A conserved checkpoint pathway mediates DNA damage--induced apoptosis and cell cycle arrest in C. elegans.

To maintain genomic stability following DNA damage, multicellular organisms activate checkpoints that induce cell cycle arrest or apoptosis. Here we show that genotoxic stress blocks cell proliferation and induces apoptosis of germ cells in the nematode C. elegans. Accumulation of recombination intermediates similarly leads to the demise of affected cells. Checkpoint-induced apoptosis is mediated by the core apoptotic machinery (CED-9/CED-4/CED-3) but is genetically distinct from somatic cell death and physiological germ cell death. Mutations in three genes--mrt-2, which encodes the C. elegans homolog of the S. pombe rad1 checkpoint gene, rad-5, and him-7-block both DNA damage-induced apoptosis and cell proliferation arrest. Our results implicate rad1 homologs in DNA damage-induced apoptosis in animals.

A mechanistic analysis of carrier-mediated oral delivery of protein therapeutics.

This article summarizes the results of a theoretical analysis of protein absorption into the systemic circulation from the small intestine, with and without molecular 'carriers' designed to enhance absorption. The predictions are compared with experimental systemic protein concentrations following intraduodenal delivery of insulin, interferon alpha-2b, and human growth hormone. The results show that, from the standpoint of improving oral absorption, the primary consequence of carrier molecules is to increase epithelial membrane permeability, thereby leading to higher bioavailability. Several possible mechanisms of this permeability enhancement are discussed.

Thymosin alpha1 treatment of chronic hepatitis B: results of a phase III multicentre, randomized, double-blind and placebo-controlled study.

Previous clinical trials have suggested that thymosin alpha1 (Talpha1), an immunomodulatory peptide, may be effective in the treatment of chronic hepatitis B (CHB). The aim of this study was to determine the efficacy of Talpha1 in a multicentre, placebo-controlled and double-blind study of 97 patients with serum hepatitis B virus (HBV) DNA- and hepatitis B e antigen (HBeAg)-positive CHB. Patients who had been hepatitis B surface antigen (HBsAg) positive for at least 12 months entered a 3-month screening period prior to randomization. Forty-nine patients received Talpha1 (1.6 mg) and 48 patients received placebo, twice weekly for 6 months, and were followed-up for an additional 6 months. At inclusion, both groups were comparable for age, gender, histological grading, and aminotransferase and HBV DNA levels. A complete response to treatment, defined as a sustained serum HBV DNA-negative status (two negative results at least 3 months apart) during the 12-month study, with negative HBV DNA and HBeAg values at month 12, was seen in seven (14%) patients given Talpha1 and in two (4%) patients treated with placebo (P = 0.084). Five (10%) patients given Talpha1 and four (8%) patients given placebo exhibited a delayed response (defined as sustained serum HBV DNA negativity achieved after the 12-month study period with negative HBV DNA and HBeAg values at the last assessment). A total of 12 (25%) patients given Talpha1 and six (13%) patients given placebo showed a sustained loss of HBV DNA with a negative HBeAg value during or following the 12-month study period (P < 0.11). These results do not confirm observations of treatment efficacy reported in other clinical studies.

Partially unfolded proteins efficiently penetrate cell membranes--implications for oral drug delivery.

We have previously reported on the biological activity of members of a library of low molecular weight compounds (carriers) that enable the oral delivery of proteins (Milstein, Proceedings of the 1995 Miami Bio/Technology Winter Symposium on Protein Engineering and Structural Biology, IRL Press at Oxford University Press, 1995, p. 13; Leone-Bay et al., J. Med. Chem. 38 (1995) 4263-4269; Leone-Bay et al., J. Med. Chem. 39 (1996) 2571-2578; [1-3]). When rats or primates are orally administered a solution of carrier and either recombinant human alpha-interferon (rhIFN), insulin or recombinant human growth hormone (rhGH) significant serum concentrations of the proteins are detectable. The transport activity of these compounds is positively correlated with their structural effects on the protein molecules. Direct measurement of the interaction of these carrier molecules with the proteins indicates that they reversibly destabilize the native state of the molecule favoring a partially unfolded conformation. Apparently these intermediate protein conformations are transport competent and are able to be absorbed through the intestinal tissue and into the bloodstream. Since the measured binding of the carriers to the partially unfolded proteins is relatively weak (Kb = 100 M(-1)) and the systemic activity of the proteins appears to be unaffected, the changes in the structure of the proteins are manifestly reversible.

