Decreased expression of DMPK: correlation with CTG repeat expansion and fibre type composition in myotonic dystrophy type 1

Abstract Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a trinucleotide repeatexpansion, cytosine-thymine-guanine (CTG)_n, in the 3′ untranslated region of a gene encoding the myotonic dystrophy protein kinase (DMPK). To correlate CTG expansion and protein expression, we studied muscle specimens from 16 adult DM1 patients using three anti-DMPK antibodies for immunoblotting. We estimated the amount of the full-length DMPK (85 kDa) in muscle biopsies from normal controls and from DM1 patients carrying different (CTG)_n expansions. We found that DMPK concentration was decreased to about 50% in DM patients’ muscles; the protein decrease did not seem correlated with the CTG repeat length. However, the fibre type composition in skeletal muscle seemed somehow to affect DMPK decrease, as the lowest level of the enzyme was found in patients with the lowest content of type 1 fibre.     

Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology.

As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all alpha-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.     

A quantitative dot immunobinding assay for coagulation factor XII in plasma

A dot immunobinding assay on nitrocellulose (NC) membranes has been developed for the quantification of human coagulation factor XII (F XII). Plasma samples were dotted on to NC filters and F XII was detected using a polyclonal antiserum followed by a radiolabelled antigen overlay. Dilutions of either pooled normal human plasma (NHP) or purified F XII in F XII deficient plasma were used as standards. Quantification was performed by measuring the radioactivity of bound 125I-F XII. Precise measurements of F XII antigen (F XII: Ag) were possible with a sensitivity down to 0.12 ng. Thus, dotting samples containing 0.5 microliter of plasma permitted detection of a F XII concentration corresponding to 1% of the level in NHP. The intra-assay coefficient of variation (CV) was less than 5% and the interassay CV was less than 16%. F XII:Ag in plasma samples of 50 healthy adults ranged from 12 micrograms/ml to 47 micrograms/ml. A good correlation (r = 0.93) existed between F XII:Ag and F XII clot promoting activity (F XII:C) in these samples. NHP contained 24.1 micrograms/ml F XII:Ag confirming earlier results obtained by other methods. In 16 pregnant women levels of F XII:Ag as well as of F XII:C were elevated, but F XII:Ag was disproportionately higher compared with F XII:C. The immunobinding assay has the following advantages: (1) rapid quantification of large numbers of samples is possible, (2) the sensitivity down to 1% of NHP is better than that of several other methods, (3) only very small amounts of both test material and reagents are needed.     

Microsatellite instability and p53 expression in gallbladder carcinomas.

We studied the MSI (microsatellite instability) status and p53 expression in a series of 71 gallbladder cancers (GCs) of different histologic type. All neoplasms were examined combining a microsatellite analysis at mononucleotide locus BAT-26 and an immunohistochemical study for hMSH2, hMLH1, and p53 proteins and markers of gastric and intestinal differentiation. All the 71 GCs were MSS (microsatellite stable). The p53 protein was found in 100% of undifferentiated GCs, 67% of conventional gallbladder adenocarcinomas, 50% of mucinous adenocarcinomas, and 20% GCs with squamous differentiation. All 71 MSS tumors showed presence of immunohistochemical expression of both hMLH1 and hMSH2 gene products. We concluded that microsatellite instability does not play a role in the developing of GC while p53 seems to be the most important alteration found in a large proportion of these cancers, with the only exception of mucinous and squamous gallbladder carcinomas.     

Cytomegalovirus retinitis in advanced HIV-infected patients treated with protease inhibitors: incidence and outcome over 2 years

We prospectively studied the incidence of cytomegalovirus (CMV) retinitis in 93 patients treated with highly active antiretroviral therapy (HAART) containing a protease inhibitor (PI), during a median follow-up period of 24 months. The median initial CD4+ count was 22 cells/microl (range, 1-311 cells/microl), and the median plasma HIV viral load was 5.1 log10 copies/ml (range, 2.4-6.4 log10 copies/ml). The fundus was examined monthly in patients with a history of CMV retinitis or an initial CD4+ count <50 cells/microl and every 3 months in the other patients. Of patients with previously controlled CMV retinitis, 1 of 7 relapsed. In addition, 6 of 59 patients with a CD4+ count <50 cells/microl and no history of CMV retinitis before starting PI therapy developed CMV retinitis. Of them, 3 had at least one relapse during follow-up. CD4+ counts were <40 cells/microl at the time of primary or recurrent CMV retinitis, except in two cases (147 cells/microl and 203 cells/microl). In conclusion, the incidence of CMV retinitis was 0.091 per patient-year among study subjects with advanced HIV infection who were receiving HAART (95% confidence interval [CI], 0.037-0.145). The time to progression of CMV retinitis (mean, 215 days; 95% CI, 113-317 days) was longer than reported before widespread use of PIs.     

Immunohistochemical pattern of hMSH2/hMLH1 in familial and sporadic colorectal, gastric, endometrial and ovarian carcinomas with instability in microsatellite sequences

Alterations of DNA mismatch repair (MMR) genes are involved in carcinogenesis of sporadic and inherited human cancers characterised by instability of DNA microsatellite sequences (MSI). MSI tumours are usually identified using molecular analysis. In the present investigation, hMLH1 and hMSH2 immunohistochemistry was tested in order to evaluate the utility of this method in predicting MMR deficiency. Colorectal (72), gastric (68), endometrial (44) and ovarian (17) carcinomas were independently evaluated for familial history, histological type of tumour, MSI status and immunohistochemical results. Loss of expression of either hMLH1 or hMSH2 was observed in 51 of 55 (92.8%) MSI tumours, while 145 of 146 microsatellite stable (MSS) tumours expressed both the hMLH1 and hMSH2 gene products. Independently of tumour site, an overall agreement between immunohistochemical and molecular results was observed in 15 hereditary non-polyposis colorectal cancer-related tumours. Among sporadic tumours, only 2 of 60 colorectal and 2 of 66 gastric carcinomas, displaying MSI, expressed both hMLH1 and hMSH2 gene products. All 39 endometrial and 16 ovarian tumours presented a concordant molecular and immunohistochemical profile. These data show that immunohistochemistry is an accurate and rapid method to predict the presence of defective DNA MMR genes and to identify both sporadic and familial MSI tumours.