Expression, characterization and purification of soluble G-protein beta gamma dimers composed of defined subunits in baculovirus-infected insect cells. Recombinant beta 1 gamma 2 dimers of signal-transducing guanine nucleotide-binding proteins (G-proteins) carrying a mutation known to block isoprenylation of the gamma 2 subunit were expressed as a soluble protein in baculovirus-infected insect cells. The soluble beta gamma dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an S20,w value of 4.1 +/- 0.4 S. The two subunits segregated as a dimer upon sucrose density gradient centrifugation and purification by sequential ion exchange and hydroxylapatite chromatography. The results show that baculovirus-infected insect cells can be employed for high level production of pure G-protein beta gamma dimers suitable for functional and structural characterization.


The primary structure of a PYY-related peptide from chicken intestine suggests an anomalous site of cleavage of the signal peptide in preproPYY. Although the amino acid sequence of members of the pancreatic polypeptide (PP)-family of regulatory peptides has been poorly conserved during vertebrate evolution, the overall length of the peptides (36 amino acid residues) has remained constant. Nucleotide sequence analysis of cloned cDNAs and/or genomic fragments has shown the PP-related sequence immediately follows the signal peptide in the prepropeptides. A peptide tyrosine-tyrosine (PYY)-related peptide with 37 residues has been isolated from the chicken intestine, and its primary structure was established as: Ala-Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly10-Asp-Ala-Ala-Ser-P ro-Glu-Glu-Ile-Ala-Gln20-Tyr-Phe-Ser-Ala-Leu-Arg-His-Tyr-Il e-Asn30-Leu-Val-Thr-Arg-Gln-Arg-Tyr.CONH2. The presence of an additional alanine residue at the NH2-terminus of the peptide suggests that the site of cleavage of the signal peptide in chicken preproPYY is different from the site of cleavage in other PP-family prepropeptides.


Three-dimensional structure of apotransketolase. Flexible loops at the active site enable cofactor binding. The structure determination of apotransketolase and the comparison of its three-dimensional structure with that of the holoenzyme has revealed that no large conformational changes are associated with cofactor binding. Two loops at the active site are flexible in the apoenzyme which enables ThDP to reach its binding site. Binding of the cofactor induces defined conformations for these two loops at the active site. One of these loops is directly involving in binding of the cofactors, Ca2+ and ThDP. This loop acts like a flap which closes off the diphosphate binding site. After binding of the cofactor, residues of this loop form interactions to residues of loop 383-398 from the second subunit. These interactions stabilize the conformation of the two loops from a flexible to a 'closed' conformation.


The length of the interdomain region of the L7/L12 protein is important for its function. Several mutated L7/L12 proteins with changed interdomain regions were obtained. The results showed that the flexible region comprising the 39-52 amino acid residues is functionally important. Its length, but not its amino acid composition, is crucial for the function.


A laser flash absorption spectroscopy study of Anabaena sp. PCC 7119 flavodoxin photoreduction by photosystem I particles from spinach. Electron transfer from P700 in photosystem I (PSI) particles from spinach to Anabaena sp. PCC 7119 flavodoxin has been studied using laser flash absorption spectroscopy. A non-linear protein concentration dependence of the rate constants was obtained, suggesting a two-step mechanism involving complex formation (k = 3.6 x 10(7) M-1.s-1) followed by intracomplex electron transfer (k = 270 s-1). The observed rate constants had a biphasic dependence on the concentrations of NaCl or MgCl2, with maximum values in the 40-80 mM range for NaCl and 4-12 mM for MgCl2. To our knowledge, this is the first time that the kinetics of PSI-dependent flavodoxin photoreduction have been determined.


Mode of action of the Spiroplasma CpG methylase M.SssI. The cytosine DNA methylase from the wall-less prokaryote, Spiroplasma strain MQ1 (M.SssI) methylates completely and exclusively CpG-containing sequences, thus showing sequence specificity which is similar to that of mammalian DNA methylases. M.SssI is shown here to methylate duplex DNA processively as judged by kinetic analysis of methylated intermediates. The cytosine DNA methylases, M.HpaII and M.HhaI, from other prokaryotic organisms, appear to methylate in a non-processive manner or with a very low degree of processivity. The Spiroplasma enzyme interacts with duplex DNA irrespective to the presence of CpG sequences in the substrate DNA. The enzyme proceeds along a CpG-containing DNA substrate molecule methylating one strand of DNA at a time.


Interrelations of M-intermediates in bacteriorhodopsin photocycle. The photocycles of the wild-type bacteriorhodopsin and the D96N mutant were investigated by the flash-photolysis technique. The M-intermediate formation (400 nm) and the L-intermediate decay (520 nm) were found to be well described by a sum of two exponents (time constants, tau 1 = 65 and tau 2 = 250 microseconds) for the wild-type bR and three exponents (tau 1 = 55 microseconds, tau 2 = 220 microseconds and tau 3 = 1 ms) for the D96N mutant of bR. A component with tau = 1 ms was found to be present in the photocycle of the wild-type bacteriorhodopsin as a lag-phase in the relaxation of photoresponses at 400 and 520 nm. In the presence of Lu3+ ions or 80% glycerol this component was clearly seen as an additional phase of M-formation. The azide effect on the D96N mutant of bR suggests that the 1-ms component is associated with an irreversible conformational change switching the Schiff base from the outward to the inward proton channel. The maximum of the difference spectrum of the 1-ms component of D96N bR is located at 404 nm as compared to 412 nm for the first two components. We suggest that this effect is a result of the alteration of the inward proton channel due to the Asp96-->Asn substitution. Proton release measured with pyranine in the absence of pH buffers was identical for the wild-type bR and D96N mutant and matched the M-->M' conformational transition. A model for M rise in the bR photocycle is proposed.


Specific antiviral activities of the human alpha interferons are determined at the level of receptor (IFNAR) structure. Differences in activity among the family of human IFNs alpha are much reduced if these ligands are assayed on bovine cells. In particular, the activity of IFN alpha D is much higher on bovine than on human cells. To examine these differences, the bovine counterpart of the human IFNAR has been cloned and expressed in a human cell line. The transfected cell line now recognizes the human IFN alpha D as a high-specific-activity IFN subtype, indicating that the differences in sensitivity between the bovine and human cells to the human IFN alpha lie in the structure of the IFNAR chain rather than in the other components of the functional receptor.


In vitro biosynthesis of lactase in preweaning and adult rabbit. Lactase is synthesized as a high-mannose large precursor (200 kDa) which is subsequently complex-glycosylated (215 kDa) and split into the 150 kDa mature form. The regulatory mechanisms responsible for the decline of activity at weaning are not yet known. We have set up in vitro cultures of intestinal mucosa from suckling and adult rabbit and found that suckling and adult animals synthesize the same four forms of lactase-phlorizin hydrolase (LPH) but with a different distribution. In the proximal adult small intestine there is very little 180 kDa form, which is most probably a product of the 215 kDa complex-glycosylated precursor. The 180 kDa form comprises a greater percentage of total LPH in the middle of the small intestine in adult and particularly in suckling rabbits. In the latter tissue this form is apparently more stable than in the adult tissue. Posttranscriptional control of lactase synthesis is therefore different in the various parts of the adult small intestine, and it is different in the suckling as compared to adult tissue.


The levels of lactase and of sucrase-isomaltase along the rabbit small intestine are regulated both at the mRNA level and post-translationally. We determined along the small intestine of young and adult rabbits the activities of lactase (LPH) and sucrase (SI), the levels of their cognate mRNAs, and examined the in vitro biosynthesis of LPH and pro-SI. Lactase activity is low in the proximal 1/3 of the intestine, whereas the mRNA levels are high. However, the rates of biosynthesis of the LPH forms correlated well with the steady-state levels of LPH mRNA in all segments, indicating that factor(s) acting post-translationally produce a decline in brush border LPH in the proximal small intestine. These factor(s) are not involved in the processing of pro-LPH to mature LPH, since the relative amounts of the various forms of LPH are almost the same along the small intestine. Unexpectedly, we find that also for SI the ratio of activity to mRNA is low in proximal intestine. The biosynthesis of pro-SI correlates with the steady-state levels of its mRNA. Hence, the steady-state levels of LPH and SI along the small intestine are regulated both by mRNA levels and by posttranslational factor(s).


Maturation of human lactase-phlorizin hydrolase. Proteolytic cleavage of precursor occurs after passage through the Golgi complex. Maturation of human intestinal lactase-phlorizin hydrolase (LPH) requires that a precursor (pro-LPH) be proteolytically processed to the mature microvillus membrane enzyme (m-LPH). The subcellular site of this processing is unknown. Using low-temperature experiments and brefeldin A (BFA), intracellular transport was blocked in intestinal epithelial cells. In Caco-2 cells incubated at 18 degrees C, pro-LPH was complex-glycosylated but not cleaved, while at 20 degrees C small amounts of proteolytically processed LPH were observed. These data exclude a pre-Golgi proteolytic event. BFA completely blocked proteolytic maturation of LPH and lead to an aberrant form of pro-LPH in both Caco-2 cells and intestinal explants. Therefore, proteolytic processing of LPH is a post-Golgi event, occurring either in the trans-Golgi network, transport vesicles, or after insertion of pro-LPH into the microvillus membrane.


Enflurane is a potent inhibitor of high conductance Ca(2+)-activated K+ channels of Chara australis. The volatile anaesthetic, enflurane, is commonly used in surgery for inducing the state of general anaesthesia. It is assumed, that general anaesthetics act on ion channels, but little is known of how they do so and what kinds of channels are sensitive. We found, that enflurane inhibits a large conductance Ca(2+)-activated K+ channel of the green alga, Chara australis. Effects occur at clinically relevant concentrations are fully reversible. The actions of enflurane are distinct from those of charybdotoxin and tetraethylammonium, which are well known blockers of this channel type. Kinetic analysis of single-channel data demonstrates multiple effects of enflurane on the channel protein.


C-type natriuretic peptide in bovine chromaffin cells. The regulation of its biosynthesis and secretion. We report here the regulation of the biosynthesis and the secretion of C-type natriuretic peptide (CNP) in cultured bovine chromaffin cells. The combined treatment with protein kinase A and -C activators induced a 6-fold increase of intracellular levels of CNP-(1-103). The biosynthesized CNP-(1-103) was co-released with its mature forms, typically CNP-(51-103), upon stimulation by nicotine or depolarizing agents. This confirms the neuropeptidic character of this third member of the natriuretic peptide family, which might act as a neuromodulator or neurotransmitter.