Thermodynamic characterization of an intermediate state of human growth hormone.

The thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calorimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under these conditions a single two-state transition was observed between 0 and 100 degrees C. The magnitudes of the deltaH and deltaCp of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which show that significant secondary structure remains after the unfolding. Above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of alcohols such as n-propanol, 2-propanol, or 1,2-propanediol (propylene glycol), which suggests that the self-association of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in relatively high concentrations of propylene glycol (up to 45% v/v at pH 7-8 or 30% at pH 3) and that under these conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes significantly populated in 70-80% propylene glycol.

The conformational equilibrium of human growth hormone.

The structural stability of recombinant human growth hormone (rhGH) has been studied by differential scanning calorimetry, circular dichroism and by following the tyrosine and histidine chemical shifts in the 1H NMR spectrum. These studies demonstrate that the folding/unfolding equilibrium of rhGH involves a partially folded dimeric intermediate. The formation of this dimeric intermediate is a reversible process. At acid pH (pH 3) the conformational equilibrium is reversible even at high protein concentrations (10 mg/ml). At neutral pH reversibility is observed only at low protein concentrations (<0.5 mg/ml). The free energy of this intermediate conformation is only approximately 3 kcal/mol apart from the native state indicating that the conformational equilibrium can be effectively modulated by changes in solvent composition or physical conditions. According to the spectroscopic and thermodynamic results, the formation of the dimeric intermediate occurs without a major loss in helical content and is driven by the formation of substantial hydrophobic contacts between two partially folded molecules. A thermodynamic model that accounts quantitatively for the experimental data has been developed. These studies demonstrate that partially folded conformations of certain proteins are able to form stoichiometric complexes, and that the formation of these complexes provide a significant source of stabilizing Gibbs energy for conformational states that, otherwise, will be characterized by extremely unfavorable free energies.

Right atrial appendage to right ventricle accessory atrioventricular connection: a case report.

Atrial and ventricular attachments of accessory atrioventricular connections have classically been localized to adjacent atrial and ventricular tissues, and this principle is responsible for the widespread success of radiofrequency catheter ablation. We present anatomical evidence of an unusually located accessory atrioventricular connection, which bridged the atrioventricular ring epicardially, directly from the base of the right atrial appendage to the right ventricle. This observation might offer a new insight into unusual accessory atrioventricular connection locations and may explain why some endocardial radiofrequency catheter ablation procedures might fail.

The influence of molecular conformation upon the self-assembly of cyclohexane diamide diacids.

BACKGROUND: Information regarding the self-association of small peptide motifs can be used in the design of peptide microstructures. Previous work in our laboratories illustrated the self-association of certain diamide diacids into microcapsules. In this report a series of cyclohexane diamide diacids are investigated. The cyclohexylene (R-C6H10-R) system (with its axial and equatorial requirements) provided an opportunity to study the influence of molecular conformation upon the self-aggregation process. RESULTS: Condensation of the respective cis- and trans-1,2-, 1,3-, and 1,4- cyclohexane dicarboxylic acid platforms with two equivalents of a L-Phe ester followed by deprotection gave the desired diamide diacids. Basic solutions of cis-1,2-, trans-1,3-, and cis-1,4-diamide diacids generated solid microspheres when acidified to pH 2.4. Molecular modeling revealed that 1,3-diaxial interactions favor a helical turn within these diamides. CONCLUSIONS: Access to 'complementary' molecular geometries is needed to self-associate into microscopic architectures.

pH-dependent microspheres from modified soybean protein hydrolysate.

