Genetic control of melatonin synthesis in the pineal gland of the mouse.

Pineal melatonin may play an important role in regulation of vertebrate circadian rhythms and in human affective disorders. In some mammals, such as hamsters and sheep, melatonin is involved in photoperiodic time measurement and in control of reproduction. Although wild mice (Mus domesticus) and some wild-derived inbred strains of mice have melatonin in their pineal glands, several inbred strains of laboratory mice (for example, C57BL/6J) were found not to have detectable melatonin in their pineal glands. Genetic analysis suggests that melatonin deficiency in C57BL/6J mice results from mutations in two independently segregating, autosomal recessive genes. Synthesis of melatonin from serotonin in the pineal gland requires the enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Pineal glands from C57BL/6J mice have neither NAT nor HIOMT activity. These results suggest that the two genes involved in melatonin deficiency are responsible for the absence of normal NAT and HIOMT enzyme activity.

Immediate maxillary reconstruction after malignant tumor extirpation.

AIMS: Immediate maxillary reconstruction after malignant tumor extirpation differs from other types of maxillary reconstruction. Our reconstruction algorithm is described in this article. METHODS: One hundred ninety-four patients who had undergone maxillectomy for malignant tumors were reviewed, and maxillectomy defects were classified with the method of Cordeiro and Santamaria. RESULTS: Mean total blood loss was 848 ml, and 71 patients died within 2 years after surgery. For type IIIa defects of the orbital floor, titanium mesh or vascularized bone or cartilage was used for reconstruction, but the rate of postoperative complications did not differ between titanium and autografts. Therefore, to reconstruct orbital floor defects we have recently used only titanium mesh. For type I or II defects, we use autografts for only selected cases. CONCLUSIONS: We strive to perform less-invasive reconstructive surgery after resection for maxillary malignancy.

Image analysis of microvessel surface area predicts radiosensitivity in early-stage laryngeal carcinoma treated with radiotherapy.

PURPOSE: The tissue oxygenation level, which is theoretically governed by distance from blood vessels, is one of the most important modulators of the radiosensitivity of carcinoma. A computed image analysis system for the detection of tissue oxygenation was developed to establish a method of predicting radiosensitivity in early-stage laryngeal carcinoma treated by curative radiotherapy. EXPERIMENTAL DESIGN: Microvessel structures labeled with CD31 antigen were investigated in 55 patients undergoing curative radiotherapy for T1 and T2 laryngeal carcinoma. We calculated (a) microvessel density [(MVD) vessels/field] under a microscope; (b) the ratio of the total microvessel number (TN):tumor area (TA) [TN:TA; vessels/mm2]; (c) the ratio of the total microvessel perimeter (TP):TA (TP:TA; mm/mm2); and (d) the ratio of tumor tissue area >150 microm from microvessels (hypoxic ratio; %) as parameters of tissue oxygenation in each whole biopsy specimen by using an image analyzer. We compared each of these factors with radiosensitivity. RESULTS: Mann-Whitney's U test revealed that tumors with a high MVD (median, 42 vessels/field), high TN:TA ratio (median=40.9 vessels/mm2), high TP:TA ratio (median, 2.92 mm/mm2), and low hypoxic ratio (median, 30.3%) had significantly greater radiosensitivity than tumors with a low MVD, low TN:TA ratio, low TP:TA ratio or high hypoxic ratio (P = 0.002, P = 0.0004, P < 0.0001, and P = 0.004, respectively). CONCLUSIONS: Prediction of radiosensitivity on the basis of the TP:TA ratio can be used as an efficient means of avoiding ineffective radiation, complications after salvage surgery, and prolonged hospital stays.

Potential role of microvessel density in predicting radiosensitivity of T1 and T2 stage laryngeal squamous cell carcinoma treated with radiotherapy.