Inhibition by spermine of the inner membrane permeability transition of isolated rat heart mitochondria. The effect of spermine on the permeability transition of the inner mitochondrial membrane of isolated rat heart mitochondria was evaluated. The permeability transition was triggered using a series of agents (t-butyl hydroperoxide, phenylarsine oxide, carboxyatractylate, and elevated Ca2+ and inorganic phosphate concentrations), and was monitored via Ca(2+)-release, mitochondrial swelling and pyridine nucleotide oxidation. By all three criteria, spermine inhibited the transition. A C50 of 0.38 +/- 0.06 (SD) mM was measured for inhibition.


Natural history of retinopathy of prematurity: a prospective study. The natural history of retinopathy of prematurity (ROP) has been studied prospectively in 572 infants < or = 1,700 g birthweight using a protocol designed specifically to investigate the subtle features of this condition. Acute ROP developed in 50.9%. All ROP stages 1 and 2 underwent complete resolution and of the 27 (4.7%) infants with stage 3/4 disease, cicatricial sequelae developed in six. Incidence and severity increased with decreasing birthweight and gestational age. Onset was not confined to the temporal retina but exhibited a predilection to start first in the nasal retina of the most immature neonate. The vertical retinal regions were relatively spared. Retinal arteriolar tortuosity developed around three months postnatally and was related to ROP severity but not its presence. The age at onset and rate of progression of retinopathy were largely determined by the stage of development but were also modified by systemic and local factors. The relevance of these findings to ROP screening is discussed.


Ocular hypertension: correlation of anterior chamber angle width and risk of progression to glaucoma. Twenty five patients with ocular hypertension (OH) and a narrow angle and 34 patients with OH and an open angle were followed for an average of six years. One eye of each patient had been randomly assigned to treatment with topical timolol. A shallow axial anterior chamber depth and a narrow angle (van Herick Grade 2 or less) was associated with the development of angle-closure glaucoma (ACG) in the narrow angle group, but the open angle group developed significantly more visual field loss. Nine patients (36%) in the narrow angle group developed ACG, and nine patients (26.5%) in the open angle group developed glaucomatous discs and field loss. The reduction in intraocular pressure due to topical timolol was equal in the narrow angle and the open angle groups, but topical timolol did not protect against the development of angle-closure glaucoma or visual field loss.


Laser microsclerostomy for primary open angle glaucoma: a review of laser mechanisms and delivery systems. A number of different lasers and delivery systems are currently used in experimental sclerostomy procedures. These are discussed with reference to their performance in terms of cutting accuracy and trauma to the adjacent tissues. Lasers emitting wavelengths close to the absorption peaks of water in the mid-infrared region and organic polymers in the far ultraviolet region have a short absorption pathlength in the sclera and produce the least adjacent thermal tissue trauma. These ablating lasers cannot be delivered gonioscopically, and contact endoscopic techniques trap hot expanding gases within the forming sclerostomy channel causing secondary thermal and mechanical damage. Optimal results should be obtained using an ablating laser delivered either through an open mask or a modified endoscopic system incorporating an adequate exhaust mechanism.


The association of iridoschisis and primary angle-closure glaucoma. Twelve patients with iridoschisis in one or both eyes were studied to determine the clinical features of the condition and to examine the relationship of iridoschisis to primary angle-closure glaucoma. A spectrum of iris pathology, from subtle intrastromal atrophy to extensive splitting of the anterior layer of iris with fibrillar disintegration, was found in the affected and fellow eyes. Gonioscopy revealed partial or complete angle closure, particularly involving the superior angle, in all patients. Seven had glaucomatous disc damage and five had normal discs. The mean axial length and anterior chamber depth measurements in patients with iridoschisis were similar to those found in matched patients with primary angle-closure glaucoma but were significantly less than the measurements found in matched normals (p < 0.001). This study suggests that iridoschisis is an unusual manifestation of iris stromal atrophy and results from intermittent or acute elevation of intraocular pressure. Primary angle-closure glaucoma should be excluded in patients who present with iridoschisis.


Doppler carotid artery studies in asymmetric glaucoma. Doppler carotid artery studies were performed in 12 glaucoma patients with marked asymmetry in bilateral visual field loss. The resistance index and the pulsatility index of the internal carotid artery velocity waveforms were significantly greater on the same side as the eye with the greater visual field loss. The increased resistance to blood flow in the internal carotid artery on the side with advanced field loss might predispose the eye on this side to the effects of raised intraocular pressure by causing a reduction in the perfusion pressure at the optic nerve head. The role of ocular perfusion pressure in the pathogenesis of glaucoma is discussed. More extensive studies are necessary.


Comparative evaluation of the Allergan Humphrey 570 and Canon RK-1 autorefractors: I. Objective autorefraction in normal subjects. Fifty normal subjects were studied comparing objective autorefraction using the Allergan Humphrey 570 (AH 570) and Canon RK-1 autorefractors in terms of ease of operation, the time taken and their accuracy compared with clinical refraction. Both the autorefractors were equally easy to operate but the AH 570 was quicker. Objective autorefraction with the AH 570 was more accurate than using the Canon RK-1 especially with respect to spherical equivalence, sphere power and cyclinder axis: approximately 80% of the values were within 0.51 dioptres or 11 degrees of clinical refraction compared to approximately 60% for the Canon RK-1. The possible reasons for the superior performance of the AH 570 are discussed.


Comparative evaluation of the Allergan Humphrey 570 and Canon RK-1 autorefractors: II. Objective autorefraction in pseudophakes. Fifty patients (50 eyes) with posterior chamber intraocular lenses and a best corrected visual acuity of at least 6/12 were studied. The Allergan-Humphrey 570 (AH-570) and Canon RK-1 autorefractors were compared in terms of ease of operation, time taken and their accuracy relative to clinical refraction. Both the autorefractors were equally easy to operate but the AH-570 was quicker. The AH-570 had a higher percentage of eyes within 11 degrees of clinically determined cylinder axis while the Canon RK-1 had a higher percentage of patients within 0.51 diopters of spherical equivalence, sphere power and cylinder power as determined by clinical refraction. This difference in the accuracy of objective autorefraction was not statistically significant; the clinical significance is uncertain.


The effects of adrenaline, hyaluronidase and age on peribulbar anaesthesia. In a prospective, randomised, masked trial, 91 patients undergoing intraocular surgery received an anaesthetic mixture containing lignocaine hydrochloride 2% and bupivacaine hydrochloride 0.5%. In addition group 1 had hyaluronidase (50 i.u./ml) and adrenaline (1:200,000), group 2 had hyaluronidase alone, group 3 had adrenaline alone and group 4 had neither. The groups were compared regarding the quality of operating conditions. Hyaluronidase had a substantial beneficial effect though there was no significant difference related to the use of adrenaline. Anaesthesia was less effective in patients under the age of 65 years.


Peribulbar anaesthesia: failure to abolish the oculocardiac reflex. Peribulbar anaesthesia is a form of regional anaesthesia which has been used with increasing popularity in cataract surgery. Recently indications for its use have been extended to include surgery for vitreo-retinal disease and strabismus. This study shows that in nine of 11 patients in whom the oculocardiac reflex was stimulated by traction on an extra-ocular muscle, peribulbar anaesthesia failed to abolish the reflex. In each of these patients, the reflex manifested as a bradycardia. In one subject, the bradycardia was profound, showing no QRS complex for five seconds. We recommend that cardiac rhythm be monitored throughout procedures performed under peribulbar anaesthesia, and that a vagolytic agent be available for immediate use.


Extracapsular cataract extraction in diabetics with rubeosis iridis. We reviewed thirteen operated eyes (twelve diabetic patients) with rubeosis iridis who underwent extracapsular cataract extraction and intraocular lens implantation. Prior to surgery five had active proliferative retinopathy (APR), and eight had non-proliferative retinopathy (NPR), either quiescent proliferative retinopathy (QPR) or background retinopathy (BR). No case with APR was visually improved by surgery. Three cases with NPR achieved a visual acuity of 6/12. After surgery, vitreous haemorrhage or progression of proliferative retinopathy occurred in three cases with APR. Early postoperative fibrinous uveitis was severe in eyes with APR, resulting in permanent fibrin membrane formation in four. We suggest a significant prognostic indicator in diabetic cataract extraction with rubeosis iridis is the status of the underlying retinopathy. With NPR, postoperative visual acuity may be good and early postoperative complications less severe. In the presence of APR the visual outcome is poor, progression of retinopathy likely and early postoperative fibrinous uveitis may be severe enough to prevent postoperative panretinal photocoagulation. Maximum preoperative panretinal ablation is essential in these cases.


Occlusion for amblyopia: a comprehensive survey of outcome. The results of a long term follow up of all patients from a single health district started on occlusion for amblyopia in 1983 are reported. Three hundred and sixty-eight patients started treatment, their average age was four years seven months, the average amount of daily occlusion was 1.5 hours and the average length of follow up was 31 months. 37% of cases achieved a final visual acuity of 6/9 or better and another 33% a visual acuity of 6/12 or 6/18.23% did not achieve 6/18 and treatment of these patients was regarded as failure. Data for the remainder (7%) were incomplete. The success rate of occlusion treatment varied little with the age of starting treatment. The group with combined strabismus and anisometropia responded least well to treatment.


Factors related to the final visual outcome of 425 patients with traumatic hyphema. A retrospective study of the visual outcome of 425 in-patients with traumatic hyphaema has been conducted. A multivariate analysis demonstrated that after adjusting for age, sex and pre-existing poor vision, the size of hyphaema on presentation and the presence of retinal damage were significant predictors of a worse final visual outcome (p = 0.00003 and 0.00001 respectively). Topical steroid and/or cycloplegic medication, and the occurrence of secondary haemorrhage did not influence the final visual outcome after adjustment for the other variables. These data illustrate, in an unselected sequential population of patients, the role of these factors in terms of final visual outcome following hyphaema from blunt ocular trauma.


Transient vessel wall sheathing in acute retinal vein occlusions. Three cases are reported which had features similar to, and evolved in a pattern consistent with central retinal vein occlusions and a fourth case is reported which behaved as a hemispheric vein occlusion. However, they differed from classic retinal vein occlusions by having prominent sheathing of the retinal venous vasculature at presentation, which in all four cases resolved within three weeks. There was no evidence for any of these cases having an inflammatory vasculitis. The significance of this transient sheathing is uncertain.


Factors related to the incidence of secondary haemorrhage in 462 patients with traumatic hyphema. In a retrospective study of 462 in-patients with traumatic hyphema, secondary haemorrhage occurred in 8.7% of patients. A multivariate analysis demonstrated that the size of hyphaema on presentation and the presence of retinal damage did not affect the probability of secondary haemorrhage. The incidence of secondary haemorrhage was found to decrease by approximately half with the use of topical steroid (p = 0.005), but did not appear to be influenced by the use of cycloplegics. These data indicate in an unselected sequential population of patients, the therapeutic importance of topical steroid in the treatment of blunt ocular trauma.


