A positive crossmatch in liver transplantation--no effect or inappropriate analysis? A prospective study

Secondary brain damage after transient cerebral hypoxia-ischemia (HI) is caused by a cascade of cellular events. In this study, complementary methods of magnetic resonance imaging and histochemistry were used to investigate the formation of cytotoxic and vasogenic edema during secondary brain damage induced by transient HI in 7-d-old rats. To elicit injury, 21 rats underwent right common carotid artery ligation followed by 1.5 h of 8% O2 exposure. Sequential apparent diffusion coefficient (ADC) and transversal relaxation time (T2) weighted magnetic resonance imaging were recorded for up to 3 d in 13 7-d-old rats. Eight animals were killed at various intervals between the end of HI and 21 h of recovery to perform histochemical assays using neuronal and astrocytic markers. Changes of the ADC revealed a biphasic function for the evolution of cytotoxic edema during the recovery period. At the end of HI, the ADC in the ipsilateral cortex was significantly decreased. Upon reoxygenation, it returned transiently to normal followed by a secondary, although less pronounced, decline after 8-48 h. After this, the ADC rose steadily. From 8 h of recovery, the proportion of vasogenic edema steadily increased as indicated by the T2 prolongation. At 21 h, the majority of glial cells showed immunoreactivity for glial fibrillary acidic protein and were of larger size, whereas the neurons were apoptotic. These results indicate that the delayed cerebral injury is accompanied by late glial swelling in conjunction with an enlarged interstitial space due to cell damage.     

Mitochondrial respiratory chain succinate-cytochrome-c oxidoreductase deficiency and hepatic cirrhosis

OBJECTIVES: The current need to evaluate necessity and cost of diagnostic and therapeutic procedures extends to transplant services. We reviewed our experience over the past 3 years as we have moved away from routine post-transplant nuclear medicine scans, ultrasounds, and cystograms. METHODS: From January 1, 1992 to December 31, 1994, 252 kidney transplants were performed at Virginia Mason Medical Center. There were 74 live donor and 178 cadaver donor kidneys transplanted. The records of these patients were reviewed for the type and number of post-transplant imaging done during their initial hospitalization. RESULTS: During the study period, the number of post-transplant imaging studies per patient decreased from 2.7 to 1.4 (P = 0.000), the percentage of patients discharged without any studies rose from 2.8% to 24.4% (P = 0.001), and the trend in 1-year actual graft survival increased from 84.7% to 93.0% (P = 0.187). CONCLUSIONS: Post-transplant imaging studies can be safely reduced. Many patients with good initial graft function can avoid having any studies.     

Minimizing a binding domain from protein A

We present a systematic approach to minimizing the Z-domain of protein A, a three-helix bundle (59 residues total) that binds tightly (Kd = 10 nM) to the Fc portion of an immunoglobin IgG1. Despite the fact that all the contacts seen in the x-ray structure of the complex with the IgG are derived from residues in the first two helices, when helix 3 is deleted, binding affinity is reduced > 10(5)-fold (Kd > 1 mM). By using structure-based design and phage display methods, we have iteratively improved the stability and binding affinity for a two-helix derivative, 33 residues in length, such that it binds IgG1, with a Kd of 43 nM. This was accomplished by stepwise selection of random mutations from three regions of the truncated Z-peptide: the 4 hydrophobic residues from helix 1 and helix 2 that contacted helix 3 (the exoface), followed by 5 residues between helix 1 and helix 2 (the intraface), and lastly by 19 residues at or near the interface that interacts with Fc (the interface). As selected mutations from each region were compiled (12 in total), they led to progressive increases in affinity for IgG, and concomitant increases in alpha-helical content reflecting increased stabilization of the two-helix scaffold. Thus, by sequential increases in the stability of the structure and improvements in the quality of the intermolecular contacts, one can reduce larger binding domains to smaller ones. Such mini-protein binding domains are more amenable to synthetic chemistry and thus may be useful starting points for the design of smaller organic mimics. Smaller binding motifs also provide simplified and more tractable models for understanding determinants of protein function and stability.     

The integrity of the SH3 binding motif of AFAP-110 is required to facilitate tyrosine phosphorylation by, and stable complex formation with, Src

The actin filament-associated protein AFAP-110 forms a stable complex with activated variants of Src in chick embryo fibroblast cells. Stable complex formation requires the integrity of the Src SH2 and SH3 domains. In addition, AFAP-110 encodes two adjacent SH3 binding motifs and six candidate SH2 binding motifs. These data indicate that both SH2 and SH3 domains may work cooperatively to facilitate Src/AFAP-110 stable complex formation. As a test for this hypothesis, we sought to understand whether one or both SH3 binding motifs in AFAP-110 modulate interactions with the Src SH3 domain and if this interaction was required to present AFAP-110 for tyrosine phosphorylation by, and stable complex formation with, Src. A proline to alanine site-directed mutation in the amino terminal SH3 binding motif (SH3bm I) was sufficient to abrogate absorption of AFAP-110 with GST-SH3STC. Co-expression of activated Src (pp60(527F)) with AFAP-110 in Cos-1 cells permit tyrosine phosphorylation of AFAP-110 and stable complex formation with pp60(527F). However, co-expression of the SH3 null-binding mutant (AFAP71A) with pp60(527F) revealed a 2.7 fold decrease in steady-state levels of tyrosine phosphorylation, compared to AFAP-110. Although a lower but detectable level of AFAP71A was phosphorylated on tyrosine, AFAP71A could not be detected in stable complex with pp60(527F), unlike AFAP-110. These data indicate that SH3 interactions facilitate presentation of AFAP-110 for tyrosine phosphorylation and are also required for stable complex formation with pp60(527F).     