Curative radiotherapy is the first choice of therapy for T1 and T2 stage laryngeal squamous cell carcinoma (LSCC) patients to preserve their phonation. Patients with recurrent tumors who undergo salvage surgery require prolonged nasal feeding. Therefore, clinical interest has been focused on elucidating a predictive factor indicating which tumors are likely to be radiosensitive before radiotherapy. We analyzed the relations between radiosensitivity and clinicopathological factors (gender, tumor location, histological factors, and clinical tumor-node-metastasis stage), expression of apoptosis-related proteins (p53, bax, bcl-2), apoptotic index using the terminal deoxynucleotidyltransferase-mediated nick end labeling method, expression of cell proliferation-related proteins (Ki-67-labeling index and epidermal growth factor receptor overexpression) and microvessel density (MVD, vessels/field = 0.391 mm2) in biopsy specimens from 31 LSCC patients given radiotherapy (total radiotherapy dose of 52-70 Gy over 4-6.5 weeks). Univariate analysis revealed that tumors with a high MVD (> or =35 vessels/field) showed better radiosensitivity than those with a low MVD (<35 vessels/field, P = 0.008) and that a high Ki-67-labeling index (> or =40%) was weakly associated with radiosensitivity (P = 0.056). Multivariate analysis and Kaplan-Meier analysis showed that MVD alone had significant predictive power for radiosensitivity in T1 and T2 stage LSCCs after radiotherapy (P = 0.012, 0.0003, respectively). No significant association between clinicopathological factors, or of overexpression of p53, bax, bcl-2, epidermal growth factor receptor, or apoptotic index, with radiosensitivity was found. These results indicate that MVD is a potentially useful clinical factor predicting radiosensitivity for patients with early stage LSCCs before treatment.

Different responses of the circadian system to GABA-active drugs in two strains of mice.

Recent work in our laboratory has shown that sodium pentobarbital injections can induce phase-dependent phase shifts of the circadian rhythm of locomotor activity with the maximum advance at circadian time (CT) 8 and the maximum delay at CT0 in SK/Nga mice but no phase shifts in C57BL/6 mice. In the present study, the possibility that the differences in the effects of pentobarbital on the circadian rhythm may be due to different contributions of the GABA-ergic system to circadian organization in the two strains was tested by comparing the responses of SK mice with those of C57BL mice to muscimol (2 mg/kg), a GABA receptor agonist, and triazolam (25 mg/kg), which is thought to act by potentiating the action of GABA. The hypothesis that pentobarbital-induced phase shifts of SK mice are mediated by the GABA receptor system was also tested by observing whether the phase-shifting effects of pentobarbital were blocked by bicuculline (0.5 mg/kg), a selective antagonist of GABA, injected 3 min prior to pentobarbital (30 mg/kg). The results indicated that muscimol induced phase advances at CT8 and phase delays at CT0, and triazolam induced phase advances at CT8 in SK mice. No phase shifts were induced by any treatment in C57BL mice. These results suggest that the role of GABA-ergic systems in circadian organization may be different in SK and C57BL mice. In addition, bicuculline could block the phase-shifting effects of pentobarbital in SK mice, suggesting that the GABA receptor system may mediate phase-shifting effects of pentobarbital in SK mice.

Differences in circadian photosensitivity between retinally degenerate CBA/J mice (rd/rd) and normal CBA/N mice (+/+).

Using the magnitude of phase shift of circadian locomotor rhythms induced by a single pulse of white fluorescent light, we compared the sensitivity of the circadian system to light in retinally degenerate mice and in normal mice. In the first experiment, phase response curves (PRCs) for 10-lux white light were generated in CBA/J mice with retinal degeneration (rd/rd) and CBA/N mice with normal retinas (+/+). Although large phase delays early in the subjective night and small phase advances in the late subjective night were observed in CBA/N mice, CBA/J mice showed only small phase delays early in the subjective night. In the second experiment, we found that the magnitude of phase shifts at circadian time (CT) 16 for delays and CT 24 for advances in CBA/J mice became larger with increasing light intensity, and that CBA/J mice could show the same amount of phase shift as CBA/N mice when higher intensities were used. These findings indicate that the differences in the shapes of PRCs are not due to differences in the nature of the oscillating system, but to differences in circadian photosensitivity between these strains. Because the genetic background for the rd loci was not completely identical in the CBA/N and CBA/J mice, it was possible that genes other than the rd gene might have caused different photosensitivity in these mice. Therefore, in the last experiment, we studied the circadian photosensitivity in F1 hybrids between CBA/N and CBA/J mice and in the backcross progeny with different genotypes (+/rd and rd/rd) obtained from the crossing between F1 hybrids between CBA/N and CBA/J mice and in the backcross progeny with different genotypes (+/rd and rd/rd) obtained from the crossing between F1 and CBA/J mice. In these mice with heterogeneous genetic backgrounds as well, mice with retinal degeneration were always less sensitive to light, suggesting that reduced circadian photosensitivity is caused by retinal degeneration. These results are discussed in relation to recent findings in retinally degenerate C57BL mice, which have been found to have normal circadian sensitivity to light.

