BM-10 [BETA]

The exercise response to a single oral dose (25 mg) of a new beta-blocking agent that also has potent vasodilating properties, carvedilol (BM 14,190), was assessed in 15 patients with stable exertional angina, positive exercise test responses (greater than or equal to 1 mm of ST depression) and coronary artery disease. A single-blind, placebo-controlled, randomized, crossover design was used. Compared with placebo, 25 mg of carvedilol significantly reduced both heart rate (HR) and blood pressure (BP) at rest (p less than 0.01). After administration of carvedilol, 10 of 15 patients did not have angina at peak exercise (p less than 0.01) and 5 had ST shifts of less than 1 mm (p less than 0.05). Total exercise time and time to 1 mm of ST depression were prolonged and ST-segment depression at peak exercise was significantly reduced (p less than 0.01). Systolic BP was reduced both at peak exercise and at 1 mm of ST depression (p less than 0.05), whereas mean HR at peak exercise did not change significantly compared with placebo. Overall, mean HR-BP product at peak exercise was significantly reduced by carvedilol compared with placebo (p less than 0.05). However, 4 patients actually achieved a higher HR-BP product but did not have angina and had less ST depression (or no ST-segment shifts) at peak exercise. This indicates an increase in their coronary flow reserve. These results suggest that carvedilol is effective therapy for effort-induced angina, and this may be related to its combined beta-blocking and potent vasodilatory properties.

BM-10 [BETA]

In E. coli, beta lactam-induced lysis usually starts from the cell septum [10,11], suggesting that amidases, which are recruited to and activated at the site of cell division, might initiate PG cleavage associated with lysis. Supporting this idea, deletion of multiple amidases leads to a lower rate of lysis after exposure to beta-lactam antibiotics [10,12]. In contrast, there is contradictory evidence regarding the role of lytic transglycosylases in the lysis process. Mutants lacking multiple lytic transglycosylases are typically more susceptible to beta lactam antibiotics [13,14], suggesting that these enzymes promote, rather than impair, survival after inhibition of cell wall synthesis. However, overexpression of bifunctional PBPs containing an inactive transpeptidase active site, which mimics exposure to beta lactam antibiotics, results in E. coli lysis via a process that is largely dependent on LTGs [15]. None of the other predicted cell wall lytic enzymes in E. coli have been definitively linked to beta lactam-induced lysis. Efforts to define the full set of gene products that mediate bacterial lysis after inhibition of cell wall synthesis or the relative importance of their activities have been thwarted by the fact that the observed phenotype (lysis) is typically rapid, potentially masking differences between mutants, and that most lytic enzymes are highly redundant.

Likely because of the prevalence of cell-wall acting antibiotics in their natural habitats [16], bacteria employ multiple strategies to cope with the dangers associated with inhibition of cell wall synthesis. The most well-studied of these strategies is resistance e.g. by beta lactamases, which inactivate beta lactams. A more passive strategy is dormancy (e.g., formation of persister cells), which allows cells to survive exposure to any normally lethal antibiotic. Persistence is mediated by activation of multiple toxin-antitoxin modules [17,18], which stop growth of a small fraction of bacterial populations and thus confer tolerance to antibiotics that are only active on growing cells [19]. Bacteria that are not replicating due to reaching high cell densities also tend to be tolerant to cell wall-acting antibiotics [20] as do bacteria exposed to factors thought to stabilize the outer membrane [11]. It is unclear what other strategies might exist to survive exposure to cell wall synthesis inhibitors.

Here, we report that Vibrio cholerae, the causative agent of the diarrheal disease cholera, routinely tolerates antibiotic-induced inhibition of cell wall synthesis. Similar to most bacteria, V. cholerae loses the structural integrity of its cell wall following exposure to a wide variety of cell wall synthesis inhibitors. However, in contrast to many other bacteria, this treatment results in formation of viable (though non-dividing) spherical cells, rather than cell lysis. Surprisingly, genetic analyses revealed that V. cholerae sphere formation depends on the activity of M23 family endopeptidases that are required for cell elongation under conditions of normal growth; in contrast, its amidase and lytic transglycosylases are not required for formation of viable spheres. Furthermore, we found that other important pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii, also fail to respond to beta lactam exposure with lysis under certain growth conditions, suggesting that intrinsic, population-wide beta lactam tolerance may be more widespread than currently appreciated.

