Sex differences in brown adipose tissue thermogenic features during caloric restriction.

Caloric restriction (CR) studies have shown that females rats conserve energy more efficiently, showing a higher resistance to weight loss and higher protection of vital organs mass than male rats. Gender-dependent inactivation of thermogenesis in brown adipose tissue (BAT) has been proposed as one of these possible energy conserving mechanisms. To study the changes underlying this inactivation in rats, a three month study with 40% CR was undertaken to unravel the effects on BAT recruitment. Under ad libitum conditions female rats had greater BAT recruitment and greater oxygen consumption than their male counterparts. Total and mitochondrial protein, as well as triglyceride and DNA content were more reduced in restricted female rats than in restricted males. Similarly, the levels of key BAT functional proteins (UCP1, LPL, HSL, TFAM) were more reduced in restricted females, whereas no changes were found in mitochondrial DNA levels (mtDNA) and OXPHOS activities in males and females. Furthermore, alpha (2A)/beta (3) adrenergic receptor ratio remained constant in male rats whereas in female rats CR increased 60%. In conclusion, our results suggest that female rats, whose BAT thermogenic activity is higher in ad libitum conditions, is depressed during CR. This inactivation involves the mitochondrial differentiation process and lipolytic system and could be due, at least in part, to the unfavourable adrenergic receptor balance for thermogenic activation.     

2-methoxyestradiol,an endogenous metabolite of 17b-estradiol,inhibits adipocyte proliferation

The effects of 2-methoxyestradiol (2ME), a naturally occurring mammalian metabolite of 17 -estradiol, on adipocyte growth has been investigated in mouse brown adipose tissue precursor cells developed in primary culture. 2ME inhibits brown adipocyte proliferation in a dose-response manner (IC50 = 1.7 × 10-6 M for DNA synthesis), with much higher potency than its hormone precursor 17-estradiol, and cells acquire the typical differentiated morphology - more round with a higher content of triglycerides. 2ME causes similar effects in the immortal brown adipocyte tumor-derived hibernoma cell line HIB 1B and the immortal 3T3-F442A white adipocyte line. These findings suggest a possible role for 2ME in adipocyte proliferation, and probably in the differentiation process, entering the cells in the adipogenic program.     

Modification of ricin A chain, by addition of endoplasmic reticulum (KDEL) or Golgi (YQRL) retention sequences, enhances its cytotoxicity and translocation

 A pKK expression system in Escherichia coli was used to produce recombinant ricin A chain (rRTA) and rRTA modified by addition of organelle-specific amino acid retention sequences, including KDEL (an endoplasmic reticulum, ER, lumen retention signal), KKMP (an ER membrane retention signal), YQRL (a trans-Golgi network retention signal) and KFERQ (a lysosome-targeting signal) to the C terminus of rRTA. The toxicities of these RTA mutants were assessed in Jurkat cells following fluid-phase endocytosis. rRTA-KDEL and rRTA-YQRL were significantly more cytotoxic for Jurkat cells than rRTA, rRTA-KKMP or rRTA-KFERQ. This difference did not result from signal(KDEL or YQRL)-mediated binding of these RTA mutants to the cell surface. Reconstituted ER and Golgi vesicles have been employed to assess translocation of rRTA and mutant rRTA. RTA-KDEL and RTA-YQRL respectively exhibited 6.7-fold and 6.1-fold more protection against papain digestion in reconstituted ER vesicles and 2.2-fold and 1.8-fold more protection in reconstituted Golgi vesicles, than unmodified rRTA. These mutants were reassociated with ricin B chain to form holotoxins. The mutant RTA-KDEL and RTA-YQRL holotoxins were 3.8-fold and 1.5-fold more cytotoxic for target cells, respectively, than ricin produced using unmodified rRTA. Our results suggest that both ER and the trans-Golgi network may play important roles in the intracellular trafficking and translocation of ricin A chain. Received: 14 August 1997 / Accepted: 14 October 1997     

Refinement of genetic localization of the Alström syndrome on chromosome 2p12-13 by linkage analysis in a North African family

