Crystal structure of the extracellular segment of integrin alpha Vbeta3.

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

Radiological studies in the hot spring region of Mahallat, Central Iran.

Five hot springs called 'Abegarm-e-Mahallat', located in the central part of Iran, have a mean water temperature of 46 +/- 1 degrees C and are used by visitors as spas. This is an area of high natural radiation background due to the presence of (226)Ra and its decay products in the deposited travertine (CaCO(3)). The mean concentration of (226)Ra in these hot springs, measured by the emanation method, ranged from 0.48 +/- 0.05 to 1.35 +/- 0.13 Bq l(-1). (222)Rn concentrations measured in the hot springs using a liquid scintillation counter ranged from 145 +/- 37 to 2731 +/- 98 Bq l(-1). Mean radon concentrations in air were 487 +/- 160 and 15.4 +/- 2.7 Bq m(-3) for indoor and outdoor, respectively. Radiation levels above that of normal background ( approximately 100 nGy h(-1)) were mainly limited to the Quaternary travertine formations in the vicinity of the hot springs. The results of environmental radiological studies in this region are presented and discussed.

Immunohistochemical identification of T-lymphocytes in the central nervous system of patients with multiple sclerosis and subacute sclerosing panencephalitis.

T-lymphocytes were identified in frozen brain sections derived from patients with chronic inflammatory disorders of the CNS by using a specific heteroantiserum and the unlabelled antibody enzyme method. Clusters of T-cells were found in post-mortem material of cases with multiple sclerosis (MS) and subacute sclerosing panencephalitis (SSPE). The results suggest that T-lymphocytes are involved in the pathogenesis of both MS and SSPE.

Kinetics of horseradish peroxidase migration through cerebral cortex.

This study provides a semiquantitative description of the migration of the extracellular space marker, horseradish peroxidase (HRP), through the cerebral cortex. Following a continuous subarachnoid infusion of HRP, this marker was fixed rapidly within the cortical extracellular space (ECS) by intravascular aldehyde perfusion fixation. Microscopic measurements of the maximum depth of penetration of HRP into the cortex perpendicular to the pial surface were taken from coronal whole brain sections of rabbits that had been exposed to HRP for varying periods of time. The depth of penetration plotted as a function of time of exposure to HRP produced a 'diffusion profile'. The failure of experimental points to conform to an ideal diffusion curve indicates that simple diffusion alone is an inadequate explanation of the rate of movement of large molecular solutes from the subarachnoid space through the cerebral cortical ECS. The complex pattern of migration velocity of HRP through cortex may be due to alterations of flux of this solute which result from variations in the volume of ECS in different cortical laminae and to the presence of bulk flow of extracellular fluid in the deeper cortical regions.

Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells.

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.

Interhemispheric approach with callosal resection for distal anterior cerebral artery aneurysms. Technical note.

Distal anterior cerebral artery aneurysms are commonly found near the genu of the corpus callosum. While these aneurysms may be surgically obliterated through a variety of approaches, exposure via the interhemispheric fissure is used by many surgeons. Early identification of the afferent artery may be difficult with this approach, however, particularly if the aneurysm lies just beneath the genu of the corpus callosum. The authors have modified the interhemispheric approach to distal anterior cerebral artery aneurysms by electively exposing the feeding artery through a small anterior callosotomy. While this maneuver is not necessary for all distal anterior cerebral artery aneurysms, it can greatly enhance exposure in the region just below the genu of the corpus callosum. Experience with this technique in five patients is reported. In all cases, the limited anterior callosotomy enhanced surgical exposure. No morbidity could be attributed to the callosotomy in any patient. It is concluded that, when the interhemispheric approach is used, anterior callosotomy improves exposure of the region just below the genu of the corpus callosum and may be a useful maneuver when treating distal anterior cerebral artery aneurysms.

Surgical anatomy of the proximal anterior cerebral artery.

The authors present this study of proximal anterior cerebral arteries in the normal human to provide a clearer basis for strategy in aneurysm surgery. They describe patterns of origin of branches, their subarachnoid course, and parenchymal distribution. Branches that originate from the anterior cerebral artery at the internal carotid bifurcation perfuse the genu and contiguous posterior limb of the internal capsule and the rostral thalamus. Proximal 4-mm branches supply the anterior limb of the internal capsule, the neighboring hypothalamus, anteroventral putamen, and pallidum. The remaining anterior cerebral artery proximal to the communicating artery sends branches to the optic chiasm, the adjacent hypothalamus, and the anterior commissure. Heubner's artery arises directly opposite the anterior communicating artery to supply much of the striatum and internal capsule rostral to the anterior commissure. The anterior communicating artery branches supply the fornix, corpus callosum, septal region, and anterior cingulum. The parenchymal distribution of these end arteries may be surmised from the site of origin named vessels. With this anatomical information one can avoid interruption of blood supply to vital structures when dealing with the anterior cerebral artery and its branches.

