ARECOLINE CANNOT ALTER PINEAL-TESTICULAR RESPONSES TO METABOLIC STRESS IN WISTAR RATS.

CONTEXT: Betel nut is consumed by millions of people for stress reduction and increased capacity to work. One of its components is arecoline which is useful for Alzheimer and schizophrenia; it also influences endocrine and gonadal functions. OBJECTIVE: Objective is to examine whether arecoline can influence pineal-testicular function in metabolic stress. DESIGN: Rats were deprived of food or water or treated them with arecoline, each separately for 5 days. SUBJECTS: Pineal and testis with sex accessories were studied. METHODS: Ultrastructural (pineal, testis, Leydig cells and prostate), hormonal (melatonin and testosterone) and other parameters (fructose and sialic acid) were examined. Pineal indoleamines were quantitated by fluorometric method; testosterone by ELISA, and carbohydrate fractions by spectrophotometric methods. RESULTS: Inanition/ water deprivation caused pineal stimulation ultrastructurally (with enlarged synaptic ribbons) and elevation of melatonin level, but reproductive dysfunction by ultrastructural degeneration of Leydig cells and prostate with fall of testosterone, fructose and sialic acid concentrations. Arecoline treatment showed reversed changes to those of metabolic stress, but arecoline treatment in metabolic stress showed same results as in metabolic stress. CONCLUSION: The findings suggest that arecoline cannot alter the action of metabolic stress on pineal-testicular activity in rats.

All-trans retinoic acid protects against arsenic-induced uterine toxicity in female Sprague-Dawley rats.

BACKGROUND AND PURPOSE: Arsenic exposure frequently leads to reproductive failures by disrupting the rat uterine histology, hormonal integrity and estrogen signaling components of the rat uterus, possibly by generating reactive oxygen species. All-trans retinoic acid (ATRA) was assessed as a prospective therapeutic agent for reversing reproductive disorders. EXPERIMENTAL APPROACH: Rats exposed to arsenic for 28 days were allowed to either recover naturally or were treated simultaneously with ATRA for 28 days or treatment continued up to 56 days. Hematoxylin-eosin double staining was used to evaluate changes in the uterine histology. Serum gonadotropins and estradiol were assayed by ELISA. Expression of the estrogen receptor (ERα), an estrogen responsive gene vascular endothelial growth factor (VEGF), and cell cycle regulatory proteins, cyclin D1 and CDK4, was assessed by RT-PCR, immunohistochemistry and western blot analysis. KEY RESULTS: ATRA ameliorated sodium arsenite-induced decrease in circulating estradiol and gonadotropin levels in a dose- and time-dependent manner, along with recovery of luminal epithelial cells and endometrial glands. Concomitant up regulation of ERα, VEGF, cyclin D1, CDK4 and Ki-67 was also observed to be more prominent for ATRA-treated rats as compared to the rats that were allowed to recover naturally for 56 days. CONCLUSIONS AND IMPLICATIONS: Collectively, the results reveal that ATRA reverses arsenic-induced disruption of the circulating levels of gonadotropins and estradiol, and degeneration of luminal epithelial cells and endometrial glands of the rat uterus, indicating resumption of their functional status. Since structural and functional maintenance of the pubertal uterus is under the influence of estradiol, ATRA consequently up regulated the estrogen receptor and resumed cellular proliferation, possibly by an antioxidant therapeutic approach against arsenic toxicity.

The role of MRI in the diagnosis of proximal femoral fractures in the elderly.

The diagnosis of an undisplaced hip fracture cannot always be made on plain radiographs alone. The use of MRI scanning in detecting occult hip fractures is well documented. However, no previous studies have suggested which specific patient group would benefit most from this investigation. Thirty-five patients with hip pain and normal plain radiographs underwent MRI scanning. Pathology was detected in 29 of the patients, of which 21 involved a neck of femur fracture. Patients were divided into two groups based on age. In patients over 70 years, pathology detected resulted in surgical intervention in 13 cases. This is in contrast with those below the age of 70 years, in whom no neck of femur fractures were found and no surgical intervention was indicated (p<0.001). We recommend that an MRI scan be performed on such patients, above 70 years of age. These are the patients in whom management is significantly altered due to the imaging process used.

Sequence parameters that determine specificity of binding of the replication-associated protein to its cognate site in two strains of tomato leaf curl virus-New Delhi.

