First test of the theory of reasoned goal pursuit: predicting physical activity.

OBJECTIVE: This study applied the theory of reasoned goal pursuit (TRGP) in predicting physical activity among Australian undergraduate students, providing the first empirical test of the model.Methods: The research comprised an elicitation study (N = 25; MAge= 25.76, SDAge= 11.33, 20 female, 5 male) to identify readily accessible procurement and approval goal beliefs and behavioural, normative, and control beliefs; and, a two-wave prospective online survey study (N = 109; MAge = 21.88, SDAge = 7.04, 63 female, 46 male) to test the tenets of the TRGP in relation to meeting World Health Organization physical activity guidelines during the COVID-19 pandemic among first year university students.Results: A linear PLS-SEM model displayed good fit-to-data, predicting 38%, 74%, and 48% of the variance in motivation, intention, and physical activity, respectively. The model supported the majority of hypothesised pattern of effects among theory constructs; in particular, the proposition that beliefs corresponding to procurement and approval goals would be more consequential to people's motivation and, thus, their intentions and behaviour, than other behavioural and normative beliefs, respectively.Conclusions: Results lend support for the TRGP and sets the agenda for future research to systematically test the proposed direct, indirect, and moderation effects for different health behaviours, populations, and contexts.Supplemental data for this article is available online at https://doi.org/10.1080/08870446.2022.2026946 .

Increased enzymatic activity of the T-cell antigen receptor-associated fyn protein tyrosine kinase in asymptomatic patients infected with the human immunodeficiency virus.

The immune system of patients infected with human immunodeficiency virus (HIV) is in a state of chronic activation; however, the nature of HIV-related immune activation is unknown. As normal T-cell activation involves early tyrosine phosphorylation induced by the T-cell antigen receptor-associated src-family protein tyrosine kinase p59(fyn(T)) (Fyn), we examined a potential role for this kinase in HIV-related immune dysfunction. We determined the relative specific kinase activity of Fyn in lysates of peripheral blood mononuclear cells from 47 normal control individuals tested negative for HIV-1 and -2, human T-cell lymphotropic virus Type I, hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis; 14 asymptomatic HIV-infected patients having near-normal CD4+ T-cell counts (350 to 980 CD4+ cells/microL); 4 patients with symptomatic acquired immunodeficiency syndrome (AIDS) (<30 CD4+ cells/microL); 13 patients having chronic infection with HBV (6 patients) or HCV (7 patients); and 6 patients with systemic lupus erythematosis (SLE). All patients with asymptomatic HIV disease were shown to have a profound increase (mean increase of 19-fold; range threefold to 56-fold increase; p = 1.33 x 10(-9)) in the relative specific kinase activity of Fyn compared to uninfected controls or patients with hepatitis or SLE. In contrast, patients with AIDS had an Fyn-specific kinase activity that was much less affected (mean increase of threefold; range onefold to sevenfold increase; p = 1.30 x 10(-5)). It was further shown that HIV infection affects the Fyn-specific kinase activity in CD8+-enriched cells, suggesting abnormal Fyn activity in both CD8+ as well as CD4+ T lymphocytes. Initial results implicate a role for the CSK protein tyrosine kinase as responsible for the abnormal Fyn kinase activity observed in HIV-infected patients. These data indicate early and chronic activation of Fyn as a unique HIV-related effect that has the potential to be diagnostic for early HIV infection and/or may serve as a prognostic indicator for advancement to full-blown AIDS. More importantly, sustained activation of the protein tyrosine kinase associated with T-cell antigen receptor function may result in, or contribute to, the immunopathogenic effects associated with HIV infection.

HIV infection in vitro enhances the activity of src-family protein tyrosine kinases.

OBJECTIVE: We examined the effect of HIV infection on src-family protein tyrosine kinase (PTK) activity to determine if alterations in src-family PTK activity could contribute to the HIV-related chronic immune system activation observed in patients infected with HIV. METHODS: Jurkat, a CD4+ human T lymphocyte cell line was infected with HIV IIIB. Kinase activity was determined by in vitro immune complex kinase assays using antibodies specific for the src-family PTKs, p56lck, p59fyn and p60c-src expressed in T lymphocytes. PTK protein and total phosphotyrosine levels were assessed by Western blotting. The role of the gp120-CD4-Lck interaction in HIV-related PTK activation was determined using gp 120-treated Jurkat cells and HIV-infection of JCaM 1.6 cells, a Jurkat-derived cell line that lacks p56lck. RESULTS: Cells infected with HIV for 24 h exhibited increased levels of total tyrosine phosphorylation and enhanced src-family PTK activity without altered levels of expression of src-family kinases. The activity of Lck and Fyn was enhanced within 30 min of infection. HIV-related src-family PTK activation was not a function of the gp120-CD4-Lck interaction and occurred in the presence of 10 mmol/l zidovudine indicating that reverse transcriptase and activation of the HIV genome is not required. CONCLUSIONS: HIV-related activation of src-family PTK is a response of the cell to early stages of the virus life cycle, possibly either membrane fusion or viral uncoating. These results indicate that endogenous src-family PTKs may play a role in HIV-related immune activation and dysfunction. Moreover, activation of src-family PTK may be a mechanism used by the virus to facilitate some aspect of its own life cycle.

