De novo germline mutations of the p53 gene in young children with sarcomas

Germline p53 mutations are associated with cancer predisposition in Li-Fraumeni families as well as in individuals with component tumors of the syndrome. In the majority of cases these mutations have been shown to be inherited rather than de novo. We screened 59 children with primary bone or soft tissue sarcomas. Germline p53 mutations were identified in 2 patients. Interestingly, analysis revealed that both mutations were de novo. Although the frequency of germline p53 mutations in primary pediatric sarcoma patients is low, there is evidence for the importance of considering pediatric patients for testing for de novo mutations.     

The effects of visual input on open-loop and closed-loop postural control mechanisms

In an earlier posturographic investigation (Collins and De Luca 1993) it was proposed that open-loop and closed-loop control mechanisms are involved in the regulation of undisturbed, upright stance. In this study, stabilogram-diffusion analysis was used to examine how visual input affects the operational characteristics of these control mechanisms. Stabilogram-diffusion analysis leads to the extraction of repeatable center-of-pressure (COP) parameters that can be directly related to the resultant steady-state behavior and functional interaction of the neuromuscular mechanisms underlying the maintenance of erect posture. Twenty-five healthy male subjects (aged 19–30 years) were included in the study. An instrumented force platform was used to measure the time-varying displacements of the COP under each subject's feet during quiet standing. The subjects were tested under eyes-open and eyes-closed conditions. The COP trajectories were analyzed as one-dimensional and two-dimensional random walks, according to stabilogram-diffusion analysis. Using this technique, it was found that visual input affects the performance of the postural control system in one of two different ways — either it significantly modifies the steady-state behavior of the open-loop postural control mechanisms, or it significantly alters the characteristics of the other closed-loop feedback mechanisms that are involved in balance control. This result is interpreted as an indication that the visual system is integrated into the postural control system in one of two different ways. The experimental population was roughly evenly divided between these two schemes. For the first group (13 of 25 subjects), visual input principally caused a decrease in the effective stochastic activity of the open-loop control mechanisms in both the mediolateral and anteroposterior directions. For the second group (12 of 25 subjects), visual input caused an increase in the effective stochastic activity and uncorrelated behavior of the closed-loop control mechanisms in the anteroposterior direction only. On the basis of these results, it is hypothesized that visual input, in both schemes, serves to decrease the stiffness of the musculoskeletal system. In the former case, this may be accomplished by decreasing the level of muscular activity across the joints of the lower limb, whereas, in the latter case, reduced stiffness may be achieved by reducing the gain(s) of the other postural feedback mechanisms, i.e., the proprioceptive and/or vestibular systems. Using stabilogram-diffusion analysis, it was also found that the two groups of subjects behaved similarly under eyes-closed conditions. This result suggests that the open-loop postural control mechanisms and reflex-based feedback systems, respectively, of healthy, young individuals are organized in functionally equivalent ways.     

Stereoselective effects of mexiletine enantiomers on sodium currents and excitability characteristics of adult skeletal muscle fibers

The effects of the enantiomers of mexiletine were tested on sodium currents of frog skeletal muscle fibers recorded by means of the three vaseline gap voltage clamp method and compared with the effects produced by tocainide enantiomers. The R-( – ) mexiletine produced a tonic block of the sodium current, elicited by single depolarizing test pulses from the holding potential of – 100 mV to – 20 mV, with an IC₅₀ of 43.9 ± 1 M, whereas the corresponding S-( + ) enantiomer produced the same effects at about twofold higher concentrations. A similar stereoselectivity was observed with tocainide enantiomers, but at about 5 fold higher concentrations. Both the R-( – ) and S-( + ) enantiomers of mexiletine and tocainide produced a further use-dependent block of sodium currents when the test pulse was applied repetitively at a frequency of 2 Hz. The use dependent behaviour led to a significant lowering of the IC₅₀ values with respect to the tonic block but the eudismic ratios ([IC₅₀S-( + )]/[IC₅₀R( – )]) and the relative potency between mexiletine and tocainide were maintained. All the tested compounds produced a left shift of the steady state inactivation curves (h) , suggesting a high-affinity interaction with the inactivated sodium channels. Again a stronger potency of R-( – ) vs. S-( + ) enantiomers and of mexiletine vs. tocainide was observed. The excitability characteristics recorded from the semitendinosus muscle by the two microelectrode technique were modified by the tested drugs in agreement with their ability to block sodium current. Thus a concentration-related increase in the threshold current required to elicit an action potential was observed along with a decrease in the amplitude and a shortening of the latency of action potential and a decrease in the firing capability of the membrane. Again the R-( – ) isomers were more potent than the S-( + ) ones and mexiletine was more effective than tocainide. These data corroborate the presence of a stereospecific site for these drugs on adult skeletal muscle sodium channels. The constant eudismic ratios between the enantiomers during both tonic and use-dependent block suggest that the increase in the apparent affinity of the receptor during statedependent conformational changes of the channel does not enhance its stereospecificity. The decrease in effective concentration upon high frequency stimulation supports the potential usefulness of low doses of R-( – ) mexiletine in the treatment of the abnormal hyperexcitability of the myotonic muscles, with a likely reduction of unwanted side effects.     

