Molecular dynamics simulations reveal that apo-HisJ can sample a closed conformation.

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP2 , to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo "Venus flytrap" movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all-atom molecular dynamics simulations of HisJ starting from four different conformations: apo-open, apo-closed, apo-semiopen, and holo-closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo-closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter-hinge strand and interdomain polar interactions contributed to the adoption of the closed apo-conformations although they are up to 2.5-fold less prevalent compared with the holo-closed simulations. The overall sampling of the closed form by apo-HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters.

Diagnostic value of cerebral vasospasm by transcranial doppler ultrasound in Vietnamese patients with subarachnoid hemorrhage.

OBJECTIVE: Although the application of transcranial Doppler (TCD) ultrasonography in clinical diagnosis of cerebral vasospasm is popular in clinical practice in Vietnam, available evidence of the predictive value of vasospasm on TCD in the literature was mostly reported from large institutions in developed countries. Hence, this study was conducted to evaluate the value of TCD ultrasonography in the diagnosis of vasospasm in patients with subarachnoid hemorrhage (SAH) in Vietnam. PATIENTS AND METHODS: This is a prospective observational study of all aneurysmal SAH patients consecutively admitted to a single center between 2008 and December 2011. TCD and 64-slice computed tomographic angiography (CTA) were used to cerebral vasospasm in SAH patients. RESULTS: 316 patients were analyzed (mean age = 52.97±12.27 years, 52.2% males). There were statistically significant difference rates of the cerebral vasospasm by Hunt and Hess Classification and Fisher classification (p <0.01). The proportion of the patients with cerebral vasospasm who were diagnosed exactly by TCD was 95.2%, while the proportion of the patients without cerebral vasospasm diagnosed exactly was 91.5%. TCD predictive diagnostic value was the highest, with the sensitivity of 0.95 (95% CI: 0.91-0.98), specificity of 0.91 (95% CI: 0.85-0.96), positive predictive value of 0.94 (5% CI: 0.90-0.97) and negative predictive value of 0.93 (95 CI: 0.87-0.97). Hemiplegia was the clinical symptom with the highest diagnostic value with the sensitivity of 0.34 (95% CI: 0.27-0.41), specificity of 0.92 (95% CI: 0.86-0.96), positive predictive value of 0.86 (95% CI: 0.76-0.93) and negative predictive value of 0.49 (95% CI: 0.41-0.54). CONCLUSIONS: Evidence of vasospasm diagnosis on TCD ultrasonography was found with high accuracy. Current study enables to suggest the wide application of TCD in Vietnam health facilities from central to grassroots levels instead of the CTA use.

Control of solid tumor metastases with a high-molecular-weight derivative of methotrexate.

Methotrexate (MTX) covalently bound to bovine serum albumin (MTX-BSA), injected ip (10 mg/kg) once every 4 days for a total of 4 doses, was more effective than an equivalent dose of free MTX in reducing the number of metastases observed in female (C57BL/6 X DBA/2)F1 mice bearing the sc implanted Lewis lung carcinoma. Treatment with the high-molecular-weight derivative of MTX in addition caused a decreased rate of growth of the primary tumor and a modest increase in the life-span of the tumor-bearing animal. When tumor-bearing mice were killed after receiving injections of [3H]MTX or [3H]MTX-BSA, no difference in the amount of drug was found at the tumor site after 1 hour; however, after 8 or 24 hours, twice as much radioactivity was found in the tumors of mice treated with carrier-bound drug. Analysis of this radioactivity indicated a ratio of 60--80% carrier-bound to 20--40% free MTX.

Activity of free and carrier-bound methotrexate against transport-deficient and high dihydrofolate dehydrogenase-containing methotrexate-resistant L1210 cells.

