Active and passive case detection strategies for the control of leishmaniasis in Bangladesh.

SETTING: Two subdistricts in Bangladesh, Fulbaria and Trishal, which are hyperendemic for leishmaniasis. OBJECTIVE: To determine 1) the numbers of patients diagnosed with visceral leishmaniasis (VL) and post-kala azar dermal leishmaniasis (PKDL) using an active case detection (ACD) strategy in Fulbaria and a passive case detection (PCD) strategy in Trishal, and 2) the time taken from symptoms to diagnosis in the ACD subdistrict. DESIGN: A cross-sectional descriptive study of patients diagnosed from May 2010 to December 2011. The ACD strategy involved community education and outreach workers targeting households of index patients using symptom-based screening and rK-39 tests for suspected cases. RESULTS: In the ACD subdistrict (Fulbaria) and PCD sub-district (Trishal), respectively 1088 and 756 residents were diagnosed with VL and 1145 and 37 with PKDL. In the ACD subdistrict, the median time to diagnosis for patients directly referred by outreach workers or self-referred was similar, at 60 days for VL and respectively 345 and 360 days for PKDL. CONCLUSION: An ACD strategy at the subdistrict level resulted in an increased yield of VL and a much higher yield of PKDL. As PKDL acts as a reservoir for infection, a strategy of ACD and treatment can contribute to the regional elimination of leishmaniasis in the Indian sub-continent.

Contexte : Deux sous-districts du Bangladesh, Fulbaria et Trishai, où la leishmaniose est hyper-endémique.Objectif : Déterminer 1) le nombre de patients ayant eu un diagnostic de leishmaniose viscérale (VL) et de leishmaniose dermique post-kala azar (PKDL) grâce à une stratégie de détection active des cas (ACD) à Fulbaria et à une stratégie de détection passive (PCD) à Trishai, et 2) le temps écoulé entre les symptômes et le diagnostic dans le sous-district à ACD.Schéma : Etude descriptive transversale des patients diagnostiqués entre mai 2010 et décembre 2011. La stratégie ACD comportait une éducation des communautés et des stratégies avancées ciblant les foyers des patients index grâce à un dépistage basé sur les symptômes et au test rK-39 pour les patients suspects.Résultats : Dans les districts de stratégie ACD (Fulbaria) et le sous-district de stratégie PCD (Trishai), 1088 et 756 patients respectivement ont eu un diagnostic de VL et 1145 et 37 respectivement ont eu un diagnostic de PKDL. Dans ce sous-district, le délai médian de diagnostic était de 60 jours pour tous les patients atteints de VL, qu'ils soient référés par du personnel des stratégies avancées ou viennent d'eux-mêmes. Il était respectivement de 345 et 360 jours pour la PKDL.Conclusion : Une stratégie ACD au niveau d'un sous-district permet de dépister un nombre accru de VL et encore plus de PKDL. Comme la PKDL constitue un réservoir d'infection, la stratégie d'ACD et le traitement des cas dépistés peuvent contribuer à l'élimination régionale de la leishmaniose du sous-continent indien.

