Coexistent oral cryptococcosis and Kaposi's sarcoma in acquired immunodeficiency syndrome. As experience with the acquired immunodeficiency syndrome has grown during the last decade, it has become important to recognize interactions between the opportunistic infections and malignancies that complicate the course of patients with acquired immunodeficiency syndrome. Our recent experience as described in this paper reveals that Kaposi's sarcoma and cryptococcal infection can associate in such a manner. The first reported case of oral Cryptococcus neoformans infection within a Kaposi's sarcoma lesion in a patient with acquired immunodeficiency syndrome is described. Based on the clinical course of this patient, it is an important consideration that the sarcoma may have harbored and even protected the fungus during systemic amphotericin B therapy.


Acrokeratosis paraneoplastica of Bazex: report of a case in a young black woman. Acrokeratosis paraneoplastica of Bazex is a rare cutaneous syndrome associated with malignant neoplasms of the pulmonary and upper gastrointestinal tract, or cervical metastatic adenopathy, usually seen in middle-aged white men. We present a unique case of Bazex syndrome in that the patient was young, black, and a woman.


Cutaneous sarcoidal granulomas in a patient with primary biliary cirrhosis. We report here the case of a fifty-year-old Japanese woman with a history of primary biliary cirrhosis who presented with a sarcoidal granulomatous plaque on the face. Although the occurrence of extra-hepatic granulomas and primary biliary cirrhosis has been documented, this is the first case report of cutaneous sarcoidal granulomas in the absence of demonstrable sarcoidosis occurring in a patient with primary biliary cirrhosis. Because primary biliary cirrhosis and sarcoidosis are systemic granulomatous diseases of unknown cause, the coexistence of findings compatible with both entities has led to speculation that they are related. We provide a brief review of these two diseases to help examine this hypothesis.


Pigmentation of the gums following hydroxychloroquine therapy. The authors describe a case of pigmentation of the gums occurring after therapy with hydroxychloroquine. A twenty-eight-year-old woman with discoid lupus erythematosus was treated only with hydroxychloroquine. After seven months of this therapy, a diffuse bluish-gray pigmentation was apparent on the vestibular surface of the gums of both dental arches. No pigmented lesions were noticed on either the skin or the nails.


Plaque-type intertriginous cutaneous calcification. Cutaneous calcification is classified into four types: dystrophic, idiopathic, tumoral, and metastatic. We present a patient with systemic lupus erythematosus undergoing hemodialysis who noted large plaque-like cutaneous calcifications in the axillae and groin. Some plaques occurred in association with striae related to prior corticosteroid therapy for the patient's underlying systemic disease. This case is unusual because of the clinical presentation, its demonstration of both dystrophic and metastatic types of calcification, and histologic calcification of elastic fibers simulating pseudoxanthoma elasticum.


Multiple scattered granulomatous skin lesions in cat scratch disease. We report a patient with cat scratch disease who presented with multiple scattered nodular lesions on the legs. Examination of skin biopsy specimens revealed a granulomatous pattern. In our opinion, this is a previously undescribed secondary cutaneous reaction of cat scratch disease. The pathogenesis of this reaction is unclear but some data suggest that the eruption might be caused by a hematogenous spread of cat scratch disease bacteria to the skin. Pathogenetic relationships with so-called bacillary angiomatosis, recently described in patients with acquired immunodeficiency syndrome, are reviewed here.


Pruritus and the parakeet.

Pruritus and the parakeet. Pruritus can be due to a variety of factors and can often be difficult for a physician to manage. Often the etiology is psychosomatic, wherein a careful history is the key to proper diagnosis. Such a case is presented.


Benign symmetric lipomatosis in a patient with severe scalp psoriasis. Benign symmetric lipomatosis, or Madelung's disease, is a rare condition of undetermined cause characterized by symmetrical subcutaneous masses that consist of normal adipose tissue and are located predominantly in the head and neck areas. A case of benign symmetric lipomatosis in a patient with unusual psoriasis and excessive alcohol intake is presented.


Treatment of notalgia paresthetica with capsaicin. Twenty-four patients with notalgia paresthetica were treated with topical capsaicin (Zostrix) in a concentration of 0.025 percent for four months. In approximately 70 percent of the patients who remained in the study, relief of pruritus was achieved up to the 90 percent level. In the majority of these patients pruritus returned when use of the medication was stopped. The history, causes, and prevalence of the condition are reviewed. This treatment is the first described that is relatively effective.


Oral lesions in patients with psoriasis: a controlled study. Stomatitis areata migrans was found in 5.4 percent of patients with psoriasis compared to 1 percent of control patients, while benign migratory glossitis was identified in 10.3 percent of patients with psoriasis and 2.5 percent of control patients. The association of these disorders with psoriasis indicates that they may be manifestations of psoriasis of the oral mucosa.


Juvenile xanthogranuloma.

Juvenile xanthogranuloma. Juvenile xanthogranuloma is a benign cutaneous growth presenting as papules or nodules. It is characterized by an intradermal collection of lipid-laden macrophages and varying degrees of fibroblastic proliferation. We have recently observed two patients with xanthogranulomas: one was found to have a papular type and the second patient had multiple nodular growths. We present these cases, which should be considered in the differential diagnosis of skin nodules.


Flashlamp-pumped pulsed dye laser treatment for pyogenic granuloma. Pyogenic granuloma is a frequently diagnosed, benign vascular lesion common in children and young adults. Treatment of pyogenic granuloma consists of removal of the lesion for therapeutic as well as diagnostic purposes. Current treatment modalities include curettage with electrodesiccation, cryosurgery, chemical cauterization, and argon laser therapy. However, these methods are not without the risk of complications such as scarring and pigmentary changes. The recently reported success of the flashlamp-pumped pulsed dye laser for treating telangiectasias, hemangiomas, and port-wine stains led us to hypothesize that this same device, using different variables, such as multiple overlapping pulses, would be of benefit in the treatment of pyogenic granuloma. We describe three patients with pyogenic granuloma treated with the pulsed dye laser, with the total number of treatments and energy densities varying according to the size of the initial lesion. All three patients demonstrated complete resolution of lesions with no evidence of scarring, atrophy, or pigment changes. This preliminary report suggests that pulsed laser therapy should be considered in the treatment of pyogenic granuloma, especially in cosmetically sensitive areas.


Generalized elastophagocytic granuloma.

Generalized elastophagocytic granuloma. The case of an eighty-three-year-old woman with the sudden onset of a generalized pruritic eruption is reported. The skin lesions resembled disseminated subacute lupus erythematosus on clinical examination, but actinic granuloma or annular elastolytic giant cell granuloma was seen in biopsy specimens of the lesions. Our case was differentiated from generalized granuloma annulare by the distinct zoning of elastolysis and the distribution of giant cells.


Amphibian oocytes and sphere organelles: are the U snRNA genes amplified? The sphere organelles (spheres) of Xenopus and other amphibian oocytes are known to contain small nuclear ribonucleoprotein particles (snRNPs) and have been suggested to play a role in snRNP complex assembly. Coupled with the similarities that exist between spheres and nucleoli and the quantitative and kinetic aspects of snRNA synthesis in the Xenopus oocyte, we have investigated whether or not the U snRNA encoding genes are amplified in Xenopus oogenesis, the spheres being possible sites for the location of such extrachromosomal gene copies. By applying a number of quantitative nucleic acid hybridization procedures to both total and fractionated oocyte and somatic DNA, employing both homologous and heterologous U snRNA gene probes and suitable amplification and non-amplification control probes, we show that the U snRNA genes do not undergo any major amplification in Xenopus oogenesis. Therefore, the analogy between the sphere organelles and nucleoli appears to be limited. The role of the spheres and their relationship to other snRNP containing structures, specifically B snurposomes, and the sphere organizer loci remains obscure.


Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei. Fluorescent in situ hybridization with chromosome specific probes was used in conjunction with laser scanning confocal microscopy to assess the three-dimensional distribution of chromosomes in human T-lymphocyte nuclei. Cells in the G1-phase of the cell cycle exhibit a distinctly non-random chromosome organization:centromeric regions of the ten chromosomes examined are localized on the nuclear periphery, often making contact with the nuclear membrane, while telomeric domains are consistently localized within the interior 50% of the nuclear volume. Chromosome homolog pairing is not observed. Transition from the G1 to G2 cell cycle phase is accompanied by extensive chromosome movement, with centromeres assuming a more interior location. Chromosome condensation and chromatin depleted areas are observed in a small subset of G2 nuclei approaching mitosis. These results demonstrate that dynamic chromosome rearrangements occur in non-mitotic nuclei during the cell cycle.


Enhanced services and stipends for foster parents: effects on retention rates and outcomes for children. Current national trends show that although the number of available foster homes is shrinking, the number of children and adolescents being cared for in the family foster care system is growing. This study demonstrates the significant benefits to both foster parents and the children in their care of providing enhanced services and stipends to foster parents.


The gene structure of Xenopus nuclear lamin A: a model for the evolution of A-type from B-type lamins by exon shuffling. Nuclear lamins are intermediate filament (IF) type proteins that form a fibrillar network underlying the inner nuclear membrane. The existence of multiple subtypes of lamins in vertebrates has been interpreted in terms of functional specialization during cell division and differentiation. The structure of a gene encoding an A-type lamin of Xenopus laevis was analysed. Comparison with that of a B-type lamin of the same species shows remarkable conservation of the exon/intron pattern. In both genes the last exon, only 9-12 amino acids in length, encodes the complete information necessary for membrane targeting of lamins, i.e. a ras-related CaaX motif. The lamin A specific extension of the tail domain is encoded by a single additional exon. The 5' boundary of this exon coincides with the sequence divergence between human lamins A and C, for which an alternative splice mechanism had previously been suggested. Arguments are presented suggesting that B-type lamins represent the ancestral type of lamins and that A-type lamins derived there from by exon shuffling. The acquisition of the new exon might explain the different fates of A- and B-types lamins during cell division.