Migrating scleral explants.

Migrating scleral explants. Anterior migration of an extrascleral explant is an uncommon complication following buckling procedures. We report five patients where the explant cheesewired through the rectus muscle insertion following retinal detachment surgery. Muscles were not disinserted at the time of surgery in any case. Ocular motility problems were only seen in two patients. The probable mechanisms of this hitherto unreported complication are discussed.


The Purkinje vascular entoptic test: a halogen light gives better results. The Purkinje vascular entoptic test is a test of macular function which employs a light directed through the sclera illuminating the fundus. This light casts shadows of retinal blood vessels on to posterior pole photoreceptors. When the light source is moved, a patient with a good macular function should be able to see a negative image of his or her retinal blood vessels. We evaluated the Purkinje test in eyes with clear ocular media. A bright 3.5 watt halogen rechargeable transilluminator was used instead of a pen torch as previously described. Sixty-eight patients (129 eyes) attending a diabetic eye clinic, were tested. The test correctly identified 91% of eyes with good macular function and 77% of eyes with poor macular function (visual acuity 6/24 or poorer). If a vascular pattern was seen, it was probable (0.89) that good macular function was present. If no vascular pattern was seen, it was probable (0.80) that the eye had poor macular function (chi 2 = 60.14, P = < 0.001). Our results were superior to those previously reported. We attribute the increased accuracy of our test to the brighter light source used.


Primary lymphoplasmacytoma of the conjunctiva. We report the case of an 80-year-old patient with isolated lymphoplasmacytoma of the conjunctiva. Only five other such cases have been previously reported in the literature. In all six patients, the disease remained limited and no sign of systemic disease could be found after prolonged follow-up. As isolated plasmacytoma of the conjunctiva seems to be benign, treatment of such tumours should be conservative.


Ocular manifestations of Noonan syndrome. Noonan syndrome is a genetic condition inherited in an autosomally dominant manner, characterised by congenital heart disease, short stature, abnormal facies and the somatic features of Turner's syndrome, but a normal Karyotype. The ophthalmological and orthoptic findings on 58 patients with Noonan syndrome are reported. External features were hypertelorism (74%), downward sloping palpebral apertures (38%), epicanthic folds (39%) and ptosis (48%). The orthoptic examination revealed strabismus in 48%, refractive errors in 61%, amblyopia in 33%, and nystagmus in 9% of cases. Sixty-three per cent of cases had anterior segment changes consisting of: Prominent corneal nerves (46%), anterior stromal dystrophy (4%), cataracts (8%) and panuveitis (2%). Fundal changes occurred in 20% of the study group, including optic nerve head drusen, optic disc hypoplasia, colobomas and myelinated nerves. Forty-seven per cent required non surgical treatment and a further 16% had undergone surgery for strabismus or ptosis. Only three patients had no visual defects. With such a high incidence of ophthalmic abnormalities it is clearly important that children with Noonan syndrome are screened by an ophthalmologist at an early age.


Prostaglandin E2 (PGE2) differentially regulates the production of IL-2 and IL-3 by murine immune T-cells. Eicosanoids are important mediators of inflammation, and have been shown to have potent, and usually suppressive immunoregulatory activities. In the paper, we have examined the role of prostaglandin (PGE2) production in the regulation of two cytokines, IL-2 and IL-3, which both play a key role in contact sensitivity and delayed type hypersensitivity reactions. In agreement with previous studies, we demonstrate that prostaglandins down-regulate IL-2 production in the system. Unexpectedly, however, IL-3 levels are enhanced in the presence of the prostaglandin PGE2 and conversely, are inhibited by treatment with aspirin, a potent inhibitor of prostaglandin metabolism. The implications of this result in terms of the immunoregulatory role of PGs will be discussed.


Enzyme ultracytochemical demonstration of Ca(++)-ATPase in the rat cerebral cortex. The localization of Ca(++)-ATPase activity was investigated in the rat cerebral cortex using an ultracytochemical method. Our cytochemical procedure for the detection of enzyme activity is based on an incubation medium consisting of tricine buffer, ATP-disodium salt as substrate, cerium chloride as capturing agent, CaCl2, and levamisole as a nonspecific alkaline phosphatase inhibitor. Final pH was 7.4. Reaction products showing Ca(++)-ATPase activity were localised on the pre- and postsynaptic plasma membrane in association with synaptic vesicles, postsynaptic dendrites and on the axolemma in myelinated nerve fibres. The verification of the main enzymatic properties of Ca(++)-ATPase localization activity is the subject of the following report.


In vitro enhanced destruction of erythrocytes as a result of their chlorination. Subarachnoidal haemorrhage is followed by accumulation of granulocytes and macrophages in cerebrospinal fluid (CSF). As a result erythrocytes are destroyed and removed from CSF. Activated granulocytes chlorinate many species including erythrocytes. It has been shown that chlorinated erythrocytes are more sensitive to phagocytosis by macrophages than the native ones.


Differentiated reaction of different types of antheridial cells in Chara vulgaris to the changes in light conditions during 24 hours. Circadian rhythm of activity of 3H-leucine incorporation into antheridial cells of Chara vulgaris, in natural photoperiod was compared with changes in mitotic activity of antheridial filament cells which form spermatozoids. Three types of cells functionally connected with each other i.e. manubria, capitular cells and antheridial filaments indicate high amplitude (80-90%) changes in circadian translational activity and some similarities in their course. The shield cells are characterized by small circadian changes in translational activity in the range of 15-30% and their different rhythm. Manubria, which are the secretive cells indicated the highest dependence of the dynamic of translational activity on the time of day. Their high activity overlaps light phase, low activity--dark phase. The reaction of capitular cells to day/night change is delayed in comparison with the reaction of manubria, and that of antheridial filaments is delayed in comparison with the capitular cells reaction. The assumption was set forth that manubria play the role of oscillators (starttercells) which induce the wave of changes translocating to the other cells functionally and spatialy connected with them. The course of the wave of antheridial filaments mitotic activity suggests that a distinct drop in MI in the morning may be the result of the lack of the factors necessary for initiation of the mitosis, dependent on light-induced high translational activity of antheridial cells.


A comparison of the proliferative activity of astrocytes and astrocyte-like cells expressing vimentin in the injured mouse cerebral hemisphere. After an unilateral injury of the cerebral hemisphere, 28 nice were injected with 3H thymidine at different intervals following the injury. Thereafter, the distribution of autographically labelled astrocytes expressing glial fibrillary protein (GFAP) and astrocyte-like cells expressing vimentin were recorded within the region of injury. Proliferative activity of the two cell types started at the same time, i.e. 24 h. after injury, reached its maximum on day 4, and returned to normal level after the 8th posttraumatic day. However, on day 4 the number of proliferating GFAP-positive astrocytes was about 50% higher than that of vimentin-positive cells. This was regarded as a proof of the concept that a significant number of astrocytes did not express vimentin during its mitotic cycle. Those facts were considered as an evidence against the hypothesis that a reactive astrocyte division induces a two-stage increase in the cytoskeletal proteins level with the elevated synthesis of vimentin preceding that of GFAP.


Transfected endometrial cultured cells: a system to study gene-regulation by estrogens. Glandular epithelial (GE) and stromal cells were isolated from guinea-pig endometrium, cultured and subcultured separately. At the end of subculture, the purity of each cell population was higher than 95% and cells displayed a high level of estrogen receptors. Calcium phosphate transfection conditions were defined using a control plasmid containing the bacterial CAT gene driven by viral promoter and enhancer sequences. Transfection experiments were performed with other plasmids in which CAT gene was linked to different estrogen response elements (EREs) derived from those of vitellogenin genes. CAT activity was significantly increased by estradiol-17 beta treatment only when GE or stromal cells were transfected with plasmids containing EREs previously reported as functional EREs in other cell types. This induction was abolished by ICI 164,384 diethylstilbestrol was as effective as estradiol-17 beta for CAT induction and estradiol-17 alpha was ineffective. Transiently transfected endometrial cells in subculture are a suitable system to study the estrogen effect on gene regulatory elements.


Single and dual hormone secretors in GH3 cultures respond differently to hypothalamic factors. Recent studies using both normal and tumoral pituitary cell cultures have demonstrated that growth hormone (GH) and prolactin (PRL) secreting populations contain cells which release either one or both of these hormones. In order to determine whether these two cell types can be differentially regulated by hypothalamic factors we performed the following study employing plaque assays for GH and PRL. Using cultures of GH3 cells, a rat tumor cell line which contains both of these cell types, we found that the hypothalamic factors vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) when used together had a greater influence on plaque formation than when each was used individually. This suggested that cells were present in culture that responded to one peptide but not the other. Estradiol-treated cultures (which contain only dual-secreting cells) were then evaluated for VIP and TRH responsiveness and found to respond to TRH but not VIP. Finally, we assessed the peptide sensitivity of cultures that were exposed to a conjugate of VIP and the A-chain of ricin (a potent cytotoxin). In addition to eliminating VIP-responsive cells, this treatment markedly reduced the proportions of cells secreting GH-only while having no appreciable influence on dual-hormone secretors. When taken together, our findings indicate that single and dual secretors respond differently to at least two hypothalamic secretagogues and suggest that regulatory differences between these cell types may be important in the control of GH and PRL secretion.


Autoinduction of thyroid hormone receptor during metamorphosis is reproduced in Xenopus XTC-2 cells. To determine if the autoinduction of thyroid hormone receptor (TR) alpha and beta mRNAs during metamorphosis in Xenopus tadpoles can be reproduced in cultured cells, we have screened four Xenopus cell lines (XTC-2, XL-177, XL2 and Kr) for receptor transcripts and their response to thyroid hormone. Exposure of XTC-2 cells to 10(-9) M triiodothyronine (T3) for 24 h upregulated TR alpha and beta mRNAs by 2-4- and 10-40-fold, respectively. In view of the marked similarity of the differential distribution of the two transcripts and their upregulation by T3 to the pattern of autoinduction seen in whole tadpoles, the process was studied in greater detail in XTC-2 cells. The time-course of autoinduction of TR alpha and beta mRNAs in these cells also resembled that in vivo, the two transcripts being significantly induced by 3-6 h after T3. Dose-response to T3, and the relative responses to its active and inactive analogs, confirmed that the process of autoinduction was initiated by thyroid hormone receptor with the same functional characteristics as that found in all amphibian and mammalian tissues. Experiments performed with cycloheximide suggested that intermediary protein(s) were involved in autoinduction, so that TR genes cannot be considered as 'immediate early' genes for this process. The possible advantages of studying thyroid hormone action in metamorphosis in XTC-2 cells are briefly discussed.


