Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis

The effect of Mycobacterium tuberculosis infection on the viability of healthy (control) human alveolar macrophages was evaluated by staining with ethidium homodimer and calcein to discriminate live from dead cells. Infection with M. tuberculosis H37Ra or H37Rv increased macrophage mortality at 6 days from the control level of 3.8% +/- 0.7% to 28.7% +/- 6.9% or 12.6% +/- 3.1%, respectively (P < 0.001 for comparisons of all conditions). A role for tumor necrosis factor alpha (TNF-alpha) in the M. tuberculosis-induced cytolysis of alveolar macrophages was demonstrated by increased cytotoxicity following the addition of exogenous TNF-alpha to the cultures and by enhancement of macrophage survival when M. tuberculosis-infected alveolar macrophages were treated with pentoxifylline or anti-TNF-alpha antibody. The cytolytic mechanism was determined to be apoptosis by the demonstration of a characteristic internucleosomal ladder of genomic DNA by agarose gel electrophoresis, by finding nuclear fragmentation and condensation by electron microscopy, and by in situ terminal transferase-mediated nick end labeling of fragmented DNA in alveolar macrophages infected with M. tuberculosis in vitro. The latter technique was employed to reveal extensive apoptosis within caseating granulomas from lung tissue samples from clinical tuberculosis cases. The induction of apoptosis in alveolar macrophages by M. tuberculosis may play a role in the macrophage-pathogen interaction of tuberculosis in vivo.     

Identification of potential ferric binding residues in the iron-binding protein of pathogenic Neisseria meningitidis through structure-based multiple sequence alignments

Toxic effects of hyperglycemia-induced advanced glycosylated end products (AGEs) may explain some vasculopathic complications of diabetes. Aminoguanidine, a known inhibitor of AGE formation, was administered by gavage to Sprague-Dawley streptozotocin-induced diabetic rats made azotemic by surgical reduction of renal mass. All rats became hyperglycemic. Renal ablation caused renal insufficiency, as evidenced by markedly reduced endogenous creatinine clearances at days 7 and 14. Aminoguanidine-treated rats had significantly (P < 0.04) superior survival to that of untreated azotemic diabetic rats. We infer from the extended life in a rat model of uremia in diabetic nephropathy that aminoguanidine may prove beneficial in human diabetes.     

Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans

Despite the association of increasing age with chronic wound-healing disorders and an impaired rate of healing of acute cutaneous wounds, the role of matrix metalloproteinases (MMPs) is unknown. To determine the spatial and temporal patterns and activities of MMP-1, -2, -3 and -9, 132 healthy humans aged between 19 and 96 years underwent 4-mm punch biopsies followed by wound excision between day 1 and day 180 post-wounding. Wounds showed an age-related increase in MMP-2 and MMP-9 immunostaining from day 3; this was associated with degradation of gelatin as shown by zymograms and with increased proteinase activity as shown by azocoll assays. Distinct spatial localisations for each MMP were observed: MMP-2 was found in epidermal structures; MMP-9 was observed in inflammatory cells up to day 21; MMP-1 was localised to keratinocytes at the wound margin. Normal old skin showed pro-MMP-2 bands on zymography and increased MMP-2 immunostaining. These results indicate that: (1) intrinsic ageing is associated with the up-regulation of MMPs previously associated with chronic wound healing; (2) wound-tissue proteinases are essentially active up to day 21 postwounding; and (3) intrinsic ageing may predispose to tissue breakdown disorders because of MMP-2 up-regulation in normal skin.     

Separation of gating properties from permeation and block in mslo large conductance Ca-activated K+ channels