Prognosis of luxated non-vital maxillary incisors treated with calcium hydroxide and filled with gutta-percha. A retrospective clinical study. A material of 885 luxated, non-vital incisors was evaluated radiographically with respect to healing of periodontal tissues including inflammatory root resorption and occurrence of ankylosis and cervical root fractures. The results were assessed after completion of calcium hydroxide treatment and 4 years after filling of the root canal with gutta-percha. After treatment with calcium hydroxide, periapical healing occurred in 95% of the teeth. Four years after filling with gutta-percha, periapical healing was present in 91% of the teeth. In the remaining teeth, recurrent or persistent periapical radiolucency was more frequent in overfilled than adequately filled teeth (P = 0.0001). There was no difference between immature and mature teeth. Inflammatory root resorption healed in 192 of 197 teeth (97%); in 5 teeth it developed into ankylosis. Ankylosis occurred in 13 teeth, all of which were intruded into the alveolar bone at the time of injury. The frequency of cervical root fractures was markedly higher in immature than mature teeth (P greater than 0.0001). Among immature teeth, the frequency of fractures was dependent on the stage of root development (chi 2 = 31.6) and ranged from 77% in teeth with the least to 28% in teeth with the most developed roots. The frequency of fractures was also related to the defects after healing of inflammatory root resorption in the cervical area of the root, significant at P less than 0.0001.


Caffeine demethylase activity in human and Dark Agouti rat liver microsomes. Comparison with aminopyrine N-demethylase activity. Human and Dark Agouti rat liver microsomes were analyzed for caffeine metabolism focusing on demethylase activity by using a simplified isocratic HPLC method. Results showed that, despite interspecies quantitative and qualitative differences on caffeine metabolism, mainly demethylated caffeine metabolites were detected in both species. Humans showed interindividual quantitative differences in caffeine demethylase activity in contrast to rats and, in both cases, the proportion of metabolites remained constant. In addition, aminopyrine N-demethylase activity was assayed, showing a direct correlation with caffeine demethylase activity in humans (r = 0.71) that is reported here for the first time, but not in rats (r = 0.21). These results indicate that, in human liver microsomes, caffeine and aminopyrine could be demethylated by the same or by closely related enzymes; whereas in Dark Agouti rats, there is no apparent relation between caffeine and aminopyrine demethylation pathways.


Induced periapical lesions in ferret canines: histologic and radiographic evaluation. Ferrets have been utilized in endodontic research to study periapical responses. However, various aspects of induction and healing of periapical pathosis in ferrets are unclear. This study evaluated the radiographic and histopathologic features of induced periapical lesions. Periapical pathosis was consistently produced in 8 canines of 2 ferrets by extirpating the pulp, leaving the cavities open for a week, then sealing for 12 weeks. The resulting lesions were evaluated radiographically every two weeks. The animals were then killed and the induced lesions evaluated histologically and histobacteriologically. Four untreated canines from 2 other ferrets were used as controls. Results showed that periapical rarefactions were visible in all teeth in the experimental group within 4 weeks. Histologically, the induced lesions consisted principally of a mononuclear inflammatory infiltrate, dominated by macrophages but with numerous lymphocytes. With Brown & Brenn staining, bacteria were demonstrated in the pulp space of teeth in which the lesions were induced and in the apical arborization of the canal system. Only one specimen had bacteria within the periapical lesion.


Hydrolysis of the 2',3'-allylic epoxides of allylbenzene, estragole, eugenol, and safrole by both microsomal and cytosolic epoxide hydrolases. 2',3'-Allylic epoxide derivatives of allylbenzene and its analogs estragole, eugenol, and safrole were synthesized, and their enzymatic conversion to dihydrodiols by cytosolic and microsomal epoxide hydrolases was examined. All four epoxides were good substrates for both epoxide hydrolases, with Michaelis constants in the low micromolar range. Two putatively selective inhibitors of cytosolic and microsomal epoxide hydrolases, trichloropropylene oxide and nordihydroguaiaretic acid, were used to inhibit the hydrolysis of these allylic epoxides. Minimal selectivity toward either hydrolase was seen with either inhibitor, suggesting that the "selectivity" of these inhibitors is highly substrate-dependent. The susceptibilities of these epoxides to rapid hydrolysis by both epoxide hydrolases may explain their low genotoxic potencies in vivo.


Sealing ability of sectional injection thermoplasticized gutta-percha technique with varying distance between needle tip and apical foramen. The purpose of this investigation was to evaluate the influence of the distance of the needle tip from the apical foramen on the apical seal and the length control of the root filling material when thermoplasticized gutta-percha is injected using a sectional injection technique. Ninety-nine human single-rooted mandibular incisors and permanent premolars with straight canals were divided into three equal Groups A, B and C. In Group A the teeth had relatively the wider root canal, in Group C the narrowest, while in Group B root canal size was intermediate. The root canals of all teeth were prepared with a step-back technique. Roots of Group A were instrumented to a Size 70 4 mm short of the working length, while roots of Groups B and C to a Size 70 6 mm and 8 mm short of the working length, respectively. The teeth of all groups were covered with aluminium foil and embedded in acrylic blocks. After the placement of sealer (Roth 811) the root canals of all teeth were obturated with injection-molded thermoplasticized gutta-percha using a three-stage sectional technique. The roots remained in India ink for three days and then cleared by successive immersion in nitric acid, ascending grades of alcohol and finally in xylene. The linear dye penetration and the linear apical underextentionion of the root canal filling were measured by two independent examiners under a stereo microscope. There was no significant difference in the linear dye leakage between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Physiology of growth hormone secretion and action. Growth hormone secretion by the pituitary gland is dependent on the dual regulation exerted by growth hormone-releasing hormone and somatostatin, which are the final integrative pathways for several signals reaching the hypothalamus. Regulatory signals include neurotransmitters, neuropeptides, and peripheral signals such as hormones or nutrients. In the circulation, growth hormone is bound to specific binding proteins and, by activating specific receptors, exerts a variety of peripheral actions.


Clinical actions of growth hormone.

Clinical actions of growth hormone. Although biotechnology has provided physicians with essentially unlimited supplies of growth hormone (GH), there are currently only a few clear-cut indications for exogenous GH therapy. Data now support the use of GH in the treatment of children with GH deficiency and short girls with Turner syndrome. Tantalizing preliminary data suggest that GH therapy has a role in the management of short, poorly growing children with other causes for their growth failure. Recent studies have examined the utility of GH therapy in GH-deficient adults, whereas other studies suggest that GH improves the clinical status of GH-sufficient older adults. This article explores the recent data underlying these claims.


Etiology of pituitary acromegaly.

Etiology of pituitary acromegaly. Pituitary tumors secreting growth hormone are clonal neoplasms that may arise following a somatic mutation within the somatotroph cell. Promotion of tumor growth also appears to be regulated by hypothalamic growth hormone-releasing hormone, which exerts a trophic action on the pituitary cell. Unraveling the mechanisms of pituitary tumorigenesis will facilitate the application of novel therapeutic techniques, genetic screening, and follow-up of tumor responses.


Pituitary pathology in acromegaly.

Pituitary pathology in acromegaly. This review summarizes the morphologic findings in patients with acromegaly correlated with data on hormone secretion and regulation in vivo and in vitro. The importance of careful morphologic analysis that allows recognition of the varied diseases that have similar manifestations is emphasized. The gaps in our knowledge of pathogenesis are glaring, but continued dedication to the investigation of structure-function correlations combined with a greater understanding of the molecular basis of disease will, no doubt, lead to new discoveries and elucidate the factors underlying pituitary tumorigenesis.


Ectopic acromegaly.

Ectopic acromegaly. Ectopic acromegaly is a rare syndrome (less than 1% of acromegalic patients) caused by ectopic growth hormone-releasing hormone (GHRH) or growth hormone (GH)-producing tumors. Its recognition is clinically important because acromegaly may be a symptom of an aggressive tumor, and different therapeutic approaches are required. Most cases are caused by either extra- or intracranial GHRH-producing tumors, whereas in rare instances the underlying disease is an ectopic GH-secreting tumor. The routine evaluation of circulating GHRH in all acromegalic patients may allow its early recognition, because plasma levels greater than 0.3 ng/mL are virtually diagnostic of a GHRH-producing tumor (frequently a bronchial or pancreatic carcinoid), whereas suppressed levels may suggest an ectopic GH-producing tumor. In addition to classic imaging techniques, whole body scintiscan with labeled octreotide may help in the localization of ectopic tumors. Surgical removal of the ectopic tumor is the therapy of choice, but it is not always feasible because patients often present with widespread metastases. Patients with GHRH-induced acromegaly benefit from the administration of the long-acting somatostatin analog, octreotide, which reduces GH, IGF-I, and GHRH, and may shrink the ectopic tumor, its metastases, and the secondary pituitary enlargement.


Clinical manifestations of acromegaly.

