Embden-Meyerhof-Parnas and Entner-Doudoroff pathways in Thermoproteus tenax: metabolic parallelism or specific adaptation?

Genome data as well as biochemical studies have indicated that--as a peculiarity within hyperthermophilic Archaea--Thermoproteus tenax uses three different pathways for glucose metabolism, a variant of the reversible EMP (Embden-Meyerhof-Parnas) pathway and two different modifications of the ED (Entner-Doudoroff) pathway, a non-phosphorylative and a semi-phosphorylative version. An overview of the three different pathways is presented and the physiological function of the variants is discussed.

Archaeal fructose-1,6-bisphosphate aldolases constitute a new family of archaeal type class I aldolase.

Fructose-1,6-bisphosphate (FBP) aldolase activity has been detected previously in several Archaea. However, no obvious orthologs of the bacterial and eucaryal Class I and II FBP aldolases have yet been identified in sequenced archaeal genomes. Based on a recently described novel type of bacterial aldolase, we report on the identification and molecular characterization of the first archaeal FBP aldolases. We have analyzed the FBP aldolases of two hyperthermophilic Archaea, the facultatively heterotrophic Crenarchaeon Thermoproteus tenax and the obligately heterotrophic Euryarchaeon Pyrococcus furiosus. For enzymatic studies the fba genes of T. tenax and P. furiosus were expressed in Escherichia coli. The recombinant FBP aldolases show preferred substrate specificity for FBP in the catabolic direction and exhibit metal-independent Class I FBP aldolase activity via a Schiff-base mechanism. Transcript analyses reveal that the expression of both archaeal genes is induced during sugar fermentation. Remarkably, the fbp gene of T. tenax is co-transcribed with the pfp gene that codes for the reversible PP(i)-dependent phosphofructokinase. As revealed by phylogenetic analyses, orthologs of the T. tenax and P. furiosus enzyme appear to be present in almost all sequenced archaeal genomes, as well as in some bacterial genomes, strongly suggesting that this new enzyme family represents the typical archaeal FBP aldolase. Because this new family shows no significant sequence similarity to classical Class I and II enzymes, a new name is proposed, archaeal type Class I FBP aldolases (FBP aldolase Class IA).

Thermal balloon and rollerball ablation to treat menorrhagia: two-year results of a multicenter, prospective, randomized, clinical trial.

STUDY OBJECTIVE: To evaluate 2-year follow-up results in patients participating in a randomized, clinical trial comparing uterine balloon therapy with rollerball endometrial ablation. STUDY DESIGN: Prospective, randomized, clinical trial (Canadian Task Force classification I). SETTING: Fourteen university-affiliated and private practice sites. PATIENTS: Two hundred fifty-five women with menorrhagia. INTERVENTIONS: Patients were randomized to rollerball or uterine balloon endometrial ablation. MEASUREMENTS AND MAIN RESULTS: Patient satisfaction with both treatments was consistently high. Only 15 hysterectomies had been performed (6 for menorrhagia) at the end of 2 years, 11 in the rollerball group, 4 in the balloon therapy group. CONCLUSION: Endometrial ablation by both procedures was highly successful in avoiding hysterectomy and relieving symptoms of menorrhagia. Additional benefits were reduction in dysmenorrhea and premenstrual syndrome.

Pyruvate kinase of the hyperthermophilic crenarchaeote Thermoproteus tenax: physiological role and phylogenetic aspects.

Pyruvate kinase (PK; EC 2.7.1.40) of Thermoproteus tenax was purified to homogeneity, and its coding gene was cloned and expressed in Escherichia coli. It represents a homomeric tetramer with a molecular mass of 49 kDa per subunit. PK exhibits positive binding cooperativity with respect to phosphoenolpyruvate and metal ions such as Mg(2+) and Mn(2+). Heterotropic effects, as commonly found for PKs from bacterial and eucaryal sources, could not be detected. The enzyme does not depend on K(+) ions. Heterotrophically grown cells exhibit specific activity of PK four times higher than autotrophically grown cells. Since the mRNA level of the PK coding gene is also accordingly higher in heterotrophic cells, we conclude that the PK activity is adjusted to growth conditions mainly on the transcript level. The enzymic properties of the PK and the regulation of its expression are discussed with respect to the physiological framework given by the T. tenax-specific variant of the Embden-Meyerhof-Parnas pathway. T. tenax PK shows moderate overall sequence similarity (25 to 40% identity) to its bacterial and eucaryal pendants. Phylogenetic analyses of the known PK sequences result in a dichotomic tree topology that divides the enzymes into two major PK clusters, probably diverged by an early gene duplication event. The phylogenetic divergence is paralleled by a striking phenotypic differentiation of PKs: PKs of cluster I, which occur in eucaryal cytoplasm, some gamma proteobacteria, and low-GC gram-positive bacteria, are only active in the presence of fructose-1,6-bisphosphate or other phosphorylated sugars, whereas PKs of cluster II, found in various bacterial phyla, plastids, and in Archaea, show activity without effectors but are commonly regulated by the energy charge of the cell.

