Diode laser cyclophotocoagulation: histopathology in two cases of clinical failure

We report an unusual case of a 55 year old Japanese woman with a seminoma but relatively normal menses. The patient was a phenotypic female with late onset menarche (18 years of age), who was amenorrhoeic for the first year, followed by menses of one to three days' slight flow with dysmenorrhoea, but an otherwise normal menstrual history. A typical seminoma was removed from the left adnexal region and an immature testis was identified separately as an associated right adnexal mass. Repeated karyotypic studies on peripheral blood lymphocyte cultures showed only 46,X,-Y,t(Y;15)(q12;p13). Cytogenetic examination of the patient's younger brother, who had fathered three healthy children, showed an identical karyotype. Mosaicism of 46,X,-Y,t(Y;15)(q12;p13)/45,X cell lines was found in skin samples from the patient's elbow and genital regions, although there were no clinical stigmata of Turner syndrome. An androgen receptor binding assay of cultured genital skin fibroblasts was negative. Molecular analysis using Southern blot hybridisation, PCR, and direct DNA sequencing showed that neither the patient nor her brother had a detectable deletion or other abnormalities of Y chromosome sequences, including the SRY (sex determining region of the Y chromosome) gene sequence. These findings suggest that Turner mosaicism of the 45,X cell line may have contributed to this atypical presentation in an XY female, although we cannot exclude abnormalities of other genes related to sex differentiation.     

Successful innominate thromboembolectomy of a paradoxic embolus

A 54 year-old man had symptoms of acute right hemispheric cerebral ischemia. He was initially considered for participation in a trial of early thrombolysis in stroke, but an innominate artery embolus was found with no apparent arterial source. The embolus was removed by means of a combined brachial and carotid bifurcation approach to protect the cerebral vasculature from embolic fragmentation during extraction. Further investigation revealed deep venous thrombosis, evidence of pulmonary emboli, and a patent foramen ovale, supporting a diagnosis of paradoxic embolus. Additional treatment included anticoagulation and placement of an inferior vena caval filter. The unusual condition of paradoxic embolus is reviewed, and the management of this patient is discussed.     

Perimetric motion thresholds are elevated in glaucoma suspects and glaucoma patients

The purpose of this study was to determine if a clinically feasible perimetric motion test utilizing random-dot kinematograms could identify glaucomatous visual field defects. Using a staircase procedure, an automated perimetric motion test and a larger foveally presented target were given to normal (n = 30), glaucoma suspects (n = 31) and primary open-angle glaucoma patients (n = 19). Motion thresholds at specific locations throughout the whole visual field were significantly elevated in glaucoma patients (P < or = 0.001). Perimetric motion testing identified 84.2% of the primary open-angle glaucoma patients and 25.8% of the glaucoma suspects as abnormal. A larger foveal stimulus was unable to distinguish between the different subject groups (P < or = 0.185). Perimetric motion thresholds were significantly correlated with Humphrey standard visual field thresholds in the glaucoma and glaucoma-suspect patients (P < or = 0.0002).     

Comparison of the pharmacological profile of S-nitrosothiols, nitric oxide and the nitrergic neurotransmitter in the canine ileocolonic junction

1. In organ bath experiments, hydroquinone (30-100 microM) and hydroxocobalamin (30-100 microM) concentration-dependently inhibited the relaxations induced by NO (0.3-30 microM) but not those by nitroglycerin (GTN, 1 microM) in the canine ileocolonic junction (ICJ). Hydroxocobalamin reduced the relaxation to low frequency (2 Hz) stimulation of the non-adrenergic, non-cholinergic (NANC) nerves, whereas hydroquinone only reduced the NANC nerve-mediated relaxations to electrical stimulation at 16 Hz, 0.5 ms. 2. Relaxations to S-nitroso-L-cysteine (CysNO, 1-30 microM), or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1-30 microM) were not inhibited by hydroquinone (30-100 microM), hydroxocobalamin (30-100 microM), pyrogallol (30-100 microM) or L-cysteine (1-3 microM). Hydroquinone (100 microM) only reduced the relaxation to 10 microM CysNO. Hydroxocobalamin, but not hydroquinone, pyrogallol or L-cysteine, potentiated the relaxations to the lowest concentration (1 microM) of S-nitrosoglutathione (GSNO, 1-30 microM). 3. In the superfusion bioassay, hydroquinone (100 microM) and hydroxocobalamin (1 microM) concentration-dependently inhibited the biological activity of authentic NO (1-4 pmol) to the same extent as that of the transferable nitrergic factor, released from the canine ICJ in response to NANC nerve stimulation (8-16 Hz, 2 ms). Responses to GTN (10 pmol) or adenosine 5'-triphosphate (10 nmol) were not affected. 4. In conclusion, the nitrosothiols CysNO, SNAP and GSNO relax the canine ileocolonic junction, but these relaxations, pharmacologically, behave differently from the NANC nerve-mediated relaxations. From the bioassay experiments, we conclude that the nitrergic factor, released in response to NANCnerve stimulation of the canine ICJ, behaves pharmacologically like NO but not like a nitrosothiol.Therefore, we suggest NO, and not CysNO, SNAP or GSNO as the inhibitory NANC neurotransmitter in the canine ICJ.     

