Analysis of ABCA4 in mixed Spanish families segregating different retinal dystrophies

Genotype-phenotype correlations highlighted the function of ABCA4 in retinitis pigmentosa (RP),cone-rod dystrophy (CRD) and Stargardt/Fundus Flavimaculatus disease (STGD/FFM). Initial screening of ABCA4 variants showed a correlation between the type of mutation and the severity of the disease. In the present study we have undertaken mutational and haplotype analysis of ABCA4 in three mixed pedigrees segregating different retinal dystrophies. In family I, we have shown cosegregation of different ABCA4 alleles with CRD (homozygosity for L1940P) and three subtypes of STGD/FFM. The first, a mild form, consisting on fundus flavimaculatus-like distribution of flecks, but good visual acuity and absence of dark choroid, was found to cosegregate with alleles R1097C and F553L; the second, a conventional Stargardt phenotype was associated to alleles L1940P/R1097C and the third, displaying severely reduced visual acuity and dark choroid (named FFM), was associated to L1940P/F553L. In family II, segregating STGD and RP phenotypes, while the involvement of ABCA4 in STGD seems clear this is not the case for RP. Finally, in family III, also segregating STGD and RP, ABCA4 fails to explain either phenotype. Our data highlight the wide allelic heterogeneity involving this gene and support the genetic variability (beyond ABCA4) of mixed STGD/RP pedigrees.     

Serum hyaluronate levels reflect disease activity in experimetal arthritis models

Serum hyaluronate (HA) levels were measured in rats subjected to adjuvant or type II collagen induced arthritis. As the arthritic lesions developed, both models showed an increase in serum HA levels of approximately 5 times, from a baseline level of 61–126 ng/ml (range). Furthermore a positive correlation was found between HA level and arthritic score. The increase in HA was not related to metabolic impairment, as the half life of serum HA in adjuvant arthritic rats was similar to that of normal rats. Serum HA may thus serve as a useful variable for evaluation of the severity of experimental arthritis.     

Differences in adsorption of serum proteins and production of IL-1ra by human monocytes incubated in different tissue culture microtiter plates

In vitro cell culture models can be of great value in order to further analyze the regulatory mechanisms underlying the inappropriate function of the immune system in diseases such as autoimmunity and cancer. Cell culture conditions have to be well controlled in a way that they mirror the in vivo situation. The objective of this study was to compare tissue culture microtiter plates from different manufacturers with respect to their ability to support monokine production by human monocytes cultured in human serum. Tissue culture ware, made of polystyrene, undergoes treatment by the manufacturers to make the surface more suitable for culture of adherent cell populations. It is possible that quality differences in this treatment can lead to variations in protein binding properties and thereby influence the adherence and functional properties of monocytes. We measured spontaneous interleukin-1 receptor antagonist (IL-1ra) production by peripheral blood monocytes, cultured in human serum, in five different microtiter plates made for adherent cell culture. Culture in plates from two of the five manufacturers resulted in significantly lower amounts of secreted IL-1ra. IL-1ra release by human monocytes can be induced by adherent IgG cross-linking membrane receptors for the Fc part of IgG (FcgammaR). We found that reduced IL-1ra production coincided with a reduced capacity for binding of serum IgG in one case. Furthermore, this brand of microtiter plate also displayed the lowest level of adsorption of human albumin. We conclude that the protein adsorption properties of the plastic tissue culture ware have to be taken into consideration when assessing monokine production by human monocytes in vitro.     