Properties of K+ and Cl- channels and their involvement in proliferation of rat microglial cells.

Essentially pure (>95%) cultures of microglia were established from neopallia of newborn rats and used for whole-cell patch-clamp recording of electrophysiological properties and for proliferation studies. Two types of cultures were examined: 1) "Primary" cultures were grown in culture medium with serum and used within 3 weeks of isolation; 2) and "Colony-stimulating factor (CSF)-1-stimulated" cultures were derived from 3-week-old "primary" cultures by passaging and culturing them for several weeks longer in the presence of conditioned medium enriched in CSF-1. Microglia in the "primary" cultures expressed: 1) an inwardly rectifying K+ current (Kir) that was inhibited by Ba2+; 2) an outwardly rectifying K+ current (Kv) with many similarities to the cloned Kv1.3 channel of lymphocytes, including block by nanomolar concentrations of charybdotoxin (ChTX) and margatoxin (MgTX); and 3) an outwardly rectifying anion current with time- and voltage-independent gating. The anion current is activated reversibly under cell swelling conditions, i.e., after exposure to a hypo-osmotic bathing medium. The anion channels are highly permeable to Cl-, measurably permeable to gluconate (P(gluconate)/ PCl = 0.34), and blocked by flufenamic acid, 4-nitro-2-(3-phenylpropylamino)- benzoic acid (NPPB), and 6, 7-dichloro-2-cyclopentyl-2, 3-dihydro-2-methyl-1-oxo-1H-inden-5-yl (oxy) acetic acid (IAA-94). Microglia in the "CSF-1-stimulated" cultures expressed Kir and Cl- current, but not Kv current. Proliferation in the latter type of cultures could be slowed by omission of the CSF-1 enriched supernatant for 2 days and stimulated by adding back the conditioned medium. This "CSF-1-stimulated" proliferation was inhibited by Ba2+ (Kir blocker), and the Cl(-)-channel blockers flufenamic acid, NPPB, and IAA-94, whereas the Kv blockers ChTX and MgTX had no effect. Thus, Kir and Cl- channels appear to be necessary for "CSF-1-stimulated" proliferation of rat microglia, and there is no evidence that even a transient activation of Kv is necessary.

Chloride-channel block inhibits T lymphocyte activation and signalling.

Both large- and small-conductance chloride (Cl-) channels have been found in human T lymphocytes; however, apart from possible roles in mediating regulatory volume decrease, their functions are not understood. We have used patch-clamp electrophysiology, Ca2+ spectrofluorometry, and Western blot assay for phosphotyrosine to investigate the effects of blocking Cl- channels on proliferation and on specific events in the activation of normal human T cells. Four chemically distinct Cl- channel blockers inhibited both the small-conductance Cl- channels and phytohemagglutinin (PHA)-induced lymphocyte proliferation in a similar dose-dependent manner; their order of potency was 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB) > 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS) > flufenamic acid >> IAA-94. The Cl- channel blockers inhibited both the PHA-induced mobilization of Ca2+ and the rapid tyrosine phosphorylation of several polypeptides. Cell proliferation was not rescued by the Ca+ ionophore ionomycin or by addition of exogenous interleukin-2 (IL-2). Moreover, the blockers also inhibited phosphotyrosine expression in IL-2-treated, activated lymphoblasts. Thus, our results support a role for Cl- channels in early, PHA-evoked signalling and in later, II-2-dependent stages of lymphocyte activation and proliferation.

Small-conductance chloride channels in human peripheral T lymphocytes.

