Cystine storage in cultured myotubes from patients with nephropathic cystinosis.

Sorted muscle cells, cultured from a patient with nephropathic cystinosis, stored 100 times normal amounts of cystine. Subcellular fractionation and density-gradient centrifugation confirmed that the cystine was located in a lysosomal compartment. 2. Myoblasts from cystinotic patients in culture underwent fusion to myotubes in a normal fashion. 3. The free thiol cysteamine effectively depleted cystinotic-muscle cells of cystine. 4. Cultured myoblast and myotubes offered a unique system for investigating the effects of lysosomal storage on differentiated cell functions.     

The Salmonella wien virulence plasmid pZM3 carries Tn1935, a multiresistance transposon containing a composite IS1936-kanamycin resistance element

Tn1935, a 23.5-kb transposon mediating resistance to ampicillin, kanamycin, mercury, spectinomycin, and sulfonamide was isolated from pZM3, an IncFIme virulence plasmid from Salmonella wien. Tn1935 possesses the entire sequence of Tn21 and contains two additional DNA segments of 0.95 and 2.7 kb carrying the ampicillin and kanamycin resistance genes, respectively. The latter is part of a composite element since it is flanked by two IS15-like insertion sequences (IS1936) in direct orientation. IS1936 is about 800 bp long and is closely related to IS15 delta, IS26, IS46, IS140, and IS176. Functional analysis of IS1936-mediated cointegrates shows that both insertion sequences are active and able to form cointegrates at the same frequency. Resolution of the cointegrates requires the presence of the host Rec system. The presence of the composite IS1936-element within Tn1935 supports the hypothesis that multidrug resistance transposons evolved by insertion of antibiotic determinants which are themselves transposable.     

Eye factors and lens-forming transformations of outer cornea in Xenopus laevis larvae

Outer cornea of lensectomized Xenopus laevis tadpoles at state 50 (according to Nieuwkoop, P.D. and Faber, J., ('56) Normal Table of Xenopus laevis, Daudin, North-Holland, Amsterdam, pp. 1-243) was removed 3, 7 and 10 days after lensectomy and implanted between the outer and the inner cornea of larvae of the same species at stage 51-52. In these conditions, the implanted outer cornea remained isolated from the retinal factor of the vitreous chamber, although it received the nutritional factors normally reaching the outer cornea. Results show that lens-forming transformation process of the outer cornea is arrested, and lens-forming structures undergo regression at speed which increases with increasing precocity of the stage of lens-forming transformation undergone by the implanted cornea. These data suggest that the process of lens-forming transformation is not a single-step process, but a sequence of interactions extending over a long period of time requiring the continuous presence of the retinal factor in the vitreous chamber until complete differentiation of the lens is achieved.     

Analysis of lumped and distributed elements models of cut muscle fibers in vaseline or sucrose gap preparations

A general method of finding the time course and the steady state distribution of potential in Vaseline or sucrose gap preparations is given by making use of the linear cable equation. The general solution has been found analytically in terms of its Laplace transform and then numerically inverted. Two particular experimental situations, namely the single gap and the double gap preparations, have been analyzed. The results have been compared with the solutions of the commonly used lumped elements models. While for the double gap no large errors are introduced by the lumped model, for the single gap there are significant differences. The dependence of the voltage distribution on various electrical and geometrical parameters has been examined. It is suggested that the proposed mathematical treatment might be used by experimenters as a reference to assess the validity of simplified lumped models.     

Neuroendocrine modulation of haloperidol-induced catalepsy

Evidence has been accumulated implicating sex hormones as possible modulators of extrapyramidal motor function. In the present study we have investigated the effects of estrogens, progesterone, testosterone, prolactin and calcitonin on behavioral parameters related to nigro-striatal dopaminergic system, such as haloperidol-induced catalepsy in male rats. It was found that 7-days estradiol benzoate treatment (5 micrograms/rat/day) significantly increases haloperidol-induced catalepsy, suggesting a possible antidopaminergic activity of estrogens. On the other hand, prolactin facilitates nigro-striatal dopaminergic transmission. Interestingly, 7 day treatment with medroxy-acetate progesterone (MAP, 5 mg/Kg, i.p.) brings about a trend to a decrease in haloperidol-induced catalepsy, while no significantly effect was observed following acute MAP administration at the same dose. So, it is tempting to speculate that chronic progestinic treatment may result in an increase in dopaminergic tonus. Testosterone, acutely administered (5mg/kg.s.c.) induces changes similar to those observed following progesterone administration. Finally, also calcitonin is able to influence haloperidol-induced catalepsy by markedly increasing it.     

