Ion-pairs in proteins

A "working definition" for an ion-pair has been derived based upon analysis of the distance distributions for like- and oppositely charged groups in 38 proteins. Ion-pairs defined according to this criterion (less than or equal to 4 A between charged groups) have been analysed in respect of: (1) the frequencies of different pair types; (2) the residue separations and secondary structural locations of the residues involved; (3) the flexibility of the side-chains involved; (4) their conformation; (5) their environment (accessibility to solvent and proximity to active site or ligand binding regions); and (6) their conservation in related proteins. The results obtained indicate that on average one-third of the charged residues in a protein are involved in ion-pairs and 76% of these are concerned with stabilizing the tertiary (rather than the secondary) structure. Only 17% of ion-pairs are buried, and conservation of the interactions is generally low unless the residues involved have more specific functions to perform. In the light of the results obtained, the role of ion pairs in globular proteins is discussed.     

Spontaneous output of acetylcholine from rat diaphragm preparations declines after treatment with botulinum toxin

A gas chromatographic spectrometric assay was used to measure tissue and released acetylcholine and choline in diaphragm preparations of rats previously injected with botulinum toxin type A. Botulinum intoxication was found not to alter the acetylcholine content of rat diaphragms in vivo or in fully paralyzed muscles in vitro. This result provides direct support for the hypothesis that botulinum toxin blocks transmitter release without affecting acetylcholine synthesis. However, in diaphragm preparations in vitro, this toxin was found to inhibit not only the evoked release of acetylcholine but also the spontaneous "leakage" of acetylcholine that is measured at rest. Additional experiments were performed to characterize this action of the toxin. The magnitude of the decline in resting acetylcholine output appears to be too large to be accounted for solely by the known effect of botulinum toxin to reduce the frequency of miniature endplate potentials. The mechanism of this action of botulinum toxin remains an enigma.     

Synthesis in vitro of intrinsic membrane proteins by free, membrane-bound, and Golgi apparatus-associated polyribosomes from rat liver

A fraction of intrinsic membrane proteins was prepared from the major membranous cell components of rat liver by extraction of the membranes with KCl and deoxycholate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the compositions of the intrinsic protein fractions from rough and endoplasmic reticulum, smooth endoplasmic reticulum. Golgi apparatus, plasma membrane, and nuclear envelope were similar to each other but distinct from that of mitochondria. Among endomembranes, differences were in the ratios of protein constituents plus a few protein bands of Golgi apparatus and plasma membranes not found in endoplasmic reticulum or nuclear envelope. The abilities of total rough endoplasmic reticulum, polysomes released from rough endoplasmic reticulum, and free polysomes to incorporate amino acids into the intrinsic protein fraction were tested in vitro. Polysomes bound to endoplasmic reticulum has the greatest capacity to synthesize proteins of this fraction as shown by co-purification of radioactive products and by immunoprecipitation. Although the majority of the radioactive products synthesized by bound polysomes were distinct from those synthesized by free polysomes, certain radioactive products synthesized by free polysomes also co-purified with intrinsic membrane proteins. The results show no absolute segregation between free and bound polysomes in the synthesis of intrinsic membrane proteins. However, the majority of these proteins appear to be synthesized by polysomes bound to the endoplasmic reticulum. Several intrinsic proteins found in plasma membranes do not appear in rough endoplasmic reticulum. To determine where these proteins were synthesized, the ability of other endomembrane components to support in vitro incorporation of [14C]leucine into protein was examined. In contrast to plasma membranes, isolated Golgi apparatus fractions did incorporate [14C]leucine to an extent greater than could be explained by contamination with rough endoplasmic reticulum. Golgi apparatus in situ and isolated from rat liver have polyribosomes associated with a zone of cytoplasm at the Golgi apparatus periphery occupied by tubules and vesicles. The polysomes are not directly attached to membranes as with rough endoplasmic reticulum and may represent a special class of "Golgi apparatus-associated" polysomes. The polysomes, when associated with Golgi apparatus membranes, incorporated amino acids in vitro. The products synthesized in vitro were analyzed by treatment with KCl and deoxycholate and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Certain proteins synthesized by the Golgi apparatus-associated polysomes remained insoluble after the treatment with KCl and deoxycholate. The proteins synthesized by the Golgi apparatus fraction had mobilities similar to proteins in plasma membranes which were absent from endoplasmic reticulum, and which were relatively minor components of Golgi apparatus...     

Diagnosis and epidural hematoma by brain scan and perfusion study: case report

By using the arterial and venous phases of an anterior cerebral perfusion study, which showed downward displacement of the sagittal sinus, and the finding of a "rim" on the delayed scans, the specific diagnosis of epidural hematoma was established.     

Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer

Adenovirus (Ad) vectors are promising for gene therapy of glioma due to their ability to achieve efficient gene transfer upon intratumoral administration. Yet in this context, Ad mediates widespread gene transfer to both tumor and surrounding parenchyma. Ad entry is dependent upon the expression of fiber receptors, such as coxsackie/adenovirus receptor, and alpha(v) integrins on the target cells for binding and internalization, respectively. We hypothesized that the susceptibility of human gliomas to Ad would likely be heterogeneous due to variable expression of these receptors. It was found that established human glioma cell lines exhibited differential susceptibility to Ad-mediated gene transfer, which correlated directly with the level of radiolabeled Ad binding and with the expression of coxsackie/adenovirus receptor but not with the expression of alpha(v) integrins. To circumvent the lack of fiber receptors and to target Ad gene transfer specifically to tumor cells, we used a bispecific antibody conjugate to ablate Ad binding to fiber receptors and retarget binding to the epidermal growth factor receptor (EGFR), a tumor-associated marker negligibly expressed in normal, mitotically quiescent neural tissues. The results demonstrate that EGFR-targeted Ad gene transfer was EGFR specific and independent of fiber-fiber receptor interactions. Furthermore, EGFR targeting significantly enhanced Ad gene delivery to 7 of 12 established glioma cell lines and to 6 of 8 cultured primary gliomas. Interestingly, EGFR-targeted Ad gene transfer did not correlate with EGFR expression across cell lines, suggesting the importance of other factors. This study establishes that fiber receptor expression limits the utility of Ad vectors for gene transfer to glioma cells and suggests that targeting Ad via EGFR may prove valuable for tumor-specific gene transfer to high-grade gliomas. These findings have key relevance in the context of Ad vector-based approaches for glioma gene therapy.     

Coarse particulate matter and airborne endotoxin within wood stove homes

Emissions from indoor biomass burning are a major public health concern in developing areas of the world. Less is known about indoor air quality, particularly airborne endotoxin, in homes burning biomass fuel in residential wood stoves in higher income countries. A filter‐based sampler was used to evaluate wintertime indoor coarse particulate matter (PM_10‐2.5) and airborne endotoxin (EU/m³, EU/mg) concentrations in 50 homes using wood stoves as their primary source of heat in western Montana. We investigated number of residents, number of pets, dampness (humidity), and frequency of wood stove usage as potential predictors of indoor airborne endotoxin concentrations. Two 48‐h sampling events per home revealed a mean winter PM_10‐2.5 concentration (± s.d.) of 12.9 (± 8.6) μg/m³, while PM_2.5 concentrations averaged 32.3 (± 32.6) μg/m³. Endotoxin concentrations measured from PM_10‐2.5 filter samples were 9.2 (± 12.4) EU/m³ and 1010 (± 1524) EU/mg. PM_10‐2.5 and PM_2.5 were significantly correlated in wood stove homes (r = 0.36, P < 0.05). The presence of pets in the homes was associated with PM_10‐2.5 but not with endotoxin concentrations. Importantly, none of the other measured home characteristics was a strong predictor of airborne endotoxin, including frequency of residential wood stove usage.     

Effects of plasma total ammonia content and pH on urea excretion in Nile tilapia

Nile tilapia (Oreochromis niloticus) were infused with ammonium salts, acid, and base to investigate the effects of changes in arterial plasma total ammonia content (Tamm) and pH (pHa) on plasma urea-nitrogen (urea-N) levels and urea-N excretory fluxes (Jurea-N). The tilapia did not possess a functional hepatic ornithine urea-cycle (no significant carbamyl phosphate synthetase III activity). Infused substances were dissolved in a saline vehicle and injected twice (5 mL kg-1), the first infusion to "prime" the animal and promote a more marked response to the second infusion, given 2.5 h later. The results reported are those of the second infusion. Infusion of 200 mM NH4Cl increased Tamm, reduced pHa, and increased plasma urea-N and Jurea-N. Two hundred mM NH4HCO3 increased Tamm and arterial plasma total CO2 content (TaCO2), reduced pHa, and increased Jurea-N. Fifty mM HCl reduced pHa but had no effects on urea dynamics. Fifty mM NaOH increased pHa, plasma urea-N levels, and Jurea-N. Two hundred mM NaHCO3 increased pHa, TaCO2, plasma urea-N levels, and Jurea-N. Infusion of the saline vehicle was without effect. The results indicate that ammonia loading and plasma alkalosis both stimulate urea excretion in uricolytic fish. The responses to hyperammonemia or alkalosis were not modified when combined with elevated plasma bicarbonate levels.     

Hinged Ilizarov external fixation for correction of antebrachial deformities

OBJECTIVE: To evaluate hinged circular external fixation for correction of antebrachial deformities in dogs. STUDY DESIGN: Uncontrolled clinical trial. ANIMAL POPULATION: Seven client-owned dogs. METHODS: Six dogs had one radius corrected and one dog had both radii corrected. Preoperative planning included measurement of the craniocaudal and mediolateral angular deformities, rotational deformity, length deficit, origin of deformity, graphical or mathematical determination of the amplitude and direction of the actual limb deformity, and frame assembly. RESULTS: Preoperatively, function and cosmesis were assessed to be fair to poor in all dogs. Deformity correction started 48 to 60 hours postoperatively and ranged from 0.46 mm to 1.36 mm twice daily. Hospitalization time ranged from 4 to 6 days. Corrections were mostly made by the owners, at home. Lengthening and angular correction ranged from 3 to 38 mm and 18 degrees to 48 degrees. Mean residual deformities were 2.7% of radial length and 2.7 degrees. The time duration with the circular external fixators in place ranged from 29 to 71 days. Two additional surgeries were necessary in one dog because of wire breakage. Mean follow-up was 40 months. Long-term function and cosmesis were good to excellent in all dogs. CONCLUSION: Although complications were present in six of seven dogs, the outcome of hinged Ilizarov external fixation was successful in all dogs treated for deformities of the antebrachium. CLINICAL RELEVANCE: Despite complex preoperative planning, the placement of hinged circular external fixators is straightforward, and allows precise correction of complex antebrachial deformities with minimal tissue trauma.