Cerebral vasoconstriction as a complication of carotid endarterectomy. Case report.

The case is presented of an elderly man with an acute confusional state occurring soon after he had undergone a right carotid endarterectomy. Angiography demonstrated segmental areas of cerebral vasoconstriction and an electroencephalogram revealed periodic lateralized epileptiform discharges, both involving the right hemisphere. Cerebral hyperperfusion has been implicated in the genesis of several transient neurological syndromes following carotid endarterectomy. This case suggests that cerebral vasoconstriction may also be associated with impairment of cerebrovascular autoregulation observed after this procedure.

Traumatic atlanto-occipital dislocation. Case report.

Traumatic atlanto-occipital dislocation is a serious injury that is usually fatal. The number of patients surviving this injury, however, appears to be increasing, and most of these survivors are children. This may reflect an improvement in emergency transport services. Seventeen previously reported cases of patients surviving atlanto-occipital dislocation for more than 48 hours are reviewed and an additional case is presented. Many of these patients had an excellent neurological outcome. The radiographic criteria necessary for the diagnosis of atlanto-occipital dislocation are discussed. Cervical computerized tomography may confirm the diagnosis when necessary. It is suggested that there are three types of atlanto-occipital dislocation; utilizing this new classification, a rationale for treatment is described. Fusion is favored for long-term stability.

Implementation of the MARSSIM philosophy to evaluate the remedial status of a radioactive waste disposal site at the Idaho National Engineering and Environmental Laboratory. Multi-Agency Radiation Survey and Site Investigation Manual.

Implementation of the MARSSIM remedial-verification protocol at a radioactive waste disposal site is described in some detail to provide a record of the utility of this process. The selected site was the Stationary Low-Power Reactor No. 1 burial ground at the Idaho National Engineering and Environmental Laboratory. Evaluation was restricted to 137Cs in the uppermost 10 cm of potentially contaminated soils. According to the MARSSIM, this site warranted a "Class 1" designation based on previous remedial activities within the burial ground, its status as a radioactive disposal facility, and the anticipated presence of discrete radioactive particles. Nine survey units within the confines of the burial ground were selected, based primarily on the presence of physical boundaries and disparate histories. Surface scans with 100% coverage were performed using a hand-held plastic scintillator and rate meter with audible output. In situ gamma-ray spectrometry was not used for the individual stationary measurements due to the limited area and proximity of engineered barriers. Instead, individual soil samples were obtained using a standard hand-held coring device. The number of soil samples taken from the background reference area and each survey unit were determined with the MARSSIM protocol, which resulted in a total of 160 (including quality-control samples). Two of the nine regions exhibited elevated radiation levels and the null hypothesis could not be rejected in one survey unit, thereby indicating the need for additional remediation. The MARSSIM process proved to be flexible, scientifically rigorous, and cost effective in this field application. Several modifications to the procedure are discussed and offered as recommendations for enhancement of the MARSSIM.

Interspecies comparison of in vitro plasma degradation of dynorphin A 1-13.

Enzymatic cleavage of peptides might release metabolites with distinct pharmacological profile. Interspecies differences in the metabolism of peptides might represent a potential source of error when animal data are intended to predict the human situation. Therefore, the metabolism of dynorphin (Dyn) A1-13 was investigated in the plasma of various species used in experimental practice (monkey, rabbit, rat, guinea pig) and compared to previously reported human data. The metabolic pathways were evaluated by enzyme inhibition and kinetic analysis, both yielding almost identical results. Rapid metabolism was observed with all dynorphin fragments across all species (half-lives from 0.3 min for Dyn A1-12 in guinea pig plasma to 2.6 min for Dyn A2-12 in monkey plasma). The metabolism of Dyn A1-13 differed mainly with respect to the half-life (from 0.5 min in guinea pig plasma) to 1.1 min in monkey plasma), while the metabolic routes were similar across the species (Dyn A1-12 major metabolite of Dyn A1-13: from 88% in guinea pig plasma to 71% in rabbit plasma). The metabolic fate of Dyn A1-12, the most important metabolite of Dyn A1-13 is much more heterogeneous across the species then the one observed for Dyn A1-13. For a first assessment, the metabolic routes and rates of Dyn A1-13 in plasma of all investigated species, except from guinea pigs, resemble those in human plasma sufficiently.

Initial investigation OF 222Rn in the Tbilisi urban environment.

