Mutant GABA(A) receptor gamma2-subunit in childhood absence epilepsy and febrile seizures.

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants. We have found a mutation in a gene encoding a GABA(A) receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS). There is a recognized genetic relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the gamma2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.

Tumor protease-activated, pore-forming toxins from a combinatorial library.

We describe a library of two-chain molecular complementation mutants of staphylococcal alpha-hemolysin that features a combinatorial cassette encoding thousands of protease recognition sites in the central pore-forming domain. The cassette is flanked by a peptide extension that inactivates the protein. We screened the library to identify alpha-hemolysins that are highly susceptible to activation by cathepsin B, a protease that is secreted by certain metastatic tumor cells. Toxins obtained by this procedure should be useful for the permeabilization of malignant cells thereby leading directly to cell death or permitting destruction of the cells with drugs that are normally membrane impermeant.

Restoration of calcium handling properties of adult cardiac myocytes from hypertrophied hearts.

Reductions in cardiac sarcoplasmic reticulum calcium-ATPase (Serca2a) levels are thought to underlie the prolonged calcium (Ca(2+)) transients and consequent reduced contractile performance seen in human cardiac hypertrophy and heart failure. In freshly isolated cardiac myocytes from rats with monocrotaline-induced right ventricular hypertrophy we found reduced sarcoplasmic reticulum Serca2a expression and prolonged Ca(2+)transients, characteristic of hypertrophic cardiac disease. Modulation of intracellular Ca(2+)levels, Ca(2+) kinetics or Ca(2+)sensitivity is the focus of many current therapeutic approaches to improve contractile performance in the hypertrophic or failing heart. However, the functional effects of increasing Serca2a expression on Ca(2+) handling properties in myocytes from an animal model of cardiac hypertrophy are largely unknown. Here, we describe enhancement of the deficient Ca(2+) handling properties evident in myocytes from hypertrophied hearts following adenoviral-mediated transfer of the human Serca2a gene to these myocytes. These results highlight the importance of Serca2a deficiencies in the hypertrophic phenotype of cardiac muscle and suggest a simple, effective approach for manipulation of normal cardiac function.

Partial functional correction of xeroderma pigmentosum group A cells by suppressor tRNA.

Genetic diseases are often caused by nonsense mutations. The resulting defect in protein translation can be restored by expressing suppressor tRNA in the mutant cells. Our goal was to demonstrate both protein restoration and phenotypic correction using these small transgenes. Functional activity of an arginine opal suppressor tRNA in cells expressing a nonsense mutated GFP gene was demonstrated by restored fluorescence. This suppressor tRNA was expressed in xeroderma pigmentosum group A cells, containing a homozygous nonsense mutation at Arg-207 in the XPA complementing gene. The transfected XPA cell population showed a twofold increase in cell survival after UV irradiation as determined by colony-forming assays compared with cell populations without the suppressor tRNA gene. The UV doses required for 37% survival of XP cells and XP cells expressing the suppressor tRNA were 0.6 and 1.2 J/m2. A similar twofold increase in the reactivation of UV-irradiated plasmid DNA was observed in XP cells expressing the suppressor tRNA. However, there was no detectable increase in XPA protein levels. Several potential limitations of this approach exist, including the availability of mutant RNA transcripts, the efficiency of suppression by the suppressor tRNA, and the abundance and availability and continued expression of the suppressor tRNA. The unique feature of this study is the relatively small size (88 bp) of the suppressor tRNA. Small-sized suppressor tRNAs can be synthetically constructed and subcloned into different viral vectors for delivery into the target cells. This approach may be useful for other genetic diseases caused by nonsense mutations.

Combinatorial RNA splicing alters the surface charge on the NMDA receptor.

Transcripts encoding four NMDA receptor subunits, generated from the NMDAR1 gene by alternative RNA splicing, have been demonstrated in adult rat brain. RNA transcripts derived from cDNAs encoding each form direct the formation of functional NMDA receptors in Xenopus oocytes. The two amino acid cassettes of 21 and 37 amino acids found in the splice variants increase the positive extracellular surface charge on the subunits and may thereby modulate the functional properties of the receptor.

Novel therapeutic strategies to selectively kill cancer cells.

Tumor invasion, metastasis, and resistance to chemotherapeutic drugs or radiation are major obstacles for the successful treatment of cancer. To overcome some of these limitations, therapeutic strategies that increase the specificity and efficacy and reduce the toxicity of the anti-cancer drugs or toxins are being explored. Cancer cells overexpress specific protein antigens and carbohydrate structures that may function as cell surface receptors. These cancer cell specific markers can be exploited while designing new cancer therapies. Monoclonal antibodies that have been humanized to reduce immunogenicity and targeted to specific antigens on cancer cells, enzyme-monoclonal antibody/prodrug conjugates that will selectively kill the target cells following drug activation, and recombinant toxins are some of the novel classes of agents in development. Another novel approach being investigated to treat cancers is the use of inactive pore-forming toxins with built-in biological "triggers" that will activate the toxin following a biological stimulus. These pore-forming cytolytic toxins can be rendered active by tumor-specific proteases, that are often overexpressed in cancer cells, thereby targeting the toxic effects. Such pore-forming or membrane-acting toxins may serve as novel cytolytic agents against solid tumors, which, to date, have proved to be more resistant to conventional toxins.

Pore-forming proteins and their application in biotechnology.

