Dietary vitamin A supplementation in rats: suppression of leptin and induction of UCP1 mRNA

All-trans-retinoic acid (RA), an active metabolite of vitamin A, induces the gene expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) and suppresses leptin gene expression in white adipose tissue (WAT) when given as an acute dose. These contrasting effects of RA leave in doubt the overall effect of chronic RA or vitamin A supplementation on energy homeostasis. To investigate the effects of dietary vitamin A supplementation on leptin and UCP1 gene expression, rats were fed either a normal diet (2.6 retinol/kg diet) or a vitamin A-supplemented diet (129 mg retinol/kg diet) for 8 weeks, and adiposity, serum leptin levels, leptin mRNA levels in perirenal WAT, UCP1 and UCP2 mRNA levels in BAT, and beta3-adrenergic receptor mRNA levels in BAT and WAT were examined. Rats on both diets gained a similar amount of weight, but there was a small 9% decrease in the adiposity index in the vitamin A-supplemented rats. Dietary vitamin A supplementation increased UCP1 gene expression in BAT by 31%, but suppressed leptin gene expression by 44% and serum leptin levels by 65%. UCP2 and beta3-adrenergic receptor gene expression in BAT and perirenal WAT were unchanged by the vitamin A diet. These data suggest that dietary vitamin A has a role in regulating energy homeostasis by enhancing UCP1 gene expression and decreasing serum leptin levels.     

Split flexor carpi radialis muscle

A detailed anatomic and intramuscular neural staining study in 22 human and 5 monkey upper limbs revealed that the flexor carpi radialis can be raised on its proximal neurovascular pedicle and that the muscle can be split along its tendon into two independently functioning neuromuscular compartments, each with its own nerve and blood supply. A study of the muscle architecture in the human specimens found the radial compartment to have significantly longer fiber length and a larger physiologic cross-sectional area than the ulnar compartment. Independence of function of each compartment was demonstrated in electrical stimulation studies in six monkeys (Macaca fascicularis), but no significant difference was noted in the peak isometric load between the two compartments (p = 0.68) in the monkey. The extra functioning muscle units become important in local transfers for restoring function in multiple nerve palsies as in Hansen's disease, severe traumatic loss of muscle in crush injuries and compartment syndromes, and after wide resection in infective and neoplastic conditions in the forearm and hand.     

Follicular variant of papillary carcinoma of the thyroid. A cytologic study of 15 cases

OBJECTIVE: To study the cytologic findings of follicular variant of papillary thyroid carcinoma (FVPTC) and to compare them with the cytologic findings on other thyroid lesions. STUDY DESIGN: The study group consisted of aspirate smears from 15 cases of histologically proven FVPTC. The control group consisted of 152 cases, including adenomatous colloid goiter (70), usual papillary carcinoma (40), follicular adenoma (30), Hürthle cell neoplasm (7) and medullary carcinoma (5). RESULTS: The smears of FVPTC revealed numerous colloid balls in the background, multilayered microfollicles (rosettes), numerous nuclear grooves and inclusions in the monolayer sheets of follicular cells, very rare giant cells, absence of calcification and papillary clusters. Rosettelike microfollicles and numerous colloid balls were not seen in the control group. CONCLUSION: The combination of numerous colloid balls and rosettelike microfollicles was frequently seen in FVPTC. This combination was not observed in the control group.     

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.     

Influence of Disulfide Bonds on the Induction of Helical Conformation in Proteins

The effect(s) of TFE (2,2,2-trifluoroethanol) on three different conformational states (native, denatured, and carboxymethylated) of CTX III and RNase A has been examined. Contrary to the general belief, the results of the present study reveal that TFE can induce helical conformation in a protein which has no sequence propensity to form a helix. It is found that the helix induction in TFE is intricately related to the destabilization of the tertiary structural conformation in proteins. More importantly, the disulfide bonds in proteins are found to have significant influence on the TFE-mediated helix induction. The results obtained in this study strongly suggest that information pertaining to the influence of disulfide bonds on helix induction need to be considered to improve the accuracy of secondary structure prediction algorithms.     

Development of special hyphal branches of Phyllactinia corylea on host and non-host surfaces

Hyphae of Phyllactinia corylea produce two kinds of special branches on the host surface: adhesion bodies which serve as fungal attachment and stomatopodia which enter the leaf through stomata. Conidial germination on host and non-host surfaces was examined with a scanning electron microscope to explain the stimuli responsible for development of the special branches, and the involvement of host recognition in the process. Conidia germinated within 4 h on host and non-host surfaces, but on non-host surfaces the emergence of the germ tube was not always directed towards the substratum. Adhesion bodies were formed from the tips of germ tubes at the first contact point on host and non-host surfaces. Development of stomatopodia was more specific and they were formed precisely over stomata on the host surface. Stomatopodia-like structures were occasionally formed over finely ridged leaf veins on the host surface and over some fine scratches on synthetic surfaces. The experiments showed that while conidial germination and development of adhesion bodies are in response to contact stimuli, the development of stomatopodia is a response to precise topographical signals, and the directional emergence and attached growth of germ tubes involve host recognition.This revised version was published online in October 2005 with corrections to the Cover Date.     

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present studyexamined the intestinal uptake of thiamine (vitaminB₁) using the human-derivedintestinal epithelial cells Caco-2 as an in vitro model system.Thiamine uptake was found to be 1)temperature and energy dependent and occurred with minimal metabolicalteration; 2) pH sensitive;3)Na⁺ independent;4) saturable as a function ofconcentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mgprotein¹ · 3 min¹;5) inhibited by the thiaminestructural analogs amprolium and oxythiamine, but not by unrelatedorganic cations tetraethylammonium, N-methylnicotinamide, and choline; and6) inhibited in a competitive mannerby amiloride with an inhibition constant of 0.2 mM. The role ofspecific protein kinase-mediated pathways in the regulation of thiamineuptake by Caco-2 cells was also examined using specific modulators ofthese pathways. The results showed possible involvement of aCa²⁺/calmodulin (CaM)-mediatedpathway in the regulation of thiamine uptake. No role for proteinkinase C- and protein tyrosine kinase-mediated pathways in theregulation of thiamine uptake was evident. These results demonstratethe involvement of a carrier-mediated system for thiamine uptake byCaco-2 intestinal epithelial cells. This system isNa⁺ independent and is differentfrom the transport systems of organic cations. Furthermore, aCaM-mediated pathway appears to play a role in regulating thiamineuptake in these cells.

     

Molecular heterogeneity among north Indian isolates of Group A Streptococcus

AIM: To monitor molecular heterogeneity among the clinical isolates of group A Streptococcus (GAS) from north India by Vir and emm typing. METHODS AND RESULTS: GAS isolates, 31 from pharyngitis and nine from rheumatic fever (RF)/rheumatic heart disease (RHD) patients were differentiated into 16 Vir types (VT). These isolates were further discriminated into 23 emm types. Most of emm types were Vir type specific, except few (7.5%), which revealed different Vir types within same emm type. The most prevalent emm type found was emm 49 (15%) followed by 7.5% of emm 69, emm 71 and emm 75 which were different from emm type distribution reported from south India. CONCLUSIONS: Analysis of data revealed 40% heterogeneity by Vir typing and 57.5% by emm typing among GAS isolates which is significant in view of small number of isolates studied. SIGNIFICANCE OF IMPACT OF THE STUDY: The molecular study for the first time demonstrates different emm types prevalent and circulating in northern region of India and such data may help in selection of types for vaccine development.     

An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.