During whole-cell patch-clamp recording from normal (nontransformed) human T lymphocytes a chloride current spontaneously activated in > 98% of cells (n > 200) in the absence of applied osmotic or pressure gradients. However, some volume sensitivity was observed, as negative pressure pulses reduced the current. With iso-osmotic bath and pipette solutions the peak amplitude built up (time constant approximately 23 sec at room temperature), a variable-duration plateau phase followed, then the current ran down spontaneously (time constant approximately 280 sec). The anion permeability sequence, calculated from reversal potentials was I-, Br- > NO3-, Cl- > CH3SO3-, HCO3- > CH3COO- > F- > aspartate, gluconate, SO4(2-) and there was no measurable monovalent cation permeability. The Cl- current was independent of time during long voltage steps and there was no evidence of voltage-dependent gating; however, the current showed intrinsic outward rectification in symmetrical Cl- solutions. The conductance of the channels underlying the whole-cell current was calculated from fluctuation analysis, using power-spectral density and variance-vs.-mean analysis. Both methods yielded a single channel conductance of about 0.6 pS at -70 mV (close to the normal resting potential of T lymphocytes). The power spectral density function was best fit by the sum of two Lorentzian functions, with corner frequencies of 30 and 295 Hz, corresponding to mean open times of 0.54 and 5.13 msec. The pharmacological profile included rapid block by external application of flufenamic acid (50 microM), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 microM), [6,7-dichloro-2-cyclopentyl-2,3- dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy] acetic acid (IAA-94, 250 microM) or 100 microM 1,9-dideoxyforskolin. The stilbene derivatives DIDS (4,4'-diisothiocyano-2,2' disulphonic acid stilbene, 500 microM) and SITS (4-acetamido-4'-isothiocyano-2,2'-disulphonic acid stilbene, 500 microM) prevented buildup of Cl- current after a 30-min preincubation at 500 microM. When tested in a mitogenic assay, DIDS, flufenamic acid, NPPB and IAA-94 all inhibited T-cell proliferation, suggesting a physiological function in addition to the observed volume sensitivity.

An octapeptide analogue of HIV gp120 modulates protein tyrosine kinase activity in activated peripheral blood T lymphocytes.

Following infection with HIV, patients exhibit lymphocyte dysfunction before the loss of CD4+ T cells. The major HIV surface glycoprotein, gp120, can modulate lymphocyte function in vitro; however, the mechanism by which gp120 affects T lymphocyte signal transduction is controversial. We have used Peptide T, a synthetic octapeptide derived from a conserved, CD4 binding region of gp120, to examine gp120-related modulation of lymphocyte signal transduction. Activation of lymphocytes through the T cell receptor (TCR) in collaboration with cell surface accessory molecules results in rapid increases in tyrosine phosphorylation, probably through the recruitment and activation of src-family protein tyrosine kinases (PTK) such as lck and fyn which have been implicated in mediating the proximal signalling events mediated through the TCR. To identify potential mechanisms by which gp120 could modulate the function of T lymphocytes, we determined the effect of Peptide T on normal, activated peripheral blood lymphoblasts. Treatment of normal, activated peripheral blood lymphoblasts with Peptide T (10(-9) M) for 60 min transiently reduced levels of protein tyrosine phosphorylation (ptyr). Reduction in levels of cellular ptyr was associated with transient inhibition of the activity of total cellular and CD4-associated p56lck kinase activity (80%). Peptide T also induced a small delayed reduction in the p59fyn activity (up to 42%). Despite the decrease in total cellular ptyr levels, pp60c-src kinase activity was increased 11-fold following treatment with Peptide T. Peptide T pretreatment also induced tyrosine phosphorylation of a 48-kD CD4-associated protein, indicating that Peptide T may have multiple effects. Peptide T did not alter the levels of total cellular p56lck enzyme, nor did it directly inhibit the activity of purified p56lck. These results are consistent with a Peptide T-dependent modulation of PTK regulation, and support the potential of gp120 to interfere with T lymphocyte signal transduction in activated T lymphocytes.

Gallysin-1, an antibacterial protein isolated from hemolymph of Galleria mellonella.

An inducible hemolysin with antibacterial properties was isolated from the hemolymph of immune Galleria mellonella larvae. The Galleria-derived lysin, named Gallysin-1, was shown to have an apparent molecular weight of 75,000 and to be relatively heat stable at 56 degrees C. Although Gallysin alone was not bactericidal it caused sufficient damage of the outer cell membranes of Pseudomonas aeruginosa RP4 and Escherichia coli K176 to release beta-lactamase from the periplasm. In the presence of either purified Galleria lysozyme or egg white lysozyme Gallysin-1 had potent antibacterial activity against gram-negative bacteria. Gallysin-1 killed osmotically shocked P. aeruginosa and E. coli that suggests that it can also attack exposed inner cell membranes of gram-negative bacteria. The identification of Gallysin-1 recognizes another distinct member of the bactericidins involved in insect immunity.

