One-step heminested PCR for amplification of Neisseria meningitidis DNA in cerebrospinal fluid.

A one-step polymerase chain reaction (Heminested-PCR) was designed to target the 16S rRNA fragment simultaneously using a set of primers for the universal bacterial group and a Neisseria meningitidis species-specific sequence for diagnostic purposes. The diagnostic features of the Heminested-PCR were evaluated in the study of 168 cerebrospinal fluid (CSF) specimens from 84 patients with a N. meningitidis infection, meningitis caused by unrelated bacteria and other etiologies (57 patients), or suspicious cases (27 patients) with clinical symptoms of bacterial meningitis but with negative results from bacteriological procedures. About 90% of patients with bacterial meningitis, including those suspicious cases, had prior antibiotic therapy. The sensitivity, specificity, positive, and negative predictive values found in relation to culture and/or microscopy were 91.7, 100, 100, 100, and 90.5%, respectively. In patients suspected of having bacterial meningitis, the Heminested-PCR revealed 51.9% (14 patients) positive for N. meningitidis infection and 40.7% (11 patients) positive for unrelated bacterial infections. The agreement of the Heminested-PCR with culture and/or microscopy was high and ranked as almost perfect (kappa indices > 0.856), in contrast to its agreement with other techniques. These findings speak in favor of the molecular diagnosis of meningococcal meningitis in patients who are culture- and/or microscopy-negative, due to their prior antibiotic treatment.

The effect of methamphetamine on the release of acetylcholine in the rat striatum.

We examined the effect of methamphetamine on the release of acetylcholine in the striatum of freely moving rats, using an in vivo microdialysis method. The basal level of acetylcholine was 3.67+/-0.47 pmol/30 microl per 15 min in the presence of neostigmine (10 microM). Tetrodotoxin (1 microM), a selective blocker of voltage-dependent Na+ channels, markedly inhibited the release of acetylcholine in the striatal perfusates. Apomorphine (1.0 mg/kg, i.p.), a dopamine receptor agonist, also significantly attenuated acetylcholine release. Methamphetamine (0.1 and 0.5 mg/kg, i.p.) did not immediately affect acetylcholine release in the striatum, but a dose of 1.0 mg/kg (i.p.) induced an increase of acetylcholine release in the striatum at 15-60 min. Striatal infusion of methamphetamine (5 and 10 microM) did not influence acetylcholine release. The increase following intraperitoneal administration of methamphetamine was slightly diminished by haloperidol (0.5 mg/kg). After microinjection of the neurotoxin, 6-hydroxydopamine (6 microg/3 microl), in the substantia nigra 7 days before, the increase of acetylcholine induced by the administration of methamphetamine (1.0 mg/kg) was slightly attenuated, whereas the administration of reserpine (2 mg/kg, i.p.) 24 h before, combined with alpha-methyl-p-tyrosine (300 mg/kg, i.p.) 2.5 h before, completely blocked the increase in release of acetylcholine. These findings suggest that methamphetamine exerts an excitatory influence on striatal acetylcholine release in freely moving rats, and that this excitatory effect involves the dopaminergic system and the catecholaminergic system.

Analyses of mixed melanogenesis in tyrosinase cDNA-transfected human amelanotic melanoma cells.

In the pigment cell the synthesis of tyrosinase and the formation of premelanosomes are independent, yet coordinated, processes. However, the interrelationship between the two processes has not been elucidated previously. In this study, an expression plasmid for human tyrosinase cDNA was constructed and transfected into a human amelanotic melanoma cell line, G-361. Stable transfected cells (G-CMHT-3) were obtained with high tyrosinase activity and distinct melanization occurred. As for the type of melanin, both pheo- and eumelanin contents increased in G-CMHT-3 cells. Interestingly, catalase activity as one of the other melanogenic enzymes was decreased in G-CMHT-3 cells. The decrease of catalase activity was considered to play a role in melanin-polymer formation, resulting in the increase of both pheo- and eumelanin contents. Under electron microscopic observation, dopa-oxidase-positive Golgi-associated endoplasmic reticulum of lysosome, coated vesicles, and premelanosomes were observed in pigmented G-CMHT-3 cells, and the expressed tyrosinase was considered to be well translocated to these organelles. In addition, the number of premelanosomes (stages I-III) as well as melanosomes (stage IV) increased in G-CMHT-3 cells compared to those in G-361 cells. It is also noted that G-CMHT-3 cells showed more normal phenotype premelanosomes with occasional transitional premelanosomes exhibiting partial melanin polymer formation within their concentric whorl-like internal membranes. Furthermore, the number of eumelanosomes in G-CMHT-3 cells was much larger than that in G-361 cells. These results suggest that the tyrosinase introduced by its cDNA transfection induced active and structurally non-aberrant premelanosome formation resulting in the upregulated pheo- and eumelanogenesis.

An investigation into the effects of apomorphine on the release of acetylcholine in the striatum of freely-moving rat using in vivo microdialysis.

1. Using in vivo microdialysis, we studied the effects of apomorphine on the release of acetylcholine (ACh) in the striatum of freely-moving rats. 2. Basal release of ACh was 3.01 +/- 0.51 pmol/15 min in 30 microliters (containing of 10 microM neostigmine) of the striatal perfusate. 3. Ringer containing tetrodotoxin (1 microM), hemicholinium-3 (5 mM), or Ca(2+)-free Ringer was perfused into the striatum. Each of these treatments produced a significant attenuation in release content of ACh. High K+ (30 mM) produced a significant increase in ACh release. 4. In normal rats, apomorphine (0.05 mg/kg, s.c.) increased striatal ACh release. A high dose (1.0 mg/kg) significantly attenuated release. 5. In 6-OHDA-pretreated rats, although the lower dose of apomorphine did not increase striatal ACh release, the high dose produced a more significant attenuation of release. 6. It is concluded that apomorphine enhanced or attenuated the striatal ACh release and that this effect was regulated by nigro-striatal dopaminergic system in the freely moving rats.