisson distribution to calculate donor percentages in a model where each donor and competitor PSC contributed equally.     

Visual and auditory evoked responses in acute severe hepatitis

Evoked responses have not been studied in patients with acute severe hepatitis (ASH) with or without hepatic encephalopathy. This prospective study was undertaken to find out diagnostic as well as prognostic value of visual evoked responses (VER), and brain stem auditory evoked responses (BAER) in patients with ASH with or without encephalopathy. Visual evoked responses and BAER were studied in 20 patients (14 males and six females) with ASH. The patients were diagnosed as having severe hepatitis if acute hepatitis was associated with raised serum bilirubin and serum transaminases, and if they had a prothrombin time index of < 50%. After a detailed neuropsychiatric examination of each patient, the study sample was divided into two groups of 10 patients: ASH without encephalopathy (ASH-WOE), and ASH with encephalopathy (fulminant hepatic failure, FHF). The median P100 latencies of FHF patients were significantly increased compared with controls and patients in the ASH-WOE group. Abnormal P100 latencies, exceeding 95th percentile values of the controls, were present in one patient in the ASH-WOE group and six patients in the FHF group. The median interpeak latencies I-III, III-V and I-V were significantly prolonged in the FHF group. Interpeak latencies III-V were also increased significantly in patients in the ASH-WOE group. While abnormal BAER were seen frequently in both groups, VER abnormalities were largely confined to patients in the FHF group. In the FHF group, six out of 10 patients survived and exhibited clinical improvement in the status of hepatic encephalopathy. Evoked responses were repeated after 2-3 weeks of recovery in these patients and VER abnormalities showed a tendency to normalize, thereby suggesting a prognostic implication. The incidence of abnormal VER in hepatic encephalopathy complicating ASH far exceeded that of abnormal BAER. Markedly prolonged P100 latencies in FHF patients indicate poor prognosis.     

A role for amino acid residues in the third cytoplasmic loop in defining the ligand binding characteristics of the alpha2D-adrenergic receptor

In this report, 5 amino acid residues (aa) in the third cytoplasmic loop of the alpha 2D-adrenergic receptor are identified which (individually or together) alter its ligand-binding characteristics. An important structural discrepancy exists in the third cytoplasmic loop of the alpha 2D-ARs encoded by the rat cDNA and the rat gene--five aa are different. The newly identified bovine receptor as well as the mouse receptor contained the 5 aa identical to that encoded by the rat cDNA. Site-directed mutation of these residues to those of the rat gene encoded receptor resulted in alteration of binding characteristics: significant changes in the ability of the mutant receptor to bind to a number of agonists and antagonists were observed--ranging from a decrease by half in the case of oxymetazoline, to near total loss of binding in the case of prazosin. Thus, the mutant receptor was no longer pure alpha 2D-AR. This indicated a hitherto unrealized role of the third cytoplasmic loop in defining the ligand-binding characteristics of the receptor, and also suggested that the rat gene sequence was most probably in error.     

Membrane guanylate cyclase signal transduction system

The suspect role of the receptor-mediated cyclic GMP signaling pathway was dispelled by the discovery of a membrane guanylate cyclase that was also an atrial natriuretic factor receptor. It is now established that the membrane guanylate cyclase transduction system is linked to the signaling of natriuretic factors, guanylin/enterotoxin, and emerging evidence suggests that a new neural tissue-specific subfamily of membrane guanylate cyclases exists whose mechanism of signal transduction is different from those of the other membrane cyclases. This review will briefly discuss the fascinating, albeit turbulent, history of this signal transduction field, which will be followed by its current status and finally the direction it is heading.     

Role of major histocompatibility complex class I antigens in modulating the performance of murine tumour cells in cold target competition assays

The role of class I major histocompatibility complex (MHC) antigen levels on the ability of five murine tumour cell lines (YAC, P815, EL4, SP20 and L929) to competitively inhibit their own lysis, as well as the lysis of other targets by lymphokine-activated killer (LAK) effector cells was examined. Basal LAK susceptibilities of the cell lines were in the order P815 > YAC > SP20 > EL4 > L929, whereas the basal class I MHC antigen levels were in the order P815 > SP20 > L929 > YAC > EL4. Treatment with interferon-gamma (IFN-gamma) induced augmentation of class I MHC antigen levels on all cell lines. A concomitant decline in LAK susceptibility was seen for P815, YAC, SP20 and L929 cells, but not for EL4 target cells. On the basis of competition results, tumour cells appear to fall into two groups (group 1: P815, YAC and SP20; group 2: EL4 and L929). Members of each group could in general competitively inhibit the lysis of cell lines of their own group only. Treatment with IFN-gamma suppressed the ability of all tumour cell lines, except EL4, to cause competitive inhibition. These results support the proposition that class I MHC antigens may interfere with the recognition of target cells by effector LAK cells.     

Simultaneous determination of benzene, toluene, ethylbenzene, and xylenes in urine by thermal desorption-gas chromatography

Amphotericin B (AmB)-resistant Leishmania donovani promastigotes were selected by increasing drug pressure, and their biological features were compared with those of the wild-type parent strain. The 50% inhibitory concentration for resistant cells was 20 times higher than that for the wild-type. Resistance was stable after more than 40 passages in drug-free medium, and resistant promastigotes were infective to macrophages in vitro but lost their virulence in vivo. They had 2.5 times longer generation time, decreased AmB uptake, and increased AmB efflux in comparison to the wild type. Fluorescence measurement with a specific plasma membrane probe, 1-[4-(trimethylammonio)-1,6-diphenylhexa]-1,3,5-triene, showed increased membrane fluidity in drug-resistant promastigotes. Analysis of lipid composition showed that in resistant cells saturated fatty acids were prevalent, with stearic acid as the major fatty acid, and the major sterol was an ergosterol precursor, the cholesta-5, 7, 24-trien-3beta-ol and not ergosterol as in the AmB-sensitive strain.     

Comparison of rocuronium and suxamethonium for use during rapid sequence induction of anaesthesia

Past investigations of in vivo arterial behavior have concentrated on determining material properties based upon the maximum and minimum pressure and diameter measured over a pulse cycle. A new in vivo technique, based upon continuous measurement of pressure and flow, has been developed to study arterial compliance throughout the pulse cycle. Compliance in the abdominal aorta of rats showed different behavior during the rising and falling portion of the pressure pulse. Previous investigations of canine arteries which used different methods are consistent with these findings. This study demonstrates the utility of a new measurement technique and shows some trends in compliance within the pulse cycle which have neither been revealed by static tests nor by dynamic tests which focused on pulse averaged values.     

Presence and modulation of interleukin-12 in seminal plasma of fertile and infertile men

A case of bilateral facial atrophy diagnosed as atrophic connective tissue panniculitis is presented. Reconstruction of both cheeks was performed with two staged latissimus dorsi muscle flaps. The initial good result on the right cheek deteriorated as the disease continued to progress after surgery. The good result on the left cheek, however, remained stable. Detailed clinical examinations, laboratory analysis, and deep biopsies from the affected areas are important for accurate diagnosis. Reconstructive procedures should be delayed while the disease is still active.     

The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.