Soybean hydrolysate is a hydrophilic mixture of amino acids and low molecular peptides which are soluble over the whole pH range. Chemical modification of soybean hydrolysate with aromatic acyl chlorides resulted in a product that yielded pH-dependent microspheres. Investigation into the physiochemical properties of the microsphere forming material indicated that acylation had alerted the hydrophobic/hydrophilic ratio as evidenced by an increased column retention time on reverse phase HPLC. This was further confirmed by analysis of the amino acid composition of the modified material. The data indicated that the hydrophobic/hydrophilic ratio and low molecular were critical factors in the formation of this supramolecular complex. An estimation based on sedimentation rate revealed an average molecular weight of these microspheres as 10(7)-10(8) Daltons. Light scattering experiments indicated that the microspheres have discrete chambers in the interior. Included among the properties of the microspheres that have been determined are the pH range of the phase transition, size distribution and zeta-potential. The physiochemical characteristics of the microspheres prepared from modified soybean protein are similar to the microsphere forming material produced by thermal condensation of amino acids. Formation of microspheres in solution containing either porcine insulin or salmon calcitonin resulted in the encapsulation of nearly 60% of the dissolved proteins. Oral gavage of encapsulated porcine insulin or salmon calcitonin into the stomach of rats resulted in significant titers of either protein in the systematic circulation.

Radiation doses during pediatric radiofrequency catheter ablation procedures.

Because RF catheter ablation procedures may be lengthy and are commonly performed in young patients, concern has arisen about radiation dose in this group of patients. This article investigates radiation doses in pediatric patients undergoing RF catheter ablation. Standard fluoroscopic equipment used for diagnostic electrophysiological catheterization studies is technologically capable of dose rates as high as 90 milligray (mGy) per minute to skin and adjacent lung and 260 mGy/min to vertebral bone. Dose rates of this magnitude when used for extended periods of time have been known to cause erythema, pneumonitis, and retardation of bone growth. We measured skin dose rates of nine pediatric patients undergoing RF catheter ablation for tachycardia and calculated doses to the skin using standard dosimetric methods. Fluoroscopic techniques and equipment were studied using a patient simulating phantom. Overlap of fluoroscopic fields was checked using radiotherapy portal verification film, and regions in which doses overlapped from multiple angle exposures were verified using a treatment planning computer. Patient skin dose rates ranged from 6.2-49 mGy/min for patients ranging in age from 2-20 years. Maximum skin doses ranged from 0.09-2.35 Gy. Our data demonstrate the need to directly measure dose rates for individual fluoroscopic equipment and procedural techniques in order to determine whether limitations need to be set for procedural times.

Neural influence on protein kinase C isoform expression in skeletal muscle.

Protein kinase C (PKC) is a family of enzymes involved in synapse formation and signal transduction at the neuromuscular junction. Two PKC isoforms, classical PKC alpha and novel PKC theta, have been shown to be enriched in skeletal muscle or localized to the endplate. We examined the role of nerve in regulating the expression of these PKC isoforms in rat skeletal muscle by denervating diaphragm muscle and measuring PKC protein expression at various postoperative times. nPKC theta protein levels decreased 65% after denervation, whereas cPKC alpha levels increased 80% compared with control hemidiaphragms. These results suggest that innervation regulates PKC theta and alpha isoform expression in skeletal muscle. To explore further how nerve regulates PKC expression, we characterized PKC isoform expression in rat myotubes deprived of neural input. Myoblast expression of nPKC theta was low, and the increase in nPKC theta expression that occurred during differentiation into myotubes resulted in levels of nPKC theta significantly below adult skeletal muscle. cPKC alpha expression in myoblastic increased during differentiation to levels that exceeded expression in adult skeletal muscle. Coculturing myotubes within neuroblastoma X glioma hybrid clonal cell line (NG108-15) increased nPKC theta expression, but not cPKC alpha, suggesting that nPKC theta in skeletal muscle and myotubes is regulated by nerve contact or by a factor(s) provided by nerve. Treating myotubes with tetrodotoxin did not affect either basal- or NG108-15 cell-stimulated nPKC theta expression. Together these results suggest that expression of nPKC theta in skeletal muscle is regulated by a transynaptic interaction with nerve that specifically influences nPKC theta expression.