Modulation of Starling forces and muscle fiber maturity permits adenovirus-mediated gene transfer to adult dystrophic (mdx) mice by the intravascular route.

Duchenne muscular dystrophy (DMD) and other inherited myopathies lead to progressive destruction of most skeletal muscles in the body, including those responsible for maintaining respiration. DMD is a fatal disorder caused by defects in the dystrophin gene. Recombinant adenovirus vectors (AdV) are considered a promising means for therapeutic delivery of a functional dystrophin gene to DMD muscles. If AdV-mediated dystrophin gene replacement in DMD is to be successful, development of a systemic delivery method for targeting the large number of diseased muscles will be required. In this study we investigated two major factors preventing efficient AdV-mediated gene transfer to skeletal muscles of adult animals after intravascular AdV administration: (1) an inability of AdV particles to breach the endothelial barrier and enter into contact with myofibers, and (2) a relatively nonpermissive myofiber population for AdV infection due at least in part to insufficient levels of the coxsackie/adenovirus attachment receptor (CAR). On the basis of established principles governing the transendothelial flux of macromolecules, we further hypothesized that an alteration in Starling forces (increased hydrostatic and decreased osmotic pressures) within the intravascular compartment would facilitate AdV transendothelial flux via convective transport. In addition, experimental muscle regeneration was employed to increase the prevalence of immature myofibers in which CAR expression is upregulated. Here we report that by employing the above-described strategy, high-level heterologous reporter gene expression was achievable in hindlimb muscles of normal rats as well as dystrophic (mdx) mice (genetic homolog of DMD) after a single intraarterial injection of AdV. Microsphere studies confirmed enhanced transport into muscle of fluorescent tracer particles in the size range of AdV, and there was a high concordance between CAR upregulation and myofiber transduction after intraarterial AdV delivery. Furthermore, in mdx mice examined 10 days after intraarterial AdV delivery, the aforementioned procedures had no adverse effects on the force-generating capacity of targeted muscles. These findings have implications for eventual AdV-mediated gene therapy of generalized skeletal muscle diseases such as DMD using a systemic intraarterial delivery approach.

Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles.

Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.

Differential effects of dystrophin and utrophin gene transfer in immunocompetent muscular dystrophy (mdx) mice.

Duchenne muscular dystrophy (DMD) is a fatal disease caused by defects in the gene encoding dystrophin. Dystrophin is a cytoskeletal protein, which together with its associated protein complex, helps to protect the sarcolemma from mechanical stresses associated with muscle contraction. Gene therapy efforts aimed at supplying a normal dystrophin gene to DMD muscles could be hampered by host immune system recognition of dystrophin as a "foreign" protein. In contrast, a closely related protein called utrophin is not foreign to DMD patients and is able to compensate for dystrophin deficiency when overexpressed throughout development in transgenic mice. However, the issue of which of the two candidate molecules is superior for DMD therapy has remained an open question. In this study, dystrophin and utrophin gene transfer effects on dystrophic muscle function were directly compared in the murine (mdx) model of DMD using E1/E3-deleted adenovirus vectors containing either a dystrophin (AdV-Dys) or a utrophin (AdV-Utr) transgene. In immunologically immature neonatal animals, AdV-Dys and AdV-Utr improved tibialis anterior muscle histopathology, force-generating capacity, and the ability to resist injury caused by high-stress contractions to an equivalent degree. By contrast, only AdV-Utr was able to achieve significant improvement in force generation and the ability to resist stress-induced injury in the soleus muscle of immunocompetent mature mdx animals. In addition, in mature mdx mice, there was significantly greater transgene persistence and reduced inflammation with utrophin compared to dystrophin gene transfer. We conclude that dystrophin and utrophin are largely equivalent in their intrinsic abilities to prevent the development of muscle necrosis and weakness when expressed in neonatal mdx animals with an immature immune system. However, because immunity against dystrophin places an important limitation on the efficacy of dystrophin gene replacement in an immunocompetent mature host, the use of utrophin as an alternative to dystrophin gene transfer in this setting appears to offer a significant therapeutic advantage.

Application of in vivo microdialysis to pineal research in birds: measurement of circadian rhythms of melatonin.