Isolation of luteinising hormone receptor binding inhibitor from bovine corpus luteum. A luteinising hormone receptor binding inhibitor (LHRBI) has been purified from bovine corpus luteum (CL). Steroid-free extract of the CL was subjected to successive chromatographies on Sephadex G-50, Q-Sepharose, Orange A dye and metal chelate affinity columns followed by high performance-reverse phase and gel filtration columns. Purification was monitored by the ability of the fractions to inhibit the binding of 125I-human chorionic gonadotropin (hCG) to porcine granulosa cells in vitro. The final isolate showed an 8000-fold enrichment of activity. It was also capable of inhibiting porcine granulosa cell secretion of estradiol and progesterone (P) in vitro. Administration of LHRBI into follicle-stimulating hormone (FSH)-stimulated, immature rats strongly inhibited the ovarian ovulatory response to hCG as revealed by decreased P levels and the number of ova released. The M(r) of LHRBI as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was ca. 15 kDa and the pI was between 5.0 and 5.5.


Identification of G protein alpha subunit mutations in human growth hormone (GH)- and GH/prolactin-secreting pituitary tumors by single-strand conformation polymorphism (SSCP) analysis. We have applied the polymerase chain reaction (PCR) and single-strand conformation polymorphism analysis (SSCP) to detect activating mutations in the Gs alpha subunit gene, amplifying genomic DNA extracted from growth hormone (GH)- and GH/prolactin (PRL)-secreting human pituitary tumors. Of 15 tumors tested six contained mutations in the analyzed regions of the Gs alpha. SSCP analysis revealed band shift in exon 8 in four GH- and in one GH/PRL-secreting tumors, and in exon 9 in one GH/PRL-secreting tumor. Direct sequencing of PCR reaction products identified the mutations as R201-H, R201-S and R201-C in exon 8 and Q227-L in exon 9. These results show the efficacy of PCR/SSCP analysis in the detection of G protein mutations and extend the generalization that these sites are hot spots in tumor-inducing mutations.


Thyroid hormone stimulates glucose transport and GLUT1 mRNA in rat Sertoli cells. The transport of 2-deoxyglucose (dGlc) by cultured rat Sertoli cells was stimulated by L-triiodothyronine (T3) in a time and dose-dependent manner. The lag-time was of about 6 h, the half-maximal dose (ED50) was 0.47 nM, which correlates with the Kd of the nuclear T3 receptor of rat Sertoli cells (Kd = 1-2 nM), and the stimulation was maintained up to 24 h. The effect was specific, as judged by the order of potency of T3 analogs. Cycloheximide prevented the stimulatory effect without affecting the basal uptake. T3 stimulated the uptake of the glucose analog 3-O-methylglucose (MeGlc) with the same order of potency as that of dGlc. The ontogenetic profile of the T3 effect coincides with that of T3 nuclear receptors in rat Sertoli cells. Northern blot analysis demonstrated that Sertoli cells express the erythrocyte/brain glucose transporter isoform (GLUT1) but not the adipose/muscle isoform (GLUT4). T3 treatment (10(-7) M for 24 h) induces an increase of GLUT1 mRNA level comparable to that of glucose analog uptake. These results suggest that thyroid hormone stimulates glucose transport by increasing the synthesis of new glucose transporter units and give further evidence for a direct effect of thyroid hormone in the modulation of Sertoli cell functions.


Membrane transport of thyroid hormone in the human choriocarcinoma cell line, JAR. We studied uptake of L-triiodothyronine (T3) by the human choriocarcinoma cell line, JAR. Uptake was time dependent with a half-time of 56.2 +/- 7.2 min (mean +/- SEM, n = 4). A non-saturable component accounted for about 24% of total uptake. We found a single saturable uptake mechanism with a calculated Michaelis constant (Km) of 586 +/- 206 nM (n = 9) and a corresponding maximum velocity of 17.0 +/- 5.7 pmol/min per mg protein (n = 9), values similar to those we have described recently in cultured normal human trophoblast cells. Uptake was dependent on temperature and intracellular energy, being reduced at lower temperatures and in the presence of potassium cyanide. It was independent of the Na+ gradient across the cell membrane and the presence of Na+ in the external medium, but was affected by the cell membrane potential.


A distal region enhances the prolactin induced promoter activity of the rabbit alpha s1-casein gene. Casein gene expression is induced in the rabbit mammary gland by prolactin (PRL). alpha s1-casein is the major casein secreted into milk. In order to define the position of the DNA sequences involved in the control of rabbit alpha s1-casein gene regulation by PRL, chimeric genes were constructed between upstream regions of the rabbit alpha s1-casein gene and the chloramphenicol acetyl transferase (CAT) reporter gene. A series of 5'-deleted fusion genes was obtained by nuclease digestion of the alpha s1-casein gene upstream region. These gene constructs were transfected into rabbit primary mammary cells, or cotransfected in CHO cells with the plasmid coding for the rabbit mammary receptor (PRL-R). A regulatory region has been located between nt -3768 and -3155. This region enhances the prolactin induced promoter activity of the alpha s1-casein gene. It might possess or cooperate with prolactin responsive elements located further downstream in the alpha s1-casein gene.


Role of local environmental factors in determining tissue-specific effects of estrogen: examination of uterine tissues transplanted to brain. Estrogen stimulates uterine epithelial cells to divide, but not estrogen-concentrating neurons in the adult brain. This effect correlates with recent evidence that estrogen can induce the expression of certain growth-related genes in uterus which are not directly induced by estrogen in the adult brain. The possibility that local diffusible factors play a major role in determining tissue-specific effects of estrogen was examined by transplanting uterine tissues into the brain, muscle and kidney of adult rats and then comparing the effects of estrogen on the incorporation of [3H]thymidine and the expression of Fos-, cdc2- and Rb-like immunoreactivity (IR) on native and transplanted uterine tissues, as well as in estrogen-concentrating regions of the brain adjacent to the uterine grafts. In native uteri, estrogen treatment stimulated Fos-, cdc2-, and Rb-like IR, as well as [3H]thymidine incorporation, within lumenal and glandular epithelial cells. All of these effects were estrogen responsive--no immunoreactive staining within uterine epithelial cells and no signs of epithelial cell proliferation were observed in the native uteri of non-estrogen-treated animals. When uterine tissues were transplanted to brain, Fos-, cdc2-, and Rb-like IR epithelial cells, as well as many [3H]thymidine-incorporating uterine epithelial cells, were observed in all estrogen-treated animals and in some non-estrogen-treated animals as well. Identical results were obtained when uterine tissues were transplanted to skeletal muscle, but not to kidney (in the kidney, transplanted epithelial cells expressed all four parameters but only in estrogen-treated animals, comparable to the native uterus). In contrast, estrogen did not stimulate cell division and did not induce Fos-, cdc2-, or Rb-like IR within estrogen-concentrating neuronal regions of the ventromedial hypothalamus. In addition, the presence of uterine tissue in the brain did not confer the ability of estrogen to stimulate any of these parameters within nearby, estrogen-concentrating regions. These data suggest that there are factors in brain and muscle which can allow uterine epithelial cells to divide in the absence of estrogen. There was no evidence of a diffusible factor in brain which inhibits uterine epithelial cell division, nor of a diffusible factor in uterus which can confer estrogenic stimulation of growth-related genes and cell division to central nervous system neurons. In addition, the data provide the first evidence for estrogen regulation of cdc2 and Rb expression in normal uterus.


Thyroid hormone response of slow and fast sarcoplasmic reticulum Ca2+ ATPase mRNA in striated muscle. The thyroid status markedly influences the contractile function of muscle, and changes in the activity of the Ca2+ ATPase of the sarcoplasmic reticulum (SR) contribute to these alterations. Two separate genes encode the major isoforms of SR Ca2+ ATPase. In fast skeletal muscle, sarcoplasmic endoplasmic reticulum Ca2+ ATPase type 1 (SERCa1) presents the major isoform, whereas in slow skeletal muscle SERCa type 2 (SERCa2) predominates. Cardiac muscle contains only SERCa2. To examine the mechanisms responsible for changes in contractile function, we quantitated SERCa1 and SERCa2 mRNA levels in fast extensor digitorum longus muscle (EDL), slow soleus muscle, and cardiac muscle in rats of different thyroid status. Hypothyroidism led in soleus to a marked decrease in SERCa1 mRNA and SERCa2 mRNA levels, in cardiac muscle SERCa2 mRNA decreased markedly, as previously shown by us, and in EDL SERCa1 mRNA decreased. These findings are compatible with a hypothyroidism induced decrease in SR Ca2+ ATPase activity and a delay in muscle relaxation. In contrast, SERCa2 mRNA of EDL, representing only a small percent of total SERCa mRNA in this muscle, increased to 175% of control values. Muscle specific and SERCa gene specific changes also occur after acute triiodothyronine (T3) administration to hypothyroid rats. T3 does not induce a significant change in SERCa1 or SERCa2 mRNA levels in soleus, but in the heart SERCa2 mRNA increases about 3-fold. In EDL, T3 increases SERCa1 mRNA from a hypothyroid level of 59 +/- 6% to 138 +/- 4% of control values but SERCa2 mRNA is decreased to 75 +/- 5% of control levels.(ABSTRACT TRUNCATED AT 250 WORDS)


A synthetic peptide corresponding to amino acids 9-30 of the extracellular domain of the follitropin (FSH) receptor specifically binds FSH. As deduced on the basis of cloning experiments, the putative extracellular domain of pituitary glycoprotein hormone (lutropin (LH), thyrotropin (TSH), and FSH) receptors (rec) is sufficiently large to suggest involvement in hormone binding. Comparison of the amino acid sequences of the extracellular domains of the glycoprotein hormone receptors indicates that the FSH receptor has a peptide sequence in the external domain close to the amino terminus (residues 9-30) which has no sequence homology to receptors for LH or TSH. To examine whether this region is involved in FSH-receptor interaction, we studied the hormone-binding properties of a corresponding synthetic peptide in several systems. (1) Binding of 125I-hFSH to receptor-containing bovine testis membranes was inhibited by preincubation with FSH rec-(9-30) peptide amide in a concentration-dependent manner. (2) 125I-labeled rec-(9-30) peptide amide bound to ovine, bovine, or human FSH preparations, and the binding was inhibited by solubilized bovine FSH receptor. 125I-labeled rec-(9-30) peptide amide, however, did not bind to LH or TSH. (3) 125I-hFSH bound to unlabeled rec-(9-30) peptide amide, and the binding was inhibited by excess unlabeled FSH, but not by LH or TSH. (4) Scatchard analysis indicated that the FSH rec-(9-30) peptide amide contained a single class of FSH binding sites with a Ka = 1.1 x 10(6) M-1. (5) The binding of 125I-labeled rec-(9-30) peptide amide to hFSH, bFSH or oFSH was effectively inhibited by rabbit polyclonal antibodies raised against rec-(9-30) peptide amide but not by preimmune rabbit serum.(ABSTRACT TRUNCATED AT 250 WORDS)


Perspectives in diabetes. Islet transplantation with immunoisolation. Immunoisolation is a potentially important approach to transplanting islets without need for immunosuppressive drugs. Immunoisolation systems have been conceived in which the transplanted tissue is separated from the immune system of the host by an artificial barrier. These systems offer a solution to the problem of human islet procurement by permitting use of islets isolated from animal pancreases. The devices used are referred to as biohybrid artificial organs because they combine synthetic, selectively permeable membranes that block immune rejection with living transplants. Three major types of biohybrid pancreas devices have been studied. These include devices anastomosed to the vascular system as AV shunts, diffusion chambers, and microcapsules. Results in diabetic rodents and dogs indicate that biohybrid pancreas devices significantly improve glucose homeostasis and can function for more than a year. Recent progress made with this approach is discussed, and some of the remaining problems that must be resolved to bring this technology to clinical reality are addressed.


