Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.     

Induction of NF-KB during monocyte differentiation by HIV type 1 infection

The production of human immunodeficiency virus type 1 (HIV-1) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA, and in purified human monocytes and macrophages. Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat. PMA treatment, and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei. In nuclear extracts from monocytes or macrophages, induction of NF-KB occurred only if the cells were previously infected with HIV-1. When U937 cells were infected with HIV-1, no induction of NF-KB factor was detected, whereas high level of progeny virions was produced, suggesting that this factor was not required for viral replication. These results indicate that in monocytic cell lineage, HIV-1 could mimic some differentiation/activation stimuli allowing nuclear NF-KB expression.     

In vivo administration of anti-CD3 monoclonal antibodies or immunotoxins in murine recipients of allogeneic T cell-depleted marrow for the promotion of engraftment.

The role of host anti-donor cells in rejection of fully allogeneic donor T cell-depleted marrow was investigated by using mAb or immunotoxins directed against T cell or NK cell determinants. Immunotoxins consisting of mAb conjugated to a low oligosaccharide-containing fraction of purified ricin toxin A chain (RTA) facilitated in vivo-depletion of target cell populations. BALB/c and DBA/1 donors were selected based upon their expression (BALB/c) or lack of (DBA/1) hemopoietic histocompatibility (Hh1) Ag, which may serve as targets for donor rejection in C57BL/6 hosts. When studies directed toward eliminating CD3+ cells were performed in both systems, injections of intact anti-CD3 mAb or anti-CD3-RTA reproducibly produced the highest engraftment values. The fact that engraftment values obtained with anti-CD3 or anti-CD3-RTA therapy in allogeneic systems were substantially higher than in syngeneic controls suggested that engraftment stimulatory proteins were released upon TCR engagement. Elevated levels of cytokines and a high mortality rate in allogeneic recipients confirmed that this was the case. Nonstimulatory preparations of anti-CD3F(ab')2 fragments and anti-CD3F(ab')2-RTA promoted engraftment of both types of allogeneic marrow, as measured by short term 125I-IUdR assays, suggesting that stimulation was not a prerequisite for engraftment. Recipients of anti-CD3F(ab')2 or anti-CD3F(ab')2-RTA showed a marked reduction of host CD3+ cells as measured by immunofluorescence and flow cytometry. In long term chimerism studies, recipients of Hh1-disparate marrow and anti-CD3F(ab')2 had a dramatic increase in donor cell engraftment as compared to controls, indicating that positive effects on engraftment were long lived. Studies further showed that BALB/c donor cells exhibiting an Hh1 disparity were rejected by host cells expressing NK1.1 or Ly-1 (NK cells and T cells). In contrast, DBA/1 donor cells that were not Hh1-disparate were rejected by cells expressing Ly-1, but not NK1.1 (T cells only). These studies provide definitive data that CD3+ cells participate in the rejection of either Hh1+ or Hh1null T cell-depleted allografts and offer new strategies for alloengraftment using regimens containing nonmitogenic anti-CD3.     

Biogenesis of the yeast vacuole (lysosome). Proteinase yscB contributes molecularly and kinetically to vacuolar hydrolase-precursor maturation.

The vacuolar proteinase yscB (PrB) has been implicated in the final maturation of procarboxypeptidase yscY (pro-CpY) to the mature wild-type form CpYb of 61 kDa. In PrB-deficient mutants, only the proteinase yscA processed form CpYa of 62 kDa is found [Mechler, B., Müller, H. & Wolf, D. H. (1987) EMBO J. 6, 2157-2163]. We report now that, akin to CpY, two forms of mature proteinase yscA (PrA) can be distinguished. In PrB-deficient mutant cells, PrAa, migrating at about 43 kDa in SDS/PAGE, is found, whereas PrAb, found in wild-type cells, had the known molecular mass of 42 kDa. In the PrB-deficient strain, pro-PrA and pro-CpY matured only to the higher-molecular-mass forms, PrAa and CpYa, and the maturation of both precursors was slower than in the isogenic wild-type strain. Pulse-labeling experiments indicated that the mature forms, PrAb or CpYb, are generated directly in the PrB-containing wild-type strain in vivo. In vitro experiments showed that PrB is able to trigger maturation of its 42-kDa pro-PrB precursor to mature PrB in the absence of PrA. Mature PrB and its proteolytic activity, however, shows a higher stability in the presence of mature PrA. The data indicate a molecular and kinetic participation of proteinase yscB in vacuolar hydrolase precursor maturation.     