Xenopus laevis sperm proteins, previously identified as surface proteins with egg coat binding capability, are indeed histone H4, histone H3, and sperm specific protein SP2.

Recently, four Xenopus sperm proteins thought to be involved in binding to the egg envelope were identified (Lindsay and Hedrick, J. Exp. Zool., 245:286-293, '88). We have studied the three more abundant ones of apparent molecular weight of 14, 19, and 25 kd in SDS-PAGE. We have shown that these proteins are indeed nuclear basic proteins: the 14 kd is the histone H4, the 19 kd is the histone H3, and the 25 kd is the sperm-specific protein SP2.     

WIP Regulates Persistence of Cell Migration and Ruffle Formation in Both Mesenchymal and Amoeboid Modes of Motility

The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.     

Altered Expression Pattern of Molecular Factors Involved in Colonic Smooth Muscle Functions: An Immunohistochemical Study in Patients with Diverticular Disease

Background

The pathogenesis of diverticular disease (DD) is thought to result from complex interactions among dietary habits, genetic factors and coexistence of other bowel abnormalities. These conditions lead to alterations in colonic pressure and motility, facilitating the formation of diverticula. Although electrophysiological studies on smooth muscle cells (SMCs) have investigated colonic motor dysfunctions, scarce attention has been paid to their molecular abnormalities, and data on SMCs in DD are lacking. Accordingly, the main purpose of this study was to evaluate the expression patterns of molecular factors involved in the contractile functions of SMCs in the tunica muscularis of colonic specimens from patients with DD.

Methods and Findings

By means of immunohistochemistry and image analysis, we examined the expression of Cx26 and Cx43, which are prominent components of gap junctions in human colonic SMCs, as well as pS368-Cx43, PKCps, RhoA and αSMA, all known to regulate the functions of gap junctions and the contractile activity of SMCs.The immunohistochemical analysis revealed significant abnormalities in DD samples, concerning both the expression and distribution patterns of most of the investigated molecular factors.

Conclusion

This study demonstrates, for the first time, that an altered pattern of factors involved in SMC contractility is present at level of the tunica muscularis of DD patients. Moreover, considering that our analysis was conducted on colonic tissues not directly affected by diverticular lesions or inflammatory reactions, it is conceivable that these molecular alterations may precede and predispose to the formation of diverticula, rather than being mere consequences of the disease.     

Gene expression in Xenopus laevis embryos after Triadimefon exposure

The triazole derivative Triadimefon (FON) is a systemic fungicide used to control powdery mildews, rusts, and other fungal pests. Some data have already been published about the teratogenic activity of this compound: craniofacial malformations were found in mouse, rat, and Xenopus laevis embryos exposed to FON. These alterations were correlated to defective branchial arch development possibly caused by abnormal neural crest cell (NCC) migration into the branchial mesenchyme. As the migration of NCCs is controlled by the HOX code and by an anteroposterior retinoic acid (RA) gradient, we analyzed the expression of CYP26, a key enzyme in RA metabolism, following FON exposure. The increased expression of this gene and the ability of citral (a RA inhibitor) to reduce the teratogenic effects of the fungicide support the notion that endogenous RA is involved in the mechanism of action of FON. Moreover, by in situ hybridization, we studied the effects of FON exposure at gastrula stage on the expression of some genes involved in craniofacial development, hindbrain patterning, and NCC migration. We observed abnormal localization of xCRABP, Hoxa2 and Xbap signal expression at the level of migrating NCC domains, whereas in the hindbrain, we did not find any alteration in Krox20 and Hoxa2 expression.