Vitamin A-sensitive tissues in transgenic mice expressing high levels of human cellular retinol-binding protein type I are not altered phenotypically.

The suggested function of cellular retinol-binding protein type I [CRBP(I)] is to carry retinol to esterifying or oxidizing enzymes. The retinyl esters are used in storage or transport, whereas oxidized forms such as all-trans or 9-cis retinoic acid are metabolites used in the mechanism of action of vitamin A. Thus, high expression of human CRBP(I) [hCRBP(I)] in transgenic mice might be expected to increase the production of retinoic acid in tissues, thereby inducing a phenotype resembling vitamin A toxicity. Alternatively, a vitamin A-deficient phenotype could also be envisioned as a result of an increased accumulation of vitamin A in storage cells induced by a high hCRBP(I) level. Signs of vitamin A toxicity or deficiency were therefore examined in tissues from transgenic mice with ectopic expression of hCRBP(I). Testis and intestine, the tissues with the highest expression of the transgene, showed normal gross morphology. Similarly, no abnormalities were observed in other tissues known to be sensitive to vitamin A status such as cornea and retina, and the epithelia in the cervix, trachea and skin. Furthermore, hematologic variables known to be influenced by vitamin A status such as the hemoglobin concentration, hematocrits and the number of red blood cells were within normal ranges in the transgenic mice. In conclusion, these transgenic mice have normal function of vitamin A despite high expression of hCRBP(I) in several tissues.     

The role of pRb2/p130 protein in diagnosing lung carcinoma on fine needle aspiration biopsies

The retinoblastoma gene family is composed of three members: the retinoblastoma gene, one of the most studied tumor suppressor genes, and two related genes: p107 and pRb2/p130. These proteins are also known as the pocket proteins due to a unique structural and functional domain composed of subdomains A and B separated by a spacer region that is highly conserved among each of the proteins. These proteins exhibit unique growth suppressive properties that are cell type specific, suggesting that although the pocket proteins may complement each other, they are not fully functionally redundant. With the development of antibodies recognizing these three proteins it is now possible to detect expression in formalin-embedded specimens. Recent studies on 235 lung cancers, using immunohistochemical techniques, suggested an independent role for Rb2/p130 in the development and/or progression of human lung carcinoma. We found a statistically significant inverse relationship between the histological grading (degree of malignant potential) and the expression of pRb/p105, p107 and pRb2/p130 in squamous cell carcinomas, meaning that an increase in grading resulted in a significant decrease in protein expression. This phenomenon was particularly evident for pRb2/p130 (p < .0001) which had the highest percentage of undetectable levels in all the specimens examined and the tightest inverse correlation (p value) with both the histological grading and PCNA expression in the most aggressive tumor types, suggesting an important role for pRb2/p130 in the pathogenesis and progression of certain lung cancers. We further explored the expression of pRb2/p130 protein in routine archival FNAB cytological material from 30 Patients with lung cancer using immunocytochemical techniques, comparing protein expression with tumor type. Two pathologists evaluated the staining pattern and scored the percentage of positive cells. Of the 30 neoplasms, 27 displayed a positive staining for pRb2/p130. In particular, we detected pRb2/p130 in 9 (100%) squamous carcinomas, 11 (84%) adenocarcinomas, 5 (100%) BAC, and 2 (66%) SCC. The percentage of positive nuclei varied in different tumors with the highest expression level in adenocarcinomas. Immunocytochemistry represents a sensitive method for detection of pRb2/p130 expression in cytological or archival specimens, and the level of detection seems to be comparable to paraffin sections. Therefore, this methodology could be used in the preoperative evaluation of routine cytological specimens in order to improve the diagnostic and prognostic evaluation of lung cancer patients.     

Clinical study on the treatment of differentiated carcinoma of the thyroid gland

A study on 88 patients operated for a differentiated thyroid carcinoma (63 papillary and 25 follicular cancer) is reported. In 66 cases a total thyroidectomy was performed (in 16 associated with an ipsilateral lymphadenectomy), in 11 a subtotal thyroidectomy and in 11 a lobar isthmectomy. In the follow-up there were 3 deaths and 4 recurrences. Notwithstanding the limited number of cases, the Authors registered a worse diagnosis for the follicular carcinomas (2 deaths and 2 recurrences on 25) compared to the papillary (1 death and 2 recurrences on 63).     

