Stoichiometry of molecular complexes at adhesions in living cells

We describe a method to detect molecular complexes and measure their stoichiometry in living cells from simultaneous fluctuations of the fluorescence intensity in two image channels, each detecting a different kind of protein. The number and brightness (N&B) analysis, namely, the use of the ratio between the variance and the average intensity to obtain the brightness of molecules, is extended to the cross-variance of the intensity fluctuations in two channels. We apply the cross-variance method to determine the stoichiometry of complexes containing paxillin and vinculin or focal adhesion kinase (FAK) in disassembling adhesions in mouse embryo fibroblasts expressing FAK, vinculin, and paxillin-tagged with EGFP and mCherry. We found no complexes of these proteins in the cytoplasm away from the adhesions. However, at the adhesions, large aggregates leave, forming a hole, during their disassembly. This hole shows cross-correlation between FAK and paxillin and vinculin and paxillin. From the amplitude of the correlated fluctuations we determine the composition of the aggregates leaving the adhesions. These aggregates disassemble rapidly in the cytoplasm because large complexes are found only in very close proximity to the adhesions or at their borders.     

The effect of maternal dexamethasone treatment after the 12th week of pregnancy on fetal genital development in adrenogenital syndrome with 21-hydroxylase deficiency

Prenatal treatment with dexamethasone starting with gestation week 5 has been proposed to prevent virilization of female fetuses with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. We report dexamethasone treatment in a mother during her third pregnancy; this treatment could not be started before the 12th week of gestation. The second child (index case) had a simple virilizing 21-hydroxylase deficiency CAH and Prader IV genitalia. Because after amniocentesis a normal female karyotype and HLA identity with the index case were found, the dexamethasone treatment (3 x 0.5 mg/die) was continued until delivery.-In contrast to patients with salt-wasting CAH, the 17-alpha-hydroxyprogesterone level in the amniotic fluid was within the normal range. Decreased maternal plasma and urine estriol concentrations, as well as the plasma cortisol values, demonstrated adequate suppression of the fetal and maternal adrenal gland. No side effects were found in the mother as a result of the dexamethasone treatment. The newborn had virilization of the external genitalia according to Prader III but without hypertrophy of the clitoris. The degree of rugated scrotum was less marked in relation to the index case and the sinus urogenitalis was more distally shifted. Thus, surgery on the clitoris could be avoided. The conditions for further surgery (vaginoplasty) could probably be improved. Therefore, dexamethasone treatment of a mother with a female CAH fetus due to 21-hydroxylase deficiency seems to be justified starting at the 12th week of gestation. However, the optimal beginning of therapy is in early pregnancy.     

Gangrene of the intestine with pneumatosis intestinalis in a child with meningomyelocele

A 13-year-old girl with meningomyelocele and a cauda-equina-paraplegia-syndrome, with resulting long-standing ileus, developed an extensive gangrene of the stomach, the small bowel and parts of the colon. As pathogenesis a non-occlusive-ischemia is suspected; the exceptional neurological situation is discussed.     

Aortic dissection in childhood. Occurrence and diagnostic procedure

Aortic dissections of the aorta thoracalis are rare in childhood, and mostly cause a life-threatening situation in which an immediate diagnosis is the key for survival. Main causes are an aortic vitium or a traumatic disruption. The conclusive diagnosis can only be obtained by a chest-X-ray, the two-dimensional echocardiography and the dynamic computed tomography scanning of the thorax. It must be taken into account, that patients with a polytrauma could possibly suffer from a traumatic aortic dissection, which sometimes can only be diagnosed at a later date.     

Determinants of pediatric injuries

Injuries are an important health issue for children. Previous research, however, has presented confusing and conflicting results on the determinants of childhood injuries, particularly psychosocial predictors, largely due to methodologic problems. The purpose of this analysis, based on a prospective follow-up study of 532 children, was to identify factors related to injuries encountered in a prepaid group practice during a 12-month period. Using logistic regression, we found four factors independently associated with the risk of at least one treated injury: high activity level, high rate of pediatric utilization for non-injury-related visits during the follow-up period, occurrence of a treated injury during the year preceding the follow-up period, and negative attitude toward medical care providers by the child's mother. In addition, four factors were found to be independent predictors of injuries judged severe enough to always warrant medical care: occurrence of a treated injury in the preceding year, high rate of pediatric utilization for non-injury-related visits during the follow-up period, working more than 15 hours a week outside the home by the child's mother, and more life events reported by the mother for the year preceding the follow-up period. Since family stressors are related specifically to the risk of more severe injuries, which are unlikely to escape medical attention, we conclude that these factors probably are related to the occurrence of common injuries of early childhood and not exclusively to utilization behavior. We therefore suggest that children from families with these characteristics be targeted for injury prevention strategies.     

Preclinical efficacy evaluations of XK-469: dose schedule,route and cross-resistance behavior in tumor bearing mice

XK-469 is advancing to Phase I clinicaltrials. Preclinical studies were carriedout to assist in clinical applications.Dose-schedule route testing: Singledose IV treatment with XK-469 producedlethality (LD20 to LD 100) above 142 mg/kg.Optimum treatment required total dosages of350 to 600 mg/kg. Furthermore, highindividual IV dosages (100 to 142 mg/kg)were poorly tolerated, producingsubstantial weight loss (8 to 18% of bodyweight), poor appearance, and slow recovery(8 to 12 days). A 1-hour infusion ofdosages more than 140 mg/kg, or BIDinjections 6 hrs apart, did not reducelethality. However, lower individualdosages of 40 to 50 mg/kg/injection IV werewell tolerated and could be given daily toreach an optimum total dose with minimaltoxicities. Likewise, 75 mg/kg/injection IVcould be used every other day to reachoptimal treatment. The necropsy profiles ofdeaths from toxic dosages were essentiallyidentical regardless of schedule (deaths 4to 7 days post treatment). The profileswere: paralytic ileus or gastroparesis; GIepithelial damage; and marrow toxicity.Interestingly, the key lethal events wererapidly reversible and simple to overcomewith lower dosages given daily or everyother day. Based on these results, the highdose, Q21day schedule should be avoided inclinical applications. Instead, a splitdose regimen is recommended (e.g., daily,every other day, or twice weekly). XK-469was also well tolerated by the oral route,requiring 35% higher dosages PO to reachthe same efficacy and toxicity as producedIV. Cross-resistance studies: XK-469resistance was produced by optimumtreatments of IV implanted L1210 leukemiaover seven passage generations. Thisleukemia subline (L1210/XK469) had reducedsensitivity to VP-16 (with a 4.0 log killin IV implanted L1210/XK469 compared to an8.0 log kill against IV implanted L1210/0). It also had a reduction in the sensitivityto 5-FU (with a 2.0 log kill in theimplanted L1210/XK469 compared to a 4.0 logkill against IV implanted L1210/0). Otheragents were approximately as active againstthe resistant tumor, including: Ara-C,Gemzar, Cytoxan, BCNU, DTIC, and CisDDPT. No case of collateral sensitivity wasobserved; i.e., no agent was markedly moreactive against the resistant sublineL1210/XK-469 than against the parent tumorin mice.