Georgia has geological formations with high uranium content, and several buildings are built with local materials. This can create potentially high radon exposures. Consequently, studies to mitigate these exposures have been started. This study presents a preliminary investigation of radon in Tbilisi, the capital of Georgia. An independent radiological monitoring program in Georgia has been initiated by the Radiocarbon and Low-Level Counting Section of I. Javakhishvili Tbilisi State University with the cooperation of the Environmental Monitoring Laboratory of the Physics/Health Physics Department at Idaho State University. At this initial stage the E-PERM systems and GammaTRACER were used for the measurement of gamma exposure and radon concentrations in air and water. Measurements in Sololaki, a densely populated historic district of Tbilisi, revealed indoor radon (222Rn) concentrations of 1.5-2.5 times more than the U.S. Environmental Protection Agency action level of 148 Bq m(-3) (4 pCi L(-1)). Moreover, radon-in-air concentrations of 440 Bq m(-3) and 3,500 Bq m(-3) were observed at surface borehole openings within the residential district. Measurements of water from various tap water supplies displayed radon concentrations of 3-5 Bq L(-1) while radon concentrations in water from the hydrogeological and thermal water boreholes were 5-19 Bq L(-1). In addition, the background gamma absorbed dose rate in air ranged of 70-115 nGy h(-1) at the radon test locations throughout the Tbilisi urban environment.

Polycationic polypeptides: a possible model for the penetration-enhancing factor in the invasion of host cells by Toxoplasma gondii.

The effect of polycationic polypeptides (polylysine, polyarginine and polyhistidine) on the invasion of mammalian cells and plant protoplasts by Toxoplasma gondii was studied. In JM cells, a human lymphoblastoid cell line with T cell characteristics, all polycationic polypeptides used increased the invasion rate in a concentration-dependent manner. This effect and the morphological changes revealed by electron microscopy resembled the action of the penetration-enhancing factor previously described by E. Lycke and co-workers. Plant protoplasts of Catharanthus roseus, which are resistant to T. gondii invasion, showed the same morphological changes in the presence of polycationic polypeptides as observed for JM cells, but were not invaded.

Tubular subviral structure produced in adenovirus-infected KB cells.

At 40 to 90 h after infection with high multiplicities of adenovirus type 2, 4 to 15% of KB cells produced relatively few intranuclear virions detectable by electron microscopy. The nuclei of these cells were found to contain long tubular structures which were made up at least in part by adenovirus structural proteins. The ends of these tubular structures were frequently terminated by morphologically normal adenovirions in varying degrees of completeness. Circumstantial evidence suggests that the production of these aberrant virus structures results from the malfunction or absence of some essential host-provided function and not from a defect in the infecting virus.

DNA of Bacillus subtilis bacteriophage SPP1: physical mapping and localization of the origin of replication.

The genome of Bacillus subtilis bacteriophage SPP1, a linear, 28.5-megadalton DNA duplex, was mapped by analysis with the restriction endonucleases endo R.Sal I, Sma I, Xba I, Bgl I, Bgl II, and EcoRI. The SPP1 genome, like that of the Salmonella typhimurium phage, P22, was found to be a terminally repetitious, circularly permuted molecule. 6-(p-Hydroxyphenylazo)uracil, a selective, reversible inhibitor of SPP1 DNA synthesis, was exploited to synchronize the initiation of genome replication and to selectively label the site of its initiation with radioactive thymidine. Restriction endonuclease analysis of the distribution of the label located the origin of replicative synthesis at an area approximately 0.2 genome length from one molecular terminus.

Reversible inhibition of poxvirus replication by cycloheximide during the early phase of infection.

Infection of primary chick embryo fibroblasts with Vaccinia WR, IHD-W, and cowpox virus even at high m.o.i. does not cause drastic early inhibition of host cell protein synthesis. This contrasts with the infection by these viruses of many eucaryotic cells. Cellular protein synthesis of mouse L cells is also only partially inhibited after infection with cowpox virus up to a m.o.i. of 2500 e.b. As predicted by Moss and Filler (1970, J. Virol. 5, 99-108) no irreversible inhibition of poxvirus replication is observed in these cells following the addition of cycloheximide early after infection. The viral cores which accumulate in chick embryo fibroblasts in the presence of cycloheximide are further uncoated after removal of the protein synthesis inhibitor. These poxvirus host cell systems can be used to identify in vivo immediate and putative delayed early viral gene products. Formation of progeny virus, viral DNA synthesis, the sequential formation of viral proteins, and sensitivity to interferon has been demonstrated in chick embryo fibroblasts after reversal of the cycloheximide block. These studies indicate a synchronized replication cycle of poxvirus after reversal of the cycloheximide block.