XIII. Yeast sequencing reports. Cloning and sequencing of the NES24 gene of Saccharomyces cerevisiae

We have cloned NES24 using a temperature-sensitive nes24-1 mutant as a host and sequenced a 3162 bp XhoI-EcoRI DNA fragment containing the NES24 gene. Computer analysis revealed that this segment contains a 1806 bp open reading frame which is needed for complementation of the nes24-1 mutation. We found SUP8 in the region upstream of the NES24 gene, placing the NES24 gene on chromosome XIII. A protein homology search indicated that NES24 encodes a new protein. The disruption of the NES24 gene resulted in temperature-sensitive growth. The sequence has been deposited in DDBJ/EmBL/GenBank data bases under Accession Number D15052.     

Antigenic and genetic diversities of Babesia ovata in persistently infected cattle

Exploring the antigenic and genetic diversities of Babesia ovata, we obtained several field isolates from grazing cattle in the Okushiri island, Japan. Parasite isolation was greatly facilitated by using bovine red blood cell-substituted SCID mice (Bo-RBC-SCID mice), into which the blood samples of the cattle were inoculated. Isolates from different individuals within a herd of cattle were compared in immunoblot analysis with an anti-B. ovata serum and also in Southern blot analysis with a probe for the small subunit ribosomal RNA gene. In both analyses, the isolates exhibited banding patterns that were significantly different from each other. We were also able to obtain a series of parasite isolates from a single cow in different seasons of a nine months period, including winter when active vector ticks were not in the field environment. Different seasonal isolates showed different banding patterns in both immunoblot and Southern blot analyses. By contrast, these analyses detected little difference among the parasites that had been passed various times in Bo-RBC-SCID mice, where no specific immune responses should be generated. These results indicate that individual animals within a herd of cattle were infected with antigenically and genetically diversified populations of B. ovata, and that the parasites could persistently infect a single animal with dynamic change in their predominant subpopulations.     

Development of a Trans-Mucosal Controlled-Release Device for Systemic Delivery of Antianginal Drugs Pharmacokinetics and Pharmacodynamics

Oral mucosa is well-known to be one of the best routes for drug absorption. But very few R & D works have been initiated to investigate the feasibility of using this site to control drug delivery. A transmucosal controlled-release device, which is capable of achieving excellent absorption and controlled release of drugs, has been developed. The device is a tabletshaped mucoadhesive system which is composed of two layers. The upper layer is a fast-release layer and the lower layer is a sustained-release layer, and designed to be applied between buccal and gingival mucosae. Both layers are formulated from synthetic polymers to control the release of drugs.

Isosorbide dinitrate(ISDN), a well-documented antianginal drug, is known to be susceptible to extensive presystemic elimination when taken orally. It was used as the candidate drug and the systemic bioavailability was studied in human and observed to be improved by as much as 5 fold when compared to a marketed oral sustained-release tablet; On the other hand, much smaller amount of metabolites was formed. The plasma profile of ISDN has also been observed to be substantially prolonged (12 hrs as compared to less than 1 hr for sublingual tablet and spray product on the market). These observations have demonstrated that this device is capable of not only bypassing hepatic “first-pass” metabolism but also having a sustainedrelease property of prolonging the release of ISDN.

Clinical studies performed in the anginal patients for up to one year have demonstrated the therapeutic benefits of this device in achieving a substantial reduction in the frequency of anginal attacks.

This type of device was also applied to the systemic delivery of another antianginal drug, Nifedipine, by employing a formulation with longer sustained drug release property. Again, the clinical results demonstrated that a prolonged duration of therapeutic plasma concentration has also been accomplished.     