Experimental transplantation with principal islets of teleost fish (Brockmann bodies). Long-term function of tilapia islet tissue in diabetic nude mice. Certain teleost fish have macroscopically visible islets called BBs that are anatomically discrete. BBs were harvested from Oreochromis nilotica (tilapia) with microscissors, divided, and cultured overnight at 37 degrees C before transplantation into STZ-induced diabetic nude mice. Each mouse received BB fragments from 3-5 fish weighing in aggregate approximately 1.7 kg. Non-FPGs were monitored 5 days/wk. Recipients remained normoglycemic (plasma glucose < 11.1 mM) for 50 days posttransplantation. Mice bearing 50-day-old grafts had essentially normal GTTs. Left nephrectomies then were performed to remove the grafts, and plasma glucose levels in recipient mice rose to > 22.2 mM. Histological examination of graft-bearing kidneys showed viable, vascularized islet tissue containing numerous well-granulated beta-cells; examination of recipient native pancreases revealed small islets composed predominantely of non-beta-cells.


Induction of resistance to endothelin-1's biochemical actions by elevated glucose levels in retinal pericytes. Because retinal pericytes have contractile properties and are affected by diabetes, we have studied the responsiveness of pericytes to ET-1, a potent vasoconstrictor, in the presence of various concentrations of glucose. Cultured calf retinal pericytes were exposed to glucose levels of 5.5 or 25 mM for up to 8 days. Radioreceptor studies that used [125I]ET-1 showed that pericytes contained high-affinity binding sites with Kd of 3 x 10(-10) M, and these binding affinities were unaffected by glucose concentration. Receptor number appears to be elevated, but this increase was NS. Responsiveness of pericytes to ET-1 was studied with respect to stimulation of DAG and IP3 levels and PKC activities. In contrast to receptor binding, exposure to 25 mM glucose for > 6 days blunted pericyte responsiveness to ET-1. The time course of ET-1 stimulation as measured by [3H]glycerol labeling, and IP3 level showed a 98% increase in [3H]DAG at 10 min and a fourfold increase for IP3, respectively. Cells exposed to 25 mM glucose only had a 32% increase for DAG, and no increase for IP3 was observed. Dose-response studies on the stimulation of [3H]DAG increase showed the range of ET-1's effect to be between 10(-9) and 10(-7) M. At maximum, cells exposed to 5.5 mM glucose had a 70% increase versus only a 30% increase in those exposed to 25 mM glucose. Similarly, ET-1 only increased the total DAG levels in pericytes exposed to 5.5 mM glucose by 41%. PKC activity also was measured because DAG is one of its cellular activators.(ABSTRACT TRUNCATED AT 250 WORDS)


Pathogenic factors responsible for glucose intolerance in patients with NIDDM. To define the pathogenic factors responsible for glucose intolerance in NIDDM, we estimated insulin secretory capacity, SI, and SG in 11 healthy, nondiabetic subjects and 9 NIDDM patients who had no SI impairment. All subjects studied were nonobese and normotensive. Each underwent a 75-g OGTT and a modified FSIGT: glucose was administered (300 mg/kg body weight), and insulin was infused (20 mU/kg over 5 min) from 20 to 25 min after the administration of glucose. SI and SG were estimated by Bergman's minimal-model method. The insulin response to oral glucose was significantly lower in NIDDM patients than in normal control subjects. First-phase insulin secretion expressed as the integrated area of plasma insulin above the basal level during the first 20 min was much smaller in NIDDM subjects (214 +/- 112 pM.min) than in control subjects (4643 +/- 885 pM.min, P < 0.01). SI was not statistically different in normal control subjects (1.27 +/- 0.18 x 10(-4) min-1.pM-1) versus diabetic patients (1.62 +/- 0.33 x 10(-4) min-1.pM-1). However, SG was significantly lower in diabetic subjects (1.11 +/- 0.17 x 10(-2) min-1) than in control subjects (2.35 +/- 0.26 x 10(-2) min-1, P < 0.01). These results suggest that impaired insulin secretion and decreased SG are the factors responsible for glucose intolerance of Japanese NIDDM patients with normal insulin sensitivity. Because SI and SG are the factors responsible for glucose intolerance of NIDDM patients with insulin resistance, it is conceivable that decreased SG is common in NIDDM patients regardless of their SI index.


Lactate production and pyruvate dehydrogenase activity in fat and skeletal muscle from diabetic rats. This study was initiated to explore the possibility that an increase in the supply of gluconeogenic precursors contributes to the overproduction of glucose by the liver in NIDDM patients. To address this issue, a form of experimental NIDDM was produced in rats by injecting a low dose (38 mg/kg) of STZ and comparing lactate and alanine production and PDH activity in skeletal muscle and isolated adipocytes from normal and diabetic rats. Skeletal muscle lactate production was measured by using a hindlimb perfusion technique and was significantly greater (P < 0.01) in the diabetic rats compared with two groups of control rats: one perfused at normal glucose levels and the other perfused at glucose concentrations comparable with those observed in diabetic rats. Alanine production by hindlimb from diabetic rats was 46% greater than hindlimbs from control rats perfused at normal glucose levels (P < 0.01) but was not significantly greater than control rats perfused at diabetic glucose levels. The percentage of glucose converted to lactate by muscle from both control groups was 4-5%, significantly lower than the 18% conversion rate observed in diabetic animals (P < 0.001). An increase in the ratio of lactate produced/glucose transport by isolated adipocytes from diabetic rats also was observed when measured in both the basal state (0.65 +/- 0.12 vs. 0.15 +/- 0.03, P < 0.01) and in the presence of maximal amounts of insulin (0.15 +/- 0.02 vs. 0.04 +/- 0.01, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)


Role of cytosolic Ca2+ in impaired sensitivity to glucose of rat pancreatic islets exposed to high glucose in vitro. Sustained exposure to high concentrations of glucose selectively impairs the ability of pancreatic islets to secrete insulin in acute glucose stimulation. In order to evaluate the interrelationship between impaired insulin secretion and the dynamics of the cytosolic free Ca2+ level ([Ca2+]i), we have investigated the effect of high glucose exposure on both [Ca2+]i dynamics in single rat beta-cells and insulin release from rat pancreatic islets. Islets cultured at a high glucose concentration (16.7 mM) for 24 h showed significant reductions of the 16.7 mM GSIR compared with islets cultured at a normal glucose concentration (5.5 mM) (3.38 +/- 0.24 vs. 4.26 +/- 0.34%, respectively, P < 0.05). The capacity of glucose to raise the [Ca2+]i level also was significantly reduced in the beta-cells maintained for 24 h at 16.7 mM glucose (P < 0.001). An additional culture in the medium with 5.5 mM glucose for 16 h restored both the GSIR and the [Ca2+]i response of islets cultured at high glucose. On the other hand, insulin release and [Ca2+]i rise in response to 20 mM L-Arg were well preserved. These observations confirm that exposure of pancreatic beta-cells to high glucose concentrations induces a selective reduction of the GSIR and, further, shows that this impaired response is reversibly restored by an additional culture with normal glucose. We also suggest that the inability of glucose to provoke a [Ca2+]i rise, which is observed in the beta-cells exposed to high glucose, may be responsible for the selective impairment of the GSIR.


Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle. Skeletal muscle surface membrane is constituted by the PM domain and its specialized deep invaginations known as TTs. We have shown previously that insulin induces a rapid translocation of GLUT4s from an IM pool to the PM in rat skeletal muscle (6). In this study, we have investigated the possibility that insulin also stimulates the translocation of GLUT4 proteins to TTs, which constitute the largest area of the cell surface envelope. PM, TTs, and IM components of control and insulinized skeletal muscle were isolated by subcellular fractionation. The TTs then were purified further by removing vesicles of SR origin by using a Ca-loading procedure. Ca-loading resulted in a five- to sevenfold increase in the purification of TTs in the unloaded fraction relative to the loaded fraction, assessed by immunoblotting with an anti-DHP-receptor monoclonal antibody. In contrast, estimation of the content of Ca(2+)-ATPase protein (a marker of SR) with a specific polyclonal antibody revealed that most, if not all, SR vesicles were recovered in the Ca-loaded fraction. Western blotting with an anti-COOH-terminal GLUT4 protein polyclonal antibody revealed that acute insulin injection in vivo (30 min) increased the content of GLUT4 (by 90%) in isolated PMs and markedly enhanced (by 180%) GLUT4 content in purified TTs. Importantly, these insulin-dependent changes in GLUT4 content of PM and purified TTs were seen in the absence of changes in the alpha 1-subunit of the Na(+)-K(+)-ATPase, a surface membrane marker.(ABSTRACT TRUNCATED AT 250 WORDS)


Improved specificity of ICA assays in the Fourth International Immunology of Diabetes Serum Exchange Workshop. The goal of the Fourth International Workshop for Standardization of ICA Measurements was to determine the specificity of ICA assays and their ability to distinguish between control sera (n = 57) and sera from IDDM-related individuals--representing relatives of IDDM patients (n = 21), healthy individuals who later developed IDDM (n = 8), or newly diagnosed IDDM patients (n = 23). Results from 28 laboratories were analyzed. The mean specificity (percentage of control sera reported as negative) among 27 laboratories was 91%, including 6 laboratories with 100% specificity. Nevertheless, 78% of laboratories found at least one control sample > 0 JDF U. Among samples from first-degree relatives, the mean concordance was 86%, including three sera found negative (0 JDF U) by all laboratories. Among individuals who later developed diabetes, the mean concordance was 93%, with two sera found positive by 100% of laboratories. In sera from newly diagnosed IDDM patients, the mean concordance was 82%. Three sera were found positive and one serum negative by all laboratories. The JDF U of the sera considered to be positive were significantly greater than each laboratory's average for the controls. In conclusion, the results from laboratories participating in the Fourth International ICA Workshop demonstrated excellent specificity, good concordance, and an ability to separate control sera from defined, IDDM-related subjects.


