Microtubule-dependent PKC-alpha localization in A7r5 smooth muscle cells.

Using laser scanning confocal, fluorescence resonance energy transfer (FRET), and atomic force (AFM) microscopy, we investigated association of protein kinase C (PKC)-alpha with microtubules during stimulus-induced relocalization in A7r5 smooth muscle cells. Confocal microscopy with standard immunostaining techniques confirmed earlier observations that colchicine disruption of microtubules blocked PKC-alpha localization in the perinuclear region of the cell caused by phorbol 12,13-dibutyrate (PDBu; 10-6M). Dual immunostaining suggested colocalization of PKC-alpha and beta-tubulin in both unstimulated and PDBu-treated cells. This finding was verified by FRET microscopy, which indicated that association of PKC-alpha was heterogeneous in distribution and confined primarily to microtubules in the perinuclear region. FRET analysis further showed that association between the molecules was not lost during colchicine-induced dissolution of microtubules, suggesting formation of tubulin-PKC-alpha complexes in the cytosol. Confocal imaging indicated that perinuclear microtubular structure was more highly sensitive to colchicine dissolution than other regions of the cell. Topographic imaging of fixed cells by AFM indicated a well-defined elevated structure surrounding the nucleus that was absent in colchicine-treated cells. It was calculated that the volume of the nuclear sleevelike structure of microtubules increased approximately fivefold in PDBu-treated cells, suggesting a probable increase in microtubular mass. In light of PKC-alpha localization, increased colchicine sensitivity, and their volume change in stimulated cells, the results suggest that perinuclear microtubules form a specialized structure that may be more dynamically robust than in other regions of the cell. PKC-alpha could contribute to this dynamic activity. Alternatively, perinuclear microtubules could act as a scaffold for regulatory molecule interaction at the cell center.

Secondary effects of the pesticide Bacillus thuringiensis kurstaki on chicks of spruce grouse (Dendragapus canadensis).

The objective of this study was to determine if application of the biological pesticide Bacillus thuringiensis kurstaki (B.t.k.) had secondary effects on spruce grouse (Dendragapus canadensis) chicks in the southern boreal forest of Ontario, Canada. B.t.k. kills larvae of Lepidoptera that might otherwise be food. This may affect types of food ingested and influence chick growth rates by reducing the availability of a high-protein food. Amount of ingested grit, which is diet-dependent, may also be influenced, which could alter exposure to anthropogenic toxic metals, such as cadmium (Cd) and lead (Pb), associated with the grit. Chick growth rate from 0 to 14 days of age was determined, and gizzard contents were analyzed for types of foods and levels of macronutrients and trace metals in chicks raised in a plot treated with B.t.k. compared to chicks raised on a control plot. Chick growth rates were significantly different (p < 0.05); chicks from the treatment plot increased body mass 30% less as compared to chicks raised on the control plot. Control chicks contained on average 2.5-fold the mass (in g) of grit and nearly twofold the mass of food, which included larvae, in their gizzards compared to those exposed to B.t.k. whose diets were dominated by spiders and ants. Concentrations of magnesium (Mg), calcium (Ca), phosphorous (P), iron (Fe), manganese (Mn), sulfur (S) and the trace metals zinc (Zn), copper (Cu), nickel (Ni), and chromium (Cr) did not differ between experimental and control birds (p > 0.05). In contrast, control chicks had higher levels of aluminum (Al), Cd, cobalt (Co), and Pb in their gizzards than chicks from the sprayed area (p < 0.05), attributed to greater amounts of consumed grit. Although birds from the sprayed area had lower levels of the toxic metals Pb and Cd in their gizzards, a protein-deficient diet could have been a factor that contributed to decreased rates of growth in B.t.k.-exposed birds as compared to those birds held on nonsprayed sites. Reduced growth during the first 2 weeks of avian development has important consequences at the population level, and it is recommended that this secondary effect be considered for all species who rely on Lepidoptera larvae as a food resource prior to wide spread application of B.t.k. for insect control.

Hereditary hemochromatosis.

Genetic (hereditary) hemochromatosis is probably the most common autosomal recessive disorder found in white Americans, of whom about 5/1,000 (0.5 percent) are homozygous for the associated gene. The hemochromatosis gene is probably located close to the HLA-A locus on the short arm of chromosome 6. Homozygous individuals may develop severe and potentially lethal hemochromatosis, especially after age 39. Hereditary hemochromatosis involves an increased rate of iron absorption from the gut with subsequent progressive storage of iron in soft organs of the body. Excess iron storage eventually produces pituitary, pancreatic, cardiac, and liver dysfunction and death may result from cardiac arrhythmias, congestive heart failure, and/or hepatic failure or cancer. Early diagnosis can prevent these excess iron-induced problems. Iron overload owing to HLA-linked hereditary hemochromatosis can be distinguished from other causes of hemochromatosis by liver biopsies and interpretations. Patients at risk for genetic hemochromatosis should be screened, identified, and treated as early as age 20 to prevent or minimize the deadly complications of hemochromatosis. Population screening should include measurements of serum iron concentration, total iron binding capacity (TIBC), percent saturation of transferrin, and serum ferritin concentrations. Family members of hereditary hemochromatosis patients are at increased risk and should be tested. Screening, identification and early treatment (phlebotomies, sometimes in combination with the use of Desferal or other iron-chelating agents) may help prevent or reduce iron-related organ damage and premature deaths. Early diagnosis and treatment will reduce the population of aging individuals with severe, complicated hemochromatosis and dramatically reduce medical costs (billions of U.S. dollars per annum) associated with the management of this disease.

