Altered somatosensory barrel cortex refinement in the developing brain of Mecp2-null mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein 2 (MeCP2) gene. In previous studies, monoaminergic dysfunctions have been detected in patients with RTT and in a murine model of RTT, the Mecp2-null mouse. Therefore, the pathogenesis of RTT is thought to involve impairments in the monoaminergic systems. However, there have been limited data showing that the impairment of monoamines leads to early symptoms during development. We used histochemistry to study the somatosensory barrel cortex in the B6.129P2(C)-Mecp2(tm1.1Bird) mouse model of RTT. The barrel cortex is widely used to investigate neuronal development and its regulation by various neurotransmitters including 5-HT. 5-HT levels were measured by high performance liquid chromatography with electrochemical detection (HPLC/EC), and serotonin transporter (SERT) and 5-HT1B receptor mRNAs were measured in the somatosensory cortex, thalamus and striatum on postnatal days (P) 10, P20 and P40. Mecp2-null mice (Mecp2-/y) had significantly smaller barrel fields than age-matched wild-type controls (Mecp2+/y) on P10 and P40, but the topographic map was accurately formed. Levels of 5-HT, and SERT and 5-HT1B receptor mRNA expression in the somatosensory cortex did not differ significantly between the Mecp2-null and wild-type mice on P10. However, thalamic 5-HT was reduced in Mecp2-null mice. Our data indicate that a lack of MeCP2 may disturb the refinement of the barrel cortex in the early postnatal period. Our findings suggest that a decrease in thalamic 5-HT might be involved in this phenomenon.

Postnatal changes in serotonergic innervation to the hippocampus of methyl-CpG-binding protein 2-null mice.

Rett syndrome is a progressive neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MeCP2) gene. Previous reports have revealed serotonergic function to be altered in the medullas of patients with Rett syndrome and in an animal model of the disease. However, it has remained unclear whether a genetic loss of MeCP2 disrupts serotonergic innervation to the forebrain. In this study, we measured levels of monoamines by high-performance liquid chromatography with electrochemical detection in selected regions of the forebrains of Mecp2-null mice (Mecp2-/y) and wild-type mice (Mecp2+/y) on postnatal day (P) 14, P28, P42 and P56. The levels of hippocampal serotonin (5-HT) and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were significantly lower in Mecp2-null mice than in age-matched wild-type mice on P28, P42 and P56. Immunohistochemical analysis revealed a loss of 5-HT-immunoreactive fibers in the Mecp2-null hippocampus on P56. By contrast, in the raphe region of Mecp2-null mice, there were significant decreases in 5-HT and noradrenaline levels, but these differences later disappeared and there was no change in the number of 5-HT-immunoreactive neuronal cell bodies. Furthermore, we conducted an experiment comparing HPLC measurements in presymptomatic heterozygous females (Mecp2+/-) and wild-type female littermates (Mecp2+/+) on P56. Significant decreases in hippocampal 5-HT and 5-HIAA contents in Mecp2-heterozygous mice were revealed, and these were not accompanied by changes in 5-HT or noradrenaline contents in the raphe region. Therefore, these results indicated decreases in serotonergic innervation to the hippocampus in Mecp2-null males and Mecp2 heterozygous females. We speculate that disturbances in serotonergic neurotransmission in the hippocampus may be linked to the behavioral abnormalities seen in Rett syndrome, such as increased anxiety-like behaviors and reduced exploratory locomotion. MeCP2 may be required for stable serotonergic homeostasis and serotonergic innervation to the hippocampus during postnatal development.

Total sleep deprivation induces an acute and transient increase in NK cell activity in healthy young volunteers.

