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The molecular basis of beta-thalassemia is predominantly point mutations in the beta-globin gene. Frameshift 41-42 (-CTTT), IVS-2 position 654 (C-->T) mutation, nonsense codon 17 (A-->T), TATA box position -28 (A-->G) mutation and frameshift 71-72 (+A) account for more than 95% of beta-thalassemia alleles in the population of South China. We have developed a polymerase chain reaction (PCR)-mediated restriction fragment length polymorphism (RFLP) method for the identification of these alleles. In this method, artificial mispairing bases in PCR-amplified products were created to distinguish normal from mutant alleles on the basis of RFLPs. The size of the five PCR-amplified DNA fragments that may potentially contain the above five types of mutations is 93 or 89 bp (codons 41-42), 221 bp (IVS-2 nt 654), 110 bp (codon 17), 123 bp (TATA box nt -28), and 97 or 98 bp (codons 71-72). After these fragments were digested with Hinc II, Mae III, Nhe I, EcoR I, and Dde I, respectively, the allele-specific RFLPs produced were analyzed by gel electrophoresis. DNA samples of 24 patients with the above five types of beta-thalassemia were investigated with the present method and allele-specific oligonucleotide (ASO) probing simultaneously. We used this method in the prenatal diagnosis of 14 Chinese families for beta-thalassemia. The results obtained by the present method correspond well with those by the ASO probe test.

Prostaglandin H synthase (PGHS) occupies a central position in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. Expression of this enzyme is enhanced by inflammatory cytokines and phorbol esters. Because of the low abundance of PGHS, PGHS mRNA levels in tissues and cells were difficult to measure accurately. In this study, we quantified vascular PGHS-1 mRNA levels by a competitive polymerase chain reaction assay. The mean basal PGHS-1 level in cultured human umbilical vein endothelial cells (HUVECs) was 24.3 +/- 10.6 amol/microgram RNA. Treatment of the HUVECs with recombinant human interleukin-1 beta at 37 degrees C for 4 hours increased the mRNA to 49.1 +/- 9.1 amol/microgram RNA, a 2.1-fold increase. Treatment of the cells with phorbol 12-myristate 13-acetate at 37 degrees C for 4 hours caused a 1.7-fold increase in the mRNA level. This procedure was utilized to measure PGHS-1 mRNA levels in rabbit abdominal aorta, inferior vena cava, liver, kidney, and lung. The aortic mRNA level (9.2 +/- 1.9 amol/microgram RNA) was about 5 times higher than the inferior vena caval level (2.4 +/- 1.3 amol/microgram RNA) but only slightly higher than the level of other organs. These results reveal a significant difference in PGHS-1 mRNA levels between arterial and venous tissues. The synthase mRNA levels in organs such as liver, kidneys, and lungs do not differ significantly. The PCR assay should be valuable for studying the role of PGHS expression in disease states.

The gene of human prostaglandin H synthase-1 (PGHS-1,EC 1.14.99.1) was isolated from a human cosmid library and was contained in two overlapping clones. Multiple transcriptional start sites were identified. The major one located 136 bases upstream from the ATG initiation codon. The promoter region contains no canonical TATA box but possesses a high G+C content. These observations suggest that PGHS-1 gene has the characteristics of a housekeeping gene. Both 0.8 and 0.4 kb of the 5'-flanking sequence of the PGHS-1 gene can confer transcriptional activity.