Sperm-surface chymotrypsin-like protease activity required for fertilization in ascidians.

During fertilization, species-specific gamete binding must be followed by sperm penetration of egg vestments before gamete fusion can occur. Sperm proteases, called lysins, aid this process. Sperm from Ascidia ceratodes, Ascidia callosa, and Ascidia paratropa were found to have a surface-mounted chymotrypsin-like protease when studied by enzymology, biotinylated, immunolabeling, and histochemistry. Chymotrypsin substrates and inhibitors blocked fertilization in a concentration-dependent manner in A. ceratodes and decreased the number of sperm heads which penetrated the egg's vitelline coat, but had no effect on sperm binding to follicle cells. Sperm bound to agarose beads coated with the chymotrypsin inhibitor alpha 2-macroglobulin. Chymotrypsin-like enzyme activity, assayed fluorimetrically using N-succinyl-leucinyl-leucinyl-valinyl-tyrosinyl-7 -amido-4-methyl-coumarin as the substrate, was associated with head fractions prepared by differential centrifugation. Biotinylation of live sperm followed by detergent extraction showed that chymotrypsin-like activity could be removed from the detergent extract using avidin-agarose beads. Indirect immunofluorescence of unreacted and reacted sperm heavily labeled membrane domains overlying the mitochondrion and at the base of the head with occasional labeling of the sperm tip. Histochemical studies, which used N-succinyl-alanyl-alanyl-prolyl-phenylalanyl-beta-naphthylamide as the substrate, colocalized enzyme activity in head regions of unreacted and near the mitochondrion of reacted sperm. Thus, we conclude that in ascidian sperm a chymotrypsin-like protease is exposed on the external surface of the plasma membrane of the head, is required for fertilization, and plays a role in sperm penetration but not binding.

Evaluating the patient with a difficult airway for anesthesia.

Patients with difficult airways present a challenge when they must undergo anesthesia. This article examines the problems inherent in evaluating patients with difficult airways for surgery. The authors believe that these patients are best evaluated in a Difficult Airway Clinic. The structure and organization of such clinics are examined.

Medical computers from the manufacturer's viewpoint.

The recent surge in the scope of computer applications in medical care has brought with it great strides in patient care, but it has also created a variety of concerns to manufacturers of medical software. The spate of product liability suits, with its potentially crippling effects in terms of product development, makes it increasingly vital that manufacturers engage in prospective planning, to anticipate problems and take steps before legal action ensues, so that all parties--the manufacturer, the hospital, and ultimately, the patient--benefit from the availability of new technology. The Food and Drug Administration (FDA) and Congress are similarly grappling with the implications of this new technology, to assure that patient care is not compromised by products that have not yet been fully tested. The tension between the desire to make technology available to the public and the realities of the present regulatory and litigious climate is the key dilemma facing manufacturers of medical software.

Atlantoaxial instability revisited. An alert for endoscopists.

Atlantoaxial instability in rheumatoid arthritis patients presents a particular hazard for endoscopists. The basic pathology, while well known to orthopedists, is not common knowledge among bronchoesophagologists or anesthesiologists. The paper describes the syndrome, potential complications and possible approaches to avoiding serious neurologic sequellae.

Endotracheal intubation and Venturi (jet) ventilation for laser microsurgery of the larynx.

Meeting the exacting requirements for microsurgery of the larynx is a challenge for the anesthesiologist. To accomplish, the necessary dissection, the otolaryngologist has several requirements. They are a quiet relaxed field, excellent illumination with magnification, binocular vision for depth perception, and, above all, an unobstructed field. The management of anesthesia for suspension microsurgery on the larynx presents many problems, the most vexing of which is the fact that the otolaryngologist and anesthesiologist are in competition for access to the patient's airway. In sharing this, neither has been able to perform with the degree of control that he would like due to either inadequate operating conditions or insufficient access to ventilatory mechanisms. Several anesthetic techniques have been used for inspection or operative laryngoscopy: topical anesthesia, apneic techniques, translaryngeal topical anesthesia, chest respirator, neuroleptanalgesia, and general endotracheal anesthesia with muscle relaxants. The latter has proven most popular, particularly in children, because ventilation and surgical conditions are considered to be most controllable. However, the presence of the requisite endotracheal tube obscures the full view of the larynx and vocal cords, and the tube may itself become obstructed. Additionally, use of the laser involves the further risk of heat effects on the endotracheal tube if the beam hits the tube. This report presents our experience and development of the combined technique of endotracheal intubation and Venturi (jet) ventilation. We believe it represents the safest available approach while providing near ideal working conditions for the otolaryngologist during laser microsurgery of the larynx.