STUDY OBJECTIVES: To investigate the effects of one night's total sleep deprivation (TSD) on NK cell activity, with rigorous control of circadian phase of sampling points as well as physical exercise level in association with sleep deprivation. DESIGN: The mean sleep onset time of each subject before starting the study was defined as his 0000 h. This study was composed of a Sleep-Sleep session (sleep times, 00:00 h - 08:00 h and 24:00 h - 32:00 h) and a Sleep-Wake session (sleep time, 00:00 h - 08:00 h) with TSD (24:00 h - 32:00 h) placed in a cross-over design with 2-week interval between each session. In each session, the subjects were rested in the supine position under dim light from - 06:00 h to 36:00 h (for 42 hours). SETTING: University-based sleep and chronobiology laboratory PARTICIPANTS: 10 healthy adult men (mean age, 20.9 y; age range, 19-23 y) INTERVENTIONS: NA. MEASUREMENTS AND RESULTS: NK cell activity was measured every 4 hours from 12:00 h. NK cell activity during TSD (at 28:00 h) has been revealed to significantly increase (p=0.01) compared with the corresponding value in the Sleep-Sleep session. This effect was weaker at their usual waking time 32:00 h (p=0.07), and disappeared until 36:00 h (4 hours after awakening). The circadian rhythm phases (dim light melatonin onset time) were coincident between the 2 sessions. CONCLUSIONS: The present findings suggest that one night TSD induces an acute and transient increase in NK cell activity that is not influenced by the effects of circadian rhythm or the amount of physical exercise undertaken during TSD.

[Influences of uncommon isoenzymes on determination of alkaline phosphatase activity by dry-chemistry analyzers].

Dry-chemistry(DC) analysis may be influenced by some matrix effects for measuring uncommon isoenzyme forms. Placental and intestinal alkaline phosphatase(AP) are overestimated by the VITROS DC, compared with results obtained with the wet-chemistry(WC) method of Bretaudiere, et al. using 2-amino-2-methyl-1-propanol (AMP) buffer, however, no such discrepancy between AP results in any DC method and that with a routine WC method recommended by Japanese Society of Clinical Chemistry in that 2-ethylaminoethanol(EAE) buffer is used, has been demonstrated. The type of buffer used affects differently the rates of AP isoenzymes activities. We therefore examined whether the presence of uncommon AP isoenzyme forms in serum caused aberrant DC results for AP in comparison with a routine WC method using EAE buffer. Here, serum samples with only liver AP and bone AP(n : 32); high-molecular-mass AP(n : 11); placental AP(n : 12); intestinal AP(n : 13) and immunoglobulin (Ig) bound AP(n : 12) were analyzed for total AP activity on three different DC analyzers: VITROS 700XR, FUJIDRYCHEM 5000, SPOTCHEM 4410 and a WC analyzer: HITACHI 7350. Values obtained in all of the DCs for sera containing only liver/bone AP agreed with those with the WC method. For sera containing placental AP, the VITROS values were higher than those with the WC method, while the FUJIDRYCHEM values and the SPOTCHEM values were lower. The VITROS values and the FUJIDRYCHEM values for sera containing intestinal AP were lower than those with the WC method, while the SPOTCHEM values were higher. All of the DCs did not affect high-molecular-mass AP and Ig bound liver/bone AP types of macro AP, but underestimated Ig bound intestinal type. Ig bound intestinal AP may be sieved by DC multilayer elements.

Melatonin secretion rhythm disorders in patients with senile dementia of Alzheimer's type with disturbed sleep-waking.

BACKGROUND: There is growing evidence that the dysregulation of circadian rhythms may play an important role in irregular sleep-waking in demented elderly. In this study, we investigated daily variation of the pineal hormone melatonin, which has been reported to possess hypnogenic and synchronizing effects, in patients with senile dementia of Alzheimer's type. METHODS: Serum melatonin secretion rhythms in inpatients with senile dementia of Alzheimer's type (SDAT group, n = 10, average age = 75.7 years) with disturbed sleep-waking and nondemented elderly (ND group, n = 10, age = 78.3 years) without clinical sleep disorders in the same facility were monitored under a dim light condition without excessive physical exercise. RESULTS: The SDAT group showed a significantly higher degree of irregularities in actigraphically recorded rest-activity (R-A) rhythm during the 7-day baseline period compared with the ND group. The SDAT group simultaneously showed significantly reduced amplitude, larger variation of peak times, and diminished amount of total secretion in the melatonin secretion rhythm compared with the ND group. There were significantly positive correlations between the severity of R-A rhythm disorder and the reduced amplitude as well as diminished amount of total melatonin secretion. CONCLUSIONS: The SDAT patients with disturbed sleep-waking possessed melatonin secretion rhythm disorders that may play an important role in irregular sleep-waking in demented elderly.