We have used the retrograde transport of Fast blue (FB) to study the origins of supraspinal projections to the lumbar and cervical spinal cord at different stages of development in the Brazilian, short-tailed opossum, Monodelphis domestica. Monodelphis was chosen for study because its young are born in a very immature state, 14-15 days after copulation, making it possible to manipulate its nervous system in an embryonic state without intra-uterine surgery. When injections of FB were made into the lumbar cord at postnatal day (PD) 1, neurons were labeled within several areas of the reticular formation (the retroambiguus nucleus, the ventral and dorsal reticular nuclei of the medulla, the gigantocellular reticular nucleus, the lateral paragigantocellular reticular nucleus, and the pontine reticular nucleus), the presumptive coeruleus complex, and the lateral vestibular nucleus. In many cases, labeled neurons were also found within the caudal raphe and the presumptive interstitial nucleus of the medial longitudinal fasciculus. The results of immunocytochemical studies provided evidence for catecholaminergic and serotoninergic neurons in the brainstem at PD1 and for axons of both phenotypes in the spinal cord. By PD3, labeled neurons were found within the ventral gigantocellular and ventral pontine nuclei of the reticular formation, the spinal trigeminal nucleus, and the presumptive paraventricular nucleus of the hypothalamus. When injections were made at PD4, neurons were also labeled within the medial and inferior vestibular nuclei, the red nucleus, the mesencephalic nucleus of the trigeminal nerve, the presumptive nucleus of Edinger-Westphal and the lateral hypothalamus. By at least PD7, the pattern of supraspinal labeling was similar to that obtained at older ages and in the adult animal. When FB was injected into the cervical cord at PD1, neurons were labeled in all of the areas labeled by lumbar injections at the same age and in larger numbers. In addition, labeled neurons were found within the ventral gigantocellular and spinal trigeminal nuclei. When cervical injections were made at PD15, labeled neurons were found within the deep cerebellar nuclei and amygdala and by PD17 they were also present within the superior colliculus and cerebral cortex. In some cases, cortical labeling was present outside the areas labeled by comparable injections in adult animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Rubral axons can grow around a lesion of their pathway in the thoracic spinal cord of developing opossums and a critical period exists for that plasticity. The critical period probably begins when rubral axons first grow into the thoracic cord, and it extends until approximately postnatal day 30. We previously noted that most rubrospinal neurons die after transection of their axon during the critical period, suggesting that plasticity results primarily from growth of axons not damaged by the lesion (Xu and Martin, J. Comp. Neurol. 279, 368-381, 1989). That observation led us to study the response of rubrospinal neurons to axotomy in more detail and at additional stages of development, using a prelabeling paradigm. We first injected fast blue (FB) into the caudal thoracic or rostral lumbar spinal cord in animals ranging from estimated postnatal day 9 to 50 and, about 4 days later, lesioned the rubrospinal tract several segments rostral to the injection. Approximately 30 days later, the animals were killed so that the red nucleus could be searched for labeled neurons. During the critical period for plasticity, rubrospinal neurons showed signs of degeneration 1 week after their axon was cut. When animals were killed 2-3 weeks after lesioning, there was an obvious decrease in axotomized neurons within the red nucleus, and by 4 weeks, more than 75% of them had degenerated. The marked susceptibility of rubrospinal neurons to axotomy during the critical period for plasticity is consistent with the hypothesis that developmental plasticity of the rubrospinal tract results primarily from growth of axons that were not damaged by the lesion. Our results also suggest that survival of axotomized rubrospinal neurons increases with age.

To elucidate the differentiation-associated expression of enzymes catalyzing arachidonic acid metabolism, we measured arachidonate metabolites by reverse-phase high pressure liquid chromatography in monocytoid leukemia (ML-1, THP-1, and U937) and myeloid leukemia (KG-1) cell lines. Undifferentiated ML-1 or THP-1 cells produced trace amounts of eicosanoids via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. Upon differentiation induced by phorbol ester (phorbol 12-myristate 13-acetate [PMA]), metabolites via the COX pathway were increased by 100-fold in ML-1 and THP-1 cells, while the LOX products remained barely detectable. All the COX metabolites were elevated, but thromboxane A2 (TXA2) formation was threefold higher in ML-1 cells than in THP-1 cells. Similar time-related increases in COX metabolites were observed in THP-1 cells induced to differentiate with retinoic acid. Undifferentiated U937 cells were capable of generating a much higher quantity of COX products than ML-1 or THP-1 cells, but, upon PMA-induced differentiation, COX products were increased by only two-fold to threefold over the undifferentiated cells and the total COX products in differentiated U937 cells were only one-seventh of those produced by differentiated ML-1 or THP-1 cells. KG-1 cells had an entirely different metabolic profile. They produced a large quantity of a metabolite coeluted with prostaglandin D2, and PMA had no effect on inducing changes in arachidonic acid (AA) metabolism. Increased COX metabolite formation in differentiated THP-1 and ML-1 cells was due to an enhanced level of prostaglandin H synthase enzyme mass, as measured by Western blot analysis. The TXA synthase activity was also increased by approximately 100-fold in PMA-induced ML-1 cells and 10-fold in THP-1 cells. These findings indicate that increased expression of prostaglandin H and TXA synthase enzymes is a feature of differentiated monocytoid leukemia cell lines.

We have combined injections of Fast Blue with lesions of the rubrospinal tract rostral and contralateral to them to determine if an ipsilateral rubrospinal projection exists in adult or developing opossums and, if so, to characterize the neurons giving rise to it. Although the results indicate that some rubral neurons project ipsilaterally, they are very few in number. Using quantitative and image analysis techniques, we have shown that 0.6% of the rubral neurons that project to the lumbar cord in adult opossums do so ipsilaterally and that such neurons are comparable in location and size to those that project contralaterally. Similar results were obtained in developing opossums. Our results are discussed in light of rubrospinal development and ongoing experiments related to rubrospinal plasticity.