Clinical manifestations of acromegaly. Acromegaly is an uncommon disorder and may present in a variety of ways, leading to considerable delay in diagnosis. Unlike other pituitary tumors, tumors associated with acromegaly tend to be fairly large in most patients. Thus, symptoms may be commonly due to the tumor mass as well as to hormone oversecretion. Mortality is two- to threefold increased due to cardiovascular, respiratory, and neoplastic causes. An increase in diabetes mellitus and hypertension may contribute to the first of these. Early treatment may reverse the diabetes, soft tissue changes, sleep apnea, cardiovascular disease, and neuromuscular disease. The effect of early treatment on neoplasia is unclear, and patients probably should continue to be screened, especially for colon neoplasia, even after appropriate therapy for the acromegaly. Hypopituitarism may be present initially as a result of tumor mass but may also develop as a result of ablative therapy.


Rheumatologic and skeletal changes in acromegaly. The anabolic functions of growth hormone (GH) and insulin-like growth factor-I (IGF-I) in cartilage and bone metabolism are important in the normal physiology of these tissues. The effects of chronic elevation of GH and IGF-I levels on bony structures produce the typical physical changes associated with acromegaly, whereas the effects on cartilage result in arthropathy, which is usually degenerative. This article presents an overview of the physiologic roles of GH and IGF-I in cartilage and bone metabolism, the clinical features of the degenerative arthropathy and other rheumatologic syndromes associated with acromegaly, effects of acromegaly on bone and mineral metabolism, and an unusual bone disease that is occasionally associated with acromegaly, the McCune-Albright syndrome.


Pituitary gigantism.

Pituitary gigantism. Pituitary gigantism is a rare condition whose association with McCune-Albright syndrome suggests that mutations in alpha-subunit of a Gs protein are an important cause of this condition. In addition to somatotroph adenoma, it is now recognized that somatotroph hyperplasia can also result from increased levels of growth hormone-releasing hormone. Transgenic rats with hypersomatotrophism are prone to renal and hepatic pathology.


Diagnosis and endocrine testing in acromegaly. Acromegaly is a characteristic clinical syndrome resulting from excessive production of GH and SmC/IGF-I generally from a GH-producing pituitary tumor. Once the diagnosis is suspected on clinical grounds, it should be established based on persistent elevation of the basal GH, along with an increased SmC/IGF-I level; it is confirmed by a lack of suppressibility of GH levels following a glucose load. Other tests, including the paradoxic GH elevation in response to TRH and LHRH, are helpful in establishing the diagnosis, as well as determining and monitoring the outcome of surgical and medical therapy. The past few years have been enriched with new knowledge in the area of IGF-binding proteins. Evidence of clinical correlation exists regarding GH status, IGF-I levels, and the IGFBPs particularly IGFBP-1 and IGFBP-3. The value of IGFBPs in clinical practice as a diagnostic tool in disorders of GH secretion appears promising, but further studies are required.


Surgical management of acromegaly.

Surgical management of acromegaly. Surgery is generally the primary therapy of choice in acromegaly, and the predominantly used transsphenoidal approach has proved to be efficient and safe. Surgical results are dependent on preoperative tumor size and extension and preoperative growth hormone levels. Invasion appears to be the most important factor in predicting surgical outcome. Growth hormone levels can be normalized in 71% of all cases and in more than 80% of microadenomas. Persistent growth hormone excess may require reoperation, radiotherapy, or medical treatment. With close interdisciplinary cooperation of neurosurgeons, endocrinologists, and radiotherapists, nearly all acromegalic patients can be successfully treated today.


Radiation therapy of acromegaly.

Radiation therapy of acromegaly. Conventional megavoltage irradiation of GH-secreting tumors has predictable effects on tumor mass, GH, and pituitary function. 1. Further growth of the tumor is prevented in more than 99% of patients, with only a fraction of a percent of patients requiring subsequent surgery for tumor mass effects. 2. GH falls predictably with time. By 2 years GH falls by about 50% from the baseline level, and by 5 years by about 75% from the baseline level. The initial GH elevation and the size and erosive features of the sella turcica do not affect the percent decrease in GH from the baseline elevation. 3. With prolonged follow-up, further decrease in GH is seen at 10 and 15 years, with the fraction of surviving patients achieving GH levels less than 5 ng/mL approaching 90% after 15 years in our experience. Gender, previous surgery, and hyperprolactinemia do not seem to affect the response to treatment. Patients with initial GH greater than 100 ng/mL are significantly less likely to achieve GH values less than 5 ng/mL during long-term follow-up. 4. Hypopituitarism is a predictable outcome of treatment, is delayed, and may be more likely in patients who have had surgery prior to irradiation. There is no evidence that this complication is more common in patients with acromegaly than in patients with other pituitary adenomas receiving similar treatment. 5. Vision loss due to megavoltage irradiation--using modern techniques and limiting the total dose to 4680 rad given in 25 fractions over 35 days, with individual fractions not exceeding 180 rad--is extremely rare. The reported cases have occurred almost entirely in patients who have received larger doses or higher fractional doses. The theory that patients with acromegaly are prone to radiation-induced injury to the CNS and optic nerves and chiasm because of small vessel disease is not supported by a review of the reported cases. 6. Brain necrosis and secondary neoplasms induced by irradiation are extremely rare. 7. Although anecdotal evidence raises the question of changes in intellectual function following irradiation, this has not been studied in adults receiving pituitary irradiation.


Living with acromegaly.

Living with acromegaly. Acromegaly is an unusual insidious but potentially serious chronic pituitary disorder. The delay in diagnosis results not only in unique somatic disfigurement but is also associated with significant mental and emotional dysfunction. This article reviews and describes some of these nonclassic and often overlooked features of acromegaly. Particular emphasis is placed on presenting the patients' perspectives of the condition. The author offers some suggestions on the care and counseling of the acromegalic patient.


Concepts and strategies for human gene therapy. Methods of modern molecular genetics have been developed that allow stable transfer and expression of foreign DNA sequences in human and other mammalian somatic cells. It is therefore no surprise that the methods have been applied in attempts to complement genetic defects and correct disease phenotypes. Two decades of research have now led to the first clinically applicable attempts to introduce genetically modified cells into human beings to cure diseases caused at least partially by genetic defects. We discuss here some of the strategies being followed for both in vitro and in vivo application of therapeutic gene transfer and summarize some of the technical and conceptual difficulties associated with somatic-cell gene therapy.


Regulation of expression of a Xenopus borealis embryonic/larval alpha 3 skeletal-actin gene. We have isolated a genomic clone, related in sequence to the skeletal-actin gene sub-family. It is expressed in the skeletal muscle of embryos from the neurula stage onwards and in tadpoles, but not in adults. The equivalent Xenopus laevis gene is expressed as a major transcript in adult muscle, as well as at earlier stages. The intron/exon structure is typical of vertebrate skeletal-actin genes, as is the possession of multiple copies of three serum-response elements in the promoter of this gene. The Xenopus actin and beta-globin genes were fused in their second introns. This construct, which contained 2.4 kb of upstream sequence, was injected into fertilized eggs at the two-cell stage. It showed the normal pattern of tissue-specific transcription. Thus all of the information necessary for appropriate expression of this actin gene in the embryo is contained in the region that extends from a point 2.4 kb upstream of transcription initiation to the centre of the second exon. A series of enhancer constructs were made in which upstream regions of the actin gene were placed upstream of a X. laevis beta-globin gene. The region immediately adjacent to the promoter, containing the three serum-response elements, was able to drive muscle-specific expression, and there was also a general enhancement of transcription by regions further upstream.


Site-directed mutagenesis of elongation factor Tu. The functional and structural role of residue Cys81. A Cys residue located in the second consensus sequence element (DCPG) of the GTP-binding region is highly conserved in bacterial elongation factors (EF) Tu. Chemical modification of this Cys81 in EF-Tu from Escherichia coli by N-tosyl-L-phenylalanine chloromethane [Jonák, J., Petersen, T. E., Clark, B. F. C. & Rychlík, I. (1982) FEBS Lett. 150, 485-488], and of homologous Cys residues in other bacterial EF-Tu, selectively blocks the binding of Xaa-tRNA. We have substituted Cys81 with Gly using site-directed mutagenesis of the EF-Tu-encoding tuf A gene. This substitution induces a partial inhibition (20-70%) of: (a) poly(U)-directed poly(Phe) synthesis; (b) EF-Tu/Xaa-tRNA interaction, determined as protection by EF-Tu of the non-enzymic deacylation of Xaa-tRNA; (c) EF-Tu-dependent binding of Xaa-tRNA to the mRNA/ribosome complex and (d) the intrinsic GTPase reaction, that is also less sensitive to stimulation by Xaa-tRNA. Our results thus provide evidence that Cys81, though important, is not essential for the binding of Xaa-tRNA to EF-Tu. The accuracy in poly(Phe) synthesis, measured as misincorporation of Leu, was increased. Both the binding affinity of [C81G]EF-Tu for the nucleotide and the resistance against thermal denaturation are more strongly decreased in the case of the GDP-bound state than in the case of the GTP-bound state, suggesting that Cys81 plays a more specific role in the former conformation. The sensitivity to N-tosyl-L-phenylalanine chloromethane is decreased by 80% but not totally lost. The inhibition by N-tosyl-L-phenylalanine chloromethane treatment of the function of EF-Tu appears to be a consequence of steric hindrance and/or of an altered conformation of EF-Tu.GTP. The lower activities of [C81G]EF-Tu are probably due to long-range effects, mediated by an overall destabilization of the molecule that is particularly pronounced for the GDP-bound state.