[Effect of macro-creatine kinase in serum on dry chemistry methods results for total creatine kinase activity].

Most enzymes in serum that are measured in clinical laboratories can occur in macro-molecular forms in a significantly number of patients. Within dry chemistry (DC) multilayer film, physical barriers may prevent contact macro-molecular enzyme forms with the active reagent ingredients. Here, serum samples with macro-creatine kinase (macro-CK) type 1: CK-immunoglobulin complex or type 2: oligomer mitochondrial CK (CKm) were analyzed for total CK activity on three different DC analyzers: VITROS 700XR, FUJIDRYCHEM 5000, SPOTCHEM SP4410 and a classic wet chemistry (WC) analyzer: HITACHI 7350. Macro-CKs were detected and identified by electrophoresis on cellulose acetate. Serum with high amounts of oligomer CKm gave CK values by all of DC methods significantly lower than that by the WC method (p < 0.05). Oligomer CKm gradually converts into monomer forms in serum after storage. With increase in day after storage at 4 degrees C, there was a gradual shift in which percent of total CK activity for oligomer CKm decreased while the ratio of total CK activity, DC method/WC method increased. The principle of analytical method for CK activity determination is commonly to all of the DC methods, the WC method and the electrophoretic analysis. These suggest that oligomer CKm is sieved by DC multilayer film elements. In contrast, each of DC method produced highly corrected CK activities for sample containing CK-immunoglobulin complex. This difference in the effects of macro-CKs may depend upon physicochemical characteristics of analytical DC elements.

[Evaluation of direct identification testing from positive blood culture bottles with MicroScan rapid identification panels].

We evaluated the direct identification method from growth-positive blood culture bottles using MicroScan Rapid ID panels (DADE BEHRING) for the purpose of rapid identification. The inoculum for Rapid ID panels were prepared using an isolation method from blood culture bottles by VACUTAINER (BD). McF 1.0 had a better result than McF 0.5 as the inoculum concentration for Rapid ID panels. Rapid ID panel identification results were effected by blood contamination for > or =0.3% of S. aureus and 0.9% of a strain of E. coli. Blood contamination from the bottle may cause an issue to the identification results. The accuracy of this direct identification testing was 72.0% (36 out of 50) for gram positives organisms and 88. 9% (80 out of 90) for gram negatives organisms. Although some strains including S. pyogenes, coagulase-negative staphylococci and non-Fermentative Gram Negative Rods had not identified correctly, this method provides a preliminary result within 3 hours and provides a fast turn around time. In conclusion, this method was considered as an effective method for routine testing.

Determination of uric acid in scalp hair for non-invasive evaluation of uricemic controls in hyperuricemia.

The uric acid concentration in blood has been widely accepted as a diagnostic indicator of hyperuricemia and gout, and its assay method is well established. In the present study, we developed a simple and rapid method for the determination of uric acid in hair, which can be obtained non-invasively. The concentration (nmol/mg hair) of uric acid extracted from 10-20 mg hair with 0.1 M potassium hydroxide was determined by an enzymatic method using uricase. The concentration of uric acid (nmol/mg hair, mean+/-S.D.: 0.49+/-0.157, n=16) in hair from hyperuricemic patients was significantly higher than that (0.26+/-0.107, n=8) in healthy volunteers (p<0.01). The within-run and between-day precision (CVs) of the assay was 9.6-10.3% (n=10 each) and 11.6-16.3% (n=7 each), respectively. The concentration (nmol/mg hair, y) of uric acid in hair correlated well with that in serum (mg/l, x): y=0.09x-0.12 (r=0.75, Syx=0.122, n=23). Changes in the concentration of uric acid in the hair of antihyperuricemic drug-treated patient paralleled that in serum, suggesting that the concentration of uric acid in hair is a reliable indicator of the metabolic control in hyperuricemia.

[Determination of uric acid in scalp hair for non-invasive estimation of uricemic control in hyperuricemia].