Clinic-based primary care of frail older patients in California.

We surveyed medical directors of primary care clinics in California to learn how those clinics cared for their frail older patients. Of 143 questionnaires sent, 127 (89%) were returned. A median of 30% of all patient encounters were with persons aged 65 or older, and a median of 20% of older patients were considered frail. A total of 20% of the clinics routinely provided house calls to homebound elderly patients. Of clinics involved in training medical students of physicians (teaching clinics), 70% had at least one physician with an interest in geriatrics, compared with 42% of nonteaching clinics (P less than .005). For frail patients, 40% of the clinics routinely performed functional assessment, while 20% routinely did an interdisciplinary evaluation. Continuing education in geriatrics emerged as a significant independent correlate of both functional assessment and interdisciplinary evaluation. Among the 94 clinics with a standard appointment length for the history and physical examination, only 11 (12%) allotted more than 60 minutes for frail patients. The data suggest that certain geriatric approaches are being incorporated into clinic-based primary care in California but do not provide insight into their content or clinical effects.     

Spatial and temporal distribution of pheromone biosynthesis-activating neuropeptide in Helicoverpa (Heliothis) armigera using RIA and in vitro bioassay.

A [3H]-PBAN (pheromone biosynthesis-activating neuropeptide) analog was synthesized, and binding of the radioligand to a specific PBAN-antiserum was achieved. The inhibition of binding of the radioligand by unlabeled PBAN, several PBAN analogs, and other competitors was studied and a specific radio-immunoassay was developed. Using this radioimmunoassay we found PBAN-like immunoreactivity in methanol extracts of hemolymph and neural tissues from females. Higher levels of PBAN-like immunoreactivity in extracts of brain-suboesophageal ganglion complexes, corpora cardiaca, thoracic ganglia, and abdominal ganglia were observed during the 4-5th h scotophase when compared to the PBAN-like immunoactivity levels during the 6-11th h photophase. On the other hand, the concentrations of PBAN-like immunoreactivity, in the terminal abdominal ganglion were higher during the photophase relative to minimal levels observed during the scotophase, indicating an accumulation before the onset of pheromone production. These differences in concentrations of PBAN were also reflected in the stimulation of in vitro pheromone glands, whereby significant stimulations were obtained by scotophase and photophase brain extracts, scotophase thoracic ganglia extracts, and photophase terminal abdominal ganglia extracts. No detectable levels of PBAN were found in hemolymph extracts during the sampling periods.     

Adenylate cyclase uncoupled beta-adrenergic receptors in salamander proximal tubules

The isolated perfused proximal tubule of the neotenic salamander Ambystoma tigrinum responds with either a hyperpolarization or depolarization of both the basolateral cell membrane and transepithelial potentials following the addition of 10(-5) M isoproterenol to the bath superfusate. Both responses were blocked by 10(-6) M propranolol but neither response was mimicked by 10(-4) M cAMP. beta-Adrenergic binding studies of individual microdissected proximal tubules using (-)-[3H]CGP-12177 as a hydrophyllic radioligand and (+/-)-timolol (0.1 mM) as the displacer drug revealed two distinct populations of proximal tubules possessing either low (KD = 153.8 nM; Bmax = 110.2 fM/mm) or high affinity (KD = 12.0 nM: Bmax = 3.9 fM/mm) binding characteristics. Competition studies indicated that the bound (-)-[3H]CGP-12177 behaved as a typical beta-adrenergic ligand, being displaced by (-)-isoproterenol but not by (+)-isoproterenol or (-)-phenylephrine. However, neither appeared to be coupled to the adenylate cyclase system. These data suggest the presence of functional beta-adrenergic receptors that do not appear to be coupled to the adenylate cyclase system.     