4-[4-[(2-Hydroxybenzoyl)amino]phenyl]butyric acid as a novel oral delivery agent for recombinant human growth hormone.

A series of N-acetylated, non-alpha, aromatic amino acids was prepared and shown to promote the absorption of recombinant human growth hormone (rhGH) from the gastrointestinal tract. Seventy compounds in this family were tested in vivo in rats. Of the compounds tested, 4-[4-[(2-hydroxybenzoyl)amino]phenyl]butyric acid was identified as a preclinical candidate and was used to demonstrate the oral delivery of rhGH in primates. A significant positive correlation was found between the relative log k' of the delivery agents, as determined by HPLC on an immobilized artificial membrane (IAM) column, and serum rhGH concentrations following oral or colonic dosing in rats. Structure-activity relationships have also been developed on the basis of electronic effects and hydrogen-bonding characteristics of the aromatic amide substituents.

A single procedure effectively treats concurrent atrioventricular reciprocating tachycardia and AV node reentry tachycardia

We report our experience regarding definitive treatment for concurrent atrioventricular reciprocating and AV node reentry tachycardia in 184 consecutive patients undergoing accessory connection ablation. 18 patients also had inducible sustained AV node reentry tachycardia. Sixteen out of 18 patients underwent accessory connection albation followed by AV node modification. There were no complications related to the ablation procedures. Fluoroscopy time for the combined procedures was 29 +/- 12 minut. There were no tachycardia recrrences during follow-up of 21 +/- 11 months. Our data show that both accessory connection ablation and AV node modification can be effectively and safely performed in a single procedure.

Therapeutic and diagnostic utility of adenosine during tachycardia evaluation in children.

Adenosine has become the drug of choice for termination of regular, normal QRS tachycardia. Initial studies in adult and pediatric patients have shown that the drug is effective for tachycardias using the atrioventricular (AV) node as an integral part of the tachycardia circuit and has few serious side effects. Experience with adenosine administration in children was reviewed to examine the diagnostic and therapeutic usefulness, effective dose, and adverse effects of adenosine. Adenosine was administered to 38 children during 50 separate electrophysiologic evaluations. Eleven patients had structural or acquired heart disease. Tachycardia mechanisms included orthodromic-reciprocating tachycardia using an accessory AV connection (23 patients), primary atrial tachycardia (6 patients), AV node reentrant tachycardia (3 patients), ventricular tachycardia (2 patients), postoperative junctional tachycardia (1 patient), and antidromic-reciprocating tachycardia (1 patient). Adenosine successfully terminated 51 of 53 episodes (96%) of tachycardia using the AV node, 5 of 10 primary atrial tachycardias, 1 of 1 junctional tachycardia, and 1 of 3 ventricular tachycardias. Reinitiation of tachycardia was seen after 16 of 58 successful terminations (28%), reducing the effectiveness to 39 of 53 (74%) for tachycardia requiring the AV node. Average effective dose was 132 micrograms/kg, range 50 to 250 micrograms/kg, and was slightly higher for peripheral (147 micrograms/kg) than for central (120 micrograms/kg) administration. Significant complications occurred in 4 of 38 patients, including atrial fibrillation, accelerated ventricular tachycardia, apnea, and 1 minute of asystole. Although adenosine is useful therapeutically and diagnostically in children with tachycardia, its effectiveness is limited by tachycardia reinitiation and adverse effects. Higher doses may be required for peripheral intravenous administration.

