The effects of phosphorylation of cardiac troponin-I on its interactions with actin and cardiac troponin-C

It is known that the phosphorylation of two serine residues on the NH2-terminal extension specific to cardiac troponin-I (Tn-I) modulates the calcium-dependent activation of the myofilaments. The process by which this occurs remains an unsolved puzzle. We have applied a dissective approach to study the effect of this phosphorylation on the interactions between Tn-I and its partner proteins, actin and troponin-C (Tn-C). Using N-[14C]ethylmaleimide-labelled Tn-I in sedimentation assays with F-actin, we found that the dephosphorylated Tn-I binds to F-actin with pronounced positive cooperativity, both in the absence and the presence of tropomyosin. Phosphorylation of the protein slightly weakens the interaction in the absence of tropomyosin, but the cooperativity remained. In the presence of tropomyosin, phosphorylation of the Tn-I also appeared to slightly weaken the interaction as well but, more significantly, the cooperativity was eliminated. These data can only be explained simply by a cooperative interaction between the monomer units in the actin filament. The interactions between cardiac Tn-I and Tn-C were studied by labelling the Tn-C with the fluorescent probe dansyl aziridine. As expected, the binding of the dephosphorylated Tn-I to Tn-C was strengthened by over 20-fold upon the addition of calcium to the assay. Phosphorylation of the protein, however, had a dramatic effect on the interaction in that it appeared to desensitise the complex to the effect of calcium: the Ka values obtained for both interactions (+/- Ca2+) were almost identical. These results clearly indicate that the phosphorylation of Tn-I in the cardiac system has dramatic effects on the isolated inter-protein interactions. We also discuss the possible significance of such an effect on the interactions of the isolated proteins in their roles within the intact cardiac regulatory complex.     

Rapid infusion system for neurosurgical treatment of massive intraoperative hemorrhage

Using an illustrative case of severe closed head injury that resulted in a posterior fossa epidural hematoma (EDH) and supratentorial epidural/subdural hematomas (SDH), the massive blood losses associated with operative repair of the torn sigmoid sinus and the significant fluid losses associated with refractory diabetes insipidus were treated by the intraoperative use of the Rapid Infusion System (RIS, Haemonetics). The RIS can rapidly infuse warm blood, crystalloid, or colloid at rates up to 1.5 L/min, thereby limiting the commonly associated hypotension, hypothermia, and coagulopathies. During the suboccipital craniectomy for evacuation of the EDH and repair of the sigmoid sinus, the patient required 18 units of blood replacement secondary to a large tear in the sigmoid sinus. During a separate craniotomy for evacuation of the SDH, the patient also developed diabetes insipidus, which increased the operative fluid replacement to 39 L. Despite these massive blood and fluid losses, the RIS limited the hypotension to less than 2 min and prevented hypothermia and the frequently associated coagulopathies. When used in a neurosurgical setting associated with massive blood and/or fluid losses, the RIS accomplishes three important objectives: (1) rapid infusion of intravenous fluids for maintaining perfusion pressure, (2) rapid warming of fluids despite high intravenous infusion rates of cold crystalloids, thereby preventing intraoperative hypothermia, and (3) continuous monitoring of infusion rates and totals.     

Adolescents and children injured or killed in drive-by shootings in Los Angeles

BACKGROUND: Drive-by shootings by violent street gangs contribute to early morbidity and mortality among adolescents and children in Los Angeles. This study attempted to determine the frequency of this problem and the population at greatest risk. We also studied the most frequently injured areas of the body, the seasons in which the most shootings occurred, the most common sites for drive-by shootings, and the types of firearms used. METHODS: We retrospectively reviewed the files of the Gang Information Section of the Los Angeles Police Department to identify all drive-by shootings in Los Angeles in 1991 in which a child or adolescent under the age of 18 was shot at, injured, or killed. RESULTS: A total of 677 adolescents and children were shot at, among whom 429 (63 percent) had gunshot wounds and 36 (5.3 percent) died from their injuries. Three hundred three of those with gunshot wounds (71 percent) were gang members. Arms and legs were the areas of the body most commonly injured. Handguns were the most frequently used type of firearm. All the homicide victims were African American or Hispanic, and 97 percent were boys. African American and Hispanic children and adolescents, especially male gang members, had a significantly higher risk than their Asian and white counterparts of injury and death from drive-by shootings in Los Angeles (P < 0.001). CONCLUSIONS: Drive-by shootings involving adolescents and children are frequent in Los Angeles. Although Los Angeles may be an atypical case, understanding why violent street gangs form, preventing causes of violence, and limiting access to firearms are essential steps in preventing this serious problem.     