Marco de referencia: Los subdistritos de Fulbaria y Trishal en Bangladesh, donde es hiper-endémica la leishmaniasis.Objetivo: Determinar: 1) el número de pacientes con diagnóstico de leishmaniasis visceral (VL) y de leishmaniasis cutánea pos kala azar (PKDL) mediante una estrategia de detección activa de casos (ACD) en Fulbaria y una detección pasiva (PCD) en Trishal; y 2) y el lapso entre la aparición de los síntomas y el diagnóstico en el subdistrito que aplicó la estrategia ACD.Método: Se llevó a cabo un estudio transversal descriptivo de los pacientes en quienes se estableció el diagnóstico de leishmaniasis entre mayo del 2010 y diciembre del 2011. Como parte de la estrategia ACD se impartió educación a la comunidad y participaron trabajadores extrainstitucionales que contactaban a los hogares de los casos iniciales y practicaban una detección basada en los síntomas y la prueba rK-39 de diagnóstico rápido en los casos de presunción diagnóstica.Resultados: En el subdistrito de Fulbaria la estrategia ACD permitió el reconocimiento de 1088 residentes con VL y 1145 con PKDL; en el subdistrito de Trishal se detectaron mediante la estrategia PCD 756 y 37 casos, respectivamente. La mediana del lapso necesario hasta establecer el diagnóstico de VL en los pacientes remitidos directamente por los trabajadores periféricos fue análoga a la mediana del lapso en los que se presentaron por su propia iniciativa y correspondió a 60 días en casos de VL; en los pacientes con PKDL la mediana del lapso fue 360 días en los pacientes remitidos por los trabajadores periféricos y 360 en los pacientes que acudieron por su propia cuenta.Conclusión: Una estrategia ACD a escala del subdistrito ofrece un mayor rendimiento diagnóstico de la VL y un incremento aun mayor del diagnóstico de PKDL. Dado que la PKDL representa un reservorio de la infección, una estrategia ACD y tratamiento puede contribuir a la eliminación regional de la leishmaniasis en el subcontinente indio.

Cardiorenal metabolic syndrome and diabetic cognopathy.

The prevalence of the cardiorenal metabolic syndrome (CRS) is increasing in parallel with obesity, type 2 diabetes mellitus, Alzheimer's disease, and other forms of dementia. Along with metabolic, inflammatory, and immunological abnormalities, there is maladaptive structural remodeling of the heart, kidney, and brain. The term 'diabetic cognopathy' (DC) may be used when discussing functional and structural changes in the brain of the diabetic patient. DC likely represents an advanced form of these changes in the brain that evolve with increasing duration of the CRS and subsequent clinical diabetes. We posit that DC develops due to a convergence of aging, genetic and lifestyle abnormalities (overnutrition and lack of exercise), which result in multiple injurious metabolic and immunologic toxicities such as dysfunctional immune responses, oxidative stress, inflammation, insulin resistance, and dysglycemia (systemically and in the brain). These converging abnormalities may lead to endothelial blood-brain barrier tight junction/adherens junction (TJ/AJ) complex remodeling and microglia activation, which may result in neurodegeneration, impaired cognition, and dementia. Herein, we describe the brain ultrastructural changes evolving from a normal state to maladaptive remodeling in rodent models of CRS including microglia activation/polarization and attenuation and/or loss of the TJ/AJ complexes, pericytes and astrocytes of the neurovascular unit. Further, we discuss the potential relationship between these structural changes and the development of DC, potential therapeutic strategies, and future directions.

A phenocopy of CAII deficiency: a novel genetic explanation for inherited infantile osteopetrosis with distal renal tubular acidosis.

The rare bone thickening disease osteopetrosis occurs in various forms, one of which is accompanied by renal tubular acidosis (RTA), and is known as Guibaud-Vainsel syndrome or marble brain disease. Clinical manifestations of this autosomal recessive syndrome comprise increased bone density, growth failure, intracerebral calcification, facial dysmorphism, mental retardation, and conductive hearing impairment. The most common cause is carbonic anhydrase II (CAII) deficiency. Several different loss of function mutations in CA2, the gene encoding CAII, have been described. To date, there have been no exceptions to the finding of CAII deficiency in patients with coexistent osteopetrosis and RTA. Most often, the RTA is of mixed proximal and distal type, but kindreds are reported in which either distal or proximal RTA predominates. We report the molecular genetic investigation of two consanguineous kindreds where osteopetrosis and distal RTA (dRTA) were both manifest. One kindred harbours a novel homozygous frameshift alteration in CA2. In the other, CAII levels were normal despite a similar clinical picture, and we excluded defects in CA2. In this kindred, two separate recessive disorders are penetrant, each affecting a different, tissue specific subunit of the vacuolar proton pump (H(+)-ATPase), providing a highly unusual, novel genetic explanation for the coexistence of osteopetrosis and dRTA. The osteopetrosis is the result of a homozygous deletion in TCIRG1, which encodes an osteoclast specific isoform of subunit a of the H(+)-ATPase, while the dRTA is associated with a homozygous mutation in ATP6V1B1, encoding the kidney specific B1 subunit of H(+)-ATPase. This kindred is exceptional firstly because the coinheritance of two rare recessive disorders has created a phenocopy of CAII deficiency, and secondly because these disorders affect two different subunits of the H(+)-ATPase that have opposite effects on bone density, but which have only recently been determined to possess tissue specific isoforms.

