Actions of some anions on electrical properties and mechanical threshold of frog twitch muscle

1. Using two micro-electrodes in a point-voltage clamp technique, the effects of the lyotropic anions, NO(3) (-), Br(-), I(-), SCN(-), and CH(3)SO(4) (-), and of SO(4) (2-) on the mechanical threshold and electrical properties of frog sartorius muscle were studied.2. In chloride Ringer solution the spike threshold was -59 mV, mechanical threshold -48 mV, and the threshold for delayed rectification of the total current at about 100 msec -52 mV.3. When Cl(-) was replaced by one of the lyotropic anions, the effective resistance determined at -100 mV tended to increase. But, because of the variability of the effective resistance in individual fibres, most lyotropic anions did not cause a statistically significant increase in the effective resistance. Only I(-) and SO(4) (2-) significantly increased the effective resistance.4. Most lyotropic anions had no significant effect on the spike threshold; I(-), at 58 mM, lowered it slightly. Sulphate raised the threshold.5. Tetrodotoxin (0.1 mug/ml.) abolished the spikes, but did not affect the mechanical and delayed rectification thresholds. It was, therefore, used to pre-treat all preparations for determining these thresholds.6. All lyotropic anions lowered the mechanical and the delayed rectification thresholds, the order of effectiveness being approximately SCN(-) > I(-) > NO(3) (-) > CH(3)SO(4) (-) > Br(-). As in Cl(-) Ringer, the two thresholds lay very close together in every case. Sulphate raised slightly both the mechanical and delayed rectification thresholds, again in close parallel.7. This close agreement of the mechanical and delayed rectification thresholds is not caused by movement artifact, because in fibres in which visible contractions were eliminated with hypertonic solutions the delayed rectification thresholds were the same as those in contracting fibres.8. In spite of the close agreement, reasons are given to doubt a direct causal relationship between the mechanical and delayed rectification thresholds.9. Nitrate apparently had little effect on the rate of inactivation of the outward current, or on the relation between steady-state inactivation and membrane potential.     

Genetic predetermination of quantitative expression of HLA antigens in platelets and mononuclear leukocytes

In view of the potential functional importance of quantitative expression of HLA antigens, a series of studies were conducted to determine the relative quantities of specific HLA-A and -B antigens expressed in MNLs and platelets of HLA-phenotyped family members and unrelated individuals. An mAb that reacts with a well-defined monomorphic epitope in the α₃ domain of the heavy chains of HLA molecules was developed and used to quantify each HLA-A or -B antigen on western blots of IEF gels. The results of these studies demonstrated that the relative quantities of HLA-A and -B antigens in platelets and MNLs of an individual did not change over time. Further studies showed that the relative quantities of HLA-A and -B antigens for haplotypes shared among the first-degree relatives were always the same and followed Mendelian inheritance. In contrast, the relative quantities of HLA-A and -B antigens for a haplotype shared by unrelated individuals varied significantly. All these findings support the hypothesis that the quantitative expression of HLA antigens is genetically predetermined and may play important roles in determining disease susceptibility and severity. Human Immunology 38, 243–250 (1993)     

Neonatal sepsis in the neonatal intensive care unit: characteristics of early versus late onset.

Neonatal sepsis is a major cause of death in newborns despite sophisticated neonatal intensive care. This retrospective study reviewed the clinical characteristics of cases of culture-proven sepsis in a neonatal intensive care unit from January 1992 to December 2001. Patients were divided into those with onset of sepsis in the first 7 days of life (early-onset group) and those with onset after the seventh day of life (late-onset group). A total of 270 cases with 325 episodes of sepsis and 353 isolated pathogens were identified and included in the study. The male-to-female ratio was 1.4. The majority of cases of sepsis occurred in low birth weight (75.9%) and premature babies (76.7%). Late onset occurred in 71.9% of cases. Patients with late onset had a lower mortality rate than those with early onset (11.3% vs 28.9%). Coagulase-negative staphylococci (20.1%) was the most common organism isolated, but infection with Pseudomonas aeruginosa was associated with the highest morality rate (55.0%). Late-onset sepsis was significantly more common in very low birth weight and premature infants. The most frequently encountered pathogens in the early-onset group were group B streptococci (GBS) and Escherichia coli, while in the late-onset group, the organisms were coagulase-negative staphylococci and Enterobacteriaceae, including E. coli, Klebsiella pneumoniae, and Acinetobacter baumannii. GBS infection resulted in the highest mortality when the onset of sepsis was within the first 24 hours of life.     