In this and the following paper we have examined the kinetic and steady-state properties of macroscopic mslo Ca-activated K+ currents in order to interpret these currents in terms of the gating behavior of the mslo channel. To do so, however, it was necessary to first find conditions by which we could separate the effects that changes in Ca2+ concentration or membrane voltage have on channel permeation from the effects these stimuli have on channel gating. In this study we investigate three phenomena which are unrelated to gating but are manifest in macroscopic current records: a saturation of single channel current at high voltage, a rapid voltage-dependent Ca2+ block, and a slow voltage-dependent Ba2+ block. Where possible methods are described by which these phenomena can be separated from the effects that changes in Ca2+ concentration and membrane voltage have on channel gating. Where this is not possible, some assessment of the impact these effects have on gating parameters determined from macroscopic current measurements is provided. We have also found that without considering the effects of Ca2+ and voltage on channel permeation and block, macroscopic current measurements suggest that mslo channels do not reach the same maximum open probability at all Ca2+ concentrations. Taking into account permeation and blocking effects, however, we find that this is not the case. The maximum open probability of the mslo channel is the same or very similar over a Ca2+ concentration range spanning three orders of magnitude indicating that over this range the internal Ca2+ concentration does not limit the ability of the channel to be activated by voltage.     

Validation of multifrequency bioelectrical impedance analysis in monitoring fluid balance in healthy elderly subjects

BACKGROUND: Multifrequency Bioelectrical Impedance Analysis (MFBIA) is a novel method to assess body composition in elderly subjects. However, it is unclear whether MFBIA can detect changes in body water compartments in elders. We aimed to determine the within-subject variability of MFBIA and the responsiveness to a diuretic intervention in aged subjects with a stable fluid balance. METHODS: We selected 12 healthy active elderly subjects (5 male, 7 female) with a mean age of 75 years. Total body water and extracellular fluid (ECF) were measured by deuterium oxide- and potassium bromide-dilution techniques. Within-subject variability in total body MFBIA was assessed by performing four measurements at 1, 5, 50, and 100 kHz within a 2-month period. Subsequently, responsiveness of MFBIA to the ECF loss caused by oral administration of 40 mg of furosemide was determined. RESULTS: Within-subject variability in MFBIA at 1, 5, 50, and 100 kHz expressed as standard deviations was 21, 19, 14, and 14 Ohm (omega), respectively. Furosemide caused a mean weight loss of 1.8 +/- 0.6 kg, which resulted in significant increases in impedance of 57 +/- 24 omega at 1 kHz and 37 +/- 12 omega at 100 kHz (p < .001). The responsiveness of MFBIA for the diuretic intervention was best at 5 kHz (responsiveness index = 1.98). CONCLUSIONS: Within-subject variability of MFBIA was small in healthy elderly subjects with stable fluid balance. Responsiveness of MFBIA to 9% furosemide-induced ECF loss was excellent. These data support the necessity for further clinical assessment of the value of MFBIA in monitoring fluid balance in geriatric patients.     

Immunoaffinity method to identify aggregin, a putative ADP-receptor in human blood platelets

The ADP-receptor on the surface of human platelets and cells of megakaryocytic lineage has been classified as P2T purinergic receptor for which ADP is an agonist and ATP is an antagonist. Although it is one of the earliest identified of the important cellular receptors, it has neither been purified nor cloned. We have developed an immunoaffinity method for rapidly identifying the platelet ADP-receptor and this method can be extended to the purification of the receptor. A polyclonal antibody to glutamate dehydrogenase (GDH) covalently modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) recognized neither FSBA nor glutamate dehydrogenase. Immunoblot of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized FSBA-labeled platelets showed the presence of a protein band at 100 kDa and this band was absent in the immunoblots of platelets that were preincubated with ADP and ATP or covalently modified by the chemically reactive ADP-affinity analogs, 2- and 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (2- and 8BDB-TADP) and 2-(3-bromo-2-oxopropylthio)adenosine-5'-diphosphate (2-BOP-TADP), prior to treatment with FSBA. FSBA as well as 2- and 8-BDB-TADP and 2-BOP-TADP have been previously shown to inhibit ADP-induced platelet responses by selectively and covalently modifying aggregin (100 kDa), an ADP-receptor in intact human blood platelets. The results show that polyclonal antibody to FSBA-labeled GDH is capable of recognizing FSBA-labeled aggregin on platelets and, thus, could be used to purify aggregin by immunoaffinity column chromatography. The immunoaffinity method was found to be far more sensitive than the radiochemical methods to identify aggregin previously developed in our laboratory. Since FSBA is also capable of reacting with enzymes that require ATP for their catalytic function, the polyclonal antibody may be used to identify and purify other P2-type purinergic receptors that require binding of ATP before eliciting cellular responses.