Circadian rhythms of pineal melatonin release were measured in free-moving pigeons, Japanese quails, and chickens under light-dark cycles followed by constant dim light. Although melatonin levels differed among individual birds, circadian rhythms of melatonin were observed in all of them. Using this technique, we could examine phase shifts of melatonin rhythms and suppression of melatonin release by photic stimulation in pigeons. We could also examine effects of norepinephrine infusion on melatonin release. These results indicate that microdialysis is useful for the study of pineal melatonin rhythms in birds.

A model of human sleep-related growth hormone secretion in dogs: effects of 3, 6, and 12 hours of forced wakefulness on plasma growth hormone, cortisol, and sleep stages.

Twenty-four canine GH (cGH) and cortisol secretion patterns associated with sleep stages were studied in 10 male adult dogs. Plasma samples were obtained at 30- or 15-min intervals via an indwelling catheter. Under baseline conditions, all dogs showed irregular polyphasic sleep, and the episodic cGH secretion had no apparent relationship with sleep or the light-dark cycle. Five dogs were subjected to regular sleep-wake cycles; 3, 6, and 12 h of forced wakefulness (FW) were repeated at 3-, 6-, and 12-h intervals (recovery sleep periods), respectively. Peak cGH secretion (mean +/- SD, 6.4 ng/ml +/- 2.4) occurred soon after recovery sleep onset in 25 of 40 total recovery periods. The incidence of sleep-onset cGH peaks and cGH secretion during the first hour of recovery sleep significantly increased with the length of the preceding FW, but were not affected by the time of day. Delta wave sleep increased during this hour, suggesting a possible correlation with the sleep-onset cGH peak. During the first 3 h of recovery after 6 and 12 h of FW, cGH secretion was significantly enhanced, but cortisol was not. Considering the characteristics of human sleep-related GH secretion, we suggest that this peak cGH secretion represents a model of human GH secretion. Possibly, a close association of cGH secretion with sleep is concealed under the baseline condition and uncovered by inducing longer sleep-wake cycles in dogs. No circadian cortisol variation was detected under the baseline or the experimental conditions.

Identification of rhodopsin in the pigeon deep brain.

We detected rhodopsin gene expression in the pigeon lateral septum, a photosensitive deep brain region that is responsible for the photoperiodic gonadal response. The nucleotide sequence of the deep brain rhodopsin cDNA clone exactly matched that of the retinal one, indicating that a single rhodopsin gene is transcribed in the two tissues. Immunohistochemical analysis localized rhodopsin in the cerebrospinal fluid-contacting neurons, which have been assumed to be photoreceptive cells in the deep brain. Pigeon rhodopsin seems to play dual important roles in the visual and non-visual systems, the latter of which contributes to the photoperiodic response.

The second carcinoma of the anterior two-thirds of the tongue after successful radiotherapy to the tongue.

Of 221 patients with carcinoma of the mobile tongue treated by radiotherapy, 129 survived without local recurrence for 5 years or longer after initial treatment. These 129 patients were studied to determine the incidence of second carcinoma of the tongue which first appeared more than 5 years after the initial treatment. Modalities of irradiation were radium needle implantation and intraoral electron irradiation for 105 and 24 patients, respectively. Twenty-two of the patients were found to have second carcinoma of the tongue. The incidence appeared to increase as the amount of radiation given increased. No obvious relationships were observed between the second carcinoma and modality of irradiation, T classification, presence of leukoplakia before the treatment, degree of histological differentiation, or degree of radiation injury. There were moderate to severe radiation injuries in 50% of the patients treated by radium needle implantation. It is likely that most of the second carcinomas of the tongue represent the late appearance of one of the multicentric foci of the tumor, not a regrowth of the residual primary tumor. In spite of the rather high rate of second carcinoma of the tongue, radiotherapy remains an excellent modality of treatment when patients are properly selected.

Radiotherapy of T1 glottic cancer with 6 MeV X rays.