Increased hindrance on the chiral carbon atom of mexiletine enhances the block of rat skeletal muscle Na+ channels in a model of myotonia induced by ATX

1 The antiarrhythmic drug mexiletine (Mex) is also used against myotonia. Searching for a more efficient drug, a new compound (Me5) was synthesized substituting the methyl group on the chiral carbon atom of Mex by an isopropyl group. Effects of Me5 on Na+ channels were compared to those of Mex in rat skeletal muscle fibres using the cell-attached patch clamp method. 2 Me5 (10 microM) reduced the maximal sodium current (INa) by 29.7+/-4.4 % (n=6) at a frequency of stimulation of 0.3 Hz and 65.7+/-4.4 % (n=6) at 1 Hz. At same concentration (10 microM), Mex was incapable of producing any effect (n=3). Me5 also shifted the steady-state inactivation curves by -7. 9+/-0.9 mV (n=6) at 0.3 Hz and -12.2+/-1.0 mV (n=6) at 1 Hz. 3 In the presence of sea anemone toxin II (ATX; 5 microM), INa decayed more slowly and no longer to zero, providing a model of sodium channel myotonia. The effects of Me5 on peak INa were similar whatever ATX was present or not. Interestingly, Me5 did not modify the INa decay time constant nor the steady-state INa to peak INa ratio. 4 Analysis of ATX-induced late Na+ channel activity shows that Me5 did not affect mean open times and single-channel conductance, thus excluding open channel block property. 5 These results indicate that increasing hindrance on the chiral atom of Mex increases drug potency on wild-type and ATX-induced noninactivating INa and that Me5 might improve the prophylaxis of myotonia.     

Histamine H2-receptor antagonists stimulate proliferation but not migration of human gastric mucosal cellsin vitro

Gastric mucosal cell migration and proliferation are crucial events in the repair of gastric mucosal erosions. This study was designed to test the hypothesis that the H₂ blockers roxatidine and ranitidine might stimulate migration and proliferation of gastric mucous cells derived from a human well-differentiated gastric adenocarcinoma cell line (MKN 28 cells)in vitro, in conditions independent of systemic factors and of acid inhibition. Confluent monolayers of MKN 28 cells were wounded with a razor blade and were then incubated with roxatidine or ranitidine. The number of cells migrating to the damaged area was determined 24 hr later. Cell proliferation was assessed by means of [³H]thymidine uptake and cell counts after incubation with roxatidine or ranitidine. Neither H₂ antagonist significantly stimulated cell migration. On the other hand, cell proliferation was dose-dependently and significantly enhanced by incubation with roxatidine and ranitidine. Exogenous administration of TGF- significantly stimulated MKN 28 cell division. However, incubation with roxatidine or ranitidine did not increase the steady-state mRNA expression of TGF- or EGFR as assessed by northern blot analysis. Based on thesein vitro findings, we postulate that the ulcer healing effect of these H₂ antagonistsin vivo might be due in part to stimulation of gastric mucosal cell proliferation.Data from this paper have been presented in part at the 1995 meeting of the American Gastroenterological Association and published in abstract form in Gastroenterology 108:A72, 1995.     

Germline mutations of the p53 gene in children with malignant solid tumors

Although somatic mutations of p53 are the most common genetic changes observed to date, the frequency of germline p53 mutations is found to be very low in sporadic malignant tumors. It has been postulated that de novo germline p53 mutations may occur in a substantial population of patients in pediatric age group, who die of their disease and do not propagate the mutation. To determine the frequency and type of p53 germline mutations in pediatric patients, we screened 65 children who were consecutively admitted with primary malignant solid tumors.     

The role of spontaneous portosystemic shunts in the course of orthotopic liver transplantation

Spontaneous portosystemic shunts are commonly found in cirrhotic patients. Not yet established is their role after orthotopic liver transplantation (OLTx), especially when an increase in portal pressure develops, as during early acute rejection. In this study, 34 cirrhotic patients in a series of 70 OLTx are considered. Each patient had preoperative angiographic assessment, and, in 21 (62%), large spontaneous portosystemic shunts were evident. In 12 cases the shunts were not affected by the surgical procedure and were present during the postoperative period; in 9 the hepatectomy itself involved interruption of the shunts. The patient population was divided into two groups: patients with postoperative shunts (n=12) and those without (n=22). The two groups were similar in age, sex, Child's stage, transplantation variables, and number and grade of rejection episodes. However, mean transaminases (AST) values in the first 2 weeks were significantly higher levels in shunt versus nonshunt patients (421±335 vs 183±126; P<0.025), and this was even more evident when rejection occurred (626±375 vs 195±129; P<0.001). Furthermore, during an acute rejection reaction, three cases showed a true steal phenomenon through the large reopened shunts with ischemic damage to the grafts. The data indicate a possible detrimental effect of the spontaneous shunts on graft perfusion and suggest the prophylactic surgical interruption of the residual shunts during the transplantation.