The metabolic profile of NIDDM is fully established in glucose-tolerant offspring of two Mexican-American NIDDM parents. NIDDM patients with overt fasting hyperglycemia are characterized by multiple defects involving both insulin secretion and insulin action. At this point of the natural history of NIDDM, however, it is difficult to establish which defects are primary and which are acquired secondary to insulinopenia and chronic hyperglycemia. To address this question, we have studied the glucose-tolerant offspring (probands) of two Mexican-American NIDDM parents. Such individuals are at high risk for developing NIDDM later in life. The probands are characterized by hyperinsulinemia in the fasting state and in response to both oral and intravenous glucose. Insulin-mediated glucose disposal (insulin clamp technique), measured at two physiological levels of hyperinsulinemia (approximately 240 and 450 pM [approximately 40 and 75 microU/ml]), was reduced by 43 and 33%, respectively. During both the low- and high-dose insulin clamp steps, impaired nonoxidative glucose disposal, which primarily represents glycogen synthesis, was the major defect responsible for the insulin resistance. During the lower dose insulin clamp step only, a small decrease in glucose oxidation was observed. No defect in suppression of HGP by insulin was demonstrable. The ability of insulin to inhibit lipid oxidation (measured by indirect calorimetry) and plasma FFA concentration was impaired at both levels of hyperinsulinemia. These results indicate that the glucose-tolerant offspring of two NIDDM parents are characterized by hyperinsulinemia and manifest all of the metabolic abnormalities that characterize the fully established diabetic state, including insulin resistance, a major impairment in nonoxidative glucose disposal, a quantitatively less important defect in glucose oxidation, and a diminished insulin-mediated suppression of lipid oxidation and plasma FFA concentration.


Glucose transporters of rat peripheral nerve. Differential expression of GLUT1 gene by Schwann cells and perineural cells in vivo and in vitro. Expression of GLUTs in rat peripheral nerve was first studied at the mRNA level with Northern transfer analysis with cDNAs specific for GLUT1, GLUT2, GLUT3, and GLUT4. GLUT1 mRNA was the only GLUT mRNA detectable in rat sciatic nerve. In situ hybridization localized this mRNA to the perineurium and to some endo- and epineurial capillaries. Indirect immunofluorescence stainings demonstrated that GLUT1 protein epitopes were concentrated primarily in the perineurium and endoneurial capillaries. Also, some Schwann cells, a few epineurial capillaries, and medium-sized blood vessels showed a faintly positive immunoreaction. All cell types present in primary cultures initiated from rat sciatic nerve (perineurial cells, Schwann cells, and fibroblasts) expressed GLUT1 protein in vitro. Thus, Schwann cells, which expressed GLUT1 only occasionally at a low level in vivo, have the potential to express GLUT1 at a markedly higher level under cell culture conditions. Incubation of the cultures in 25 mM D-glucose for 7 days caused a 39% reduction in the amount of immunodetectable GLUT1 protein, and a marked (34%) decrease of GLUT1 mRNA compared with cultures incubated in 5.5 mM D-glucose. Interestingly, the reduction of [3H]-2-DG uptake in the same cultures exceeded 70%, suggesting that the reduced amount of GLUT1 protein alone did not explain the marked reduction in glucose uptake in these cultures. Immunostaining of the cell cultures suggested that perineurial cells were the main target for the glucose-induced decrease of GLUT1 protein.


Intermittent hypoglycemia impairs glucose counterregulation. IDDM patients who maintain strict glycemic control have impaired counterregulatory hormone and symptomatic responses to hypoglycemia. To test the hypothesis that intermittent exposure to hypoglycemia plays an etiological role in these defective responses, we produced 4 consecutive daily episodes of hypoglycemia in 10 healthy, nondiabetic volunteers by using the insulin clamp technique. Fasting (5.3 +/- 0.1 vs. 5.4 +/- 0.1 mM) and nadir (2.3 +/- 0.1 vs. 2.4 +/- 0.1 mM) glucose levels achieved during insulin infusion did not differ on study days 1 and 4. In contrast, the glucose levels required to stimulate an increase in EPI (2.8 vs. 3.1 mM), glucagon (2.8 vs. 3.2 mM), cortisol (2.4 vs. 2.9 mM), GH (2.6 vs. 3.0 mM), and autonomic hypoglycemic symptoms (2.2 vs. 2.5 mM) were all significantly lower on study day 4 versus study day 1 (P < 0.005-0.05). Basal levels of EPI and cortisol, but not glucagon, GH, or NE also were reduced on the final study day. We conclude that intermittent hypoglycemia can result in attenuation of the hormonal and symptomatic responses to insulin-induced hypoglycemia and may contribute to the defective counterregulatory responses in patients with well-controlled IDDM.


CD8 T cells are not required for islet destruction induced by a CD4+ islet-specific T-cell clone. A panel of CD4+ T-cell clones has been isolated from the spleen and lymph nodes of diabetic NOD mice. These clones have been shown to be islet-specific both in vivo and in vitro. One of the clones, BDC-6.9, initiates extensive damage to islet tissue when placed adjacent to an NOD islet graft that has been used to reverse diabetes in (CBA x NOD)F1 recipients or when injected intraperitoneally into such animals. In this study, we show that BDC-6.9 T cells can initiate islet destruction in the absence of detectable CD8 T cells either in the periphery or in the lesion that develops after the transfer of the cloned islet-reactive T cells.


Polygenic nature of spontaneous diabetes in the rat. Permissive MHC haplotype and presence of the lymphopenic trait of the BB rat are not sufficient to produce susceptibility. We describe the phenotypic characteristics of animals in the fifth backcross-intercross generation of a breeding program in which the RT1 u haplotype and the phenotypic trait responsible for the T-lymphopenia of BB rats have been transferred to the ACI background. In this generation of animals, 24% were lymphopenic with decreased numbers of PBL expressing CD5, TCR alpha, and RT6. The PBL of the lymphopenic animals had a decreased mitogenic response to ConA. All of the nonlymphopenic animals were homozygous for RT6.2. Phenotypic analysis of intestinal IEL revealed that this was also the case for the lymphopenic animals. Moreover, IEL of the lymphopenic animals exhibited a pattern of staining (increased numbers of TCR alpha beta+CD4+CD8+ and decreased numbers of TCR alpha beta+CD4-CD8+) similar to that of BB DP animals. The ACI.1U(BB)-lymphopenic animals, although having two of the genetic traits associated with the expression of spontaneous diabetes mellitus, uniformly fail to develop diabetes. Breeding studies in which these animals were crossed with BB and hBB rats suggest that other genes are necessary for development of overt diabetes.


Reevaluation of autoantibodies to islet cell membrane in IDDM. Failure to detect islet cell surface antibodies using human islet cells as substrate. Since their demonstration in 1975, ICSAs have been proposed as serological markers and pathogenic elements in IDDM. ICSAs are detected in the sera of most newly diagnosed IDDM patients by indirect IFL that uses viable preparations of rat islet or insulinoma cells as substrate, but they also can be detected by using human insulinoma or fetal islet cells. We have tried to demonstrate ICSAs in the sera of 31 newly diagnosed diabetic patients, including 6 positive samples on human fetal islet cells, which used their natural target for the first time: normal human islet cells. In spite of using different types of preparations of these cells (i.e., freshly dispersed cell suspensions, monolayer cultures, or dispersed islets after culture), ICSAs could not be detected by IFL under the UV microscope, nor by flow cytometry. In contrast, 9 of 29 of the sera gave a positive staining on the RIN rat insulinoma cells. In an attempt to establish whether the putative ICSA autoantigen is present in the surface of human islet cells in the diabetic pancreas, the insulitis microenvironment was emulated by exposing the islets to three types of stress: 1) cytokines (IFN-gamma and TNF-alpha); 2) heat shock; and 3) hyperglycemia. However, diabetic sera failed again to recognize membrane antigens on the islet cells after either of these treatments. Neither were islet cells from a newly diagnosed diabetic patient stained by its autologous serum (ICA titer > 80 JDF U). These results suggest that ICSA autoantigen is not expressed in the membrane of human islet cells and therefore raises doubts about their proposed pathogenic role.


Sib-pair analysis of adenosine deaminase locus in NIDDM. Recently, linkage between the ADA gene locus and MODY, a subtype of NIDDM, has been reported. The possibility that the region of chromosome 20q containing the ADA locus also may play a role in susceptibility to NIDDM needs to be investigated. Therefore, we examined the linkage between the ADA locus and NIDDM in affected siblings of 50 European white diabetic pedigrees--21 Italian and 29 British. Departure from independent segregation of the disease and an Alu VpA polymorphism within the 5' flanking region of the ADA locus was tested in the affected sib-pairs with the APM statistical method. After DNA amplification by the PCR and PAGE, five alleles were identified in the ALU VpA tract at the ADA locus in the two populations. Allele frequencies did not differ significantly between the two populations (chi 2 = 2.426, P > 0.05 [NS]). Analysis of the 50 diabetic sib sets, and independently of the Italian and British groups of affected sib pairs, revealed no segregation distortion between the marker locus and NIDDM. We conclude that mutations within or around the ADA locus are unlikely to play a major role in the etiology of NIDDM.


PGE2 prevents anomalies induced by hyperglycemia or diabetic serum in mouse embryos. Both a high level of D-glucose in the medium and serum from a diabetic rat can induce neural-tube fusion defects and growth retardation in cultured mouse and rat embryos. To test our hypothesis that a deficiency of PGs may be involved in the mechanism of hyperglycemia- and diabetic serum-induced teratogenesis and growth retardation, we added PGE2 to the medium of a whole mouse embryo culture containing either normal rat serum and 52.7 mM D-glucose (hyperglycemic) or diabetic rat serum and 22.2 mM D-glucose (diabetic). After a 24-h culture, 94% of hyperglycemic embryos and 81% of diabetic embryos had neural-tube fusion defects; in addition, the number of somites, the morphological score, and the protein content of the embryos were significantly lower than those of controls. Supplementing the medium with PGE2 at concentrations of 0.028-28.4 nM (hyperglycemic) or 28.4 nM (diabetic) significantly reduced the incidence of neural-tube defects and increased the number of somites, the morphological score, and the protein content. These results strongly support the hypothesis that the teratogenicity of diabetic serum, as well as the teratogenic action of hyperglycemic culture, are mediated through a deficiency of PGs.