Functional analysis of 13 GBA mutant alleles identified in Gaucher disease patients: Pathogenic changes and "modifier" polymorphisms

Gaucher disease, the most prevalent sphingolipidosis, is caused by the deficient activity of acid beta-glucosidase, mainly due to mutations in the GBA gene. Over 200 mutations have been identified worldwide, more than 25 of which were in Spanish patients. In order to demonstrate causality for Gaucher disease, some of them: c.662C>T (p.P182L), c.680A>G (p.N188S), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1093G>A (p.E326K), c.1289C>T (p.P391L), c.1292A>T (p.N392I), c.1322T>C (p.I402T), and the double mutants [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), were expressed in Sf9 cells using a baculovirus expression system. Other well-established Gaucher disease mutations, namely c.1226A>G (p.N370S), c.1342G>C (p.D409H), and c.1448T>C (p.L444P), were also expressed for comparison. The levels of residual acid beta-glucosidase activity of the mutant enzymes produced by the cDNAs carrying alleles c.662C>T (p.P182L), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1289C>T (p.P391L), and c.1292A>T (p.N392I) were negligible. The c.1226A>G (p.N370S), c.1322T>C (p.I402T), c.1342G>C (p.D409H), c.1448T>C (p.L444P), and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]) alleles produced enzymes with levels ranging from 6 to 14% of the wild-type. The three remaining alleles, c.680A>G (p.N188S), c.1093G>A (p.E326K), and [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]), showed higher activity (66.6, 42.7, and 23.2%, respectively). Expression studies revealed that the c.1093G>A (p.E326K) change, which was never found alone in a Gaucher disease-causing allele, when found in a double mutant such as [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), decreases activity compared to the activity found for the other mutation alone. These results suggest that c.1093G>A (p.E326K) should be considered a "modifier variant" rather than a neutral polymorphism, as previously considered. Mutation c.680A>G (p.N188S), which produces a mutant enzyme with the highest level of activity, is probably a very mild mutation or another "modifier variant."     

The differential effect of lysolecithin on mycoplasmas and acholeplasmas

The cidal effect of lysolecithin on mycoplasmas and acholeplasmas was studied. Thirty-one mycoplasma strains and species were tested. The growth of all of them was inhibited by 250 g/ml of lysolecithin and the growth of most by 125 g/ml. T-mycoplasmas were as sensitive to lysolecithin as the other mycoplasmas tested. On the other hand, acholeplasmas were about four-fold less sensitive; several strains of the three acholeplasma species grew even in the presence of 500 g/ml of lysolecithin. Strain Squire of Bovine Group 6 was more susceptible to lysolecithin than the other non-sterol requiring strains tested and in this respect resembled the mycoplasmas. Mycoplasmas were found also to be more susceptible to lecithin than acholeplasmas. The influence of the serum and cholesterol content of the medium on the results of the tests is considered and the importance of maintaining standard conditions is stressed. The possibility of using the susceptibility of mycoplasmas and acholeplasmas to lysolecithin of distinguish between them is discussed. The different amounts of cholesterol in the cell membrane of the organisms of these families might account for the differences observed.     

Directly bone-anchored implants for fixation of aural epistheses

In cases of missing outer ears, generally, epistheses are attached to eye-glasses which gives insecure attachment and a fairly poor cosmetic result. A new method for stable episthesis attachment is being tested in Gothenburg, Sweden. Threaded, cylindrical titanium implants are inserted with a meticulous technique in the temporal bone of the patient. At the next stage, 3–4 months later, the implants are connected to abutments which are allowed to penetrate the skin, To these abutments a silicone episthesis is attached. Presently, 7 patients who had no outer ear because of congenital disorders, hereditary diseases or status post trauma or tumour surgery have been operated and followed for up to $\text{1}\text{2}$ have been no problems reported with the bone anchorage or the skin penetration.     