We have shown previously that rubral axons can grow around a lesion of their spinal pathway in the developing opossum and that a critical period exists for that plasticity (Martin and Xu, Dev Brain Res 39:303, 1988). Since most rubrospinal neurons degenerate after axotomy during the critical period, we have proposed that plasticity results primarily from growth of late arriving axons around the lesion rather than regeneration of cut axons (Xu and Martin, J Comp Neurol 279:368, 1989). In the present study, we used a double-labeling paradigm to test that hypothesis. Four groups of pouch young opossums received bilateral or unilateral injections of Fast Blue (FB) into the caudal thoracic or rostral lumbar cord (T12-L2) at different ages in order to label rubrospinal neurons. Three or 4 days later, the rubrospinal tract was transected unilaterally, four to five segments rostral to the injection(s). If the injection was unilateral, the lesion was made ipsilateral to it. The animals were maintained for about 1 month before a second marker, Diamidino Yellow (DY), was injected, usually bilaterally, between the FB injection(s) and the lesion. The animals were maintained for about 5 days before sacrifice and sections through the red nucleus and spinal cord were examined with a fluorescence microscope. During the critical period for plasticity, only a few rubral neurons contralateral to the lesion were labeled by FB alone, supporting our previous contention that most axotomized neurons degenerate. In contrast, many neurons were labeled by DY alone, indicating that their axons were not present in the caudal cord at the time of the FB injection and that they grew around the lesion during the 1 month survival to incorporate DY. A few double-labeled neurons were also found. One interpretation of such neurons is that they survived axotomy, as evidenced by the presence of FB, and supported axons which grew around the lesion to take up DY. Another interpretation is that they supported late growing axons which incorporated residual FB as well as DY. In order to choose between these alternatives, a similar double-labeling paradigm was carried out, but with removal of FB at the time of the lesion. Since a few neurons were still double labeled, we conclude that regeneration of cut axons also contributed to rubrospinal plasticity. Our results support our previous suggestion that developmental plasticity of the rubrospinal tract results primarily from growth of late arriving axons around the lesion, but they also suggest that regeneration of cut axons occurs.

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. Structural genes encoding the subunits of this enzyme complex constitute at least one gene cluster [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991) Biochemistry 30, 6422-6428]. The 25-kDa subunit (NQO2), which has been isolated from sodium dodecyl sulfate-polyacrylamide gels, is a polypeptide of this enzyme complex. The partial N-terminal amino acid sequence and amino acid composition of the NQO2 subunit have been determined. On the basis of the amino acid sequence, the NQO2 gene was found to be located 1.7 kilobase pairs upstream of the gene for NADH-binding subunit (NQO1). The complete nucleotide sequence of the NQO2 gene was determined. It is composed of 717 base pairs and codes for 239 amino acid residues with a calculated molecular weight of 26,122. The NQO2 subunit is homologous to the Mr 24,000 subunit of the mammalian mitochondrial NADH-ubiquinone oxidoreductase which bears an electron paramagnetic resonance-visible binuclear iron-sulfur cluster (probably cluster N1b). Comparison of the predicted amino acid sequence of the Paracoccus NQO2 subunit with those of its mammalian counterparts suggests putative binding sites for the iron-sulfur cluster. In addition, nucleotide sequencing shows the presence of two unidentified reading frames between the NQO1 and NQO2 genes. These are designated URF1 and URF2 and are composed of 261 and 642 base pairs, respectively. The possible function of the protein coded for the URF2 is discussed.

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. The NADH-binding subunit (Mr = 50,000) of this enzyme complex was identified by direct photoaffinity labeling with [32P]NADH [Yagi, T., & Dinh, T.M. (1990) Biochemistry 29, 5515-5520]. Primers were synthesized on the basis of the N-terminal amino acid sequence of this polypeptide, and these primers were used to synthesize an oligonucleotide probe by the polymerase chain reaction. This probe was utilized to isolate the gene encoding the NADH-binding subunit from a genomic library of P. denitrificans. The nucleotide sequence of the gene and the deduced amino acid sequence of the entire NADH-binding subunit were determined. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47,191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. The deduced amino acid sequence of the Paracoccus NADH-binding subunit shows remarkable similarity to the alpha subunit of the NAD-linked hydrogenase of Alcaligenes eutrophus H16. When partial DNA sequencing of the regions surrounding the gene encoding the NADH-binding subunit was carried out, sequences homologous to the 24-, 49-, and 75-kDa polypeptides of bovine complex I were detected, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex constitute a gene cluster.