Longitudinal study of plasma lipoproteins and hormones during pregnancy in normal and diabetic women. Plasma lipoproteins were studied longitudinally at the 1st, 2nd, and 3rd trimester of gestation and at postpartum and postlactation in 12 age-matched PGDM women, 9 GDM women, and 12 healthy control subjects. FPG and HbA1c were higher in every case in PGDM women than in control subjects, whereas in GDM patients, glucose was augmented only after parturition. FFA and beta-hydroxybutyrate levels were higher in both PGDM and GDM patients than in control subjects during gestation but not after parturition. Total TGs and VLDL, LDL, and HDL TGs increased with gestational time in the three groups and declined at postpartum, and although total cholesterol and VLDL, LDL, and HDL cholesterol followed a similar trend, their rise was less pronounced, and the decline after parturition was slower than that of the TGs in the three groups, with no difference among them. The VLDL TG/cholesterol ratio declined in the three groups at the 3rd gestational trimester, whereas in both LDL and HDL, the TG/cholesterol ratio, but not the cholesterol/phospholipid ratio, increased during gestation in the three groups, indicating a specific enrichment of TGs in these particles. The increase in apoA-I and apoB with gestation was parallel to the respective changes in HDL and LDL cholesterol and, again, no difference was observed between the three groups. Plasma levels of beta-estradiol, progesterone, and prolactin increased sharply with gestation and declined at postpartum in the three groups, but absolute values of beta-estradiol and prolactin, at the three trimesters of gestation, were lower in PGDM patients, but progesterone levels were lower than controls in GDM women only at the 3rd trimester. (ABSTRACT TRUNCATED AT 250 WORDS)


Intrathymic islet cell transplantation reduces beta-cell autoimmunity and prevents diabetes in NOD/Lt mice. Intrathymic transplantation of syngeneic islets into adolescent NOD/Lt mice was performed to establish whether the thymus would serve as an immunoprivileged site for beta-cell engraftment, and whether this treatment would prevent the development of diabetes by eliciting tolerance to islet antigens. Intrathymic injection of cells from 200 NOD islets into 4-wk-old female NOD/Lt mice produced a significant reduction in the severity of insulitis at 24 wk of age. Furthermore, diabetes development was strongly suppressed (11% incidence) compared with controls (100% incidence). Both thymus histology and thymic insulin content revealed a rapid loss of the implanted beta-cells with < 1% remaining 1 wk posttransplantation. Despite the rapid loss of thymus-implanted islet cells, evidence for tolerance induction to islet cell antigens was obtained by adoptive transfer of splenic leukocytes from these mice into NOD-scid/scid recipients. After adoptive transfer of splenic leukocytes from 24-wk-old untreated prediabetic donors, 4 of 5 NOD-scid/scid recipients developed diabetes within 4 wk, and none of the recipients became diabetic after transfer of splenocytes from intrathymic islet-implanted donors. Intrathymic islet transplantation did not lead to reduction of sialitis in females with reduced severity of insulitis, indicating that the protective effect was tissue specific. This also was reflected in adoptive transfer experiments, because equal severity of sialitis was observed in NOD-scid/scid recipients of spleen cells from either islet transplanted or control NOD/Lt mice. In conclusion, the data suggest that intrathymic injection of islet cells prevents diabetes by stimulating immunological tolerance to beta-cells.


[Immunogenetics of immunoglobulins in domestic mink. VII. Features of phenotypic expression of gamma-heavy chain constant region allotypes mask linkage of IgCH genes]. Reduced expressivity and penetrance of allotypes H2-H4 and the regulation of allotype H6 expression by regulatory gene concealed the linkage of mink C genes in statistical analyses. Their linkage was demonstrated by di- and polyhybrid test crosses with 3, 4 and more generations.


[Variability of polymorphic gene markers in Buryat and Russian newborns from Ulan-Ude]. Variability of ten polymorphic loci (ABO, RhD, PGD, ACP, PGM1, GLO, ESD, ADA, GC, TF) was studied in 326 Buryat and 310 Russian newborns from Ulan-Ude city. Marked differences between two groups were observed in the distributions of allelic frequencies of ABO, RhD, PGD, ACP, PGM1, GLO, ESD, GC loci. Genetic similarities between Buryat and other mongoloids were estimated. Close similarity was observed between Buryat, Mongols, Yakut and Kyzyl.


[Distribution of abnormal hemoglobins S and C in the Republic of Guinea]. Distribution of the HbS and HbC in the Guinean Republic was determined by the analysis of the excerption of 2213 inhabitants representing different ethnic groups of the country. It was found that the mean frequency of the HbS heterozygotes is 21.2 +/- 0.9% and of the HbC heterozygotes is 2.6 +/- 0.3%. Among major nationalities of the country the frequencies of the HbS heterozygotes make up 21.0 +/- 1.9% for the Fulbe, 22.2 +/- 1.6% for the Malinke and 26.5 +/- 1.6% for the Susu and frequencies of the HbC heterozygotes make up 3.0 +/- 0.8% for the Fulbe, 2.5 +/- 0.6% for the Malinke and 1.6 +/- 0.4% for the Susu. Relative viability of the HbS and HbC carriers for major Guinean nationalities is estimated. The relative fitnesses account is 1.05-1.13 for HbC heterozygotes and 1.07-1.16 for HbC heterozygotes.


[Role of activated cellular c-Ha-ras-1 oncogene in the mutagenic effect of the plasmid pEJ6.6]. The role of the activated oncogene c-Ha-ras-1 from human bladder carcinoma integrated into the pEJ6.6 plasmid in the mutagenic effect of the plasmid was studied in Chinese hamster cells. The frequency of hypoxanthine-phosphoribosyltransferase defective (HPRT-) mutants after treatment with pEJ6.6 containing an active c-Ha-ras-1 exceeded that in control dishes treated with a derivative of pEJ6.6 plasmid with an inactivated oncogene. The inactivation was achieved by introducing a deletion into the coding region of the oncogene. The mutagenic effect was rather weak but statistically significant. Thus, the data obtained show that the mutagenic activity of pEJ6.6 plasmid is determined by its oncogene. The role of mutagenic effects of activated cellular oncogenes in malignant transformation is discussed.


[Dependence of mutagenic activity of heterocyclic analogs of pyrene on their chemical structure]. Comparative mutagenic activity of 6 heterocyclic analogs of pyrene was studied. The highest activity was revealed for 2,7-dinitro-4,9-dioxy-5,10-dioxo-4,5,9,10-tetrahydro-4,9- diazapyrene (2,7-DN-DDTDP) and 2,7-dinitro-5,10-dioxo-4,9-dioxapyrene (2,7-DN-DDP) which induced mutations in the tester strain Salmonella typhimurium TA98 and TA100. High mutagenicity of 2,7-DN-DDTDP and 2,7-DN-DDP is conditioned by reduction of nitro groups in the 2,7 position. The carbonyl groups in the 5,10 position were believed to cause bifunctional activity of 2,7-DN-DDTDP and 2,7-DN-DDP and to promote deletion of two G-C in the D3052 site.


[Activity of nucleolus organizer regions (NORs) in embryonic livers from twin minks]. Ag-NOR patterns were studied in hepatocytes from nine mink embryo siblings, including a pair of monochorionic (presumably monozygotic, MZ) twins. Both the number and the size of Ag-NORs per cell were found to be identical in MZ twins. All the other sibs had the patterns different from each other and from the MZ ones. The conclusion is that the NORs activity is a strongly inherited character and the Ag-NOR pattern can be used as a reliable genetic marker to distinguish the twin zygosity.


[Influence of mutations in the suppressor of hairy wing and modifier of MDG4 loci on phenotype expression of super-unstable alleles at the yellow locus of Drosophila melanogaster]. A number of super-unstable systems were obtained earlier by the induction of P-M hybrid dysgenesis in the strains with a mobilized Stalker. One of the super-unstable mutations appeared at the yellow locus of Drosophila melanogaster on the background of preexisting y mutation. The latter had been induced by mdg4 insertion into the regulatory region of the yellow locus. The suppressor genes of Hairy wing and modifier of mdg4 are known to encode proteins which bind to the mdg4 enhancer and are involved in the control of its transcription. A large spectrum of yellow alleles was obtained in super-unstable systems which could be recognized by the intensity of pigmentation in twelve different areas of the cuticle. Combining of these alleles with su(Hw) and mod(mdg4) mutations followed by the study of phenotype changes demonstrated that the same regulatory protein may influence expression of the gene in opposite directions.


[Modification of drug mutagenicity by their immobilization. Effect of prostatilen immobilized in polyvinyl alcohol in mice]. Mutagenic drug effect of prostatilen and the possibility of modification were analysed in the sperm head anomalies (SHA) and the bone marrow cell aberrations (CA) tests in Mus musculus. It was found that intraperitoneal injection of 2.5 micrograms of prostatilen induced no significant increase in SHA and CA frequencies, the dose of 5 micrograms inducing both SHA and CA. Ultrafiltration of prostatilen led to decrease in its mutagenicity in the SHA test. Immobilization of the drug (5 and 10 micrograms) in polyvinyl alcohol reduced SHA and CA frequencies, the former decreasing to the control level.


Construction and characterisation of a yeast artificial chromosome library containing two haploid Beta vulgaris L. genome equivalents. We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded protoplasts of a sugar beet (Beta vulgaris L.) cell suspension line, Ar+. This library contains 15,000 clones with an average insert size of 140 kb. Based on a sugar beet haploid genome size of 1.1 x 10(3) Mb it should represent approximately two haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. High-density filters, each containing 864 YAC clones, have been screened in colony hybridisation experiments. In this way, sugar beet YAC clones have been identified containing chloroplast DNA, mitochondrial DNA and two satellite DNAs. Ten pools of DNA from 1500 individual YAC clones have also been prepared for rapid PCR screening of the library. Using this approach, in combination with colony hybridisation, we have been able to isolate sugar beet YAC clones containing a transcribed low-copy-number gene.