A difference in the mechanism of transmembrane transport was demonstrated for methotrexate (MTX) and MTX bound to the high molecular weight carrier bovine serum albumin (MTX-BSA) when the drug dose needed to reduce growth of cells to 50% of that of untreated cells (ID50) was compared in the sensitive L1210 leukemia and 3 L1210 sublines resistant to MTX by virtue of either deficient MTX transport or high levels of dihydrofolate dehydrogenase (DHFD). The loss of transport increased the ID50 for inhibition of growth rate by free MTX tenfold to twentyfold, whereas the elevation of DHFD levels increased the ID50 by tenfold. In contrast, deficiency of transport resulted in only a twofold increase in the ID50 for MTX-BSA, and elevation of DHFD caused a tenfold increase similar to that for free MTX. This difference was confirmed in studies of inhibition of DHFD activity by free and BSA-bound MTX. MTX-BSA but not MTX had antitumor activity against the transport-deficient L1210 line in (C57BL/6 x DBA/2)F1. These studies confirm a separate mode of cell entry for MTX-BSA and suggest a role for these complexes in overcoming resistance.

Preparation of ligation intermediates and related polynucleotide pyrophosphates.

Unprotected oligonucleotides and oligodeoxynucleotides terminated with an unhindered 5'-phosphate group react with nucleoside 5'- phosphorimidazolides in aqueous solution to give 'capped' pyrophosphates in at least 70% yield. If adenosine 5'- phosphorimidazolide is used as a substrate in the reaction, ligase intermediates are obtained as products.

A simple procedure for cross-linking complementary oligonucleotides.

A simple, efficient procedure for cross-linking two complementary oligonucleotides, which does not require chemical modification of either oligonucleotide, is described. One of the oligonucleotides is first converted to the 5'-phosphorothioate derivative with polynucleotide kinase. It is then incubated with its complement in the presence of 1 microM trans-platinum(II)diammine dichloride. After overnight incubation, 40-50% cross-linking is observed. DNA synthesis by the Klenow fragment of Escherichia coli DNA polymerase I is blocked at the cross-linked site, resulting in the formation of truncated products. Potassium platinous chloride (K2PtCl4) and cis-platinum(II)diammine dichloride form cross-links less efficiently than the trans isomer.

MR findings in spinal hemangioblastoma: correlation with symptoms and with angiographic and surgical findings.

BACKGROUND AND PURPOSE: To our knowledge, a detailed analysis of MR findings in spinal hemangioblastoma has not been conducted to date. Our purpose was to elucidate the MR features of this disease with special attention to tumor size, correlation with MR findings and clinical symptoms, and any differences between patients with and without von Hippel-Lindau disease (VHLD). METHODS: MR images in five patients with VHLD and seven patients without VHLD were reviewed retrospectively for spinal hemangioblastoma by two neuroradiologists. The MR findings were correlated with clinical symptoms and with angiographic and surgical findings. RESULTS: The MR features depended on the size of the spinal hemangioblastoma. Small (10 mm or less) hemangioblastomas were mostly isointense on T1-weighted images, hyperintense on T2-weighted images, and showed homogeneous enhancement. Larger hemangioblastomas tended to be hypointense or mixed hypo- and isointense on T1-weighted images, heterogeneous on T2-weighted images, and tended to show heterogeneous enhancement. Small hemangioblastomas were located at the surface of the spinal cord, most frequently along its posterior aspect. These were subpial in location at surgery and showed well-demarcated, intense enhancement. Symptomatic small hemangioblastomas had relatively large associated syringes, whereas asymptomatic ones did not. A hemangioblastoma larger than 24 mm was invariably accompanied by vascular flow voids. There was no difference in the MR findings between the two patient groups except for the multiplicity and higher percentage of small tumors in patients with VHLD. CONCLUSION: Knowledge of these MR features helps to differentiate spinal hemangioblastoma from other diseases that show enhancing nodules.

Increased urinary excretion of nucleic acid and nicotinamide derivatives by rats after treatment with alkylating agents.

Rats treated with di(2-chloroethyl)methylamine (HN2), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) excrete significantly larger amounts of deoxycytidine (dC) and thymidine in their urine 0-24 h after treatment. Ethyl methanesulphonate (EMS) and dimethylnitrosamine (DMN) gave negative results in this respect but all five alkylating agents increased the excretion of 1-methyl-nicotinamide (1-meNmd). In addition, a larger quantity of 7-methylguanine (7MG) and uric acid was excreted after DMN treatment. 1,4-Dimethanesulphonoxybutane (myleran), 2,2-dichlorovinyl dimethyl phosphate (dichlorvos), 5-fluorouracil (5FU), cytosine arabinoside (araC), 2-acetylaminofluorene (AAF) and 7-bromomethylbenz-[a]anthracene (7-BrMBA) gave negative results.