Penile vibratory stimulation in spinal cord injured men: optimized vibration parameters and prognostic factors

OBJECTIVE: To study the efficacy of penile vibratory stimulation (PVS) with optimized vibration parameters in spinal cord injured (SCI) men and to examine prognostic factors for success. DESIGN: Case series. SETTING: University hospital outpatient clinic. PATIENTS: Thirty-four consecutive SCI men seeking fertility treatment. INTERVENTION: PVS with optimized vibration parameters to induce reflex ejaculation. MAIN OUTCOME MEASURES: Ejaculatory response; semen analysis. RESULTS: Antegrade ejaculation was seen in 65% of patients. High rates were seen in lesions above T10 (81%) and in presence of hip flexion and bulbocavernosus reflexes (77%). Of men with lesions above T10, those with a penile prosthesis had lower ejaculation rates (40% vs 90%). Average total sperm counts were 968 million, with 26% motility. CONCLUSIONS: High rates of ejaculation are seen with optimized vibration parameters, especially in men with lesions above T10 and intact lower spinal reflexes. A penile prosthesis may impair success with PVS.     

Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 1. Tryptophanyl residues of the carrier can be mutated without impairing protein activity

During the transport process the mitochondrial adenine nucleotide carrier (Ancp) undergoes conformational changes which result in modifications of the intrinsic fluorescence of the carrier. To further study these changes by a fluorometric approach, the three tryptophanyl residues (Trp87, Trp126, and Trp235) of the Saccharomyces cerevisiae Anc2p were individually mutated to their tyrosine counterparts. The resulting mutated genes (two-Trp, one-Trp or Trp-less variants) were integrated at the ANC2 locus. A prerequisite for such studies is that all the engineered carrier molecules are still able to catalyze ADP/ATP exchange. The cellular characteristics of the strains expressing the mutated Anc2p and the biochemical properties of the variant Anc2p in mitochondria were examined. Although Trp87 is absolutely conserved in all 30 available Ancp sequences, none of the tryptophanyl residues is essential to the carrier protein folding and the transport activity. The mutated and wild-type Anc2p were expressed to the same level, as evidenced by both ligand binding and immunochemical analyses. When isolated in the presence of detergent, all the variant Anc2p preparations contained ergosterol in similar amounts (9 mol/mol of 35 kDa Anc2p) but no specific interaction was revealed. Our results show that the tryptophanmutated Anc2p are suitable for fluorescence studies, which are reported in the accompanying paper by Roux et al. [(1996) Biochemistry 35, 16125-16131].     

Radiation dose estimates of 186Re-hydroxyethylidene diphosphonate for palliation of metastatic osseous lesions: an animal model study

A common complication in patients with breast or prostate cancer is bone metastases causing pain. New radionuclide therapy methods have recently been proposed for palliation, including 186Re-hydroxyethylidene diphosphonate (186Re-HEDP). This paper reports on the local development of 186Re-HEDP and the biodistribution studied in animals for eventual use in patients. Adult dose was computed assuming a 70 kg standard man. The 186Re was labelled to HEDP using standard techniques. The biodistribution in five Chacma baboons (Papio ursinus) was studied. Doses ranging from 39.4 to 44.9 MBq kg(-1) (mean 43.6 +/- 2.8 MBq kg[-1]) were administered, corresponding to an adult human dose of 2960 MBq (80 mCi). Whole-body images of the animals were obtained with a dual-headed scintillation camera on an hourly basis for 6 h post-injection and then daily for 3 days. The bone, soft tissue, kidneys and urinary bladder were considered source organs and data from these organs were used in a compartmental model to obtain the mean residence times of the radionuclide in the different source organs. Radiation dose estimates for 186Re-HEDP were subsequently obtained with the MIRDOSE 3 program. The estimated absorbed radiation doses to some of the organs (expressed in mGy MBq[-l]) were as follows: bone surface 1.69; kidneys 0.09; liver 0.04; ovaries 0.04; red marrow 0.75; total body 0.12; urinary bladder wall 0.43. 186Re-HEDP yielded an effective dose of 0.17 mSv MBq(-1). The radiation dose delivered to the bone marrow in this study did not cause any detrimental effect to the baboons, indicating that locally produced 186Re-HEDP is suitable for clinical use.     

The role of pyridine dinucleotides in regulating the permeability of the mitochondrial outer membrane

Both NADH and NADPH reduce the permeability of the mitochondrial outer membrane to ADP. This is specific for the outer membrane and uncorrelated with the respiratory control ratio. This could result in a 7-fold difference between the concentration of ADP in the intermembrane space and that in the external environment (at 5 microM ADP). In both cases the permeability declines by a factor of 5, but NADH is more potent: KD = 86 microM for NADH versus 580 microM for NADPH. The lower apparent affinity for NADPH is partly explained by Mg2+-NADPH being the active species, and under our conditions only 30% of the NADPH is in this form. The corrected KD is 184 microM. Free NADH has the same charge as the Mg2+-NADPH complex, and thus both likely bind to the same site. The ability of NADH and NADPH to induce the closure of reconstituted VDAC channels is consistent with VDAC being the main pathway for metabolite flow across the outer membrane. Oncotic pressure, effective at inducing VDAC closure, also decreases the outer membrane permeability. Thus, in the presence of cytosolic colloidal osmotic pressure NAD(P)H may inhibit mitochondrial catabolic pathways and divert reducing equivalents to anabolic pathways.