Analysis of human papillomavirus prevalence and TP53 polymorphism in head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma is a disease associated with tobacco and alcohol abuse. There is evidence that the oncogenic human papillomavirus (HPV) may also be a risk for upper aerodigestive tract cancers. High-risk HPVs encode two early proteins, E6 and E7, that can bind to p53 and pRb, respectively, and induce its degradation or inactivation. The TP53 gene has a single polymorphism at codon 72 of exon 4 that encodes either arginine (Arg) or proline (Pro). The purpose of this study was to evaluate the role of HPV infection and TP53 polymorphism in head and neck cancer. We analyzed 50 tumors, as well swabs of oral mucosa from 142 control individuals, with a polymerase chain reaction technique. The prevalence of HPV in controls was 10.6% and in cancer specimens 16%. The frequency distribution of genotypes in controls was 50% Arg/Arg, 43% Arg/Pro and 7% Pro/Pro; in tumors, it was 52% Arg/Arg, 32% Arg/Pro, and 16% Pro/Pro. Contrary to the results of some studies on cervical cancer, no association between any TP53 genotype or allele and the development of head and neck cancer was observed, regardless of HPV status, except for the Pro/Pro genotype, which is associated with the absence of HPV. The arginine allele appears to protect against head and neck cancers. Also, the data showed that HPV infection results in no increased risk of developing head and neck tumors.     

Jejunal endocrine tumour composed of somatostatin and gastrin cells and associated with duodenal ulcer disease

Summary A case of malignant endocrine tumour of the jejunum, associated with severe duodenal ulcer is described. The tumour and a local metastasis were examined by immunohistochemistry and found to contain abundant somatostatin-immunoreactive cells together with less numerous cells displaying gastrin immunoreactivity. This is to our knowledge the first case of intestinal somatostatinoma. The presence of gastrin cells in the tumour may explain the ulcer diathesis.     

Delineation of the QRS complex using the envelope of the e.c.g.

A new algorithm for QRS delineation has been developed. Based on the envelope of the e.c.g. signal a delineation function is defined, which yields a single positive pulse for each complex. From this function the onset and end of the QRS or, alternatively, a fiducial point is determined. To remove low-frequency component such as S-T abnormalities without distortion of the QRS complex, a filter with time-varying characteristics is used. The accuracy of the method has been evaluated in a test set of different QRS complexes obtained from coronary care patients. For QRS onset, the standard deviation of the difference between automated and manual determination was 7 ms in normal beats and 14 ms in ectopic beats. With simulated noise added to each waveform an average dispersion of 7 ms was observed in the recognition of the QRS onset at a signal-to-noise ratio of 15 dB. The corresponding dispersion in the location of a fiducial point was 2 ms. Using simulated e.c.g. data, the stability of the method is demonstrated for transitions between different waveform morphologies. Presented in part at ‘Computers in cardiology’, Florence, 23rd–25th September 1981     

Force-velocity relation and rate of ATP hydrolysis in osmotically compressed skinned smooth muscle of the guinea pig

Summary Chemically skinned guinea pig taenia coli fibre bundles showed a concentration-dependent decrease in width when incubated in media containing Dextran T500 (0-0.2 g ml^–1). The maximal reduction in width, observed at 0.2 g ml^–1 dextran, was 32%. The effect was reversible upon removal of dextran. Isometric force was slightly increased (about 10%) at the lowest dextran concentration (0.025 g ml^–1) but decreased at higher concentrations (40% decrease at 0.2 gJml^–1). The energetic tension cost (ATP turnover/force) was decreased by about 40% after dextran addition. Force development and relaxation were markedly slower in 0.1 g ml^–1 and absent in 0.2 g ml^–1 dextran. In isotonic quick-release experiments 0.025 g ml^–1 dextran did not influence maximal shortening velocity (V_max) and relative stiffness, whereas 0.1 g ml^–1 markedly increased stiffness and decreased V_max to about 27%. Vanadate induced relaxation in the activated muscle (pCa 4.5) both in the absence and presence (0.1 g ml^–1) of dextran and increased the rate of relaxation (pCa 9) at 0.1 g ml^–1 dextran. The isometric rate of crossbridge turnover, as reflected by the energetic tension cost and the rate of relaxation, was decreased at all degrees of osmotic compression. Crossbridge turnover rate during shortening (V_max) was unaffected at an osmotic compression of 12% (width) but was decreased at higher compression (32%).