Characterization and expression of a NADP-malic enzyme cDNA induced by salt stress from the facultative crassulacean acid metabolism plant, Mesembryanthemum crystallinum. The facultative halophyte and crassulacean acid-metabolism plant, Mesembryanthemum crystallium shifts from C3 photosynthesis to crassulacean acid metabolism when exposed to high-salt or drought conditions. To study the molecular basis of this metabolic transition, the expression of NADP(+)-dependent malic enzyme (NADP-ME), which catalyzes the decarboxylation of malate to release pyruvate and CO2, has been investigated. The complete nucleotide sequence of a full-length cDNA clone was determined and found to contain a single open reading frame encoding a 585-amino-acid polypeptide of 64284 Da. The ice plant (M. crystallinum) NADP-ME shares amino acid identities in the range 72.5-79.0% when compared to other higher-plant enzymes and is more closely related to C3 rather than C4 forms of the enzyme. Genomic Southern-blot analysis of ice-plant DNA indicates that NADP-ME is encoded by a small gene family. Steady-state transcript levels increase 8-10-fold in response to salt stress in the leaves. Transcript levels in roots are extremely low and are unaffected by salt-stress treatment. Nuclear run-on experiments, using isolated nuclei from leaf tissue, confirm that the accumulation of NADP-ME transcripts is, in part, the result of increased transcription of this gene during salt stress.


A missense mutation Pro157 Arg in lipoprotein lipase (LPLNijmegen) resulting in loss of catalytic activity. Here we report on the molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in a proband of Dutch descent. Southern-blot analysis of the LPL gene from the patient did not reveal any major DNA rearrangements. Sequencing of polymerase-chain-reaction-amplified DNA revealed that the proband is a homozygote for G725C, resulting in a substitution of Pro157 for Arg. This substitution alters a restriction site for PvuII, which allowed rapid identification of the mutant allele in family members. Site-directed mutagenesis and transient expression of the mutant LPL in COS cells produced an enzymatically inactive protein, establishing the functional significance of this mutation. This naturally occurring mutation which alters the Pro157 adjacent to Asp156 of the proposed catalytic triad, indicates that this region of the protein is indeed crucial for LPL catalytic activity.


Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris. The crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the three-dimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed beta sheet. The active site is located at the C-edge of the central (parallel) part of the beta sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.


Temperature effects on the MgATP-induced electron transfer between the nitrogenase proteins from Azotobacter vinelandii. The temperature dependence of the pre-steady-state MgATP-dependent electron transfer from the MoFe protein to the Fe protein of the nitrogenase from Azotobacter vinelandii has been investigated between 6 degrees C and 31 degrees C by stopped-flow spectrophotometry. Below 14 degrees C, the data are consistent with a model in which interaction of MgATP with nitrogenase is fast and irreversible, and is followed by reversible electron transfer. From the extent and from the rate of the absorbance change, the rate constants for electron transfer from Fe protein to MoFe protein and of the reverse reaction were calculated. The direct rate constant increases with temperature (6-14 degrees C) from about 1 s-1 to about 26 s-1. The rate constant for the reverse reaction was found to be approximately 4 s-1 and invariant with the reaction temperature. Analysis of the data obtained in the temperature range between 6 degrees C and 12 degrees C within the framework of the transition-state theory show that electron transfer from the Fe protein to the MoFe protein occurs via a highly disordered transition state with activation parameters delta H(0) ++ = 289 kJ.mol-1 and delta S(0) ++ = 792 J.K-1.mol-1. The Eyring plot of the stopped-flow data displays an inflection point around 14 degrees C. From the stopped-flow data obtained between 18 degrees and 27 degrees C the activation parameters delta H(0) ++ and delta S(0) ++ for the reduction of the MoFe protein by Fe protein are calculated to be 90 kJ.mol-1 and 99 J.K-1.mol-1 respectively. A second inflection point in the Eyring plot could exist around 28 degrees C.


Intermediates of peroxisomal beta-oxidation of [U-14C]hexadecanedionoate. A study of the acyl-CoA esters which accumulate during peroxisomal beta-oxidation of [U-14C]hexadecanedionate and [U-14C]hexadecanedionoyl-mono-CoA. 1. The oxidation of [U-14C]hexadecanedionoyl-mono-CoA was stimulated by CoA, by carnitine in the absence of CoA and by the presence of an NAD(+)-regenerating system. 2. Substrate inhibition was observed with respect to [U-14C]hexadecanedionoyl-mono-CoA at concentrations greater than 35 microM. 3. Acetyl-CoA and the dicarboxyl-CoA esters of chain length C6-16 were detected by HPLC under standard incubation conditions. 4. In the absence of the NAD(+)-regenerating system, 2-enoyl-CoA and 3-hydroxacyl-CoA esters were detected. 5. In general, the peroxisomal beta-oxidation of dicarboxylates is very similar to that of monocarboxylates [Bartlett, K., Hovik, R., Eaton, S., Watmough, N. J. & Osmundsen, H. (1990) Biochem. J. 270, 175-180] except that chain shortening does not proceed beyond C6. 6. We conclude that the peroxisomal beta-oxidation of dicarboxylates is regulated by the redox state of the peroxisomal matrix and CoA availability.


Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA. Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-dioleoyl-sn-glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca(2+)-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glycerol in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-sn-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar fraction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme. Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.


Biosynthesis and intracellular transport of alpha-2,6-sialyltransferase in rat hepatoma cells. We investigated biosynthesis, intracellular transport and release of beta-galactoside alpha-2,6-sialyltransferase in a dexamethasone-inducible rat hepatoma cell line. Confluent cells were induced by 10 microM dexamethasone for 24 h, and metabolically labelled with [35S]methionine/cysteine, followed by immunoprecipitation of sialyltransferase and electrophoretic/fluorographic analysis. The 35S-labelled enzyme was synthesized as a 46-kDa precursor, converted to an intermediate 47-kDa form after 1 h, and gradually to a mature form of 48 kDa within the following 3 h. By means of either tunicamycin inhibition of N-glycosylation or cleavage of N-glycans from isolated sialyltransferase using N-glycosidase F, the sizes of the precursor and the mature form were reduced to 41 kDa and 43 kDa, respectively. After a 4-h chase, treatment with endoglycosidase H revealed two distinct molecular forms of sialyltransferase, bearing either two N-acetyllactosamine-type or one oligomannose-type and one N-acetyllactosamine-type N-linked sugar chain. In addition, sialyltransferase became sensitive to neuraminidase digestion after a 4-h chase. The half-life of intracellular [35S]sialyltransferase was estimated at 3 h. A soluble form was detectable in the supernatant, 2 h after the pulse. Only 12% of the initially labelled sialyltransferase was found in the medium after 12 h, while 73% of the enzyme was degraded intracellularly. To characterize a possible intracellular degradation site, we studied intracellular transport in the presence of either secretion-blocking or acidotropic agents or protease inhibitors. Degradation was significantly delayed by all treatments. Our results show that sialyltransferase follows the secretory pathway as a membrane protein and is retained at a late Golgi stage. We suggest that the bulk of sialyltransferase in rat hepatoma cells is diverted to a post-Golgi degradation pathway. This route contrasts with the post-Golgi trafficking of beta-1,4-galactosyltransferase in HeLa cells, which is constitutively secreted [Strous, G. J. A. M. & Berger, E. G. (1982) J. Biol. Chem. 257, 7623-7628].


Protein structure of pig liver 4-aminobutyrate aminotransferase and comparison with a cDNA-deduced sequence. The amino acid sequence of pig liver 4-aminobutyrate aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments. The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work [Kwon, O., Park, J. & Churchich, J. E. (1992) J. Biol. Chem. 267, 7215-7216]. Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107-142. Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work. The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli.


Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core. A large, low-density form of heparan sulfate proteoglycan was isolated from the Engelbreth-Holm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.


Dimerization and reactivation of triosephosphate isomerase in reverse micelles. The reactivation of the homodimeric enzyme triosephosphate isomerase (TPI) was studied in reverse micelles. The enzyme was denatured in conventional aqueous mixtures with guanidine hydrochloride and transferred to reverse micelles formed with cetyltrimethylammonium bromide, hexanol, n-octane and water. In the transfer step, denatured TPI monomers distributed in single micelles, and guanidine hydrochloride was diluted more than 100 times. Under optimal reactivation conditions, 100% of the enzyme activity could be recovered. The rate of appearance of the catalytic activity increased with the concentration of protein, which indicated that catalysis required the formation of the dimer. The rate of TPI reactivation also increased with increasing protein concentration in the system with denatured TPI covalently derivatized at the catalytic site with the substrate analogue 3-chloroacetol phosphate. Thus, reactivation could take place via the formation of dimers composed of an inactive and an active subunit. Reactivation critically depended on the amount of water in the reverse micelles. The plot of the extent of reactivation versus the amount of water (2.5-7.0%) was markedly sigmoidal. Less than 20% reactivation took place with water concentrations below 3.5%, due to the formation (in less than 30 s) of stable inactive structures. The results indicate that reverse micelles provide a useful system to probe the events involved in the transformation of unfolded monomers to polymeric enzymes.


Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution. The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution. Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy-atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent-flattening procedure were used for electron-density-map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit. The phenylalanyl-tRNA synthetase from T. thermophilus molecule has a 'quasi-linear' subunit organization. As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer.


