Characterization of Nine Novel Mutations in the CD40 Ligand Gene in Patients with X-Linked Hyper IgM Syndrome of Various Ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome–specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Distribution and abundance of house-dust mites (Acarina: Pyroglycphidae) in Rome,Italy

For the first time in Rome, house-dust mite infestation was studied in 90 randomly selected houses. In each house, mite infestation was assessed in three sites: mattress, bedroom and living room. In total, 87.8% of the sampled houses were positive for dust mites. In the houses infested, 11.4% showed densities of >100 mites/g of dust, 15.2% registered densities between 50 and 99, and in the remaining houses (73.4%), the densities were between 1 and 49 mites/g dust. The percentages of infested houses were positively correlated with the relative humidity (RH) values (r=0.89,P=0.02). At the lowest range of RH (between 46 and 50), the infestation was 50% and at the highest range of RH (between 73 and 78) it was 100%. The mattress was significantly the most infested (71.1%) of the tested sites. Only wool and spring mattresses were infested, and they did not show any significant differences in mite concentrations.Dermatophagoides farinae was the most abundant species (53.1%), followed byGlycyphagus domesticus (34.5%),D. pteronyssinus (5.2%), andEuroglyphus maynei (0.2%);D. farinae was also the most frequent species (56.9%). The remaining specimens (7.0%) were predator species commonly found in houses. The prevalence ofD. farinae in Rome is discussed.     

Glycosyl transferases in mouse and human milk fat globule membranes

Summary Membranes isolated from mouse and human milk fat globules were found to contain the enzymes responsible for the synthesis of dolichol monophosphate mannose and dolichol monophosphate glucose as well as those involved in the transference of the glycosyl residues from the two dolichol derivatives to dolichol diphosphate oligosaccharides. The levels of most of the enzymes were comparable to those found in mouse mammary gland microsomes. The presence of enzymes involved in protein glycosylation via dolichol derivatives in the milk fat globule membrane provides evidence in favor of an outward flow of membrane components from the rough endoplasmic reticulum, where these enzymes are active in vivo, towards the cell surface.     

Synthesis of dolichol derivatives in human erythrocyte membranes

Summary Human erythrocyte membranes contain the enzymes responsible for the synthesis of dolichol-P-glucose, dolichol-P-mannose, dolichol-PP-N-acetylglucosamine, dolichol-PP-NN diacetylchitobiose and of dolichol-PP-oligosaccharides containing NN diacetylchitobiose and mannose or the same sugar residues plus glucose. The transfer of the oligosaccharide moieties from the dolichol-PP-oligosaccharides to endogenous proteins could not be detected. These enzymes appeared to be integral membrane proteins.Abbreviation Dol dolichol Dedicated to ProfessorLuis f. Leloir on the occasion of his 70th birthday.     

Procedure for testing kinetic models of the photocycle of bacteriorhodopsin.

Given some simple kinetic models of the photocycle of bacteriorhodopsin (bR) and data taken at many wavelengths and under conditions that avoid photoselection and steady-state cycling complications, it is shown how to extract the apparent rate constants and the spectra of the intermediates. Special consideration was given to establishing the range of error of these results. There are many criteria, which we explicitly discuss, that the spectra should satisfy in order that the kinetic model be acceptable. New data for the photocycle of purple membrane fragments in dilute buffer at pH 7.0 has been obtained at 15 measuring wavelengths and four temperatures. The procedure, which can be generalized to more complex models, has been applied to these data to test two kinds of kinetic models: the unidirectional unbranched model and the undirectional model with simple branching straight back to bR from any intermediate. In these models the spectrum of the O intermediate is highly temperature sensitive, even with branching, and/or has two broad maxima. Moreover, the spectrum of the M intermediate has a secondary maximum and two M-like states appear to be required. Thus, neither model satisfies the physical criteria.     

Studies on DNA damage: Discordant responses of rate of DNA disentanglement (viscosimetrically evaluated) and alkaline elution rate,obtained for several compounds

Alkaline elution is a well-known method for detecting DNA damage. Recently we have developed a viscosimetric method that is even more sensitive than alkaline elution. Here we report that the two methods, although apparently both revealing alkaline DNA fragmentation, can give dramatically different results for a significant series of compounds. We suspect that alkaline elution might reveal not only DNA fragmentation but also the extent of disentanglement of chromatin structure, whereas this DNA disentanglement rate, when evaluated viscosimetrically, is more strictly correlated with the initiation of DNA unwinding.     