Crystal structure of the dimeric extracellular domain of human carbonic anhydrase XII, a bitopic membrane protein overexpressed in certain cancer tumor cells.

Overexpression of the zinc enzyme carbonic anhydrase (CA; EC ) XII is observed in certain human cancers. This bitopic membrane protein contains an N-terminal extracellular catalytic domain, a membrane-spanning alpha-helix, and a small intracellular C-terminal domain. We have determined the three-dimensional structure of the extracellular catalytic domain of human CA XII by x-ray crystallographic methods at 1.55-A resolution. The structure reveals a prototypical CA fold; however, two CA XII domains associate to form an isologous dimer, an observation that is confirmed by studies of the enzyme in solution. The identification of signature GXXXG and GXXXS motifs in the transmembrane sequence that facilitate helix-helix association is additionally consistent with dimeric architecture. The dimer interface is situated so that the active site clefts of each monomer are clearly exposed on one face of the dimer, and the C termini are located together on the opposite face of the dimer to facilitate membrane interaction. The amino acid composition of the active-site cleft closely resembles that of the other CA isozymes in the immediate vicinity of the catalytic zinc ion, but differs in the region of the nearby alpha-helical "130's segment." The structure of the CA XII-acetazolamide complex is also reported at 1.50-A resolution, and prospects for the design of CA XII-specific inhibitors of possible chemotherapeutic value are discussed.

Bone marrow transplantation corrects osteopetrosis in the carbonic anhydrase II deficiency syndrome.

Carbonic anhydrase II (CAII), found in renal tubules, brain, and osteoclasts, is critical in acid-base homeostasis and bone remodeling. Deficiency of CAII gives rise to a syndrome of osteopetrosis, renal tubular acidosis (RTA), and cerebral calcification with associated developmental delay. It is inherited in an autosomal recessive fashion and found most frequently in the Mediterranean region and the Middle East. We report 2 related Irish families with clinically severe CAII deficiency in whom the gene mutation has been fully elucidated. Two children, one from each family, have undergone allogeneic bone marrow transplantation because of severe progressive visual and hearing loss. The older 2 children had already developed cerebral calcification and marked visual loss at the time of diagnosis and were treated symptomatically. Post-transplantation evaluation at 2 and 3 years demonstrates histologic and radiologic resolution of their osteopetrosis with stabilization of hearing and vision. Both children remain developmentally delayed and continue to have RTA, and the older child has now developed cerebral calcification. Allogeneic bone marrow stem cell replacement cures the osteoclast component of CAII deficiency and retards the development of cerebral calcification, but it appears to have little or no effect on the renal lesions. (Blood. 2001;97:1947-1950)

Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain.

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Purification and kinetic analysis of recombinant CA XII, a membrane carbonic anhydrase overexpressed in certain cancers.