The gene encoding NADH dehydrogenase from an alkalophile, Bacillus sp., was cloned and sequenced. The cloned DNA fragment contained an open reading frame of 1,557 nucleotides which encodes a polypeptide composed of 519 amino acid residues (Mr 55,830). The predicted amino acid sequence was consistent with the partial amino acid sequences including the N-terminal and C-terminal sequences determined in a previous study. Sequence comparison with other flavoenzymes revealed high homology between the present dehydrogenase and Escherichia coli thioredoxin reductase.

We have employed immunohistochemistry and the retrograde transport of Fast blue to study the origin of serotoninergic projections to the lumbosacral spinal cord at different stages of development in the North American opossum. A few serotoninergic axons are present in the lumbosacral cord at birth, 12 days after conception, and serotoninergic neurons are numerous in the brainstem where they are present in most, if not all, of the areas which contain them in the adult animals. A few neurons of the caudal raphe and adjacent reticular formation were labeled by lumbar injections of Fast blue on postnatal day 1, and by postnatal day 3, labeled neurons were numerous within all areas which provide serotoninergic projections to the lumbosacral cord in adult animals. By postnatal day 11, it was possible to combine Fast blue labeling with immunofluorescence to show that some of the labeled neurons were serotoninergic. By postnatal day 24, neurons which provide serotoninergic projections to the lumbosacral cord were especially numerous and some of them were found in areas which do not provide comparable projections in adult animals. In developing and adult animals, few, if any, neurons were labeled in the dorsal raphe or superior central nuclei. We have shown previously that serotoninergic axons do not innervate laminae I and II of the lumbosacral cord until approximately postnatal day 50, although they are present in the marginal zone at birth and have grown into laminae III-X by postnatal day 15. Since serotoninergic axons which project to laminae I and II originate within the raphe magnus and adjacent reticular formation, and those areas provide serotoninergic projections to the spinal cord well before postnatal day 50, it is possible that serotoninergic innervation of laminae I and II is provided by late growth of collaterals from axons that have been present in the marginal zone for some time.

The energy-transducing NADH--quinone oxidoreductase (NDH-1) isolated from Thermus thermophilus HB-8 is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN and at least three iron-sulfur clusters [Yagi, T., Hon-nami, K., and Ohnishi, T. (1988) Biochemistry 27, 2008-2013]. When NDH-1 of T. thermophilus HB-8 was irradiated by short UV light in the presence of [adenylate-32P]NADH or [adenylate-32P]NAD, radioactivity was incorporated into a single polypeptide of Mr 47,000. The labeling of the Mr 47,000 polypeptide was diminished when UV irradiation of the enzyme complex with [adenylate-32P]NAD was carried out in the presence of NADH or deamino-NADH which act as substrates for the NDH-1, but not in the presence of NADP(H) or AMP which act neither as substrates nor as competitive inhibitors. These results strongly suggest that the Mr 47,000 polypeptide is an NADH-binding subunit of the NDH-1 of T. thermophilus HB-8.

Both debrisoquine hydroxylation and sulfamethazine acetylation phenotypes were studied in the same native Chinese population. Debrisoquine hydroxylation status was determined by HPLC assay of debrisoquine metabolic ratio in urine after a single oral dose of 10 mg debrisoquine. Three poor metabolizers were found in 220 subjects (1.36%). One hundred and one subjects of this population previously debrisoquine phenotyped were also tested for acetylation phenotyping on a separate occasion. Their acetylation status were determined by HPLC assay of "% acetylation" after a single oral dose of 1 g sulfamethazine. Twenty (19.8%) slow acetylators were found. There were no significant association between the 2 metabolic pathways.

This paper reports the results of 32 gossypol derivatives and analogues. Among 12 compounds screened by oral administration to male rats, only dipotassium gossypolate (NC030) exhibited an antifertility activity similar to gossypol. Among 24 compounds screened by injecting into pouches of cauda epididymides of rats, 10 exhibited spermicidal activities.

To provide endpoints for developmental studies of rubrospinal plasticity in the North American opossum, we have attempted to determine the degree to which rubrospinal neurons survive axotomy in the adult animal. Bilateral or unilateral injections of the long-lasting fluorescent marker fast blue were made into the T-10 or the T-11 segment of the spinal cord to label rubrospinal neurons, and 7 days later, the rubrospinal tract was cut unilaterally four segments rostral to the injection(s). In cases with unilateral injections, the lesion was made ipsilateral to the injection. The animals were allowed to survive for 30-60 days before being sacrificed and perfused so that sections through the red nuclei could be examined for labeled neurons. The results show that most axotomized neurons survived the lesion, suggesting that lesion-dependent cell death is not a major factor in the failure of the rubrospinal tract to regenerate in the adult animal.