The number of clonogenic cells in a mouse jejunal crypt

A study of eight deaths involving the benzodiazepine flunitrazepam was performed to assess the contribution of this drug to the fatalities. Coronial deaths in Victoria in the 2-year period to mid-1991 were selected in which either flunitrazepam or flunitrazepam and ethanol were the principal toxicological findings. All bodies were subject to a full autopsy by forensic pathologists, and a full toxicological examination. No significant pathology was found at autopsy in any case. Very high concentrations of 7-aminoflunitrazepam, a metabolite of flunitrazepam, were present in all cases. In four cases no other significant drug was detected, whereas in the other four cases there were significant concentrations of ethanol (mean 1.6 g/L). In these two groups of cases the concentrations of 7-aminoflunitrazepam were 0.45 mg/L and 0.16 mg/L, respectively. Only moderate levels of flunitrazepam were detected, suggesting that 7-aminoflunitrazepam is produced postmortem and may be an important marker of flunitrazepam usage. The causes of death in these eight cases were probably either flunitrazepam toxicity or a combination of flunitrazepam and ethanol toxicity. Only one case appeared likely to have been a suicide. Our observations in these cases suggest that flunitrazepam may cause death in the absence of other drugs or significant disease. The presence of ethanol reduces the amount of flunitrazepam needed to cause death.     

Kinetics of modification of the mitochondrial succinate-ubiquinone reductase by 5,5''-dithiobis-(2-nitro-benzoic acid)

In the present study, we examined the effect of the thromboxane/prostaglandin endoperoxide analogue U46619 on proliferation and hypertrophy in cultured rat vascular smooth muscle cells and the roles of protein kinase C and transforming growth factor-beta (TGF-beta) in the mediation of the hypertrophic response to U46619. Since an increase in basic fibroblast growth factor (bFGF) was previously shown to mediate the hypertrophic response to U46619, we also assessed the relationship between bFGF and TGF-beta in the expression of U46619 actions. U46619 increased [35S]methionine incorporation into protein and protein content of vascular smooth muscle cells but had no effect on cell number. A role for TGF-beta was supported by the following observations: (1) exogenous human TGF-beta 1 increased protein synthesis; (2) antibody to TGF-beta blocked both TGF-beta- and U46619-induced increases in protein content; (3) U46619 increased active and total TGF-beta bioactivities; and (4) the actions of U46619 on protein content and TGF-beta bioactivity were blocked by the thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548. Previous observations had demonstrated a role for bFGF in the expression of U46619 actions on protein synthesis. Results of the present study suggest that TGF-beta and bFGF interact in mediating the protein synthetic response to U46619. First, the concentration of exogenous TGF-beta (10 pmol/L) alone required to produce a protein synthetic response equivalent to that induced by U46619 was much higher than the concentration of endogenous active TGF-beta that accumulated in the media in response to U46619 (0.7 pmol/L). Second, bFGF (20 ng/mL) increased total TGF-beta bioactivity and stimulated protein synthesis. The hyper-trophic response to bFGF was blocked by anti-TGF-beta. The ability of U46619 and bFGF to increase protein synthesis and protein content in vascular smooth muscle cells was associated with TGF-beta-induced suppression of proliferation, as evidenced by the ability of antibody to TGF-beta to enhance U46619- and bFGF-induced increases in [3H]thymidine incorporation into DNA. Results of the present study also supported a role for protein kinase C in the expression of U46619 and bFGF actions. U46619 increased protein kinase C activity in the particulate fraction of vascular smooth muscle cells. Moreover, the protein kinase C inhibitors GF109203X and staurosporine blocked U46619- and bFGF-induced increases in protein synthesis as well as active and total TGF-beta bioactivities. By contrast, the protein kinase C inhibitors did not prevent the increases in protein synthesis induced by exogenous TGF-beta. The results demonstrate that thromboxane/prostaglandin endoperoxide signals increased TGF-beta bioactivity via protein kinase C. Increases in both bFGF and TGF-beta are required for an optimal hypertrophic response to U46619. The hypertrophic response to TGF-beta occurs through a protein kinase C-independent pathway.