The lysis phenotype was also observed in a ShyA-depleted ΔshyA ΔshyB ΔshyC Ptac:shyA (S8 Fig) strain, and expression of ShyC but not ShyB could at least partially prevent lysis of ShyA-depleted Δendo (S8 Fig), demonstrating that either one of the paralogues ShyA and ShyC must be present for beta lactam tolerance and formation of viable spheres, while the other M23 endopeptidases and NlpC are dispensable. We also observed penicillin G-induced lysis of Δendo ΔamiB cells when ShyA was depleted (S8 Fig), as well as in Δendo cells with additional mutations in LTG genes (Δendo ΔmltBΔmltD and Δendo ΔmltB Δslt70, which were the only strains with multiple LTG disruptions that we were able to make in the Δendo background). These results indicate that neither amidase activity nor MltB, MltD or Slt70 activity are necessary to cause lysis of Δendo cells. Lastly, ShyA-depleted Δendo cells were not more susceptible to membrane-acting agents than ShyA-replete cells (S9 Fig), suggesting that the observed lysis phenotype is not simply the consequence of a general weakness of the cell envelope.

It will be interesting to explore whether PG-degrading enzymes important for cell elongation are required for sphere formation in other organisms, as in V. cholerae. Our observations suggest that the absence of lysis after treatment with beta lactam antibiotics may be more common than currently appreciated, but the determinants of such survival have not been identified. Importantly, some reports have suggested that spherical bacteria can be isolated from patients treated with beta lactam antibiotics during chronic infections (e.g. respiratory infections caused by Haemophilus influenzae, [35]). Thus, similar to persister cells [19], population-wide tolerance and sphere formation may represent another fairly widespread way by which bacteria can evade the lethal consequences of beta lactam exposure. New antibiotics that target processes critical for sphere formation (e.g. inhibitors of ShyA) or for sphere survival might exhibit potent synergy with beta lactams and thus provide a novel approach for improved antimicrobial therapeutics.

As a dietary supplement, HMB has been commercially available as a mono-hydrated calcium salt, with the empirical formula Ca (HMB)2-H2O (HMB-Ca). The magnitude and rate of appearance of HMB following ingestion is dependent on the dose, and whether or not it is consumed with additional nutrients. Specifically, Vukovich et al. [29] found that 1 g of HMB-Ca resulted in a peak HMB level in blood two hours following ingestion, while 3 g resulted in peak HMB levels 60 minutes after ingestion at 300% greater plasma concentrations (487 vs. 120 nmolml-1), and greater losses in urine (28% vs. 14%), for 3 and 1 g HMB-Ca ingestion, respectively. Peak HMB concentrations were also delayed by an hour and significantly lower (352 nmolml-1) when the HMB-Ca dosage was combined with 75 g of glucose. It is likely that the addition of glucose slowed gastric emptying, or improved HMB clearance. Recently a new delivery method of HMB, administered as a free acid, has been investigated [30]. The free acid form is called beta-hydroxy-beta-methylbutyric acid and can be designated as HMB-free acid (HMB-FA). The initial research studies have utilized HMB-FA associated with a gel, containing a buffering mechanism (K2CO3) that raises the pH to 4.5.

Recovery from diabetes requires restoration of beta cell mass. Igf1 expression in beta cells of transgenic mice regenerates the endocrine pancreas during type 1 diabetes. However, the IGF-I-mediated mechanism(s) restoring beta cell mass are not fully understood. Here, we examined the contribution of pre-existing beta cell proliferation and transdifferentiation of progenitor cells from bone marrow in IGF-I-induced islet regeneration.

Streptozotocin (STZ)-treated Igf1-expressing transgenic mice transplanted with green fluorescent protein (GFP)-expressing bone marrow cells were used. Bone marrow cell transdifferentiation and beta cell replication were measured by GFP/insulin and by the antigen identified by monoclonal antibody Ki67/insulin immunostaining of pancreatic sections respectively. Key cell cycle proteins were measured by western blot, quantitative RT-PCR and immunohistochemistry.

Despite elevated IGF-I production, recruitment and differentiation of bone marrow cells to beta cells was not increased either in healthy or STZ-treated transgenic mice. In contrast, after STZ treatment, IGF-I overproduction decreased beta cell apoptosis and increased beta cell replication by modulating key cell cycle proteins. Decreased nuclear levels of cyclin-dependent kinase inhibitor 1B (p27) and increased nuclear localisation of cyclin-dependent kinase (CDK)-4 were consistent with increased beta cell proliferation. However, islet expression of cyclin D1 increased only after STZ treatment. In contrast, higher levels of cyclin-dependent kinase inhibitor 1A (p21) were detected in islets from non-STZ-treated transgenic mice. 041b061a72