Alström syndrome is a rare autosomal recessive disorder characterized by retinal pigment degeneration, neurogenic deafness, infantile obesity, hyperlipidemia, and non-insulin-dependent diabetes mellitus. While the disease-related gene remains unknown, studies of the genetic isolate of French Acadians provisionally locate the Alström syndrome on chromosome 2p12-13 within a 14.9-cM interval. To confirm this finding in another ethnic population and refine the candidate region we investigated by linkage analysis a consanguineous family of North African origin, in which three of seven siblings displayed all major neurological and metabolic features of Alström syndrome. Genotyping was performed on an ABI377 DNA automatic sequencer and LOD scores were obtained with the Fastlink program. Five markers previously investigated in French Acadians confirmed the involvement of the candidate region, although pairwise LOD scores were of poor significance (Z _max=2.9). To further confirm homogeneity and refine the candidate region, 20 additional markers were investigated. Haplotype analysis and allele segregation revealed that affected children shared a single haplotype and were homozygous for the eight most centromeric markers (D2S291–D2S2114), over a 6.1-cM interval. Significative multipoint LOD scores (Z _max=3.96) were obtained between markers D2S2110/145 and D2S286. Two clusters of known genes are present in this refined region of chromosome 2p, the most attractive candidate being the hexokinase II gene. However, except for several known polymorphisms, no mutations were detected in the coding region of this gene. In conclusion, the location of Alström syndrome on chromosome 2p12-13 is confirmed, reducing the genetic interval to 6.1 cM.     

Ovulation does not constrain egg parcel size in the simultaneous hermaphrodite Serranus subligarius

Among egg trading hermaphrodites, any factor which limits the number of eggs released by a female role hermaphrodite can potentially limit the mating success of the male role hermaphrodite fertilizing those eggs. This work examines the hypothesis that the timing of ovulation constrains the size of egg parcels and thereby limits male mating success in the egg parceling hermaphroditic fish Serranus subligarius. Two alternatives were evaluated: (1) Ovulation is a discrete event preceding spawning. It does not constrain the size of egg parcels and therefore does not limit mating success of male role partners. (2) Ovulation is an incremental process occurring throughout the spawning period. It limits the number of eggs available for release in each parcel and thereby limits mating success of the male role partner. Assessment of ovulation was conducted in a field stock of S. subligarius. Fish from size matched pairs were manually stripped at the onset of the spawning period or quarantined and sampled at the end of the spawning period. Fish sampled at either time point had the same number of eggs, suggesting that ovulation was a discrete event occurring at the onset of the spawning period. The diurnal cycle of ovulation was observed in naturally spawning hermaphrodites captured at intervals throughout the day. Ovulation began 2–4 h before spawning began. Some fish appeared to ovulate the entire day's clutch of eggs before spawning, while other fish released egg parcels before completing ovulation. I conclude that the pattern of ovulation is not uniform throughout the spawning stock. Because of the variability in timing of ovulation relative to parcel release, ovulation does not consistently limit the size of egg parcels and therefore is unlikely to be a physiological limit to male role mating success in S. subligarius hermaphrodites.     

Machine for Automatic Bacteriological Pour Plate Preparation

A fully automatic system for preparing poured plates for bacteriological analyses has been constructed and tested. The machine can make decimal dilutions of bacterial suspensions, dispense measured amounts into petri dishes, add molten agar, mix the dish contents, and label the dishes with sample and dilution numbers at the rate of 2,000 dishes per 8-hr day. In addition, the machine can be programmed to select different media so that plates for different types of bacteriological analysis may be made automatically from the same sample. The machine uses only the components of the media and sterile polystyrene petri dishes; requirements for all other materials, such as sterile pipettes and capped bottles of diluents and agar, are eliminated.     

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17β

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.     

Treatment of Patients with Systemic Lupus Erythematosus including Nephritis with Chlorambucil

Six female patients with systemic lupus erythematosus (S.L.E.) have been treated with chlorambucil. In five the decision was taken after failure by corticosteroids to control progressive renal disease in the face of unacceptable corticosteroid toxicity. After the introduction of chlorambucil renal function improved and all patients remain well six, six, five, three, and two-and-a-half years later, respectively. On renal biopsy five had focal proliferative glomerulonephritis. Repeat biopsy in two cases showed quantitative improvement. The sixth patient was treated with chlorambucil because of failure by corticosteroids to control peripheral vascular lesions and haemolysis and she remains well four years later. In four patients is it probable that amenorrhoea was related to chlorambucil treatment, but there were no other important side effects although one patient developed a degree of marrow depression during treatment. Chlorambucil may hold advantages over the immunosuppressive drugs normally recommended in this condition, azathioprine and cyclophosphamide, as it appears less liable to cause important marrow suppression and, unlike cyclophosphamide is not associated with alopecia and haemorrhagic cystitis.