On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids

25 aromatic carboxylic acids which are analogs of benzoic acid were tested in the rat diaphragm preparation for effects on chloride conductance (G(Cl)). Of the 25, 19 were shown to reduce membrane G(Cl) with little effect on other membrane parameters, although their apparent K(i) varied widely. This inhibition was reversible if exposure times were not prolonged. The most effective analog studied was anthracene-9-COOH (9-AC; K(i) = 1.1 x 10(-5) M). Active analogs produced concentration-dependent inhibition of a type consistent with interaction at a single site or group of sites having similar binding affinities, although a correlation could also be shown between lipophilicity and K(i). Structure-activity analysis indicated that hydrophobic ring substitution usually increased inhibitory activity while para polar substitutions reduced effectiveness.

These compounds do not appear to inhibit G(Cl) by altering membrane surface charge and the inhibition produced is not voltage dependent. Qualitative characteristics of the I-V relationship for Cl(-) current are not altered. Conductance to all anions is not uniformly altered by these acids as would be expected from steric occlusion of a common channel. Concentrations of 9-AC reducing G(Cl) by more than 90 percent resulted in slight augmentation of G(I). The complete conductance sequence obtained at high levels of 9-AC was the reverse of that obtained under control conditions. Permeability sequences underwent progressive changes with increasing 9-AC concentration and ultimately inverted at high levels of the analog. Aromatic carboxylic acids appear to inhibit G(Cl) by binding to a specific intramembrane site and altering the selectivity sequence of the membrane anion channel.

     

Extensive genomic and functional polymorphism of the complement control proteins

Using combinations of genomic markers, we describe more than 20 distinct ancestral haplotypes (AH) of complement control proteins (CCPs), located within the regulators of complement activation (RCA) alpha block at 1q32. This extensive polymorphism, including functional sites, is important because CCPs are involved in the regulation of complement activation whilst also serving as self and viral receptors. To identify haplotypes, we used the genomic matching technique (GMT) based on the pragmatic observation that extreme nucleotide polymorphism is packaged with duplicated sequences as polymorphic frozen blocks (PFB). At each PFB, there are many alternative sequences (haplotypes) which are inherited faithfully from very remote ancestors. We have compared frequencies of RCA haplotypes and report differences in recurrent spontaneous abortion (RSA) and psoriasis vulgaris (PV).     

Amino Acid Patterns Within Short Consensus Repeats Define Conserved Duplicons Shared by Genes of the RCA Complex

Complement control proteins (CCPs) contain repeated protein domains, short consensus repeats (SCRs), which must be relevant to diverse functions such as complement activation, coagulation, viral binding, fetal implantation, and self-nonself recognition. Although SCRs share some discontinuous and imperfect motifs, there are many variable positions and indels making classification in subfamilies extremely difficult. Using domain-by-domain phylogenetic analysis, we have found that most domains can be classified into only 11 subfamilies, designated a, b, c, d, e, f, g, h, i, j, or k and identified by critical residues. Each particular CCP is characterized by the order of representatives of the subfamilies. Human complement receptor 1 (CR1) has ajefbkd repeated four times and followed by ch. The classification crosses CCPs and indicates that a particular CCP is a function of the mix of SCRs. The aje set is a feature of several CCPs including human CR1 and DAF and murine Crry and appears to be associated with the success or failure of implantation inter alia. This approach facilitates genomic analysis of available sequences and suggests a framework for the evolution of CCPs. Units of duplication range from single SCRs, to septamers such as efbkdaj, to extensive segments such as MCP-CR1L. Imperfections of duplication with subsequent deletion have contributed to diversification.     

Allelic and interlocus comparison of the PERB11 multigene family in the MHC

 The major histocompatibility complex (MHC) contains at least a hundred genes over 4 megabases of DNA. Within the MHC there are several new multigene families which have been recently described. PERB11 is a multigene family which occurs over the class I and central region of the MHC. Two members of the family have been shown to be functional and share domains with members of the supergene family including HLA class I, FcRn, and Zn-α2-glycoprotein molecules. The two functional members are contained within an area of the MHC which has been associated with increased susceptibility to autoimmune diseases such as insulin-dependent diabetes mellitus and also rapid progression to AIDS following HIV-1 infection. Intralocus and interlocus differences between PERB11.1 and PERB11.2 include: (1) several nucleotide substitutions leading to amino acid changes; (2) presence and absence of potential glycosylation sites; (3) insertions and deletions leading to a frame shift resulting in diversity at the amino acid level and an early termination signal. There are ten different alleles of PERB11.1 including one allele which contains a frame shift in the transmembrane region causing a putative truncated molecule lacking the cytoplasmic tail. The significance of this polymorphism in disease associations is under investigation. The most divergent domain is the transmembrane region when PERB11.1 and PERB11.2 are compared. The results suggest that these two molecules may have different functions. Received: 23 July 1996 / Revised: 17 September 1996     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

The isolation of nucleic acid from nematodes requires an understanding of the parasite and its cuticular structure

As parasitology increasingly becomes the domain of molecular biologists, it is important to keep in mind that a fundamental understanding of the whole parasite, its structure and behaviour can help to solve complex problems of molecular biology. Hugh Dawkins and Terence Spencer discuss the preparation of DNA from filaform larvae of Trichostrongylus colubriformis and Ostertagia circumcincta and how a detailed knowledge of the morphology and life cycle of each parasite helps in this process.