Gentamicin-induced hypercalciuria in the rat: assessment of nephron site involved.

Two independent techniques were used in anesthetized rats in an attempt to locate the nephron site of the reduced tubular calcium reabsorption accompanying acute gentamicin infusion. The first technique was that of lithium clearance used to assess proximal sodium (and secondarily calcium) handling. Observations that lithium clearance was comparable in control and gentamicin-treated animals (1.83 +/- 0.39 vs. 1.46 +/- 0.14 ml.min-1 for first experimental period) suggests a lack of proximal effect of the drug. The second technique was that of tracer microinjection whereby superficial nephrons were injected with 45Ca and tubule calcium transport was assessed from the recovery of radioactivity in the final urine. 45Ca recovery values from distal microinjections were comparable in control and gentamicin-treated groups (81.1 +/- 2.0 vs. 77.7 +/- 4.6%). However, 45Ca recovery values from proximal microinjections were significantly higher in the gentamicin group (9.4 +/- 1.0 vs. 3.5 +/- 0.8%; P < .001). These data suggest that the effects of gentamicin on renal calcium handling are mediated at a nephron site proximal to the distal tubule (i.e., loop of Henle or proximal tubule itself). Closer examination of individual proximal micropuncture data may point to an effect occurring predominantly in the pars recta of the proximal tubule or loop of Henle. Taken together, the results of both parts of the present study suggest that the early physiological effects of gentamicin on the kidney occur in a different nephron segment from any subsequent nephrotoxicity.

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.

The hemolytic activity of Galleria mellonella hemolymph.

A hemolytic activity was identified in the hemolymph of normal and immune Galleria mellonella larvae. The hemolysin was active against sheep, human, guinea pig, and rabbit erythrocytes. Hemolysis occurred in the presence of 0.04M EDTA. Vaccination of the larvae with formalized Pseudomonas aeruginosa increased the hemolytic activity. The increase, and subsequent decline of this activity paralleled the pattern of induced in vivo antibacterial activity that is characteristic of the insect's immune response. The hemolytic activity was distinct from induced phospholipase A-like and phospholipase C-like activities that were detected in immune hemolymph and which were inhibited by EDTA. The hemolytically active material (HAM) was partially purified (apparent molecular weight range 69,000 to 75,000) and was found not to be antibacterial for P. aeruginosa. The physiological role of the HAM is as yet unknown. It is possible that it may act together with other hemolymph components to produce an immune state.

A simple isolation procedure for functionally pure components of the bovine alternative complement pathway (ACP) C3 convertase and bovine conglutinin (K).

A simple multicomponent isolation procedure for bovine C3, factor B, factor D and conglutinin (K) from a single serum sample is described. The components of the alternative pathway C3 convertase were isolated in milligram quantities from 800 ml bovine serum and were found to be functionally pure with respect to each other and to factors H and I.

Renal response to vasopressin and indomethacin in cisplatin-treated rats.

1. Cisplatin [6 mg/kg body weight, in 0.9% (w/v) NaCl] was injected intraperitoneally as a single dose to two groups of rats (Fischer 344 strain). Two further groups of rats, injected intraperitoneally with an equivalent volume of 0.9% (w/v) NaCl, were used as controls. The cisplatin-treated rats developed a pronounced polyuria which did not recover during an 18 week observation period. 2. After 21 weeks, one group of the cisplatin-treated animals received a 6 h infusion of 2.5% D-glucose. Vasopressin (60 mu-units min-1 100 g-1 body weight) was incorporated into the infusate for the final 2 h. A control group of animals received an identical infusion. One week later the other group of cisplatin-treated rats received a 6 h infusion of 0.9% (w/v) NaCl. Indomethacin was incorporated into the infusate for 15 min, at 3 h 52.5 min, to deliver a dose of 10 mg/kg body weight. A control group again received an identical infusion. 3. Cisplatin did not impair the antidiuretic effect of vasopressin, but it reduced the natriuretic effect of vasopressin, and also impaired the ability of the animals to produce concentrated urine. 4. Cisplatin did not alter basal PGE2 excretion, or the reduction in PGE2 excretion induced by indomethacin. However, the urine flow in the cisplatin-treated group did not fall after indomethacin, whereas there was a fall in urine flow in the control group.