Mechanism of adenosine 3′,5′-monophosphate (cAMP)-induced increase in Ca2+ uptake by the sarcoplasmic reticulum in functionally skinned myocardial fibers

The increased rate of Ca²⁺ uptake and ATPase activity in isolated cardiac sarcoplasmic reticulum (SR) by adenosine 3,5-monophosphate (cAMP) has been shown to be activated by a cAMP-dependent protein kinase (cAMP kinase). Functionally skinned myocardial fiber preparations were used to study the mechanisms of cAMP action on the SR at the same time that tension was monitored. cAMP effects were studied on Ca²⁺-activated tension of the contractile proteins, and on Ca²⁺ uptake and release from the SR using caffeine-induced tension transients. Neither cyclic AMP (0.1–5 M) nor the catalytic subunit of cAMP kinase (0.1–1 M) (PK-C) significantly changed either the maximal or the submaximal Ca²⁺-activated tension. The areas of the tension transients were unchanged when cAMP was present in the releasing solution (release phase), and were significantly increased up to a mean of about 80% when cAMP or PK-C was present in the Ca²⁺ loading solutions (uptake phase). The increased tension transient was blocked by the heat-stable inhibitor of cAMP kinase. We conclude that cAMP-induced increases in Ca²⁺ uptake by the SR could play an important role in the positive inotropic effect. cAMP kinase could thus play a crucial role in the regulation of myocardial contractility.     

AMPK as a metabolic switch in rat muscle,liver and adipose tissue after exercise

An increasing body of evidence has revealed that activation of adenosine monophosphate (AMP)‐activated protein kinase (AMPK)‐activated protein kinase increases fatty acid oxidation by lowering the concentration of malonyl coenzyme A (CoA), an inhibitor of carnitine palmitoyl transferase 1. Studies carried out primarily in skeletal muscle suggest that AMPK modulates the concentration of malonyl CoA by concurrently phosphorylating and inhibiting acetyl CoA carboxylase (ACC), the rate limiting enzyme in malonyl CoA synthesis, and phosphorylating and activating malonyl CoA decarboxylase (MCD), an enzyme involved in its degradation. We have recently observed that AMPK and MCD activities are increased and ACC activity diminished in skeletal muscle, liver and, surprisingly, in adipose tissue 30 min following exercise (treadmill run) in normal rats. In liver and adipose tissue these changes were associated with a decrease in the activity of glycerol‐3‐phosphate acyltransferase (GPAT), which catalyses the first committed reaction in glycerolipid synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. Similar changes in ACC, MCD and GPAT were observed following the administration of 5‐aminoimidazole 4‐carboxamide‐riboside (AICAR), further indicating that the exercise‐induced alterations in these enzymes were AMPK‐mediated. Conclusions: (1) AMPK plays a major role in regulating lipid metabolism in multiple tissues following exercise. (2) The net effect of its activation is to increase fatty acid oxidation and diminish glycerolipid synthesis. (3) The relevance of these findings to the regulation of muscle glycogen repletion in the post‐exercise state and to the demonstrated ability of AMPK activation to decrease adiposity and increase insulin sensitivity in rodents remains to be determined.     

Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D'', I'', and L2''.

The nucleotide sequences of variable segments (VS) 1, 2, and 4 for the major outer membrane protein gene (omp1) of Chlamydia trachomatis were determined for serologically defined subtypes D', I', and L2'. Asymmetric DNA amplification was used to produce single-stranded DNA for direct sequencing. Amino acid substitutions were detected in VS1, VS2, and VS4 for I', in VS2 for L2', and in VS4 for D'. DNA sequencing of omp1 variant regions may be an important method for evaluating the molecular epidemiology of Chlamydia spp.     

Characterization of monoclonal antibody CIBCNSH3 generated to the human EGF receptor

Monoclonal antibody CIBCNSH3 of IgG1 isotype has been generated against human epidermal growth factor receptor (EGFR) using MDA MB 468 breast carcinoma cell line as immunogen. Earlier studies have revealed that this MAb blocked growth factor-receptor interaction and thus inhibited cell proliferation and tumor growth. In the present paper, this MAb has been extensively characterized to evaluate its application in the study of human cancers. The results were compared with those obtained using a control MAb ICR 62 specific to EGFR. Competitive assay showed that this MAb bound to an epitope in the extracellular domain of the EGFR to which MAb ICR 62 also bound. This MAb immunoprecipitated the 170 kD glycoprotein. The specificity was further confirmed by the formation of a single discrete band in western blot analysis. By flow cytometric analysis this monoclonal antibody revealed high binding affinity with MDA MB 468 cells. By immunocytochemical assay, out of 35 breast tumors studied, 40% were found to exhibit strong cell membrane staining and in the case of 25 oral cancers studied, 56% were strong positive. High expression of EGFR was observed in MDA MB 468 cells and HN 5 cells. These studies clearly indicate that MAb CIBCNSH3 might prove useful to identify tumors with high level of expression of EGFR associated with poor prognosis.     