Stability study of drug-loaded proteinoid microsphere formulations during freeze-drying.

Drug-loaded proteinoid microspheres were freeze-dried to facilitate shipping and handling and to enable long term storage. Heparin was chosen as the model drug in developing the optimum lyophilization process. The factors influencing the integrity of either heparin-loaded or unloaded ('empty') proteinoid microspheres during freeze-drying were determined, with emphasis on: selecting an optimum freezing and resuspending temperature; choosing an appropriate cryoprotectant and its optimum concentration in the formulation; and, designing a suitable method for formulating the microspheres. Freezing at/below -70 degrees C was found to minimize damage to the microspheres. Addition of sugars, such as trehalose and lactose, as cryoprotectants, further increased the stability of the heparin-loaded microspheres during freeze-drying. The optimum trehalose or lactose concentrations were determined to be 5% (w/v). Using the optimumized lyophilization process described in this manuscript, microspheres remained intact during freeze-drying. The freeze-dried microspheres were stable for at least three months post-lyophilization.

Oral immunization of rats with proteinoid microspheres encapsulating influenza virus antigens.

Influenza virus antigen microspheres were prepared by a pH-dependent process using a protein-like polymer (proteinoid) made by thermal condensation of amino acids. The efficacy of these preparations to induce specific IgG responses when used as oral vaccines in rats was evaluated. A single enteric dose of M1 entrapped in proteinoid microspheres was able to induce a significant IgG response to M1 as early as 2 weeks postdosing, while rats dosed orally with the same M1 total dose (no microspheres) showed no detectable antibody response. An unencapsulated hemagglutinin and neuraminidase (HA-NA) preparation induced a moderate anti HA-NA IgG response. A single enteric dose of HA-NA spheres induced a response in 33% of the rats; this response was up to eight times higher than that observed in the rats dosed with unencapsulated antigen.

Anatomical mechanisms explaining damage to pacemaker leads, defibrillator leads, and failure of central venous catheters adjacent to the sternoclavicular joint.

The literature suggests that approximately 93% of all pacemaker lead fractures occur in the segment of the lead lateral to the venous entry, and costoclavicular compression has been implicated. While blood vessels can be compressed by movements of the clavicle, our research suggests that lead and catheter damage in that region is caused by soft tissue entrapment rather than bony contact. Dissection of eight cadavers with ten leads revealed that two entered the cephalic vein, and were not included in the study. Of the other eight leads, four passed through the subclavius muscle, two through the costoclavicular ligament, and two through both these structures before entering the subclavian, internal jugular, or brachiocephalic vein. Anatomical studies demonstrated that entrapment by the subclavius muscle or the costoclavicular ligament could cause repeated flexing of leads during movements of the pectoral girdle. Cineradiology of patients with position dependent catheter occlusion confirmed entrapment by the subclavius muscle. Soft tissue entrapment imposes a static load upon leads and catheters, and repeated flexure about the point of entrapment may be responsible for damage previously attributed to cyclic costoclavicular compression.

Adult Day Care centres in British Columbia: models, characteristics and services.

Adult Day Care (ADC) is increasingly being recognized as an important sub-system of the continuing care system. This paper reviews models developed in the United States and Britain and compares them, and the services they offer, with centres in British Columbia, Canada. Data on British Columbia are from a study in which all 49 centres in the province provided detailed information about their staffing, operating characteristics, activities and services. The study found B.C. compared favourably in providing services needed by ADC clients. Key differences between the B.C. centres and those in the U.S. and U.K. were: a larger proportion of B.C. centres were not affiliated with any other organization; B.C. centres admitted a range of clients and were less likely to cater exclusively to special needs groups; and, B.C. centres were more likely than centres in the U.S. to provide a number of services such as: dental care, transportation, bathing and physiotherapy.