Uric acid in blood has been widely accepted as a reliable indicator of hyperuricemia and gout, and its assay method has been established. In the present study, we developed a simple and non-invasive rapid method for the determination of uric acid in hair. Concentration(nmol/mg hair) of uric acid extracted from 10-20 mg of hair(95% < extractability; 0.1N KOH, 37 degrees C, 2 hr) was determined by an enzymatic method using uricase. The concentration of uric acid(nmol/mg hair, mean +/- SD: 0.489 +/- 0.157, n = 16) in hair from hyperuricemic patients was significantly higher than that(0.258 +/- 0.107, n = 8) from healthy volunteers(p < 0.01). Within-run and between-day precisions(reproducibilities, CVs) for the assay were 9.6-10.3%(n = 10 each) and 11.6-16.3%(n = 7 each), respectively. The concentration(y, nmol/mg hair) of uric acid in hair correlated well with that in blood(x, g/l): y = 8.770x-0.123(r = 0.746, Syx = 0.122, n = 23). Changes in the concentration of uric acid in hair of hyperuricemic patient treated with allopurinol paralleled to those in blood. In conclusion, it was confirmed that the concentration of uric acid in hair reflected that in blood, suggesting that measuring uric acid in hair can be available for the metabolic control in hyperuricemia.

Preparation of useful synthetic intermediates of Taxol analogs, cyclooctenone derivatives.

Stereoselective syntheses of omega-(alpha-bromoketo) octanals and nonanal with oxygenated functions and formation of the corresponding eight-membered carbocyclic aldols by subsequent samarium(II)-mediated cyclization are demonstrated. Cyclooctenones deoxygenated at the C2 or C10 position in the taxane framework are prepared by dehydration of the above aldols.

[Modification of enzyme molecules due to naturally-occurring immunoglobulins linking].

The appearance of circulating enzyme-linked immunoglobulin complexes (E-Ig), that are thought to be formed because of specific interaction between circulating immunoglobulins and normal serum isoenzymes. The presence of E-Ig in an individual is regarded as a benign phenomenon, although the presence of E-Ig may result in altered enzyme activity in serum and interfere with the measurement of isoenzymes, and is thus of diagnostic importance. The present paper deals with the isoenzyme specificities of immunoglobulins isolated from E-Igs and electrophoretically abnormal enzyme components due to E-Igs. The immunoglobulins react with a single isoenzyme, certain isoenzymes only or all the isoenzymes of a given enzyme. E-Igs thus formed are themselves heterogeneous, giving rise to many different electrophoretically abnormal enzyme components.

[The ABO blood groups-dependent reference intervals for serum alkaline phosphatase isozymes and total activity in individuals 20-39 years of age].

Reference intervals of the activity of serum alkaline phosphatase isozymes (AP-IZ: high molecular-, liver-, bone- and intestinal-types) were determined according to the ABO (blood type) system in 200 healthy subjects aged 20-39 years. AP activity was determined according to the JSCC method. AP-IZ was stained by the formazan method after isolation by TITANIII-Lipo plate electrophoresis. For the electrophoresis, treated serum with neuraminidase and untreated one were concomitantly used for detecting liver AP and bone AP respectively. As a result of comparison of mean AP-IZ activity among the groups divided according to the ABO system, total AP, intestinal AP and liver AP activities in the type B and O persons were significantly higher than in the type A and AB persons. It is well known that the activities of total AP and intestinal AP in type B and O persons are higher than in type A and AB persons, but there have been no reports showing that the activity of liver AP in type B and O persons is higher than in type A and AB persons. Furthermore, in the type B and O persons there was a low correlation (r = 0.195, p < 0.05) between the activities of liver AP and intestinal AP. The present assessment included subjects in the age group (20-39 years) considered not to show fluctuations in the activity of bone AP, which is influenced by age. The above findings should be investigated in regard to other age groups.

[Serum IgD concentrations in normal individuals 20-39 years of age].

Immunoglobulin D(IgD) concentration in serum of normal individuals varies widely, and the reference interval for the concentrations remains to be defined. In the present study, IgD concentrations were determined in serum from 637 normal individuals 20-39 years of age by the latex nephelometric immunoassay. The IgD concentrations ranged from < 0.20 to 71.5mg per deciliter, and the median and the 95% range as reference intervals were 1.99 and < 0.20 to 17.3mg/dl, respectively. There was difference of serum IgD concentration between female 20-29 years of age and female 30-39 years of age (p < 0.05), but not on sex. The correlation between serum IgD concentration and serum IgA concentration was significant (p < 0.001).