Carbonic anhydrase XII (CA XII) is a transmembrane glycoprotein with an active extracellular CA domain that is overexpressed on cell surfaces of certain cancers. Its expression has been linked to tumor invasiveness. To characterize its catalytic properties, we purified recombinant secretory forms of wild-type and mutant CA XIIs. The catalytic properties of these enzymes in the hydration of CO(2) were measured at steady state by stopped-flow spectrophotometry and at chemical equilibrium by the exchange of (18)O between CO(2) and water determined by mass spectrometry. The catalysis of CO(2) hydration by soluble CA XII has a maximal value of k(cat)/K(m) at 34 microM(-1) small middle dots(-1), which is similar to those of the membrane-associated CA IV and to soluble CA I. The pH profiles of this catalysis and the catalyzed hydrolysis of 4-nitrophenylacetate indicate that the pK(a) of the zinc-bound water in CA XII is 7.1. His64 in CA XII acts as a proton shuttle residue, as evidenced by the reduced rate constant for proton transfer in the mutants containing the replacements His64 --> Ala and His64 --> Arg, as well as by the selective inhibition of the proton transfer step by cupric ions in wild-type CA XII. The catalytic rate of CO(2) hydration by the soluble form of CA XII is identical with that of the membrane-bound enzyme. These observations suggest a role for CA XII in CO(2)/HCO(3)(-) homeostasis in cells in which it is normally expressed. They are also compatible with a role for CA XII in acidifying the microenvironment of cancer cells in which CA XII is overexpressed, providing a mechanism for CA XII to augment tumor invasiveness and suggesting CA XII as a potential target for chemotherapeutic agents.

Mitochondrial carbonic anhydrase CA VB: differences in tissue distribution and pattern of evolution from those of CA VA suggest distinct physiological roles.

A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitochondria isolated from transfected COS cells. Northern blot analyses of total RNAs from mouse tissues and Western blot analyses of mouse tissue homogenates showed differences in tissue-specific expression between CA VB and CA VA. CA VB was readily detected in most tissues, while CA VA expression was limited to liver, skeletal muscle, and kidney. The human orthologue of murine CA VB was recently reported also. Comparison of the CA domain sequence of human CA VB with that reported here shows that the CA domains of CA VB are much more highly conserved between mouse and human (95% identity) than the CA domains of mouse and human CA VAs (78% identity). Analysis of phylogenetic relationships between these and other available human and mouse CA isozyme sequences revealed that mammalian CA VB evolved much more slowly than CA VA, accepting amino acid substitutions at least 4.5 times more slowly since each evolved from its respective human-mouse ancestral gene around 90 million years ago. Both the differences in tissue distribution and the much greater evolutionary constraints on CA VB sequences suggest that CA VB and CA VA have evolved to assume different physiological roles.

Human egasyn binds beta-glucuronidase but neither the esterase active site of egasyn nor the C terminus of beta-glucuronidase is involved in their interaction.

Lysosomal beta-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine beta-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human beta-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg-GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that (i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg.

Active site residues of human beta-glucuronidase. Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue.

Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose that residues Glu(451), Glu(540), and Tyr(504) in hGUSB are involved in catalysis, Glu(451) being the acid-base residue and Glu(540) the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu(451), Glu(540), and Tyr(504) hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. The k(cat)/K(m) values (mM(-1).s(-1)) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu(451) as the acid-base catalyst. We conclude that, like their homologues in E. coli beta-galactosidase, Glu(540) is the nucleophilic residue, Glu(451) the acid-base catalyst, and Tyr(504) is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB.

Age-related changes in thyroid hormone responsive protein (THRP) expression in cerebral tissue of rats.

To determine if aging in rats is associated with insensitivity of cerebral tissue to thyroid hormones (TH), the expression of a TH responsive protein or (THRP) in cerebral tissue was studied in male Fischer rats at 4, 12 and 24 months of age during euthyroid, hypothyroid and hyperthyroid states. The basal levels of THRP mRNA was significantly increased in 24-month-old and in 12-month-old rats while THRP mass measured by Western blots was decreased compared to 4-month-old rats. Compared to euthyroid rats, hyperthyroidism in 4-month-old rats was associated with 5.1-fold increase in THRP mRNA and 3.7-fold increase in protein content while in hyperthyroid aged rats, the increase of THRP mRNA was only 1.6-fold and the increase in the protein was 2.4-fold. Hypothyroidism did not significantly alter THRP or its mRNA in either young or aged rats. It is concluded that aging in rats is associated with reduced cerebral tissue responsiveness to thyroid hormones.

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers.

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30-42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

Novel translational repressor (NAT-1) expression is modified by thyroid state and age in brain and liver.