The UDP-Glc:glycoprotein glucosyltransferase is a soluble protein of the endoplasmic reticulum.

N-linked oligosaccharides devoid of glucose residues are transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum. The reaction products have been identified, depending on the organisms, as protein-linked Glc1Man5-9GlcNAc2. Incubation of right side-sealed vesicles from rat liver with UDP-[14C]Glc, Ca2+ ions and denatured thyroglobulin led to the glucosylation of the macromolecule only when the vesicles had been disrupted previously by sonication or by the addition of detergents to the glucosylation mixture. Similarly, maximal glucosylation of denatured thyroglobulin required disruption of microsomal vesicles isolated from the protozoan Crithidia fasciculata. Treatment of the rat liver vesicles with trypsin led to the inactivation of the UDP-Glc:glycoprotein glucosyltransferase only when proteolysis was performed in the presence of detergents. The glycoprotein glucosylating activity could be solubilized upon sonication of right side-sealed vesicles in an isotonic medium, upon passage of them through a French press or by suspending the vesicles in an hypotonic medium. Moreover, the enzyme appeared in the aqueous phase when the vesicles were submitted to a Triton X-114/water partition. Solubilization was not due to proteolysis of a membrane-bound enzyme. The enzyme could also be solubilized from C. fasciculata microsomal vesicles by procedures not involving membrane disassembly. About 30% of endogenous glycoproteins glucosylated upon incubation of intact rat liver microsomal vesicles with UDP-[14C]GLc could be solubilized by sonication or by suspending the vesicles in 0.1 M Na2CO3. These and previous results show that the UDP-Glc:glycoprotein glucosyltransferase is a soluble protein present in the lumen of the endoplasmic reticulum. In addition, both soluble and membrane-bound glycoproteins may be glucosylated by the glycoprotein glucosylating activity.     

Drug metabolism polymorphisms as modulators of cancer susceptibility.

Recently, several molecular genetic bases of polymorphic enzyme activities involved in drug activation and detoxification have been elucidated. Many molecular epidemiology studies based on these premises have sought to gather information on the association of genetically determined metabolic variants with different risks of environmentally induced cancer. While rare alterations of tumor suppressor genes dramatically raise cancer risk for the single affected subjects, far more common and less dramatic differences in genes encoding for drug metabolism enzymes can be responsible for a relatively small, but rather frequent increase of cancer risk at the population level. This increase could be especially important in specific cases of occupational, pharmacological or environmental exposure. Examination of the current literature reveals that the most extensively investigated metabolic polymorphisms are those of P450 1A1 and P450 2D6 cytochromes, glutathione S-transferases (GSTs; M1 and, to a lesser extent, M3, P1 and T1) and N-acetyltransferases (NATs; NAT1 and NAT2). Making reference to these enzymes, we have assayed the current knowledge on the relations among polymorphisms of human xenobiotic-metabolizing enzymes and cancer susceptibilities. We have found intriguing models of susceptibility toward different types of cancer. We have reviewed and commented these models on light of the complex balance among different enzyme activities that, in each individual, determines the degree of each cancer susceptibility. Moreover, we have found techniques of molecular genetic analysis, more suitable than previous ones on phenotypic expression, now allowing better means to detect individuals at risk of cancer. According to the models presently available, a systematic screening of individuals at risk seems to make sense only in situations of well defined carcinogenic exposures and when performed by the polymorphism analysis of coordinated enzyme activities concurring to the metabolism of the carcinogen(s) in question. Genetic polymorphism analysis can allow for the detection of patients more prone to some types of specific cancers, or to the adverse effects of specific pharmaceutical agents. Considering the increasingly confirmed double-edged sword nature of metabolism polymorphism (both wild-type and variant alleles can predispose to cancer, albeit in different situations of exposure), individual susceptibility to cancer should be monitored as a function of the nature, and mechanism of action, of the carcinogen(s) to which the individual under study is known to be exposed, and with reference to the main target organ of the considered type of exposure.     

E-cadherin deregulation in breast cancer

E-cadherin protein (CDH1 gene) integrity is fundamental to the process of epithelial polarization and differentiation. Deregulation of the E-cadherin function plays a crucial role in breast cancer metastases, with worse prognosis and shorter overall survival. In this narrative review, we describe the inactivating mechanisms underlying CDH1 gene activity and its possible translation to clinical practice as a prognostic biomarker and as a potential targeted therapy.