Trans-splicing of an early embryo mRNA in Litomosoides carinii, coding for the major microfilarial sheath protein gp22. Both genomic and cDNA clones have been isolated encoding the major sheath glycoprotein, gp22, of Litomosoides carinii microfilariae. The mature gp22 mRNA is shown to result from both trans-splicing of a 22-nucleotide 5'-leader sequence to an acceptor site at position 313 of the pre-mRNA, immediately upstream from the start codon, and from cis-splicing of a 117-nt intron located within the coding sequence. Cis-splicing precedes the trans-splicing reaction. The gp22 reading frame of 148 codons has the inferred structure of a prepro-protein and includes a leader peptide and a pro-segment ahead of the known N terminus of the mature, extracellular protein of 105 amino acids. The N-terminal part of that protein contains five repeats of an elastin-related pentapeptide sequence, which, together with a proline-threonine segment between two Cys clusters in the center and at its C terminus, may cause an elongated conformation with an apparent molecular size of 22 kDa in contrast to the calculated M(r) of 11,200.


Conservation of gene organization and trans-splicing in the glyceraldehyde-3-phosphate dehydrogenase-encoding genes of Caenorhabditis briggsae. The genes encoding body-wall-specific glyceraldehyde-3-phosphate dehydrogenase from Caenorhabditis briggsae were sequenced and compared to the homologous genes from Caenorhabditis elegans. The direct tandem organization of these genes, gpd-2 and gpd-3, and the size and location of the two introns in each gene are the same in C. elegans and C. briggsae. Primer-extension studies demonstrated that the two genes in C. briggsae are trans-splice differentially with the same splice leader (SL) RNAs as are observed in C. elegans. The gdp-2 gene is trans-spliced with SL1 while gdp-3 is trans-spliced with SL2. Significant sequence conservation was observed within the promoter regions of each species and may indicate those regions responsible for body-wall-muscle-specific gene expression and/or differential trans-splicing. Comparisons of the sequences suggest that the tandem repeat of the genes has been subjected to concerted evolution and that C. briggsae and C. elegans diverged much earlier than would be anticipated based on morphological similarities alone. Finally, an open reading frame found several hundred nucleotides upstream from gpd-2, in both species, appears to be homologous to the ATP synthase subunit, ATPase inhibitor protein, from bovine mitochondria.


Regulatory elements and transcriptional regulation by testosterone and retinoic acid of the rat nerve growth factor receptor promoter. The low-affinity nerve growth factor receptor (LNGFR) is a membrane-associated glycoprotein which is thought to participate in some of the biological activities of nerve growth factor (NGF). Expression of the LNGFR gene is known to be regulated both during development and in response to various agents in cell culture. However, molecular mechanisms responsible for the regulation have not been described. We report here an analysis of a 4.8-kb sequence from the 5'-flanking region of the rat LNGFR gene. Several regulatory elements were identified in this region by transfection of plasmid constructs containing sequences from LNGFR fused to a bacterial cat reporter gene. The proximal part of the promoter region (0.4-kb) was shown to be sufficient to support cat expression in all cell types used. A silencer element located between -1.5 kb and -1.8 kb from the start of translation, as well as an enhancer element in more upstream regions of the promoter, were identified in the phaeochromocytoma cell line, PC12, and in the Sertoli cell line, TM4, that express the LNGFR gene. Treatment of TM4 cells with retinoic acid (RA) increases the level of LNGFR mRNA twofold, while testosterone treatment results in a tenfold decrease. Regions of the promoter responsive to testosterone and RA in TM4 cells were found at -610 to -860 bp and -1840 to -4800 bp upstream from the translation start codon, respectively. A RA-responsive element active in PC12 cells is located between bp -610 to -860 from the start codon.


Cloning of the goat beta-casein-encoding gene and expression in transgenic mice. The goat beta-casein-encoding gene (CSN2), which encodes the most abundant protein of goat milk, has been cloned and sequenced. The intron/exon organization of the 9.0-kb goat CSN2 gene is similar to that of other CSN2 genes. Expression of the goat gene was principally restricted to the mammary gland of lactating transgenic animals. A low level of expression was also observed in skeletal muscle and skin. In contrast to a rat CSN2 transgene [Lee et al., Nucleic Acids Res. 16 (1988) 1027-1041], the goat gene was expressed to a high degree in the lactating mammary gland. Differences in the content or context of regulatory elements may account for the enhanced performance of the goat relative to the rat CSN2 gene in transgenic mice.


Transgenic pigs carrying cDNA copies encoding the murine Mx1 protein which confers resistance to influenza virus infection. An important aspect of gene transfer into farm animals is the improvement of disease resistance. The mouse Mx1 protein is known to be sufficient to confer resistance to influenza viruses. Gene constructs containing the mouse Mx1 cDNA controlled by the human metallothionein IIA promoter (hMTIIA::Mx), the SV40 early enhancer/promoter region (SV40::Mx) and the mouse Mx1 promoter (mMx::Mx) were transferred into pigs. The results of the gene transfer experiments with the hMTIIA::Mx and the SV40::Mx constructs indicate that the permanent high-level synthesis of Mx1 might be deleterious to the organism: the gene transfer efficiency was surprisingly low, and all transgenic piglets born had rearrangements in their transgene copies that abolished protein synthesis. The use of the interferon (IFN)- and virus-inducible mMx::Mx construct resulted in normal gene transfer efficiency. Two transgenic pig lines could be established which expressed IFN-inducible mouse Mx1 mRNA. Extensive protein analysis did not detect mouse Mx1 in IFN-treated transgenic animals.


Combinatorial functions of two chimeric antibodies directed to human CD4 and one directed to the alpha-chain of the human interleukin-2 receptor. The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human complement, the individual Abs are negative; however, when used in combination, complement activation was performed. When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by combination of an anti-CD4 mAb with one directed against the alpha-chain of the human IL2 receptor, nearly 100% inhibition of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4 mAb and an anti-IL2R alpha chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating that this mAb cocktail could be a new strategy for immunosuppressive therapy.


Intrahelical pseudoknots and interhelical associations mediated by mispaired human minisatellite DNA sequences in vitro. The human minisatellite arrays, 33.6 and 33.15, consist of tandem reiterations of a 37-nucleotide (nt) and a 16-nt repeat unit sequence, respectively, both of which contain a majority of purine bases on one strand. Knot-like tertiary structures, which mapped to the cloned arrays, were observed by electron microscopy (EM) in homoduplex molecules produced by denaturation and reannealing in vitro. They result from a primary hybridization between misaligned repeat units of the array, forming a slipped-strand structure with staggered single-stranded DNA loops, followed by a secondary hybridization between repeat units in the two loops. Depending on the relative alignment of the loops when they hybridize, a particular form of intrahelical pseudoknot is produced. Theta-shaped, figure-of-eight, and bow-shaped structures were the most common conformational isomers observed in homoduplexes flattened into two dimensions during EM preparation. At the site of a bow-shaped structure, a conformation-dependent bend of approximately 60 degrees between the flanking DNA segments is induced; the other conformations generally do not deflect the line of the main DNA axis. Paired loops, similar to the bow-shaped structure, were apically situated in some supercoiled plasmids containing the 33.6 array. Both plasmids formed intermolecular associations, consisting of two (or more) homoduplex molecules held together at or immediately adjacent to a nexus which mapped to the minisatellite sequences. These associations might arise either by interhelical hybridization between arrays or by knot-like structures interfering with branch migration of chi-form Holliday junctions.


A human endogenous long terminal repeat provides a polyadenylation signal to a novel, alternatively spliced transcript in normal placenta. We have been investigating the impact that the long terminal repeats (LTRs) of the RTVL-H family of human endogenous retroviral-like elements may have on the expression of adjacent cellular genes. Using a differential hybridization strategy, we have screened a cDNA library from a normal full-term human placenta and have identified two clones containing non-RTVL-H-related cellular sequences that have been polyadenylated within an RTVL-H LTR. One of these clones, cPj-LTR, contains an open reading frame (ORF) of 223 amino acids. Southern analysis indicated that the corresponding gene, termed PLT, is most probably a single multi-exon locus and that related sequences are present in the mouse genome, suggesting that this gene has been evolutionarily conserved. Database searches detected no significant homology to previously published sequences, indicating that PLT is a novel gene. Northern analysis identified several PLT-related transcripts in placental RNA samples, one of which is associated with the LTR. The presence of this PLT-LTR fusion transcript in normal placenta was also confirmed by PCR. Additional hybridization studies with RNAs from various cell lines suggested that the PLT locus is differentially expressed in different cell types. To investigate the structure of the non-LTR-associated PLT-related transcripts, additional clones were isolated from the placental cDNA library. Analysis of these clones suggests that the PLT mRNA undergoes alternative splicing at its 3' end, with polyadenylation within an RTVL-H LTR occurring in one of the resulting transcripts.


Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit. To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.


Cloning and characterization of the ADH5 gene encoding human alcohol dehydrogenase 5, formaldehyde dehydrogenase. Human chi-alcohol dehydrogenase (chi-ADH) is a zinc-containing dimeric enzyme responsible for the oxidation of long-chain alcohols and omega-hydroxyfatty acids. Class-III ADHs, of which chi-ADH is the prototype, are widely produced and well conserved during evolution. This suggests that they fulfill important housekeeping roles in cellular metabolism. Recent evidence suggests that class-III ADH and formaldehyde dehydrogenase (FDH) are the same enzyme. We have isolated and characterized two overlapping genomic clones that cover the entire ADH5 (FDH) gene. ADH5 is composed of nine exons and eight introns. Two major transcription start points were identified by primer extension. The 5' nontranslated region is unusual in that it contains two additional upstream ATG codons, which would encode peptides of 20 and 10 amino acids. Neither of the upstream ATGs is in a good context for translation initiation, whereas the ATG initiating &khgr;-ADH is in a favorable context. The 5' region of ADH5 is a CpG island; it is extremely G+C rich and has many CpG doublets. It does not contain either a TATA box or a CAAT box. This is consistent with ubiquitous expression, and contrasts with the promoters of all previously cloned ADH genes, which are expressed in a tissue-specific manner. The 5' region of ADH5 contains consensus binding sites for the transcriptional regulatory proteins, Sp1, AP2, LF-A1, NF-1, NF-A2, and NF-E1. A 1.5-kb upstream fragment from ADH5 was able to drive the transcription of a cat reporter gene at high levels in monkey kidney cells (CV-1). Several processed pseudogenes were also isolated.


Affinity purification of histidine-tagged proteins transiently produced in HeLa cells. In order to produce eukaryotic proteins in a functional state, it is often necessary to use eukaryotic instead of prokaryotic expression systems. We have designed vectors which can be employed to express either N- or C-terminally histidine-tagged proteins in transiently transfected eukaryotic cells. The histidine tag allows the rapid enrichment of these proteins by metal chelate affinity chromatography in a native and functional state. Yields of up to 5 micrograms protein/5 x 10(7) cells were achieved.