The effect of changing excitation frequency on parallel conductance in different sized hearts

OBJECTIVE: An important component of the ventricular volume measured using the conductance catheter technique is due to parallel conductance (Vc), which results from the extension of the electric field beyond the ventricular blood pool. Parallel conductance volume is normally estimated using the saline dilution method (Vc(saline dilution)), in which the conductivity of blood in the ventricle is transiently increased by injection of hypertonic saline. A simpler alternative has been reported by Gawne et al. [12]. Vc(dual frequency) is estimated from the difference in total conductance measured at two exciting frequencies and the method is based on the assumption that parallel conductance is mainly capacitive and hence is negligible at low frequency. The objective of this study was to determine whether the dual frequency technique could be used to substitute the saline dilution method to estimate Vc in different sized hearts. METHODS: The accuracy and linearity of a custom-built conductance catheter (CC) system was initially assessed in vitro. Subsequently, a CC and micromanometer were inserted into the left ventricle of seven 5 kg pigs (group 1) and six 50 kg pigs (group 2). Cardiac output was determined using thermodilution (group 1) and an ultrasonic flow probe (group 2) from which the slope coefficient (alpha) was determined. Steady state measurements and Vc estimated using saline dilution were performed at frequencies in the range of 5-40 kHz. All measurements were made at end-expiration. Finally, Vc was estimated from the change in end-systolic conductance between 5 kHz and 40 kHz using the dual frequency technique of Gawne et al. [12]. RESULTS: There was no change in measured volume of a simple insulated cylindrical model when the stimulating frequency was varied from 5-40 kHz. Vc(saline dilution) varied significantly with frequency in group 1 (8.63 +/- 2.74 ml at 5 kHz; 11.51 +/- 2.65 ml at 40 kHz) (p = 0.01). Similar results were obtained in group 2 (69.43 +/- 27.76 ml at 5 kHz; 101.24 +/- 15.21 ml at 40 kHz) (p < 0.001). However, the data indicate that the resistive component of the parallel conductance is substantial (Vc at 0 Hz estimated as 8.01 ml in group 1 and 62.3 ml in group 2). There was an increase in alpha with frequency in both groups but this did not reach significance. The correspondence between Vc(dual frequency) and Vc(saline dilution) methods was poor (group 1 R2 = 0.69; group 2 R2 = 0.22). CONCLUSION: At a lower excitation frequency of 5 kHz a smaller percentage of the electric current extends beyond the blood pool so parallel conductance is reduced. While parallel conductance is frequency dependent, it has a substantial resistive component. The dual frequency method is based on the assumption that parallel conductance is negligible at low frequencies and this is clearly not the case. The results of this study confirm that the dual frequency technique cannot be used to substitute the saline dilution technique.     

Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma: identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity

The human DNA polymerase gamma catalytic subunit was overexpressed in recombinant baculovirus-infected insect cells, and the 136 000 Da protein was purified to homogeneity. Application of the same purification protocol to HeLa mitochondrial lysates permitted isolation of native DNA polymerase gamma as a single subunit, allowing direct comparison of the native and recombinant enzymes without interference of other polypeptides. Both forms exhibited identical properties, and the DNA polymerase and 3' --> 5' exonuclease activities were shown unambiguously to reside in the catalytic polypeptide. The salt sensitivity and moderate processivity of the isolated catalytic subunit suggest other factors could be required to restore the salt tolerance and highly processive DNA synthesis typical of gamma polymerases. To facilitate our understanding of mitochondrial DNA replication and mutagenesis as well as cytotoxicity mediated by antiviral nucleotide analogues, we also constructed two site-directed mutant proteins of the human DNA polymerase gamma. Substituting alanine for two essential acidic residues in the exonuclease motif selectively eliminated the 3' --> 5' exonucleolytic function of the purified mutant polymerase gamma. Replacement of a tyrosine residue critical for sugar recognition with phenylalanine in polymerase motif B reduced dideoxynucleotide inhibition by a factor of 5000 with only minor effects on overall polymerase function.     