Using axonal tracing techniques we have shown that rubral axons are capable of growing around lesions of the rubrospinal tract during early stages of development in the North American opossum and that a critical period for such growth exists. The opossum was employed for study because it is born in a very immature state 12-13 days after conception and the entire development of its rubrospinal tract occurs postnatally.

In this study the content of cantharidin in Mylabris was analyzed quantitatively by UV. Analytical results thus obtained agree well with those by the neutralization method set forth in ChP. The average recovery of cantharidin is 99.98% and the coefficient of variation is 0.719%.

The alkalophile NADH dehydrogenase (NADH: 2,6-dichlorophenolindophenol oxidoreductase) [EC 1.6.99.3] consists of two identical subunits of 65 kDa, and each subunit contains the catalytic and liposome-binding regions. On treatment with trypsin, the polypeptide exhibiting the liposome-binding property in one of the subunits was digested to form an enzymatically active hetero-dimer (40 and 65 kDa), and then the polypeptide in the other subunit was digested to form an active homo-dimer (40 and 40 kDa). The hetero-dimer bound to liposomes, but the homo-dimer did not. Kinetic analysis showed that removal of one or two of the polypeptides in the enzyme slightly affects its kinetic parameters. For all the enzyme species, NAD inhibited competitively with respect to NADH and non-competitively with respect to 2,6-dichlorophenolindophenol. The partially determined amino acid sequence of this alkalophile enzyme suggested that (i) a long random-coiled peptide (58 amino acid residues) or a portion of the peptide is located between the polypeptides with liposome-binding and catalytic properties, (ii) the polypeptide exhibiting liposome-binding property is in the amino terminal region of the enzyme, (iii) the amino acid sequences around the subtilisin and trypsin cleavage sites of the peptide are hydrophilic and on the surface of the protein molecule and therefore are susceptible to digestion, and (iv) the FAD-binding site is located near the amino terminal region of the catalytic region.

We have shown previously that rubral axons can grow caudal to a lesion of their pathway at thoracic levels of the spinal cord in the developing opossum, Didelphis virginiana. In the present report we expand on that observation and present evidence which suggests that the critical period for plasticity of the rubrospinal tract ends earlier at cervical than at thoracic levels. In addition, we show that most rubrospinal neurons die as a result of axotomy during early stages of the critical period. The opossum was chosen for study because the development of its rubrospinal tract occurs after birth. In one set of experiments the area containing the rubrospinal tract was lesioned at cervical or thoracic levels and after 30 days or more, retrograde transport techniques were used to determine if rubral axons had grown caudal to the lesion. When the lesions were made at rostral cervical levels between estimated postnatal day 26 and maturity, neurons could not be labeled in the contralateral red nucleus by injections of retrograde markers ipsilateral to the lesion and caudal to it. We were not able to obtain adequate survival after cervical lesions made prior to estimated postnatal day 26. When the lesions were made at mid to caudal thoracic levels between estimated postnatal days 19 and 26, neurons could be labeled in the contralateral red nucleus. When comparable lesions were made at estimated postnatal day 40, there was usually a decrease in the number of labeled neurons, and when they were made at estimated postnatal day 54, none was labeled. In selected cases, operated at estimated postnatal day 19, cell counts provided evidence for loss of neurons in the red nucleus contralateral to the lesion. In orthograde transport experiments performed on animals with thoracic lesions of the rubrospinal tract made between estimated postnatal days 18 and 33, rubral axons could be labeled caudal to the lesion, and they seemed to take the most direct route around it. Although they sometimes assumed abnormal positions caudal to the lesion, rubral axons appeared to reach areas of the gray matter appropriate to them. When lesions were made at estimated postnatal day 54 or in older animals, labeled axons could be traced to the lesion site but not caudal to it.(ABSTRACT TRUNCATED AT 400 WORDS)

Based on the pathological observation of 100 cases of severe deep stromal herpetic keratitis, the author suggested the classification of the disease, according to the nature and progression of the chronic inflammation, into 2 categories of the acute active stage (40%) and chronic progressive stage (60%), which were in agreement with the clinical manifestations and also satisfactorily explained the pathogenetic basis of the clinical types. Inflammatory granuloma appeared in 21% of the cases, indicating that the corneal lesions were still in progress.

By using X-ray cinemographic techniques and biomechanical methods, the open-closing movement of the human temporomandibular joint was investigated in 14 living subjects. The theory of the instantaneous center of rotation (IRC) of the mandibular movement is strongly recommended and the position and the tendency of the IRC are also roughly determined. Moreover, a two-step model of the joint is put forward.