Increased urinary excretion of labelled deoxyribonucleosides in rats with stable pre-labelled DNA treated with methyl methanesulphonate or nitrogen mustard.

After the DNA of newborn female rats had been labelled by repeated injections of [14C]orotate (totalling 36 mu Ci) during the first 3 weeks of life, approximately 1,000,000 dpm were found in the DNA of the liver, lungs, kidneys, gut, brain, heart and spleen of 8-week-old rats. Methyl methanesulphonate (MMS) (80 mg/kg) and di-(2-chloroethyl)methylamine (HN2) (5 mg/kg) injection increased the amount of 14C-labelled DNA pyrimidine nucleosides excreted in the urine to 5000 dpm from 350 dpm before injection. The effect on RNA products was much less marked.

Demonstration of the distal end of the oesophagus by transabdominal ultrasound.

This study aimed to demonstrate the distal end of the oesophagus using a transabdominal ultrasound technique (TUS) and to measure the normal oesophageal wall thickness in adults. 65 patients without oesophageal disease and 38 normal volunteers were examined by TUS. A left subcostal approach was used to demonstrate the oesophagus. The wall thickness and length were measured in both the supine and 45 degrees right side up oblique (RUO) positions. The abdominal oesophagus was visualized in 80% of patients in the supine position and 92% in the RUO position. Satisfactory demonstration was obtained in 67% of patients in the supine and 85% in the RUO position. The oesophageal wall thickness averaged 2.8 mm (range 2.0-4.0 mm, SD 0.7 mm). The visualized length in these subjects averaged 2.3 cm in the supine position and 3.0 cm in the RUO position, which included approximately 1.5 cm of the lowest portion of the thoracic oesophagus in addition to the abdominal oesophagus. TUS can demonstrate the abdominal oesophagus in the majority of patients and has the potential to provide information on disorders of structure and motility.

The lack of effect of pentoxifylline on random skin flap survival.

The effects of pentoxifylline on skin flap survival were studied in rabbits. A total of 40 rabbits had caudally based single-pedicle flaps measuring 4 x 14 cm raised on the mid dorsum of each animal. Twenty of these rabbits were given intraperitoneal injections of pentoxifylline in doses of 24 mg/kg per day beginning 48 hours prior to flap construction and continued daily for 7 days postoperatively. The remaining 20 control rabbits received intraperitoneal injections of saline in equal volumes as the experimental groups. At the end of 7 days, viable flap length was visually inspected and measured in all 40 rabbits. There was no significant difference in skin flap viability in rabbits treated with pentoxifylline compared to the control group.

Flow volume in the common carotid artery detected by color duplex sonography: an approach to the normal value and predictability of cerebral blood flow.

PURPOSE: To determine normal common carotid artery (CCA) flow volume, its relationship with age, and the predictability of cerebral blood flow (CBF) by color duplex sonography. SUBJECTS AND METHODS: Forty-five healthy subjects (18 men, 27 women, 23-86 years old) and 13 patients (3 men, 10 women, 51-88 years old) without neurological disease underwent color duplex sonography. All 13 patients also underwent xenon CT. CCA flow volume in the healthy subjects was measured to determine normal values. This volume was divided by mean brain weight to estimate CBF, which was correlated with CBF measured by xenon CT in regions of ipsilateral internal carotid arteries (ICA). RESULTS: In healthy subjects, CCA flow volume ranged from 155.0-458.8 ml/min (mean+/-SD: 267.77+/-59.91), corresponding to an estimated CBF of 12.43-32.84 ml/min/100 g brain weight (mean+/-SD: 20.63+/-4.22). No relationship was found between flow volume and age. A good correlation was found between estimated CBF and CBF measured by xenon CT in regions of both ICAs (gamma=0.713, p=0.0062 on the left; gamma=0.686, p=0.0096 on the right). CONCLUSION: By using color duplex sonography, we established a set of normal CCA flow volumes, which do not decline with age. Estimated CBF derived from flow volume can predict actual CBF.