Mechanistic studies of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase from Escherichia coli. The anomeric specificity and the steady-state kinetic mechanism of homogeneous 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase were investigated. The open-chain 4-deoxy analogue of arabinose-5-phosphate (Ara5P), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase. It was found that the analogue functions as a substrate with a similar kcat value to that of the original substrate. The kcat/Km value for the natural substrate is seven-times greater than that of the 4-deoxy analogue. However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4-deoxy analogue and Ara5P in solution, then the 'true' Km values must be in the range 31.5 microM and 0.26 microM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4-hydroxyl group of Ara5P. The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate. The steady-state kinetic study of KDO8P synthase was analyzed via inhibition using the products KDO8P and inorganic phosphate, and D-ribose-5-phosphate as a dead-end inhibitor. First, intersecting lines in double-reciprocal plots of initial-velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme-catalyzed reaction. The inhibition by D-ribose-5-phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P-pyruvate). These inhibition patterns are consistent with the model wherein P-pyruvate binding precedes that of Ara5P binding. Furthermore, this order of substrate binding was supported by the observations that KDO8P is a competitive inhibitor for P-pyruvate binding, supporting the concept that KDO8P and P-pyruvate bind to the same enzyme form, and noncompetitively with respect to Ara5P. In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both P-pyruvate and Ara5P, suggesting an apparent ordered release of products such that Pi first, followed by KDO8P. In conclusion, these data suggest a steady-state kinetic mechanism for KDO8P synthase where P-pyruvate binding precedes that of Ara5P, followed by the ordered release of inorganic phosphate and KDO8P.


Effect of substrate structure on the activity of Man9-mannosidase from pig liver involved in N-linked oligosaccharide processing. Man9-mannosidase, an alpha 1,2-specific enzyme located in the endoplasmic reticulum and involved in N-linked-oligosaccharide processing, has been isolated from crude pig-liver microsomes and its substrate specificity studied using a variety of free and peptide-bound high-mannose oligosaccharide derivatives. The purified enzyme displays no activity towards synthetic alpha-mannosides, but removes three alpha 1,2-mannose residues from the natural Man9-(GlcNAc)2 substrate (M9). The alpha 1,2-mannosidic linkage remaining in the M6 intermediate is cleaved about 40-fold more slowly. Similar kinetics of hydrolysis were determined with Man9-(GlcNAc)2 N-glycosidically attached to the hexapeptide Tyr-Asn-Lys-Thr-Ser-Val (GP-M9), indicating that the specificity of the enzyme is not influenced by the peptide moiety of the substrate. The alpha 1,2-mannose residue which is largely resistant to hydrolysis, was found to be attached in both the M6 and GP-M6 intermediate to the alpha 1,3-mannose of the peripheral alpha 1,3/alpha 1,6-branch of the glycan chain. Studies with glycopeptides varying in the size and branching pattern of the sugar chains, revealed that the relative rates at which the various alpha 1,2-mannosidic linkages were cleaved, differed depending on their structural complexity. This suggests that distinct sugar residues in the aglycon moiety may be functional in substrate recognition and binding. Reduction or removal of the terminal GlcNAc residue of the chitobiose unit in M9 increased the hydrolytic susceptibility of the fourth (previously resistant) alpha 1,2-mannosidic linkage significantly. We conclude from this observation that, in addition to peripheral mannose residues, the intact chitobiose core represents a structural element affecting Man9-mannosidase specificity. A possible biological role of the enzyme during N-linked-oligosaccharide processing is discussed.


Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase. 1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.


Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis. Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding. The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis. Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.


Matrix NADH dehydrogenases of plant mitochondria and sites of quinone reduction by complex I. In order to distinguish the pathways involved in the oxidation of matrix NADH in plant mitochondria, the oxidation of NADH and nicotinamide hypoxanthine dinucleotide (reduced form) was investigated in submitochondrial particles prepared from beetroot (Beta vulgaris L. cv. Derwent Globe) and soybeans (Glycine max L. cv. Bragg). Nicotinamide-hypoxanthine-dinucleotide(reduced form)-oxidase activity was more strongly inhibited by rotenone than the NADH-oxidase activity but both of the rotenone-inhibited activities could be stimulated by adding ubiquinone-1. The corresponding ubiquinone-1-reductase activities were inhibited by rotenone (to 69%) and further inhibited by N,N'-dicyclohexylcarbodiimide (to 79%), whilst the K3Fe(CN)6-reductase activities were not sensitive to either rotenone or N,N'-dicyclohexylcarbodiimide. Immunological analysis of mitochondrial proteins using an antiserum raised against purified beetroot complex I indicated very few differences between soybean and fresh and aged beetroot mitochondria, despite their varying sensitivities to rotenone. We confirm that there are two dehydrogenases capable of oxidising internal NADH and that only one of these, namely complex I, is inhibited by rotenone. Further, we conclude that complex I has two potential sites of quinone reduction, both sensitive to N,N'-dicyclohexycarbodiimide inhibition but only one of which is sensitive to rotenone inhibition.


H2-forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron-sulfur clusters in methanogenic archaea. A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2 = H4MPT) to methenyltetrahydromethanopterin (CH identical to H4MPT+) and H2 and was therefore named H2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H2 or CH2 = H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (Vmax) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between 3H2 and 1H+; (d) the enzyme catalyzed the reduction of CH identical to H4MPT+ with 3H2 generating [methylene-3H]CH2 = H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxin-type iron-sulfur protein. Properties of the H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.


Cytochrome P-450 induction in human lung tumor-derived cell lines. Characterisation and effects of inflammatory mediators. Cytochrome P-450 species (P-450) comprise a polymorphic multigene family of heme-containing enzymes which are essential to the phase-I metabolism of xenobiotics. Induction of P-450 species by drugs and carcinogens has been extensively studied; endogenous regulation of P-450 also occurs during normal development and disease. The aim of this project was to study the in-vitro induction of P-450 and its modulation by inflammatory mediators in the human lung tumor-derived cell lines NCI H322 and NCI H358. The cell lines expressed detectable levels of 7-ethoxyresorufin O-deethylase which could be induced by benzanthracene. After benzanthracene treatment, a protein tentatively identified as isozyme CYP1A1 was detected by Western-blot analysis and a concommitant increase in CYP1A mRNA expression was observed. Optimal induction was observed at a benzanthracene concentration of 5 micrograms/ml with cells grown in RPMI medium containing 10% fetal calf serum. The effects of endotoxin, dexamethasone and five recombinant DNA-derived cytokines, interleukin-1 beta, tumor necrosis factor, and interferons alpha, beta and gamma, on constitutive and benzanthracene-induced ethoxyresorufin O-deethylase activity were examined in NCI H322 cells. Of all the lymphokines studied, only interferon gamma had any marked effect. Administration of this lymphokine strongly suppressed ethoxyresorufin O-deethylase activity in both control and benzanthracene-treated cells.


Differentiation-inducing-factor dechlorinase, a novel cytosolic dechlorinating enzyme from Dictyostelium discoideum. Differentiation-inducing factor 1 (DIF-1) is a dichlorinated alkyl phenone (1-[(3,5-dichloro-2,6-dihydroxy-4-methoxy)phenyl]hexan-1-one) from Dictyostelium discoideum, that induces amoebae to differentiate into stalk cells. It was shown previously that DIF-1 is rapidly metabolized into a series of more polar compounds by living cells [Traynor, D. & Kay, R.R. (1991) J. Biol. Chem. 266, 5291-5297]. The first step in DIF-1 metabolism is the formation of DIF metabolite 1 (now known to be DIF-3) by a monodechlorination. We report here the discovery of the enzyme activity catalyzing this dechlorination. A very sensitive enzyme assay was developed, using [3H]DIF-1 and a TLC system to separate DIF-1 from the product, DIF-3. DIF-1 3(5)-dechlorinase is present in the high-speed supernatant of cell lysates, and uses glutathione, at physiological concentrations, as cofactor. Kinetic measurements indicate a Km for DIF-1 of about 70 nM. The enzyme activity is inhibited by DIF-2 (the pentan-1-one analogue of DIF-1), with a median inhibitor concentration (IC50) of 1 microM, and DIF-3 (IC50 = 5 microM), which presumably act as substrates, but other compounds structurally related to DIF-1 were much less effective. Aurothioglucose, an inhibitor of selenocysteine enzymes, inhibited DIF-1 3(5)-dechlorinase with IC50 = 100 nM. DIF-1 3(5)-dechlorinase activity is developmentally regulated. It is essentially absent from growing cells and increases at the end of aggregation to reach a first peak of activity at the first finger stage, with a further rise at culmination.


Analysis of glycosaminoglycan chains from different proteoglycan populations in human embryonic skin fibroblasts. 1. The structure of chondroitin/dermatan and heparan-sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]-glucosamine and 35SO4(2-), and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmström, A. & Fransson, L.-A. (1990) Biochem. J. 265, 289-300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl-Sepharose in Triton X-100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL-6B, Bio-Gel P-6, ion exchange HPLC and gel electrophoresis. 2. Three types of dermatan-sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell-associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatan-sulphate chains were very similar to those of the small proteoglycans. 3. Heparan-sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of D-glucuronate, L-iduronate (with or without sulphate) and N-sulphate groups. They all displayed heparin-lyase-resistant domains with average molecular mass of 10-15 kDa. The heparan-sulphate chains from proteoglycans with 250-kDa and 350-kDa cores were the largest greater than 50 kDa), containing an average of four or five domains, in contrast to heparan-sulphate chains from the small heparan-sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell-associated heparan-sulphate chains were heterogeneous in size (5-45 kDa). 4. These results suggest that the core protein may have important regulatory functions with regard to dermatan-sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.