The DNA binding sites for the replication-associated protein (Rep) of two strains of tomato leaf curl virus from New Delhi (ToLCV-Nde) were identified using electrophoretic mobility shift assays (EMSAs). The Rep proteins of the two strains were found to exhibit sequence specificity in recognition of their cognate repeat motifs (iterons) in the origin, despite the fact that they share 91% sequence identity. Using a series of synthetic oligonucleotides as probes in EMSAs, the interaction of Rep protein with its binding site was found to be dependent on number, size, and sequence of the two iterons. Mutations in the sequence of the repeat motifs or alteration in the arrangement of the motifs compromised the ability of Rep protein to bind the DNA sequence and reduced accumulation of viral DNA in protoplasts, suggesting that binding of Rep protein to its cognate iterons is an essential step in virus replication. In addition, a difference in sequence of two base pairs in the binding site of two ToLCV-Nde strains was found to affect DNA binding by the corresponding Rep protein and replication of the virus DNA in protoplasts.

Feline immunodeficiency virus Vif localizes to the nucleus.

Monoclonal antibodies prepared against recombinant Vif derived from the 34TF10 strain of feline immunodeficiency virus (FIV) were used to assess the expression and localization of Vif in virus-infected cells. Analyses by Western blotting and by immunoprecipitation from cells infected with FIV-34TF10 revealed the presence of a single 29-kDa species specific for virus-infected cells. Confirmation of antibody specificity was also performed by specific immunoprecipitation of in vitro-transcribed and -translated recombinant Vif. Localization experiments were also performed on virus-infected cells, using different fixation procedures. Results for methanol fixation protocols similar to those reported for localization of human immunodeficiency virus (HIV) Vif showed a predominant cytoplasmic localization for FIV Vif, very similar to localization of HIV type 1 Vif and virtually identical to the localization observed for the Gag antigens of the virus. However, with milder fixation procedures that used 2% formaldehyde at 4 degrees C, FIV Vif was strongly evident in the nucleus. The localization was distinct from the nuclear localization noted with Rev and did not involve the nucleolus. Attempts to show colocalization or coprecipitation of Vif with Gag antigens were unsuccessful. In addition, Vif was not detected in purified FIV virions. The results are consistent with the notion that the primary role of Vif in virus infection initiates in the nucleus.

Paracrine effects of a uterine agglutinin are mediated via the sialic acids present in the rat uterine endometrium.

A 32 kDa estrogen-induced, sialic acid-specific agglutinin (P-SAS) was isolated from rat endometrium in its proestrus stage. To investigate the functional importance of P-SAS in the uterine milieu, specific binding assays were carried out with 125I-labeled P-SAS and different cellular components of the uterus (epithelial, stromal and myometrial cells), that were isolated from different stages of the estrus cycle. The results indicate that although the protein is secreted from the epithelial cells in the estrogenic phase, it binds specifically to the stromal cells, especially to those isolated from the diestrus stage of the estrus cycle. The specific binding, however, is seen to decrease with the progression of pregnancy. Scatchard analysis performed with varying amounts of 125I-P-SAS in the presence of excess cold P-SAS revealed that the binding occurs with a Ka = 1.69 x 10(8) M(-1). As P-SAS binds specifically to sialic acids on the stromal cell surface, further characterization of the sialic acid molecule to which P-SAS binds was carried out by gas liquid chromatography (GLC). The studies revealed that P-SAS preferentially binds to N-glycolylneuraminic acid, which is attached to the penultimate sugar of the stromal cell surface glycoprotein chain via alpha2,6 linkage. As P-SAS is further known to be mitogenic, the effect of P-SAS on cultured stromal cells was studied in vitro. The growth regulatory assays revealed that P-SAS induced 3H-thymidine uptake by stromal cells in culture. Thus, from the above observations, paracrine effects of P-SAS on the stromal cells and on the subsequent growth and development of the uterus can be assumed.

Involvement of host factors in transcription from baculovirus very late promoters -- a review.

The baculovirus expression vector system has emerged as the system of choice for the expression of a number of heterologous genes of both prokaryotic and eukaryotic origin. This system utilizes the baculovirus very late, hyperactive polyhedrin and p10 promoters to drive the transcription of foreign genes. Regulation of transcription from these promoters is presently not well understood even though a number of viral gene products that may be important for transcription have been identified. Fresh insight into host-virus interactions during baculovirus pathogenesis is now offered by the identification of insect host factors that interact with transcriptionally essential motifs of these promoters as well as cis-acting enhancer-like elements upstream from the promoter.

Posterior skin flap numbness after total hip arthroplasty.