[The clinical application of diffusion weighted magnetic resonance imaging to acute cerebrovascular disorders].

Diffusion is a measure of motion freedom and is a sensitive parameter to characterize the tissue at the microscopic level. The methods of measuring in vivo diffusion by magnetic resonance imaging (MRI) have been based mainly on the addition of two motion-probing gradients (MPG) to the spin echo sequence to produce signal attenuation for the spins moving at random. The resultant MR images reflect the intravoxel incoherent motions (IVIM), which contain both water molecule diffusion and perfusion in the capillary network, and can be quantified by an apparent diffusion coefficient (ADC). Diffusion weighted MRI, acquired from IVIM MR imaging by the addition of the very strong MPG predicate water diffusion and anisotropy. High signal or reduced ADC can be observed in case of the slower diffusion. The anisotropy depends upon the orientation of the subjects and the gradients. Greater signal attenuation (faster diffusion) can be observed when the relative orientation of white matter tracts to the MPG is parallel as compared to that obtained with a perpendicular alignment. This anisotropy may preclude the detection or delineation of an ischemic lesion. Diffusion tensor trace has been designated to eliminate this anisotropy effect. In ischemic animal models, low signal (fast diffusion) and high signal (slow diffusion) have been noted in the vasogenic edema and cytotoxic edema, respectively. High signal appears only in case of cerebral blood flow below 15-20 ml/100 g per minute, a value identical to the threshold of tissue at high energetic metabolism and ion homeostasis. ADC value decreases following the cerebral vessel occlusion, or remains unchanged when collateral circulation develops. It has been speculated that reduction in ADC reflects the water shift from extracellular space to intracellular space due to the membrane permeability and/or intracellular osmolality increase. These results suggest that diffusion weighted MRI correlates well with the cell metabolism, and cytotoxic edema plays an important role in the acute cerebral stroke. In clinical setting of acute cerebral ischemia, diffusion weighted MRI may detect superacute infarction by showing high signal (slower ADC) over the 6 hours following the insult, whereas conventional MRI generally fails to do so. In chronic liquefied cerebral infarction, increased ADC, or attenuated signal are the most frequent findings, suggestive of an elevated diffusion. Therefore, diffusion weighted MRI improves early diagnosis of stroke and help differentiate acute from chronic stroke. One disadvantage of diffusion weighted MRI is motion artifact, which may be reduced by the introduction of a navigator echo to correct for the phase shift caused by the first imaging echo, or by the utility of ultrafast imaging technique, such as echo planar. Another shortcomings is the susceptibility artifact incorporating the diffusion weighted MRI. The eddy current may also result from the strong gradients, producing shiftlike artifact. Such artifacts can be compensated for by appropriate shaping of the current pulses sent into the gradient coils, or by use of shielded gradients. As with rapid progresses in perfusion imaging of ischemia penumbra, misery perfusion and luxury perfusion, new insight into the diffusion weighted MRI will be significant.

Optimization of the efficiency of cross-linking PtII oligonucleotide phosphorothioate complexes to complementary oligonucleotides.

We have investigated the efficiency with which PtII complexes cross-link phosphorothioates of oligonucleotides to complementary DNA targets. The A and G residues 2-5 bases downstream from the 5'-phosphorothioate group are preferred sites for cross-linking. Replacement of residues in this part of the target by T residues results in greatly decreased cross-linking when cis platinum diammine dichloride (cisPtII) or potassium platinous chloride (K2PtCl4) are used. Trans platinum diammine dichloride (transPtII) forms cross-links with T residues if A and G residues are absent from the susceptible region of the target. Oligomers containing an internal phosphorothioate group can also be linked to their templates with transPtII, but not with cisPtII or K2PtCl4. Cross-linking via an internal phosphorothioate group tends to be less efficient than cross-linking via a 5'-terminal phosphorothioate. The Sp isomers of internal phosphorothioates are cross-linked more efficiently than the Rp isomers. Preliminary experiments suggest that the efficiency of cross-linking to RNA targets will prove similar to that found for DNA targets.