The first report of a case with acute myocardial infarction showing familial deficiency of creatine kinase.

A 46-year-old male patient was diagnosed as suffering from acute myocardial infarction, but his serum creatine kinase (CK) level was extremely low and no CK isozymes were detected in the serum. The total CK activities in the skeletal muscle amounted to only 2% of that of the control. Electrophoresis of the CK isozymes in the skeletal muscle showed that CK-MM was absent but the CK-BB and abnormal isozyme bands were present. There was no evidence of myocardial ischemia, although the exercise treadmill test revealed ST segment depression in the chest leads. One of the patient's sisters had an extremely low serum CK level suggesting inheritance of this abnormality. This is the first report of a case showing familial deficiency of CK.

Separation and characterization of sialic acid-containing salivary-type amylase from patients' sera with immunoglobulin A-type myeloma.

Isoamylases, with an abnormal anodic migration, were detected by an electrophoretic technique in the sera from two patients with immunoglobulin A-type myeloma. The abnormal isoamylase bands migrate towards the anode faster than the salivary isoamylase (S2) band and were stained more strongly than the S2 sub-band. The abnormal isoamylase could be separated from patients' sera by using size-exclusion high-performance liquid chromatography. The serum abnormal isoamylases were showed to be sialic acid residues containing amylase, after the study of treatment with neuraminidase (EC 3.2.1.18), and to be salivary-type amylase, after the study of reaction with human salivary monoclonal antibody. The abnormal bands were not detected in the saliva from one patient. The two patients had no detectable malignancies except myeloma. These findings strongly suggest that the sialic acid-containing salivary-type amylases were produced ectopically from myeloma cells. In this regard the ectopic amylase production by myeloma cells is discussed.

Electrophoretic analysis of enzyme-linked immunoglobulins and their clinical significance.

The appearance of circulating enzyme-linked immunoglobulin complexes (E-Ig) is common for most enzymes used in clinical biochemical tests. The presence of E-Ig may result in altered enzyme activity in serum and interfere with the measurement of isoenzymes, and is thus of diagnostic importance. E-Ig can be identified by confirming that the binding protein is an immunoglobulin by its reaction with specific anti-human immunoglobulin antibodies. Currently, the presence of E-Ig in an individual is regarded as a benign phenomenon, not indicative of any particular disease process. However, it is becoming clear that E-IgG are closely associated with autoimmune states.

Enzyme-linked immunoglobulins and their clinical significance.

Most of the enzymes used in routine biochemical tests have been detected in circulating enzyme-linked immunoglobulin (Ig) complexes (E-Ig). The complexes often cause unexplained hyperenzymemia and give anomalous patterns on isozyme electrophoresis. However, E-Ig does not appear to be associated with any pathological conditions. The present study was undertaken to determine the clinical characteristics of patients with E-Ig. More than 42,000 patients selected at random and 10,000 blood donors were screened for lactate dehydrogenase (LDH)-linked Ig complexes (LDH-Ig), amylase (Amy)-linked Ig complexes (Amy-Ig), alkaline phosphatase (AP)-linked Ig complexes (AP-Ig) and creatine kinase (CK)-linked Ig complexes (CK-Ig). LDH-Ig and Amy-Ig were screened by electrophoresis for routine isozyme analysis and identified by precipitin reactions. AP-Ig and CK-Ig were screened and identified by counter immunoelectrophoresis. The incidence of all E-Ig ranged from 0.18% to 0.61% in patients and from 0.03% to 0.23% in blood donors. The incidence curves of E-Ig were age-related, generally increasing with age. The curves of E-IgG's for different enzymes resembled each other in shape but those of E-IgA's did not. A positive correlation between E-IgG and autoimmune diseases was present. Consequently, it is concluded that both autoimmune diseases and age are the common clinical factors involved in E-IgG. No specific diseases were found for cases with E-IgA. However, it is suggested that some CK-IgA, but not all, is caused by elevated serum CK activity.