We treated 154 patients with T1 glottic carcinoma with 6 MeV X rays through 16 cm2 parallel-opposing open fields on a free set-up delivering a median dose of 67 Gy in 6 2/3 weeks. Observed and relative 5-year survival rates for all patients were 87% and 100%, respectively. The local control rate at 5 years was 89%. Of 18 patients who clinically had local recurrence, 17 were salvaged by a secondary treatment. There were no complications requiring medical or surgical attention. A tendency toward increasing local control rates with increasing total doses was observed in the range between 57.5 Gy and 72.5. No significant correlation was found between local control rates and field size, daily dose, or the technique used. A tendency toward a lower local control rate was noted for patients whose anterior commissures were grossly involved; however, it is not known if this could be attributed to the use of 6 MeV X rays. The results are comparable to those obtained with 60Co as reported in the literature. It is concluded that 6 MeV X rays on a free set-up delivering 65-70 Gy in 6 1/2-7 weeks can be used satisfactorily for the treatment of early glottic carcinoma.

Strychnine-induced potassium current in CA1 pyramidal neurones of the rat hippocampus.

1. Direct actions of strychnine (Str) and brucine (Bru) on the dissociated hippocampal CA1 neurones of the rat have been investigated with the whole-cell mode of the patch-clamp technique. 2. At a holding potential (VH) of -20 mV, both Str and Bru elicited outward current at concentrations over 10(-5) M. The reversal potential of Str-induced current (EStr) was -77.8 mV, which was close to the K+ equilibrium potential (EK = -80.3 mV). The change in EStr for a ten fold change in extracellular K+ concentration was 58 mV, indicating that the membrane behaves like a K+ electrode in the presence of Str. 3. The concentration-response curves for Str and Bru were bell-shaped, and nearly maximum response occurred at 10(-4) M for Str and 3 x 10(-4) M for Bru. The maximum current amplitude induced by Bru was about 80% of that induced by Str. A transient 'hump' current appeared immediately after the wash-out of external solutions containing Str and Bru at concentrations higher than 10(-4) and 3 x 10(-4) M, respectively. 4. The Str-induced current (IStr) was antagonized by K+ channel blockers such as Ba2+, tetraethylammonium (TEA)-chloride, and 4-aminopyridine (4-AP) in a concentration-dependent manner. IStr was insensitive to glibenclamide, a blocker of ATP-sensitive K+ channels. 5. Internal perfusion with 10 mM BAPTA did not affect the Str-induced IK. Depletion of the intracellular Ca2+ store by caffeine had no effect, indicating that intracellular Ca2+ does not mediate the Str-induced activation of K+ conductance.6. Both guanosine-5'-0-3-thiotriphosphate (GTPyS) and guanosine-5'-O-thiodiphosphate (GDPPS) suppressed the Str-induced IK, the former action appearing more rapidly than the latter. The results suggest that the GTP binding proteins are involved in this Str response.7. When neurones were loaded with cholera toxin (CTX) or pertussis toxin (PTX) through a patch pipette, PTX suppressed the Str response whereas CTX did not, suggesting that G, and/or Go might be involved in the Str-induced IK.

Suramin and reactive blue 2 are antagonists for a newly identified purinoceptor on rat megakaryocyte.

1. The effects of purinoceptor antagonists on ATP-induced oscillatory K(+)-currents in rat isolated megakaryocytes were investigated. 2. Both reactive blue-2 (RB-2), a selective antagonist of the P2Y purinoceptor, purinoceptor, at concentrations of 0.3-10 microM and suramin, a non-selective P2 purinoceptor antagonist, at 1-30 microM blocked the ATP-induced oscillation in a concentration-dependent manner. 3. RB-2 and suramin also blocked the ADP-induced K(+)-current oscillation at the same concentration range as in the case of ATP. However, both suramin and RB-2 had no effect on thrombin- and inositol 1,4,5-trisphosphate (IP3)-induced K+ current oscillation, indicating that they act as specific purinoceptor antagonists. 4. Thus, the purinoceptors on megakaryocytes show the properties of the P2 subtype according to their blockade by antagonists.

Inhibitory action of brotizolam on circadian and light-induced per1 and per2 expression in the hamster suprachiasmatic nucleus.