The technique of reverse transcriptase-polymerase chain reaction differential display was used to identify thyroid hormone (TH) responsive mRNAs in the adult rat cerebral tissue. A partial cDNA (0.76 kb) was cloned and sequenced. Comparison of the sequence to the GenBank data base showed almost 100% homology to mouse translational repressor (NAT-1) mRNA 3'-end. In a northern blot analysis this cDNA hybridized with a mRNA whose expression in hyperthyroid rat cerebral tissue was approximately 6-fold higher than in euthyroid rats. The time course studies showed a rapid induction of this mRNA within 3 h following thyroxine administration. This mRNA is widely expressed in various tissues, and in hepatic tissue it is also TH responsive. To determine if TH responsiveness of this mRNA persists during aging, 25-month-old aged rats were studied and the results were compared with those of 4-month-old rats. Unlike young mature rats, the TH responsiveness of NAT-1 mRNA in both the cerebral and hepatic tissue of aged rats was blunted. It is concluded that cerebral tissue in aging rats beyond the developmental stages, like the hepatic tissue, is associated with altered TH responsiveness.

GLUT-1 expression in the cerebra of patients with Alzheimer's disease.

To investigate the molecular basis of reduced GLUT-1 concentration of the blood-brain barrier in patients with Alzheimer's disease (AD), the GLUT-1 mass, mRNA content, and structure were studied in eight patients with AD and seven age-matched controls. The results indicate that the 55-kDa GLUT-1 is significantly reduced in AD without a significant change in GLUT-1 mRNA concentrations. Because in some animal models changes in GLUT-1 expression is associated with changes in GLUT-1 mRNA structure, the length of the poly(A) tail of the GLUT-1 mRNA was estimated with a reverse transcription-polymerase chain reaction technique. The length of poly(A) tail of GLUT-1 mRNA in AD subjects was not significantly different from the controls. It is concluded that the AD-related change in GLUT-1 expression is not the result of altered poly(A) length of GLUT-1 mRNA.

Age-related changes in glucose transporter-one mRNA structure and function.

To determine the molecular mechanisms of age-related changes in the expression of glucose transporter 1 (GLUT-1) mRNA in cerebral tissue, male Fischer 344 rats at 4, 12, and 24 months of age were studied. The GLUT-1 mRNA in cerebral tissue was not significantly different among the various age groups. The in vitro translatability of GLUT-1 mRNA of 24-month-old rats (0.867 +/- 0.066 arbitrary units) was significantly lower than that in 4-month-old (1.403 +/- 0.153) P < 0.01 or 12-month-old rats (1.387 +/- 0.122) P < 0.01. The poly (A) tail length of GLUT-1 mRNA decreased from 200-350 nt in 4-month-old rats to only 50-100 nt in 24-month-old rats. Twelve-month-old rats also showed reduced poly (A) tail lengths. The poly (A) tail of G3PDH mRNA was not altered with age. The changes in GLUT-1 mRNA translatability did not correlate with GLUT-1 content in total cerebral tissue homogenate or in isolated cerebral microvessels, suggesting that GLUT-1 protein turnover is altered with age. It is concluded that aging is associated with significant changes in the structure and function of GLUT-1 mRNA.

Cloning and characterization of a complementary DNA for a thyroid hormone-responsive protein in mature rat cerebral tissue.