Measurement of low-molecular-weight antioxidants, uric acid, tyrosine and tryptophan in plaques and white matter from patients with multiple sclerosis. The levels of the antioxidants ascorbic acid, cysteine, reduced glutathione and alpha-tocopherol, of the free-radical marker uric acid and of the amino acids tyrosine and tryptophan were measured by means of high-pressure liquid chromatography in plaques, adjacent white matter and distant white matter from patients with multiple sclerosis, and in central nervous system tissue from patients without neurological diseases. Cholesterol and DNA were also determined, to check demyelination and cellularity. Uric acid was increased and glutathione correspondingly decreased in plaques; alpha-tocopherol was lowest in plaques and highest in distant white matter in all cases. Ascorbic acid, cysteine, tyrosine and tryptophan were not significantly changed in any tissue. The results provide evidence supporting the involvement of free radicals in multiple sclerosis.


Spontaneous body sway as a function of sex, age, and vision: posturographic study in 30 healthy adults. Detailed neurological examinations and body sway measurements with a stable force measuring platform were carried out on 30 healthy adults between 21 and 63 years of age. The results were analyzed for sex- and age-associated changes with regard to three different sway components (total sway, anterio-posterior sway, lateral sway) and two different conditions (eyes open, eyes closed). Sex-associated differences were highly significant for all sway components in the oldest age group (51-65 years) in which men exhibited more spontaneous postural sway than women in the condition eyes open. With eyes closed these differences increased. Middle-aged men (36-50 years) also exhibited significantly more postural sway than women of the same age. In the condition eyes open especially total sway and anterioposterior sway were increased, whereas in the condition eyes closed total sway and lateral sway were predominantly higher in men than in women. In the youngest age group (21-35 years) no sex-related differences in postural sway were found. Age-associated differences were significant for anterioposterior sway (eyes open) in men, increasing continuously from the young to the middle-aged, and again from the middle-aged to the older age group. Anterioposterior sway in women, on the contrary, did not change with age. Age-associated differences in women were found for total sway (eyes open) and lateral sway (eyes closed). However, the highest values for total sway and lateral sway within the female group were obtained from young women in both conditions eyes open and eyes closed.(ABSTRACT TRUNCATED AT 250 WORDS)


Intracranial paramedian hourglass-shaped dermoid associated with hereditary steatocystoma multiplex. This is the first report to describe the coexistence of two rare diseases, intracranial paramedian hourglass-shaped dermoid and steatocystoma multiplex. A 46-year-old female with a history of steatocystoma multiplex, bradydactylies and kyphosis showed oscillopsia, ataxia and hemifacial spasm. MRI findings suggested a giant dermoid cyst extending from the left middle temporal fossa to the cerebellopontine angle, and this was confirmed surgically. We propose the involvement of some genetic factor or pathological process common to both disorders, steatocystoma multiplex and dermoid. From the clinical point of view, patients with cystic skin lesion should also be checked for intracranial lesion.


Segmental myoclonus preceding herpes zoster radiculitis. Segmental myoclonus arising in the spinal cord occurs with several viral infections, including herpes zoster radiculitis. Usually, abnormal movements follow the rash and require drug treatment to suppress. We report a patient with AIDS in whom arm and shoulder myoclonus preceded herpes zoster involving the same segments contralaterally on two occasions. Myoclonus remitted promptly with antiviral treatment. Unlike in other immunosuppressed patients, encephalitis did not occur after the second episode.


Lipoma of the cauda equina selectively involving lower sacral roots. Case report. A 44-year-old male patient suffering from sexual and voiding dysfunction did not exhibit any sensorimotor deficit except for a lax anal sphincter with loss of the bulbocavernosus reflex (BCR). The absence of both the electrically induced BCR and cortical evoked responses to stimulation of the dorsal penile nerve as well as partial denervation of the pelvic floor musculature suggested damage to the lower sacral roots. The site of the lesion as indicated by electrophysiological findings was confirmed by computerized tomography and magnetic resonance imaging. The operation revealed a lipoma involving a few cauda fibers which produced a distension in the region of the conus medullaris.


Superficial siderosis of the central nervous system: neuroradiological evaluation of two cases. We present 2 cases of superficial siderosis of the central nervous system secondary to chronic subarachnoid bleeding. The diagnosis was made with MR and cerebrospinal fluid (CSF) examination. MR showed, in T2-weighted images, superficial hypointensity of the cervical cord, brainstem, cerebellum, and basal cisterns due to hemosiderin deposits. CSF was xanthochromic, with increased protein content. In 1 of the 2 cases also the CT examination was positive, showing a hyperdense rim around the brainstem. A complete neuroradiological evaluation (MR, CT, angiography and myelography) did not permit to detect the source of the bleeding.


Thermal thresholds and motor sensory conduction measurements in Guillain Barré syndrome: 12-month follow-up study. We performed serial neurophysiological tests in 22 patients with confirmed Guillain Barré syndrome (GBS) for 12 months following onset of the disease. Twenty-five age- and sex-matched healthy controls were also tested. Tests included nerve conduction and automated quantitative thermal threshold measurements. The commonest early abnormality was delayed distal motor latency, F wave abnormality and abnormal thermal threshold measurements in 1 or more nerves. Further abnormalities of all measurements were observed during maximum disability. After 12 months, all measured parameters returned to normal levels in the majority of patients except the thermal thresholds which remained abnormal in a large proportion of asymptomatic patients. The findings are suggestive of an early pathological involvement of the smaller nerve fibres with slow recovery in GBS.


Circulating autoantibody to muscle protein in a patient with paraneoplastic myositis and colon cancer. A muscle-specific autoantibody was found in a patient with paraneoplastic myositis and colon cancer. By immunoblotting, we found high titers of circulating antibody to a 34-kDa neutral protein in the soluble sarcoplasmic fraction of the rat skeletal muscle. The serum did not react at all with other tissue extracts including the central nervous tissue, liver, or kidney. The possible role of this muscle-specific autoantibody in the pathogenesis of paraneoplastic myositis is discussed.


Chronic Mycobacterium avium complex infection of the central nervous system in a nonimmunosuppressed woman. We report the case of a woman who developed chronic meningitis and a brain abscess due to Mycobacterium avium, without immunosuppressed state. She was treated with antituberculous drugs but the infection progressed and she died 4 years after first becoming ill. The occurrence of CNS infection with M. avium in the absence of acquired immunosuppression is rare. Its diagnosis is difficult, delaying clinical recognition and appropriate therapy.


Focal involvement and lateralization in Creutzfeldt-Jakob disease: correlation of clinical, electroencephalographic and neuropathological findings. Focal involvement can be assumed in the initial stage of Creutzfeldt-Jakob disease (CJD). We correlated the clinical, electroencephalographic and neuropathological findings in 13 cases of CJD with special reference to focal or lateralized changes. Eight cases showed clinical and 9 electroencephalographic signs of asymmetry. Four cases with a course of the disease shorter than 12 months presented circumscribed tissue alterations at autopsy. Cases with a clinical duration longer than 12 months had pathological changes involving both hemispheres symmetrically. The infectious agent seems to spread through the entire brain within a period of 12 months.


Lovastatin/gemfibrozil myopathy: a clinical, histochemical, and ultrastructural study. Lovastatin has been used with increasing frequency over the past few years to reduce serum cholesterol. The onset of muscle weakness, one of the most serious side effects of long-term treatment with the drug, constitutes a contraindication to the continuation of therapy and commonly occurs in patients who are also receiving gemfibrozil or cyclosporine. We report the clinical and pathologic findings in a patient treated for hypercholesterolemia with lovastatin and gemfibrozil who developed a rapidly progressive necrotizing myopathy. A 57-year-old woman with hyperlipidemia, treated with lovastatin and gemfibrozil, was admitted to the hospital for evaluation of muscular weakness in her legs and neck. Neurologic examination revealed severe proximal muscle weakness involving both upper and lower extremities as well as proximal muscle tenderness and areflexia in the lower limbs. A biopsy of the quadriceps muscle showed multiple foci of mononuclear cell infiltration with myophagocytosis and slight variation in the size and shape of muscle fibers. Electron microscopy of the affected fibers showed accumulations of subsarcolemmal autophagic lysosomes. The patient's condition dramatically improved after discontinuation of lovastatin-gemfibrozil therapy.


Herald facial numbness.

Herald facial numbness. Three unusual patients who developed subacute facial numbness as the heralding symptom of an expanding tumor that involved the trigeminal nerve fibers are reported. The first patient had clinical and electrophysiological evidence of an isolated mental neuropathy as a result of metastatic lesions with bone destruction from a renal cell carcinoma. The second patient had a sensorimotor trigeminal neuropathy caused by a direct compression of the semilunar ganglion by a cavernous hemangioma of Meckel's cave. The last patient experienced facial numbness as the unusual presenting manifestation of a primary brainstem lymphoma. Patients 1 and 3 died a few weeks after the admission, whereas patient 2 poorly recovered. Despite the availability of new techniques for early diagnosis, this report demonstrates how difficult it can initially be to differentiate a 'benign' trigeminal neuropathy from serious conditions and underscores the poor prognosis of fifth nerve fibers involvement by an expanding mass. Early referral with clinical and electrophysiological evaluation appears to be of crucial importance.


Relation of aphemia and agraphia.