Proteins and peptides that form membrane-spanning pores and channels comprise a diverse class of molecules ranging from short peptides that are unregulated and create non-selective pathways to large ion channel proteins that are highly regulated and exhibit exquisite selectivity for particular ions. The diversity of regulation and selectivity, together with recent advances in protein "re-engineering" technology, provide a strong framework on which to build custom molecules with wide-ranging biotechnological application. Here we review a selection of pore-forming peptides and proteins from a number of different species to highlight their structural and functional diversity. The current and potential uses of native and re-engineered molecules are discussed together with a novel strategy to re-engineer alpha-hemolysin to create targeted and regulable cell-killing agents termed proimmunolysins. Numerous pore-forming peptides are currently in development as antimicrobial agents with potential application as anti-tumorigenic agents. In addition to their roles as biotherapeutic agents, pore-forming proteins are also being developed as biosensors for a range of different analytes. Recent examples of this technology include the use of alpha-hemolysin with an adapter molecule to create sensors for organic molecules and gramicidin as a general-purpose sensor for a range of analytes. These approaches promise to deliver a configurable binding site for analytes encoded in a readily measured electrical signal. The number of applications for pore-forming molecules is sure to grow in both quantity and diversity with increased knowledge of the fundamental structure and function of pores.

Restriction fragment length polymorphisms of the human N-myc gene in normal healthy individuals and oral cancer patients in India.

Southern blot hybridization with N-myc oncogene probes coding for different regions of the N-myc gene demonstrated three polymorphic restriction sites in the Indian population. The SphI and PvuII polymorphic pattern due to the SphI polymorphic site in the second intron and the PvuII polymorphic site in the 3'-region of the human N-myc oncogene respectively, was similar to that reported in the Japanese population. The allelic frequency distribution for SphI polymorphism did not differ significantly for the S1 and S2 alleles representing presence (allele S1) or absence (allele S2) of a SphI site. However, the allelic frequency distribution was distorted in the case of PvuII polymorphism, as the frequency of P1 allele (0.7) indicating presence of PvuII site, was higher than the P2 allele (0.3) indicating absence of PvuII site, in the Indian population. An additional polymorphic HindIII site localised in the second intron of the N-myc gene was also observed in both the Indian oral cancer patients and the normal healthy individuals, indicating that this RFLP was not tumor associated and may perhaps represent N-myc alteration in the Indian population.

Interactions between residues in staphylococcal alpha-hemolysin revealed by reversion mutagenesis.

alpha-Hemolysin (alpha HL), a pore-forming polypeptide of 293 amino acids, is secreted by Staphylococcus aureus as a water-soluble monomer. Residues that play key roles in the formation of functional heptameric pores on rabbit red blood cells (rRBC) have been identified previously by site-directed mutagenesis. alpha HL-H35N, in which the histidine at position 35 of the wild-type sequence is replaced with asparagine, is nonlytic and is arrested in assembly as a heptameric prepore. In this study, second-site revertants of H35N that have the ability to lyse rRBC were generated by error-prone PCR under conditions designed to produce single base changes. The analysis of 22 revertants revealed new codons clustered predominantly in three distinct regions of the H35N gene. One cluster includes amino acids 107-111 (four revertants) and another residues 144-155 (five revertants). These two clusters flank the central glycine-rich loop of alpha HL, which previously has been implicated in formation of the transmembrane channel, and encompass residues Lys-110 and Asp-152 that, like His-35, are crucial for lytic activity. The third cluster lies in the region spanning amino acids 217-228 (eight revertants), a region previously unexplored by mutagenesis. Single revertants were found at amino acid positions 84 and 169. When compared with H35N, the heptameric prepores formed by the revertants underwent more rapid conversion to fully assembled pores, as determined by conformational analysis by limited proteolysis. The rate of conversion to the fully assembled pore was strongly correlated with hemolytic activity. Previous work has suggested that the N terminus of alpha HL and the central loop cooperate in the final step of assembly. The present study suggests that the key N-terminal residue His-35 operates in conjunction with residues flanking the loop and C-terminal residues in the region 217-228. Hence, reversion mutagenesis extends the linear analysis that has been provided by direct point mutagenesis.

Differential phosphorylation of neuronal substrates by catalytic subunits of Aplysia cAMP-dependent protein kinase with alternative N termini.

cAMP-dependent protein kinase (PKA) is an important participant in neuronal modulation: the ability of neurons to change their properties in response to external stimuli. In Aplysia mechanosensory neurons, PKA plays roles in both short and long term presynaptic facilitation, which is a simple model for learning and memory. PKA in Aplysia is a collection of structurally and functionally diverse regulatory and catalytic (C) subunits. We have argued that this diversity may in part account for the ability of the enzyme to take part in neuronal events that are spatially and temporally separated. Here, we add credence to this hypothesis by showing that C subunits of Aplysia PKA with alternative N termini target different substrates in subcellular fractions from Aplysia neurons, despite their similar actions on synthetic peptide substrates. Purified recombinant CAPL-AN1A1, which has an N terminus that is homologous to the myristylated sequence described in mammals, catalyzes the formation of two phosphoproteins of 24 and 8 kDa more rapidly than CAPL-AN2A1, which has a distinct N terminus weakly related to that of the yeast TPK1 gene product. The 24-kDa phospoprotein, but not the 8-kDa species, is detected in taxol-stabilized microtubules, suggesting that it is associated with the cytoskeleton. CAPL-AN2A1, in contrast, generates a 55-kDa phosphoprotein that is not observed with CAPL-AN1A1. The 55-kDa species is found in the detergent supernatant of the cytoskeleton fraction. Differential targeting of substrates by C subunits of PKA may therefore contribute to the ability of this kinase to play multiple roles in neuronal modulation.