Dynamics and reactivity of HbXL99 alpha. A cross-linked hemoglobin derivative

Resonance Raman spectroscopy, transient absorption, and fluroescence techniques have been employed to investigate the structure and dynamics of the alpha-cross-linked hemoglobin derivative, HbXL99 alpha. The resonance Raman spectra of the deoxy form of HbXL99 alpha are identical to those of native NbA (VFe-His approximately 222 cm-1), which exhibit a T-state (low affinity) structure regardless of solvent conditions. The resonance Raman spectra of the transient heme photoproduct resulting from CO photolysis from HbXL99 alpha appear to have structures intermediate between deoxy-T and ligand-bound R structures (VFe-His approximately 222 cm-1). Time-resolved resonance Raman data of HbXL99 alpha-CO show that complete CO recombination occurs after approximately 5 ms, with only a small amount of the CO-bound species reforming within approximately 200 ns (geminate recombination). Transient absorption spectra of HbXL99 alpha-O2 indicate that the extent of sub-nanosecond geminate recombination of O2 is also reduced in the cross-linked derivative relative to native HbA. The decrease in tryptophan fluorescence of HbXL99 alpha upon oxygenation further indicates that tertiary structural changes at the alpha 1-beta 2 interface upon ligation are apparently reduced, but not eliminated in the cross-linked derivative relative to HbA.     

Discrimination and consistency of five myosin ATPase stains in human normal and duchenne dystrophic muscle

Summary Limitations in the ability of the human visual system to assess accurately the relative staining densities of individual fibers in muscle tissue stained for myosin ATPase can complicate the objective evaluation of fiber type populations. In this study a novel approach is employed which utilizes human visual capabilities to provide accurate fiber classification. Using this approach, the ability of five ATPase staining techniques to discriminate fiber type categories in single samples of human normal and Duchenne dystrophic skeletal muscle is evaluated, as is the consistency of the fiber type classifications between stains. While no major discrepancies in fiber typing were observed in the sample of normal muscle, significant differences in classification, along with a decrease in the ability to discriminate fiber types were noted in the sample of Duchenne muscular dystrophy. For the most part, these discrepancies were resolved by a re-interpretation of the staining characteristics of fibers in one stain.This work was supported in part by NIH grant NS 15584, and by a grant from the Muscular Dystrophy Association     

Host modification of Sindbis virus sialic acid content influences alternative complement pathway activation and virus clearance

Previous studies have shown that Sindbis virus, an enveloped alphavirus of the togavirus group, activates the alternative complement pathway in the absence of detectable antiviral immunoglobulin. The present studies examined the role of the host-determined sialic acid content of Sindbis virus on activation of the alternative complement pathway. Purified Sindbis virus grown in baby hamster kidney (BHK-SV) and in mosquito (MOSQ-SV) cells yielded virus with 10.2 and less than 2.0 nmol sialic acid/mg viral protein, respectively. Sindbis virus deficient in sialic acid (2.0 nmol sialic/mg) was also produced by treating the BHK-SV with neuraminidase (NANase-SV). When MOSQ-SV or NANase-SV was incubated in either C4DGPS or C2DHS, each consumed significantly more C3 than did BHK-SV, indicating that the ability of Sindbis virus to activate the alternative pathway is inversely related to its sialic acid content. Studies in vivo showed that virus deficient in sialic acid (MOSQ-SV) was cleared from the blood of mice much more efficiently than was virus rich in sialic acid (BHK-SV), after i.v. inoculation. Furthermore, when animals were depleted of C3 through C9 by cobra venom factor (CoVF) treatment, no differences in the clearance of high and low sialic acid-containing viruses were observed. Thus both the activation in vitro and complement-dependent clearance in vivo are significantly affected by the host-determined sialic acid content of Sindbis virus.