Relation of aphemia and agraphia. A 49-year-old right-handed man presented with a severe impairment of motor speech output aphemia. Initially, he could make grunting sounds, but was otherwise mute. There was no disturbance of comprehension, and he preferred to communicate by writing. Writing was agrammatic with lexical errors and mispellings which improved with the speech disturbance. The writing abnormalities of aphemics emphasize the aphasic nature of this speech abnormality.


Effects of Y-20811, a thromboxane A2 synthetase inhibitor, on experimentally induced coronary thrombosis in anesthetized dogs. The effects of Y-20811, a selective inhibitor of thromboxane A2 (TXA2) synthetase, on blood flow and local levels of immunoreactive thromboxane B2 (i-TXB2) and 6-keto prostaglandin F1 alpha (i-6-keto PGF1 alpha) in the coronary artery were investigated in the canine model of coronary thrombosis. Thrombosis was induced by applying an electric current to the intraluminal surface of the coronary artery. The plasma levels of i-TXB2 and i-6-keto PGF1 alpha were measured distal to the electrode. In the control group, coronary blood flow decreased and finally stopped 207 +/- 53 min (mean +/- S.E.M., n = 5) after the start of current application. The level of i-TXB2 rose before the coronary occlusion. Coronary blood flow did not change significantly in the Y-20811-treated group (1 mg/kg i.v.). The level of i-TXB2 decreased and remained significantly lower than that in the control group. The level of i-6-keto PGF1 alpha tended to increase slightly in the Y-20811-treated, but not in the control group. The weight of the thrombus in the Y-20811-treated group was significantly less than that in the control group (P less than 0.01). These results suggest that Y-20811 prevents coronary thrombosis by the inhibition of TXA2 production around the electrically injured lumen of the coronary artery.


Cardioprotective effect of pindolol in ischemic-reperfused isolated rat hearts. The effects of pindolol and timolol on ischemia reperfusion damage were studied in isolated working rat hearts. Ischemia (15 min) decreased the mechanical function and the energy state, and increased the tissue levels of free fatty acids (FFA). During reperfusion (20 min), the mechanical function did not recover, but the energy state recovered incompletely, whereas FFA increased further. Pindolol (50 microM) accelerated recovery of the mechanical function and the energy state that had been decreased by ischemia during reperfusion, and inhibited the accumulation of FFA during ischemia and reperfusion, especially when it was applied during the whole period of reperfusion. Timolol (50 microM), however, did not accelerate recovery of the mechanical function and the energy state during reperfusion, although it attenuated FFA accumulation during reperfusion. The pindolol-induced recovery of the mechanical function during reperfusion was reduced by timolol. The results suggest that the intrinsic sympathomimetic activity of pindolol may play an important role, at least in part, in producing the cardioprotective effect, especially during reperfusion.


Vasodepressor mechanisms of 2-(1-octynyl)-adenosine (YT-146), a selective adenosine A2 receptor agonist, involve the opening of glibenclamide-sensitive K+ channels. The vasodepressor mechanism of 2-(1-octynyl)-adenosine (YT-146), a selective adenosine A2 receptor agonist, was compared with that of adenosine in spinally anaesthetized dogs whose blood pressure was kept elevated with i.v. infusion of noradrenaline. Cumulative i.v. administration of YT-146 (1-1000 nmol/kg) caused a slowly developing and long-lasting decrease in mean blood pressure (MBP) and a small decrease in heart rate (HR) at high doses, whereas single i.v. administration of adenosine caused short-lived decreases in MBP and HR. In dogs given glibenclamide (6 mumol/kg i.v.) or theophylline (30 mumol/kg i.v.) dose-response curves for decrease in MBP due to YT-146 underwent parallel rightward shifts. The ED50 values for YT-146 were increased about 5.5-fold with glibenclamide and about 11.9-fold with theophylline. However, no further rightward shifts of the curves were obtainable in dogs given glibenclamide plus theophylline; the increase in ED50 values for YT-146 was about 6.8-fold, being close to the increase obtained with theophylline alone. The ED50 values for adenosine to lower MBP increased about 1.6-fold with theophylline but not with glibenclamide. Glibenclamide and theophylline failed to antagonize the negative chronotropic effects of both drugs. These results suggest that the vasodepressor effect of YT-146 involves two mechanisms following stimulation of adenosine A2 receptors; one is probably a cyclic AMP-dependent mechanism and the other is the opening of glibenclamide-sensitive K+ channels.


Effect of DL-propranolol on nitric oxide production in perfused rat hindquarters. The effect of DL-propranolol on NO release in perfused rat hindquarters was studied by using oxyhemoglobin as a capture system to allow the quantitative assay of NO production. In some experiments the stable prostacyclin metabolite 6-keto-PGF1 alpha (6-keto) was simultaneously assayed. We observed that: (1) DL-propranolol induced an increase in NO and 6-keto release. The dextro isomer was inactive; (2) DL-propranolol-induced NO release was only slightly reduced by acetylsalicylic acid in a concentration that inhibits prostacyclin synthesis, and was abolished by the chemical removal of the endothelium with 3-3 cholamidopropyl dimethylammonium 1-propane sulphonate (CHAPS) applied before or during stimulation; (3) NG-nitro-L-arginine blocked DL-propranolol-induced NO production, an effect that was antagonized by L-arginine but not by its dextro isomer.


In vivo visualization of central muscarinic receptors using [11C]quinuclidinyl benzilate and positron emission tomography in baboons. The muscarinic antagonist, quinuclidinyl benzilate (QNB), labeled with carbon 11 was used as a radioligand to visualize in vivo by positron emission tomography (PET) the central muscarinic acetylcholine receptors (mAChR) in baboons (Papio papio). The binding characteristics of [11C]QNB showed its specific binding to central mAChR. [11C]QNB brain uptake was high in cerebral cortex and striatum, areas that are rich in mAChR, whereas it decreased rapidly in cerebellum, evidencing non-specific binding in this structure that is almost devoid of mAChR. These results are consistent with the known cerebral distribution of mAChR in primates. [11C]QNB specific cerebral binding was enhanced by pretreatment with methyl-QNB, a peripherally acting muscarinic antagonist. Specifically labeled binding sites alone were blocked by prior administration of dexetimide, a muscarinic antagonist. Specific radioactivity was driven out from mAChR-rich regions by atropine and dexetimide, drugs with high affinity for mAChR. This competition was stereospecific since only dexetimide, the pharmacologically active isomer of benzetimide, was able to compete with the radioligand on its binding sites. A relationship between the occupancy of [11C]QNB-labeled receptors by atropine or dexetimide and the concomitant induction of a pharmacological effect was also detected by simultaneous PET scanning and electroencephalographic recording. Since mAChR form an important part of choline receptors in the central nervous system, [11C]QNB appears to be a suitable radiotracer to monitor cerebral physiological or pathological phenomena linked to the cholinergic system in living subjects.


The involvement of products of the phospholipase pathway in the acute release of tissue-type plasminogen activator from perfused rat hindlegs. Using a perfused rat hindleg system, release of tissue-type plasminogen activator (t-PA) from endothelial cells could be induced by platelet-activating factor (PAF), bradykinin, substance P, thrombin, carbachol and A23187, while this release was inhibited by mepacrine and by nor-dihydroguaiaretic acid. The PAF-induced release of t-PA was inhibited by the cytochrome P-450 mono-oxygenase inhibitors, metyrapone, ketoconazole and SKF 525A and by eicosatetraynoic acid but not by indomethacin or BW 755C, suggesting the involvement of epoxygenase products. The PAF-induced release of von Willebrand factor (vWF) was also similarly inhibited by the cytochrome P-450 monooxygenase inhibitor, ketoconazole. Phorbol ester and phospholipase C induced the release of both t-PA and vWF, while phospholipase A2 did not. The release induced by PAF and bradykinin was not influenced by pretreatment with pertussis toxin.


Role of 5-HT1-like receptors in the increase in intragastric pressure induced by 5-hydroxytryptamine in the rat. The study concerned the effects of 5-hydroxytryptamine (5-HT) on intragastric pressure in bilaterally vagotomized spinal rats. Intravenous (i.v.) bolus injections of 5-HT (2.5, 5.0 and 10 micrograms/kg) produced dose-dependent increases in intragastric pressure; these effects were not modified by atropine (up to 0.2 mg/kg) or mepyramine (1 mg/kg), but were blocked by the mixed 5-HT1-like and 5-HT2 receptor antagonists, methiothepin (0.1, 0.3 and 0.5 mg/kg i.v.) and methysergide (0.5, 1 and 2.5 mg/kg i.v.). However, metergoline (0.5, 1 and 2 mg/kg i.v.) did not markedly modify this effect of 5-HT; only the response induced by 5 micrograms/kg 5-HT was significantly antagonized by the highest dose of metergoline. In contrast, neither the 5-HT2 receptor antagonist, ketanserin (0.5, 1 and 1.5 mg/kg i.v.), nor the 5-HT3 receptor antagonist, ICS 205-930 (0.5, 1 and 3 mg/kg i.v.), influenced the 5-HT-induced increase in intragastric pressure. In addition, 5-carboxamidotryptamine (25, 50 and 100 micrograms/kg i.v.) and RU 24969 (50, 100 and 200 micrograms/kg i.v.) mimicked the aforementioned effects of 5-HT but were weaker than 5-HT. These data suggest that the 5-HT-induced increase in intragastric pressure in the spinal and bilaterally vagotomized rat is mediated by an atypical 5-HT1-like receptor, which, based on the low agonist potency of 5-carboxamidotryptamine and RU 24969 and the resistance to blockade by metergoline, does not seem to correspond to either the 5-HT1A, 5-HT1B, 5-HT1C or the 5-HT1D receptor subtypes.