Diagnosis of adrenal cortical dysfunction by liquid chromatography-tandem mass spectrometry

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to measure 6 metabolic compounds of the adrenocorticosteroid pathway simultaneously on residual specimens from patients who had previously been previously diagnosed, on the basis of immunoassays, as having congenital adrenal hyperplasia (CAH), 11 beta-hydroxylase deficiency, 21-hydroxylase deficiency, or Addison disease (adrenal insufficiency). Two subjects with normal adrenal function had serum cortisol values of 13.6 and 8.9 micrograms/dL and serum cortisone values of 2.1 and 0.6 microgram/dL, but the rest of the compounds were undetectable. Two patients with 11 beta-hydroxylase deficiency had serum 11 beta-deoxycortisol values of 14.9 and 10.0 micrograms/dL and serum 11-deoxycorticosterone values of 3.9 and 1.0 microgram/dL, but their serum levels of cortisol and cortisone were diminished. A patient with 21-hydroxylase deficiency had a highly increased serum 17-hydroxyprogesterone concentration of 28.5 micrograms/dL (or 28,500 ng/dL, the traditional unit to report this assay) and a serum 21-deoxycortisol concentration of 6.9 ug/dL (this is a pathologic marker of 21-hydroxylase deficiency that is nondetectable in sera of healthy subjects). This patient also had diminished concentrations of serum cortisol and cortisone (0.9 and 0.3 microgram/dL, respectively). At 30 and 60 min after corticotropin (ACTH) stimulation, serum cortisol was the only compound that showed a dramatic increase in the normal subjects; the patient with 21-hydroxylase deficiency showed an increase of serum 17-hydroxyprogesterone level, but no increase of serum cortisol level; the patient with Addison disease showed no increase in the levels of serum cortisol or other compounds. Metyprapone, which blocks 11 beta-hydroxylase activity, increased the serum 11-deoxycorticosteroid levels and decreased the serum cortisol level. This pilot study demonstrates that it is feasible to use LC-MS/MS for the laboratory diagnosis of adrenal cortical dysfunction. The authors envision that LC-MS/MS may soon become an ideal analytical technique for the diagnosis of such endocrine diseases.     

SPECT findings in the Kleine-Levin syndrome

STUDY OBJECTIVES: The Kleine-Levin Syndrome, is a rare disorder with onset during teenage years, but little is known on etiopathogenesis. Seven subjects with Kleine-Levin Syndrome accumulated over time had systematic SPECT studies during (n=5) and out (n=7) of the symptomatic period. SUBJECTS: Seven boys with symptom onset between 11 and 17 years of age and at least 2 episodes per year were followed for a mean of 6 years. METHODS: Electroencephalogram awake-asleep, computed tomography scan, and magnetic resonance imaging studies were performed before Tc-99m ECD single photon emission tomography (SPECT) obtained during day 4 or 5 (n=5) and at least 1 month away from the symptomatic period (n=7). RESULTS: All imaging tests except SPECT were normal. Hypoperfusion of both thalami were seen during the symptomatic period that completely disappeared during the asymptomatic period. Hypoperfusion in other regions were also noted in some, but not all subjects. They persisted during the asymptomatic period in 2 cases over the temporal lobe (2/7 cases), frontal lobe (1/7 cases), and basal ganglia (1/7 cases). The largest amount of persistent hypoperfusion was seen in the subject with longest clinical evolution. CONCLUSION: Hypoperfusion of the thalamus is a consistent finding during the symptomatic period, but perfusion abnormalities may persist even during the asymptomatic period. The longer the duration of the syndrome, the more extended the hypoperfusion regions during the asymptomatic period.     

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we describe the problems and solutions of setting up the recombinant SARS-CoV S-based ELISA for antibody detection. The SARS-CoV S-based immunoglobulin M (IgM) and IgG ELISAs were evaluated and compared with the corresponding N-based ELISA for serodiagnosis of SARS-CoV pneumonia, using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS-CoV pneumonia patients in Hong Kong. Results obtained by the recombinant S (rS)-based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers, followed by addition of different regeneration buffer, identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection. The specificities of the S-based ELISA for IgG and IgM detection were 98.6% and 93.9%, with corresponding sensitivities of 58.9% and 74.7%, respectively. The sensitivity of the rN IgG ELISA (94.7%) is significantly higher than that of the rS IgG ELISA (P < 0.001), whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA (55.2%) (P < 0.01). An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS-CoV pneumonia.     

A quantitative in vitro assay of polymorphonuclear leukocyte migration through human amnion membrane utilizing 111in-oxine

A modified amnion chemotaxis assay is described for measurement of polymorphonuclear leukocyte(s) (PMNL) migration (random and directed) into a viable membrane. The primary modifications are the use of 111In-oxine-labelled PMNL and replacement of the nitrocellulose 'trap' filter with a type I collagen sponge. The modifications resulted in four important benefits: the quantification of PMNL migration was simplified; reader subjectivity was eliminated; the information gained of the migration process was enhanced; and the assay time was decreased. The amnion chemotaxis assay with the modifications reported should provide the means of evaluating several aspects of the inflammatory response of PMNL.