Mechanical stress control in a VLSI-fabrication process: a method for obtaining the relation between stress levels and stress-induced failures

An ideal fabrication process is designed to minimize mechanical stress in semiconductor devices and to improve device reliability. Mechanical stress levels were predicted by in-house simulations supported by a thin-film database. These stress levels were correlated with stress-induced defects by TEM analysis supported by fail bit addressing on matured megabit SRAMs. Amorphous-doped silicon film with various annealing temperatures were used for the gate electrode to change the mechanical stress in devices and to get the direct relationship between predicted stress levels and stress related defects. The authors describe brief guidelines for suppressing dislocations in the small geometry shallow-trench isolation process utilizing this system. Polysilicon thickness in the W-polycide gate electrode is designed to minimize mechanical stress in the gate oxide and to suppress the gate oxide failure in probe and class tests. Moreover, critical stress generates dislocations during post source/drain ion implantation anneal obtained by a ball indentation method. This indicated that lower temperature anneal is effective in suppressing the dislocations. A two-step anneal was introduced to suppress dislocations and to enable higher ion activation.     

Design of electrofusion joints and evaluation of fusion strength using fusion simulation technology

Fusion simulation is one of the key techniques in designing and producing electrofusion (EF) joints for gas distribution and in evaluating fusion joint integrity. This paper describes the result of a numerical simulation of a thermal fusion process, using the finite element method. A nonlinear heat transfer computer program was used to obtain the temperature profile of a large electrofusion joint at fusion. The effects of applied voltage, heating time, wire pitch, and ambient temperature were examined for designing a 150-mm EF joint. A method to shorten the cooling time was also investigated. The fusion condition range suitable for a 150-mm EF joint was found to be slightly narrower than that suitable for a 50-mm EF joint. Examination of the effect of wire pitch revealed that if the pitch is extremely large, thermal degradation starts in the resin close to the wire before the fusion-interface strength reaches the maximum value. We have developed a program to simulate the process of closing the gap between the pipe and the joint due to resin expansion and melting after the power is supplied.     

REM sleep deprivation potentiates the effects of imipramine and desipramine but not that of clomipramine in the forced swimming test

The human amnestic syndrome associated with lesions of the hippocampus and amygdala is characterized by a selective impairment of recent (explicit, episodic) memory. Benzodiazepine (BZ) treated normal subjects demonstrate similar, marked impairments in episodic memory, but in addition, BZ also induces sedation and inattention. Thus, the amnestic effects of BZ may be secondary to drug-induced sedation. However, when subjects were pretreated with the specific BZ receptor antagonist, flumazenil, the sedative and attentional effects of diazepam were blocked, but a marked impairment in episodic memory still occurred. This demonstrates that, using neuropharmacological methods, it is possible to produce a dissociation of memory impairment from inattention and sedation. Such distinct patterns of cognitive dysfunction may serve as models for clinical cognitive syndromes.     

Opioid receptors altered binding nature in guinea-pig brain following the development of morphine dependence

Morphine is well known to produce tolerance and dependence. The mechanisms for these phenomena are not clearly understood and there are a number of conflicting reports that chronic morphine administration decreases, increases, or leaves unchanged the number of opioid binding sites. We examined the potency of MScontin (oral controlled-release preparation of morphine) to induce morphine dependence and also determined the change of mu, delta and kappa opioid receptor types in brain homogenates obtained from morphine-dependent guinea-pigs. 1. Guinea-pigs were implanted subcutaneously with MScontin (300 mg.kg-1) and naloxone was employed to precipitate jumping behavior of withdrawal symptoms at various times. The highest degree of physical dependence was observed on the 2nd day after implantation. Therefore, this period was chosen to investigate opioid receptor binding assay. 2. Two days after implantation, the binding of 3H-DAGO (mu agonist), 3H-DPDPE (delta agonist) and 3H-U69593 (kappa agonist) to brain membranes prepared from morphine dependent and control guinea-pigs was determined. Scatchard plot of the saturation binding data revealed an increase in Bmax values (maximum specific binding) and no change in the Kd values (equilibrium dissociation constants) of 3H-opioid ligand bindings obtained from morphine-dependent animals as compared to controls. These results indicate that brain mu, delta and kappa opioid receptors are up-regulated in morphine dependent guinea-pigs.