Forms of lung lymphatics: A scanning electron microscopic study of casts

In a recent study, rats given monocrotaline underwent angiogenesis on their pleural surfaces. The rats also had novel structures in their bronchovascular bundles that were detected by scanning electron microscopy of vascular casts. These vessels could have been either new blood capillaries or dilated lymphatic capillaries. To determine if these structures were lymphatics or new blood vessels, specimens from animals that were undergoing angiogenesis were compared to those that were not. Finding similar structures in normal animals would imply that they were lymphatic. The second purpose of this work was to describe the three-dimensional anatomy of the lymphatics of the lung. Cast lymphatics were found in most lungs with edema or angiogenesis, but were rare in other conditions. The vascular structures in question were found in animals not undergoing angiogenesis and were, therefore, lymphatic. Additionally, scanning electron angiogenesis and were, therefore, lymphatic. Additionally, scanning electron microscopy of casts showed several distinct forms of lymphatics in the lung. Prelymphatics are tissues planes beneath the pleura and around bronchovascular structures. They join reservoir, conduit or tubulo-saccular lymphatics. Reservoir lymphatics are broad ribbon-like structures with textured surfaces and small laterally branching pouches. They occur on the pleural surface, are closely linked with prelymphatics, and join conduit lymphatics. Conduit lymphatics are tubular structures that may contain valves, twist and go great distances without accepting tributaries. On the pleural surface, they may wind around blood vessels and vary greatly in diamater. Sacculo-tubular lymphatics surround arteries, veins and bronchioles. They have thin walls with wide saccular segments. They may be so dense that they form cylinders around the vessels or airways. Different forms of lung lymphatics suggest different function and potential. © 1992 Wiley-Liss, Inc.     

16S Ribosomal DNA Typing for Identification of Pathogens in Patients with Bacterial Keratitis

The identification of pathogens in patients with bacterial keratitis remains problematic because standard diagnostic tests are negative for 40 to 60% of patients. A cross-sectional study was undertaken to determine if PCR and sequence analysis of 16S ribosomal DNA (rDNA) could be used to detect bacterial pathogens in patients with keratitis. Corneal specimens were collected for culture and rDNA typing. Variable segments of each rDNA specimen were amplified by PCR, sequenced, and aligned with the sequences in GenBank. Eleven patients had microbiologically documented bacterial keratitis, while 17 patients had keratitis due to other causes. Nine (82%) of 11 bacterial keratitis patients were PCR positive; each sequencing result matched the culture results. Seventeen (100%) patients with nonbacterial keratitis were PCR negative. Our data suggest that 16S rDNA typing holds promise as a rapid alternative to culture for identifying pathogens in patients with bacterial keratitis.     

Inhibition of interleukin-17 prevents the development of arthritis in vaccinated mice challenged with Borrelia burgdorferi

We showed that Borrelia burgdorferi-vaccinated interferon gamma-deficient (IFN-gamma(0)) mice challenged with the Lyme spirochete developed a prominent chronic severe destructive osteoarthropathy. The immune response underlying the development of the severe destructive arthritis involves interleukin-17 (IL-17). Treatment of vaccinated IFN-gamma(0) mice challenged with B. burgdorferi with anti-IL-17 antibody delayed the onset of swelling of the hind paws but, more importantly, inhibited the development of arthritis. Histopathologic examination confirmed that treatment with anti-IL-17 antibody prevented the destructive arthropathy seen in vaccinated and challenged IFN-gamma(0) mice. Similar preventive results were obtained when vaccinated and challenged IFN-gamma(0) mice were treated with anti-IL-17 receptor antibody or sequentially with anti-IL-17 antibody followed by anti-IL-17 receptor antibody. By contrast, treatment of vaccinated and challenged IFN-gamma(0) mice with recombinant IL-17 (rIL-17) did not alter the development and progression of arthritis found in vaccinated and challenged IFN-gamma(0) mice without treatment with rIL-17. Therapeutic intervention may be a realistic approach to prevent arthritis, especially if IL-17 is involved in the perpetuation of chronic or intermittent arthritis.     

Mast cell heterogeneity: Evidence and implications

Mast cells and basophils play a fundamental role in the pathogenesis of allergic disease, although their physiologic role is largely unknown. A large body of evidence now indicates that the properties of mast cells are dependent on the tissue and species from which they are derived. Such mast cell heterogeneity encompasses differences in morphology, development, cytochemistry, and function. The evidence for such heterogeneity, and some of its clinical implications, is discussed.     

Memory Deficits in Aged Cebus Monkeys and Facilitation With Central Cholinomimetics

Cebus monkeys of 3 different age groups were trained to perform an automated behavioral task (delayed response), intended to measure recent memory ability. In in initial study, the aged monkeys (18 years and older) exhibit prprogressively greater performance impairments (relative to young monkeys) as they were required to remember the location of a visual stimulus for increasingly longer durations (0 to 20 sec). This deficits replicated previously published results from aged Rhesus monkeys and appeared similar to the primary memory deficits reported in elderly humans and demented patients. In subsequent studies, the effects of three different cholinomimetics were evaluated for their ability to improve the aged monkey's performance on this task. Each monkey was tested under several acute doses of the cholinergic precursor, choline, the anticholinesterase, physostigmine, and the cholinergic muscarinic receptor agonist, arecoline. The results revealed clear differences in the ability of these drugs to improve performance on this task. Choline exerted no apparent effects in the aged monkeys at any dose tested. Physostigmine clearly enhanced performance in certain aged monkeys, but the optimal dose varied dramatically between subjects, replicating previously published results with aged Rhesus monkeys and humans. Arecoline produced clear improvement within a restricted dose range, with little variation in optimal dose between subjects. In addition to demonstrating differences in the effects of different cholinomimetics on memory performance in aged primates, these data also suggest a possible rationale for future investigations. Assuming that each of these drugs primarily affected cholinergic function in the manner conventionally attributed, these data suggest that, within the cholinergic system, the more directly one stimulates the receptor, the more one might expect robust and consistent effects on memory performance in aged subjects.