A gene responsive to thyroid hormone (TH) has been identified in the adult rat brain cerebral tissue. A cDNA probe differentially expressed in euthyroid, hypothyroid and hyperthyroid rat cerebral tissue, generated by reverse transcriptase-PCR differential display of mRNA, was used to screen the rat brain cDNA library. A 3.4 kb positive clone hybridized in Northern blots with a 3.8 kb mRNA that proved to be TH responsive (THR). The remaining coding sequence and a part of the 5' untranslated region of this cDNA were obtained by 5' rapid amplification of cDNA ends. The deduced amino acid sequence revealed that THR protein (THRP), a 68 kDa moiety, has 83% sequence similarity with c-Abl interactor protein (Abi-2), which is a substrate for tyrosine kinase activity of c-Abl. The extensive similarity between the two proteins suggests a potential role for THRP as a substrate for c-Abl. Northern analysis showed that the expression of THR mRNA in hyperthyroid rats is 6-fold that in euthyroid rats. There is also a 4-6-fold increase in the concentration of THRP, as analysed by Western analysis. Owing to the extensive similarity between rat THRP and human Abi-2, a polyclonal anti- (human Abi-2) antibody was successfully used for Western analysis of proteins from the rat tissues. The observed increase in both the mRNA and the protein did not decline after beta-adrenergic system blockade with propranolol, suggesting that the action of TH on the expression of this gene is not mediated through the beta-adrenergic system. Immunohistochemical studies revealed that neuronal cells were particularly rich in THRP. Both THR mRNA and THRP are rapidly induced in vivo after intravenous administration of thyroxine. Tissue distribution studies indicated that the cerebral tissue was particularly enriched with THR mRNA and 68 kDa THRP. A cDNA clone for a THR gene could provide a useful tool to study the molecular mechanisms of TH effects on cerebral tissue in adult animals.

Apolipoprotein A1 expression in young and aged rats is modulated by dietary carbohydrates.

To determine if dietary carbohydrates modulate apolipoprotein A1 (ApoA1) expression, plasma ApoA1 protein and hepatic ApoA1 mRNA levels were measured in young and aged rats maintained on a high-fructose (60% of diet weight consisting of fructose), or high-glucose (60% glucose) diet or fed regular rat chow for 10 days. Aged rats on regular chow had significantly higher plasma ApoA1 concentrations and hepatic ApoA1 mRNA than young rats maintained on this diet. Plasma ApoA1 and hepatic ApoA1 mRNA levels in young rats or aged rats maintained on the 60% fructose diet were significantly higher than in rats within the same age group maintained on regular rat chow (P < .01). Similar induction of ApoA1 protein and mRNA was found in rats maintained on the 60% glucose diet (P < .01). It is concluded that ApoA1 expression in rats is modulated by factors related to the nature of dietary carbohydrates rather than insulin resistance associated with high-fructose feeding.

Age-related changes in the blood-brain barrier.

Aging of the cerebral microcirculation results in significant alteration in the blood-brain barrier (BBB). The barrier function appears to remain intact in older animals, although it may be more susceptible to disruption by external factors (hypertension) and drugs (haloperidol). While overall transport processes do not change with age, aging animals and humans have altered BBB function of select carrier mediated transport systems including the transport of choline, glucose, butyrate and triiodothyronine. These age-related changes are the result of either alteration in the carrier molecules or the physiochemical properties of the cerebral microvessels. At the present time, it is not known whether changes in the BBB contribute to the age-related neurodegenerative diseases or are merely epiphenomena of aging.

Shortening of poly (A) tail of glucose transporter--one mRNA in experimental diabetes mellitus.

To determine the molecular mechanisms of diabetes-related changes in the expression of GLUT-1 in cerebral tissue, streptozotocin-induced diabetic rats and vehicle injected controls were studied after 4 weeks of diabetes. The GLUT-1 mass in cerebral microvessels was reduced in diabetic rats by approximately 38% (P < 0.01). The GLUT-1 concentration in insulin-treated diabetic group was not significantly different from controls. The GLUT-1 mRNA content of cerebral tissue in diabetic rats (0.064 +/- 0.007) was significantly reduced compared to control rats (0.122 +/- 0.011) or insulin-treated diabetic rats (0.122 +/- 0.015) P < 0.01. The in vitro translation of GLUT-1 mRNA of diabetic rats (0.793 +/- 0.047 arbitrary units) was also significantly lower than that in control rats (1.403 +/- 0.153) P < 0.01 or insulin-treated diabetic rats. (1.124 +/- 0.083) P < 0.01. These changes occurred in asssociation with a reduction in poly (A) tail length of GLUT-1 mRNA which decreased from a control value of 200-350 nt to only 50-100 nt in diabetic rats. Shortening of poly (A) tail of mRNAs is a novel mechanism of diabetes-related changes in the expression of specific genes which are regulated at a translational level.