The results of a study into posterior skin flap numbness in 25 scars following primary hip arthroplasty using a straight lateral incision are reported. Nineteen scars (76%) examined were found to have some degree of skin flap numbness. In none of these patients, however, was it considered to be a significant problem. This is believed to be the first description of this neurologic problem following this approach to total hip arthroplasty and that patients should be advised of it before surgery.

Bifunctionality of the AcMNPV homologous region sequence (hr1): enhancer and ori functions have different sequence requirements.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) homologous region sequence hr1 is a putative origin of replication (ori) sequence and can also function as a transcriptional enhancer for delayed-early genes. We demonstrate that this 750-bp sequence, carrying five 28-bp core palin-dromes, enhances expression from the very late polyhedrin promoter up to 11-fold in a classical enhancer fashion in transient expression assays. Enhancement is at the level of transcription, as evident from RNase protection assay analysis. It is mediated by an alpha-amanitin-insensitive RNA polymerase from the authentic polyhedrin promoter transcription start site and follows the temporal activation profile characteristic of the polyhedrin promoter. Three lines of evidence conclusively demonstrated that hr1 acts typically as an enhancer of polyhedrin gene transcription independent of its role as an ori: (i) linearized hr1-reporter plasmids, incapable of replicating in the host cell, could enhance transcription from the promoter; (ii) reporter plasmid copy number was not affected by the presence of aphidicolin during transfection; (iii) reporter plasmid DNA recovered from Sf9 cells was sensitive to Dpn I confirming its unreplicated state in the transfection regime followed by us. Molecular dissection of the hr1 sequence elements revealed that a core palindrome alone can function as an ori sequence whereas a palindrome along with flanking sequences is essential for the enhancer activity. Enhancement of luciferase expression from the polyhedrin promoter is a function of the number of core palindromes and flanking sequences. Our results demonstrate that hr1, which has several motifs for enhancer binding proteins and transcription factors, has a dual role associated with both DNA replication and transcriptional enhancement.

A recombination-efficient baculovirus vector for simultaneous expression of multiple genes.

The baculovirus system is an extremely powerful tool for expression of heterologous genes in eukaryotic environment. A multiple expression vector, pBacUCmP3, was constructed which harbored two copies of the Autographa californica nuclear polyhedrosis virus very late gene promoter and the Drosophila melanogaster 70-kDa heat-shock protein (hsp70) promoter with downstream unique restriction sites for cloning of three independent foreign genes. Co-transfection of pBacUCmP3 with Bsu36I-linearized viral DNA yields recombinant progeny viruses at very high frequencies. The utility of this multiple expression transfer vector was demonstrated using three heterologous reporter genes encoding the beta-subunit of the human chorionic gonadotropin hormone, firefly luciferase and the bacterial beta-galactosidase (beta Gal) enzyme. The expression of reporter genes, monitored at various times post-infection, confirmed that while beta-Gal synthesis was under the transcriptional control of the hsp70 promoter, the beta hCG and Luc proteins were synthesized as a function of polyhedrin promoter activation profile. This vector will be useful for multiple synthesis of proteins at different time points.

The abnormal quinine drinking aversion in the Brattleboro rat with diabetes insipidus is reversed by the vasopressin agonist DDAVP: a possible role for vasopressin in the motivation to drink.

The Brattleboro rat with hypothalamic diabetes insipidus (BDI) has an abnormal aversion to drinking quinine-adulterated water compared with normal rats of the parent Long Evans (LE) strain. This BDI animal tolerates marked hypovolemia and decreased body weight in preference to drinking the quinine-adulterated fluid, indicative of a reduced motivation to drink. Acute or chronic treatment of BDI rats with desamino-8D arginine vasopressin (DDAVP) restored to normal their drinking response to quinine solution. Partial restoration of fluid turnover in BDI rats with hydrochlorothiazide, which has an antidiuretic effect in diabetes insipidus (when vasopressin is absent), failed to abolish the abnormal drinking response to quinine-adulterated solution in 8 out of 12 animals. In contrast, induction of diabetes mellitus in LE rats, which resulted in a marked polydipsia and polyuria even though vasopressin was still present, did not impair the drinking response to quinine solutions. These results suggest that the abnormal drinking response to quinine-adulterated fluid in BDI rats is reversed by treatment with the vasopressin V2-receptor agonist DDAVP but is unlikely to be a consequence of the restoration of fluid turnover to normal levels by a renal action. A possible central action involving vasopressin and the motivation to drink is discussed.