Calcium-dependent release specificities of various cell lines loaded with different transmitters

By loading cells in culture with acetylcholine (ACh) we have characterized a calcium-dependent release mechanism and shown that it was expressed independently of synthesis or storage of ACh. (Isra?l et al., 1994, Neurochemistry International 37, 1475-1483; Falk-Vairant et al., 1996a, Proc. Natl. Acad. Sci. U.S.A. 93, 5203-5207; Falk-Vairant et al., 1996b, Neuroscience 75, 353-360; Falk-Vairant et al., 1996c, Journal of Neuroscience Research 45, 195-201). The transmitter loading procedure was applied to two other transmitters, gamma-aminobutyric acid (GABA) and glutamate (Glu). We could then study the specificity of the release mechanism for the three transmitters in a variety of cell lines, including neural-derived cells. Four different calcium-dependent release phenotypes were identified: two were specific for ACh or GABA, and two co-released two transmitters ACh and GABA but not Glu, or ACh and Glu but not GABA. We conclude that release mechanisms having different specificities are expressed by the cell lines studied, they become functional after loading the cells with the relevant transmitters. These observations will help the identification of proteins controlling the specificity of release, and provide an interesting model for pharmacological studies.     

Human immunodeficiency virus type 1 proteinase resistance to symmetric cyclic urea inhibitor analogs

Resistant virus was isolated from virus propagated in cell culture in the presence of the human immunodeficiency virus type 1 (HIV-1) proteinase inhibitor DMP 323, Ro 31-8959, or A-75925. The proteinase gene of resistant virus was sequenced, and key mutations (G48V, V82A, I84V, L90M, and G48V/L90M) were introduced into clones used for the expression, purification, and further characterization of the enzyme. The mutant enzymes were all less active than the wild-type enzyme, as judged by k(cat) and k(cat)/Km values. L90M had a lower Km than the wild type, whereas the G48V/L90M double mutant had an increased Km compared with that of the wild type, contributing to a 10-fold reduction in the k(cat)/Km. Vitality values were used to show that the enzyme of the I84V mutant is the enzyme most resistant to the two cyclic urea inhibitors DMP 323 and AHA 008. Virus with the same mutation is also resistant, although the double mutation L10F/I84V confers even greater resistance. All of these mutants are more resistant to DMP 323 than to AHA 008. The resistance of the I84V mutant may be attributed to a loss of van der Waals interactions with the inhibitor, since the larger amino acid side chain involved in the interaction is replaced by a smaller side chain. This is supported by the lower level of resistance to AHA 008 that was observed. The phenyl groups of AHA 008 should protrude deeper into the S1 and S1' subsites than those of the smaller compound DMP 323, reducing the loss of interaction energy. These results reveal that small structural modifications of inhibitors that do not affect the inhibitory effect on wild-type virus can influence the inhibition of resistant strains. This is of importance for optimizing drugs with respect to their potency and resistance.     

Interferon resistance of hepatitis C virus genotype 1b: relationship to nonstructural 5A gene quasispecies mutations

A 40-amino-acid sequence located in the nonstructural 5A (NS5A) protein of hepatitis C virus genotype 1b (HCV-1b) was recently suggested to be the interferon sensitivity-determining region (ISDR), because HCV-1b strains with an ISDR amino acid sequence identical to that of the prototype strain HCV-J were found to be resistant to alpha interferon (IFN-alpha) whereas strains with amino acid substitutions were found to be sensitive (N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, N. Izumi, F. Marumo, and C. Sato, J. Clin. Invest. 96:224-230, 1995; N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, Y. Ogura, N. Izumi, F. Marumo, and C. Sato, N. Engl. J. Med. 334:77-81, 1996). We used single-strand conformation polymorphism (SSCP) analysis, combined with cloning and sequencing strategies, to characterize NS5A quasispecies in HCV-1b-infected patients and determine the relationships between pre- and posttreatment NS5A quasispecies mutations and the IFN-alpha sensitivity of HCV-1b. The serine residues involved in phosphorylation of NS5A protein were highly conserved both in the various patients and in quasispecies in a given patient, suggesting that phosphorylation is important in NS5A protein function. A hot spot for amino acid substitutions was found at positions 2217 to 2218; it could be the result of either strong selection pressure or tolerance to these amino acid replacements. The proportion of synonymous mutations was significantly higher than the proportion of nonsynonymous mutations, suggesting that genetic variability in the region studied was the result of high mutation rates and viral replication kinetics rather than of positive selection. Sustained HCV RNA clearance was associated with low viral load and low nucleotide sequence entropy, suggesting (i) that the replication kinetics when treatment is started plays a critical role in HCV-1b sensitivity to IFN-alpha and (ii) that HCV-1b resistance to IFN-alpha could be conferred by numerous and/or related mutations that could be patient specific and located at different positions throughout the viral genome and could allow escape variants to be selected by IFN-alpha-stimulated immune responses. No NS5A sequence appeared to be intrinsically resistant or sensitive to IFN-alpha, but the HCV-J sequence was significantly more frequent in nonresponder quasispecies than in sustained virological responder quasispecies, suggesting that the balance between NS5A quasispecies sequences in infected patients could have a subtle regulatory influence on HCV replication.     