Lysophosphatidic acid induces inward currents in Xenopus laevis oocytes; evidence for an extracellular site of action. Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that can elicit platelet aggregation, smooth muscle contraction and, in fibroblasts, cell proliferation. We now report that LPA in nanomolar concentrations evokes an inward current in native, defolliculated Xenopus laevis oocytes. Extracellular application of LPA from a pressure pipette to the surface of the oocyte induced an immediate response. In contrast, intracellular injection of the same amount of LPA failed to elicit a response. These data suggest the existence of a Ca(2+)-mobilizing, endogenous LPA receptor in the Xenopus laevis oocyte cell membrane.


Computerized digital image analysis of retinal vessel density: application to normoxic and hyperoxic rearing of the newborn rat. Because of the inherent nature of existing techniques for vascular assessment of the retina, quantitative rigor has been difficult to attain. Computer imaging has now made possible measurement of oxygen-induced alterations in retinal blood vessels that is precise, accurate and repeatable. Newborn rats were exposed to 80% oxygen for 14 days as an animal model for retinopathy of prematurity (ROP). Ink perfusion, followed by retinal dissection, yielded silhouettes of the retinal vascular network. These images were digitized and enhanced by the MegaVision 1024XM imaging system in order to facilitate measuring extent of vasculature and capillary density. Mean grey levels, or 'mu', were 33.1 +/- 3.4 and 20.1 +/- 3.5 for room air- and oxygen-reared rats, respectively. These values are directly proportional to the fraction of the overall image that is vascular material, and, therefore, describe the extent of vaso-obliteration resulting from the oxygen exposure. The values were subsequently converted to square millimeters of vessel area to facilitate comparison to past and future studies. Oxygen rearing does not effect retinal area, but decreases retinal artery and vein diameter and decreases the linear extent of vessels toward the retinal periphery.


Glycation of human lens proteins: preferential glycation of alpha A subunits. Glycation of crystallins and high molecular weight (HMW) aggregates was followed during aging (16-85 years) and in diabetes (44 and 70 years old). Lens soluble and insoluble fractions were reduced with [3H]NaBH4 and separated by molecular sieve HPLC. The protein content in each HPLC peak was measured by the Lowry method. The tritium incorporation, expressed as cpm mg-1 protein, was taken as a measure of early glycation and specific non-tryptophan fluorescence (Ex: 370 nm; Em: 440 nm), expressed as relative fluorescence U mg-1 protein, was taken as a measure of advanced glycation. The youngest lenses analysed were 16 and 17 years old and these provided the baseline values. The results showed that during aging there was about a three-fold increase in tritium incorporation and fluorescence of alpha-crystallin, while the increases in beta and gamma were only two-fold from the levels seen in 16- and 17-year-old lenses. On the other hand, both the soluble and insoluble HMW aggregate fractions showed up to five-fold increase in tritium incorporation during aging. The fluorescence was about two-fold higher in the insoluble HMW aggregates as compared to the soluble HMW aggregates in 16- and 17-year-old lenses and both showed an increase of about three-fold during aging. Diabetes resulted in an approximately 10-50% increase in tritium incorporation and non-tryptophan fluorescence of various crystallins and HMW aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)


Ganglion cells in the juvenile chick retina and their ability to regenerate axons in vitro. Ganglion cells in the chicken retina fail to regenerate their axons upon mechanical injury. In order to determine whether this failure to regenerate axons is intrinsic to the neurons or is mediated by the environment, we asked whether ganglion cells possess an ability to regrow their injured axons in the absence of their natural environment, namely in vitro. Since the retina contains morphologically different types of ganglion cells, it became desirable to investigate whether all types of ganglion cells contribute to regeneration of axons. Ganglion cells were labelled post-mortem with the fluorescent dye DiI and described morphologically. Morphometric parameters like the sizes of their perikarya, their dendrites, and the patterns of dendritic ramification and stratification were considered for grouping cells. Although a strong classification of the cells could not be achieved because of the high diversity among this population of neurons, the chick retinal ganglion cells could be separated into seven regular groups which have their somata located within the ganglion cell layer and into one group with the somata located within the inner nuclear layer (displaced ganglion cells). The experimental procedure for regeneration combines crush injury of the chick optic nerve in situ with explanation of retinal pieces 1 week later for organ cultures in a serum-free medium. Under these conditions, the ganglion cells extended axons 1 day after explanation on polylysine/laminin. The densities of ganglion cells contributing to the axonal regrowth reached up to 1447 cells mm-2 (mean 1028 cells mm-2; S.D. 237). This density corresponds to 13% of the ganglion cell density in the normal retina, averaged across the total retina area. Although the dendrites of some cells whose axons had regenerated were altered in comparison with the normal ganglion cells, all morphological types of ganglion cells including those with displaced cell bodies contributed proportionally to the regrowth of axons from the explants.


An immunoelectron microscope study of the aged human lens capsule. The distribution of types I-IV collagen and laminin was studied in seven aged human lens capsules using the immunogold EM technique on LR White embedded tissue. Samples were taken from the anterior, equatorial and posterior regions. Labelling for type II collagen was not observed. Type IV collagen was evenly distributed throughout the thickness of the capsule but was absent from the zonules. However, an unexpected finding was strong labelling for types I and III collagen, again evenly distributed throughout the capsule. The presence of type III collagen makes the lens capsule unique among ocular basement membranes. Laminin was present in linear densities, zonular lamellae and zonular fibres, suggesting that linear densities are an integral part of the zonular apparatus.


Comparison of ascorbic acid levels in the eye and remainder of the chicken embryo during development. Ascorbic acid was not detectable in egg white and egg yolk of fertilized eggs prior to incubation. It was demonstrated in the embryo at 5 days of incubation and its content in the eyes and other parts of the embryo increased with the embryo's age. Ascorbic acid of the eyes rose rapidly after day 6 during maximal eye growth and reached a peak by days 10-14 when the expansion of the eyes began to level off. Ascorbic acid content of the eyes decreased thereafter, while ascorbic acid in the head and the body tissues continued to rise up to the time of hatching. Whether this rapid increase of ascorbic acid concurrent with the increase of eye diameter indicates a role for ascorbic acid in the growth of the embryonic eye remains to be determined.


Proteoglycans in the mouse interphotoreceptor matrix. V. Distribution at the apical surface of the pigment epithelium before and after retinal separation. The distribution of chondroitin sulfate proteoglycans in the mouse interphotoreceptor matrix (IPM) proximal to the retinal pigment epithelium (RPE) was evaluated with EM histochemical techniques using Cupromeronic Blue (CmB) before and after retinal separation. Densely packed, sheet-like processes surrounding vesicle-like compartments containing CmB staining were normal constituents of the IPM at the apical surface of the RPE. Most of the vesicle-like compartments, which appeared to be isolated from the IPM in single section profiles, were found to be continuous with the IPM when three-dimensional reconstructions of serial thin sections were evaluated. Analyses of stereo image pairs of semithin sections visualized by electron spectroscopic imaging (ESI) also demonstrated that the CmB stained proteoglycans in the lumen of these pseudovesicles were in continuity with the CmB stained components present in the IPM. Moreover, ESI demonstrated that the CmB stained profiles formed an elaborate interconnecting network extending from the apical border of the RPE to the level of the outer limiting membrane of the retina. After removal of the retina, rinsing of the RPE with Ringer's solution prior to fixation eliminated proteoglycan staining near the base of the apical processes, whereas staining near the tips of these processes remained. The CmB stained filaments remaining after rinsing were thicker and shorter, and made fewer interconnections than those in the non-separated preparations. These results suggest that two types of chondroitin sulfate proteoglycans are present in the IPM which differ in distribution and in the degree of aqueous solubility. Additionally, a closely associated retina appears to be required for maintenance of the normal structure of proteoglycans associated with the RPE surface. The elaborate topography at the RPE apical surface may play a role in the delivery and/or recovery of components of the IPM.


Membrane architecture as a function of lens fibre maturation: a freeze fracture and scanning electron microscopic study in the human lens. The ultrastructure of fibre membranes in human lenses, varying in age from premature to 40 years, was investigated using a strict protocol regarding their localization within the lens. The ultrastructural approaches used were scanning electron microscopy (SEM), and transmission electron microscopy (TEM) of ultrathin sections and freeze-fracture replicas. Irrespective of the age of the lens, superficial fibre membranes are characterized by a high density of intramembrane particles (IMPs) and numerous gap junctions (GJs). In contrast deep cortical fibres, at the SEM-level characterized by grooves and ridges, are largely free of IMPs but still contain numerous GJs. In between these regions a transitional zone was observed. At the SEM-level the transitional fibres are characterized by wrinkled membranes and formation of grooves and ridges. In freeze-fracture replicas the presence of numerous square arrays (SAs) associated with GJs is most remarkable. It is concluded that at all ages studied, the maturation and compaction of lens fibres results in a transformation of membrane architecture leading to clear-cut ultrastructural differences between superficial and deep cortical membranes. It is argued that this ultrastructural heterogeneity parallels the gradients observed biochemically for intrinsic membrane proteins and cholesterol:phospholipid ratios. The observations confirm the electrophysiological view that superficial membranes have an 'average' permeability and that deep cortical membranes are 'degenerate' or 'non-leaky'.