Triazolam reportedly causes phase advances in hamster wheel-running rhythm after injection during subjective daytime. However, it is unclear whether benzodiazepine affects the PER: gene expression accompanying a behavioural phase shift. Brotizolam (0.5 - 10 mg kg(-1)) induced large phase advances in hamster rhythm when injected during mid-subjective daytime (circadian time 6 or 9), but not at circadian time 0, 3 or 15. Brotizolam (5 mg kg(-1)) significantly reduced the expression of PER:1 and PER:2 in the suprachiasmatic nucleus 1 and 2 h after injection at circadian time 6, and slightly reduced them at circadian time 20. Injection of 8-OH-DPAT (5 mg kg(-1)) at subjective daytime induced similar phase advances with a reduction of PER:1 and PER:2 expression. Co-administration of brotizolam with 8-OH DPAT failed to potentiate the 8-OH DPAT-induced phase advances and reduced PER: expression. Both phase advance and rapid induction of PER:1 and PER:2 in the suprachiasmatic nucleus after light exposure (5 lux, 15 min) at circadian time 20 was strongly attenuated by co-treatment with brotizolam 5 mg kg(-1). The present results strongly suggest that reduction of PER:1 and/or PER:2 expression during subjective daytime by brotizolam may be an important step in causing a behavioural phase advance. The co-administration experiment suggests that common mechanism(s) are involved in brotizolam- or 8-OH DPAT-induced phase advances and the reduction of PER: gene expression. These results suggest that brotizolam is not only a good drug for insomnia but also a drug capable of facilitating re-entrainment like melatonin.

Stage I-II carcinoma of the anterior two-thirds of the tongue treated with different modalities: a retrospective analysis of 244 patients.

Treatment results of 244 patients with stage I-II cancer of the mobile tongue were analyzed according to the modalities employed (implantation, surgery, cryosurgery and intraoral irradiation). Overall local control rates at three years were 90 +/- 3% for implant, 89 +/- 7% for cryosurgery, and 84 +/- 9% for surgery. Local control rates in stage II patients treated with intraoral electron irradiation, however, were only 50 +/- 13%. Five-year survival rates were 72 +/- 3% with no significant differences observed in patients with either stage I or stage II regardless of treatment modality. Sixty percent (29/48) of the patients with local recurrences were salvaged by the second treatment. Since the local control and survival achieved by these modalities were similar, with the exception of patients with stage II treated by intraoral electron irradiation, we recommend interstitial implantation with iridium, intraoral electron irradiation or surgery for patients with T1 tumors, and iridium implantation or surgery for patients with T2 tumors. For those with superficial lesions measuring 5 mm or less in thickness, cryosurgery is being offered as an alternative. The patient can choose the treatment modality taking into account his/her age, sex and profession.

The locus controlling pineal serotonin N-acetyltransferase activity (Nat-2) is located on mouse chromosome 11.

Melatonin is synthesized from serotonin by the enzymes serotonin N-acetyltransferase (SNAT) and hydroxyindole-O-methyl-transferase (HIOMT). We have previously reported that C57BL/6 mice do not have SNAT activity because of a mutation in an autosomal gene which is responsible for the absence of normal SNAT activity. In the present study, we have tried to map the loci of Nat-2 (the locus controlling SNAT activity) on chromosomes using a set of the BxH recombinant inbred strains which were derived from an initial cross between C3H/He with SNAT and C57BL/6 without the enzyme. Based on strain distribution patterns (SDPs), a close linkage on chromosome 11 was found between Nat-2, Es-3 (esterase-3), Glk (the locus controlling galactokinase activity) and Myla (myosin alkali light chains expressed in cardiac atrial muscle). The linkage between Nat-2 and Es-3 was confirmed by a conventional linkage test and the recombination frequency between these loci was estimated to be 16.1 +/- 3.6% (mean +/- S.E.M.).

Clock gene expressions in the suprachiasmatic nucleus and other areas of the brain during rhythm splitting in CS mice.

The CS mouse is a mutant strain which displays spontaneous splitting in the circadian locomotor rhythm under continuous darkness. To clarify whether the rhythm splitting occurs in the suprachiasmatic nucleus (SCN) where the mammalian circadian clock is located, the circadian rhythmicities of mammalian clock genes, mPer1, mBMAL1 and mClock, were examined in the SCN and cerebral cortex during rhythm splitting. The circadian profiles of the clock genes during rhythm splitting were essentially the same as those observed under unsplit conditions. However, the mPer1 gene expression throughout the day was bimodal in the piriform and cingulate cortices, peaking in correspondence with two split components of behavioral rhythm. These results indicate that the circadian profiles of three clock gene expressions in the SCN are not consistent with the overt circadian locomotor rhythm, suggesting that the site of rhythm splitting is somewhere outside the SCN, or alternatively different subregions or other clock genes in the SCN are involved in rhythm splitting.