Follitropin (FSH) deficiency in an infertile male due to FSHbeta gene mutation. A syndrome of normal puberty and virilization but underdeveloped testicles with azoospermia, low FSH but high lutropin and normal serum testosterone concentrations

Vitamin K deficiency bleeding within the first 24 h of life is caused in most cases by maternal drug intake (e.g. coumarins, anticonvulsants, tuberculostatics) during pregnancy. Haemorrhage is often life-threatening and usually not prevented by vitamin K prophylaxis at birth. We report a case of severe intracranial bleeding at birth secondary to phenobarbital-induced vitamin K deficiency and traumatic delivery. Burr hole trepanations of the skull were performed and the subdural haematoma was evacuated. Despite the severe prognosis, the infant showed an unexpected good recovery. At the age of 3 years, neurological examinations were normal as was the EEG at the age of 9 months. CT showed close to normal intracranial structures. CONCLUSION: This case report stresses the importance of antenatal vitamin K prophylaxis and the consideration of a primary Caesarean section in maternal vitamin K deficiency states and demonstrates the successful management of massive subdural haemorrhage by a limited surgical approach.     

Macular imaging with optical coherence tomography

PURPOSE: Optical Coherence Tomography (OCT) is a novel noninvasive and noncontact imaging technique providing cross-sectional representations of the eye structures. OCT is analogous to Ultrasound B-scan, except that it analyzes the reflection of a 850 nm light wave. The aim of this study was to assess the potential of ocular coherence tomography for diagnosing and monitoring macular diseases. METHODS: Cross-sectional images were performed with the Zeiss-Humphrey OCT. Over one year period, we examined approximately 300 patients with idiopathic full thickness macular hole, lamellar hole, cystoid macular edema, choroidal new vessels, epiretinal membrane, diabetic maculopathy, and central serous chorioretinopathy. RESULTS: OCT can provide new information concerning the posterior pole diseases mentioned above. OCT can also be useful in thickness measurements. CONCLUSION: OCT allows tomographic analysis of macular diseases. The information obtained is different from that obtained by histologic study which is sometimes hard to interprete. OCT is mostly useful in studying internal layers of the retina. Further applications may be developed.     

DNA structure properties and phospholipid composition of Salmonella derby

The properties of DNA structure and the phospholipid content of Salmonella derby cells were studied with respect to their plasmid content and radiosensitivity. The role of R-plasmid in determining the qualitative and quantitative compositions of S. derby phospholipids was revealed. The radiosensitivity of plasmid-carrying S. derby mutants was shown to be most likely determined by the structure of DNA, its GC content, and the level of methylation. We suggest that the phospholipid molecules and their interaction with DNA play a key role in formation of the radio-resistance of plasmid-free S. derby cells.     

Management of childhood splenic trauma--the Jos experience

We studied retrospectively 22 children who were managed for splenic injuries at Jos University Teaching Hospital between September 1988 and October 1995. Sixteen were males and six were females (M:F = 2.7:1) with their ages between five and 15 years and a mean of 10.3 years. Eleven were knocked down by vehicles while crossing the freeway, playing or hawking goods; seven fell from slippery mango trees after a downpour of rain while two were passengers in a vehicle that was involved in a head-on collision and another two were kicked in the abdomen as a result of assault. All had surgery. Four sustained Uphadhyaya and Simpson's Type 1 injuries while six sustained Type IV injuries. Twelve had total splenectomy, seven had total splenectomy with heterotopic autotransplantation of splenic wafers while three had splenorrhaphy augmented with omentoplasty. An average of 1.5 units of blood per patient were transfused. There was only one death. Considering that the majority (18) of these children sustained their splenic injuries while crossing the freeway (playing or hawking goods) and from falls from mango trees after the rains, we believe that there are preventable causes. Mandatory primary and junior secondary school education as well as legislature against child labour will help keep these children safe out of harm's way and thus drastically reduce the incidence of childhood splenic injuries in our environment.