Enhanced involvement of endothelin in the haemodynamic sequelae of endotoxaemia in conscious, hypertensive, transgenic ((mRen-2)27) rats

1. Age-matched (3-4 months old) male, heterozygous, hypertensive, transgenic ((mRen-2)27) rats (abbreviated to TG rats) and the normotensive control animals (homozygous, Hannover Sprague-Dawley rats (abbreviated to SD rats), were chronically instrumented for the assessment of regional haemodynamic responses to continuous lipopolysaccharide (LPS) infusion (150 microg kg(-1) h(-1), i.v.) 2. The early (1-2 h) hypotension in SD rats (-11+/-3 mmHg; n=7) was significantly less than that in TG rats (-35+/-3 mmHg; n=8), but by 24 h mean arterial blood pressure (MAP) in both strains of rat was not different from the pre-LPS value (SD rats: baseline, 108+/-3 mmHg; 24 h LPS, 112+/-4 mmHg; TG rats: baseline, 171+/-2 mmHg; 24 h LPS, 169+/-3 mmHg). At this stage in the SD rats there was a renal vasodilatation (delta vascular conductance, 29+/-10 [kHz mmHg(-1)]10(3)) but not in TG rats (delta vascular conductance 2+/-3[kHz mmHg(-1)]10(3)). 3. Co-infusion of LPS and the non-selective endothelin receptor antagonist, SB 209670 (600 microg kg(-1) bolus, 600 microg kg(-1) h(-1)) between 24 and 31 h in SD rats caused a fall in MAP of 16+/-2 mmHg accompanied by hindquarters vasodilatation (delta vascular conductance 11+/-3 (kHz mmHg(-1))10(3)). In TG rats, under the same conditions, the fall in MAP was -60+/-6 mmHg, and there were renal, mesenteric and hindquarters vasodilatations (delta vascular conductance, 23+/-5, 32+/-7, and 14+/-4 (kHz mmHg(-1))10(3), respectively). All effects, except the hindquarters vasodilatation, were greater in TG than in SD rats. 4. In TG rats infused with LPS alone for 31 h, between 24 and 31 h the fall in MAP was -17+/-4 mmHg, and the changes in renal, mesenteric and hindquarters vascular conductances were 5+/-3, -4+/-5, and 12+/-4 (kHz mmHg(-1)10(3), respectively. 5. Administration of the angiotensin (AT1)-receptor antagonist, losartan (10 mg kg(-1), i.v.) following co-infusion of LPS and SB 209670 between 24 and 31 h caused similar falls in MAP in SD and TG rats (-12+/-3 and -14+/-4 mmHg, respectively). 6. These results, together with previous findings, are consistent with a relative enhancement of the contribution of endothelin to the maintenance of cardiovascular status in endotoxaemic TG rats, particularly through a mesenteric vasoconstrictor action.     

Reduced inward rectifying and increased E-4031-sensitive K+ current density in arrhythmogenic subendocardial purkinje myocytes from the infarcted heart

Astrocytes in primary culture from rat cerebral cortex were probed concerning the expression of delta-opioid receptors and their coupling to changes in intracellular free calcium concentrations ([Ca2+]i). Fluo-3 or fura-2 based microspectrofluorometry was used for [Ca2+]i measurements on single astrocytes in a mixed astroglial-neuronal culture. Application of the selective delta-opioid receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE), at concentrations ranging from 10 nM to 100 microM, induced concentration-dependent increases in [Ca2+]i (EC50 = 114 nM). The responses could be divided into two phases, with an initial spike in [Ca2+]i followed by either oscillations or a sustained elevation of [Ca2+]i. These effects were blocked by the selective delta-opioid receptor antagonist ICI 174864 (10 microM). The expression of delta-opioid receptors on astroglial cells was further verified immunohistochemically, using specific antibodies, and by Western blot analyses. Pre-treatment of the cells with pertussis toxin (100 ng/ml, 24 h) blocked the effects of delta-opioid receptor activation, consistent with a Gi- or Go-mediated response. The sustained elevation of [Ca2+]i was not observed in low extracellular Ca2+ and was partly blocked by nifedipine (1 microM), indicating the involvement of L-type Ca2+ channels. Stimulating neurons with DPDPE resulted in a decrease in [Ca2+]i, which may be consistent with the closure of the plasma membrane Ca2+ channels on these cells. The current results suggest a role for astrocytes in the response of the brain to delta-opioid peptides and that these opioid effects in part involve altered astrocytic intracellular Ca2+ homeostasis.