Purification, reconstitution, and characterization of KdpD, the turgor sensor of Escherichia coli.

In response to K+ availability or medium osmolality, the sensor kinase KdpD and the response regulator KdpE control the expression of the kdpFABC operon, coding for the high affinity K+-translocating Kdp ATPase of Escherichia coli. The stimulus for KdpD to undergo autophosphorylation is believed to be a change in turgor or some effect thereof, reflecting the role of K+ as an important cytoplasmic osmotic solute. The membrane-bound sensor kinase KdpD was overproduced as a fusion protein containing six contiguous histidine residues two amino acids before the C terminus. This KdpD-His6 protein was functional in vitro and in vivo. KdpD-His6 was purified from everted membrane vesicles by solubilization with the zwitterionic detergent lauryldimethylamine oxide followed by nickel chelate chromatography and ion exchange chromatography to >99% homogeneity. The solubilized protein was not active with respect to autophosphorylation, but retained the ability to bind 2-azido-ATP. KdpD-His6 was reconstituted into proteoliposomes in a unidirectional inside-out orientation as revealed by ATP accessibility and protease susceptibility. Purified and reconstituted KdpD-His6 exhibited autokinase activity, and the phosphoryl group could be transferred to KdpE. Furthermore, KdpD-His6 was found to be the only protein that mediates dephosphorylation of KdpE approximately P.

The effect of regular exercise on women receiving danazol for treatment of endometriosis.

OBJECTIVE: To determine the effects of regular exercise on women receiving danazol for the treatment of endometriosis. METHODS: Thirty-nine patients were randomized to a danazol-only or a danazol/exercise regimen in a prospective clinical trial carried out at tertiary care institutions. Patients in the danazol/exercise group were instructed to exercise four times per week, for 40 min per session, at an intensity of 20 metabolic units. Side effect profiles, pelvic symptoms, aerobic fitness, strength and hormone levels were compared for all subjects. The number of side effects of danazol was analyzed by the method of generalized estimating equations. RESULTS: The number of side effects reported during a 4-week period was 1.09-2.17 times greater for the danazol-only than for the danazol/exercise group. All patients had improvement of symptoms during treatment. The danazol/exercise group had significantly lower testosterone levels during treatment. The time to recurrence of endometriosis was not different between groups. CONCLUSIONS: Exercise during danazol therapy reduces the number of androgenic side effects. Relief of pain and time to recurrence are unaffected.

Polycystic ovary syndrome: relationship to growth hormone, insulin-like growth factor-I, and insulin.

Polycystic ovary syndrome is frequently encountered by the practicing gynecologist and can be a difficult and frustrating disease process to treat. The cause of this syndrome remains elusive and may be multifactorial. The role of growth hormone, insulin, and insulin-like growth factor-I, as related to polycystic ovary syndrome, has received considerable interest. The exact relationships of these factors have not been fully elucidated, though recent studies have begun to shed new light on this complex issue. A better understanding of the pathophysiology will ultimately lead to more appropriate treatment modalities.

Time-related effects of RU486 treatment in experimentally induced endometriosis in the rat.

OBJECTIVE: To evaluate the time-dependent effect of the progesterone antagonist RU486 on experimentally induced endometriosis in the rat. DESIGN: Endometriosis was induced by implanting endometrium onto the peritoneal side wall. Size of the implants was measured before and after treatment. PARTICIPANTS: Fifty-two adult female Sprague-Dawley female rats weighing 200 to 225 g. SETTING: Departmental research laboratory of university hospital. INTERVENTION: Animals treated with RU486 or vehicle only for 2, 4, 6, or 8 weeks. MAIN OUTCOME MEASURES: Size of the implant was measured before and after treatment; relative change in size of the implants was calculated. RESULTS: There was not a statistically significant difference between the treatment groups and the control groups. There was no regression of endometriosis in any of the treatment groups. CONCLUSIONS: RU486 administered for 8 weeks in the rat model was not effective therapy for endometriosis.

Superovulation with or without intrauterine insemination for the treatment of infertility.

Patients undergoing human menopausal gonadotropin (hMG) superovulation were reviewed retrospectively to determine whether fecundity was greater for intrauterine insemination (IUI) than timed intercourse. Forty patients with unexplained infertility, American Fertility Society I or II endometriosis, luteal phase defect and/or cervical factor were treated with hMG alone or hMG plus IUI. Twenty-eight underwent 52 cycles of hMG/IUI, and 19 underwent 31 cycles of hMG. The probability of pregnancy after four cycles was significantly better in the hMG/IUI group (.90) than the hMG group (.37, P = .049). There was a 54.5% multiple pregnancy rate, and one patient was admitted to the hospital for hyperstimulation. When traditional therapy fails, hMG/IUI significantly increases the pregnancy rates as compared to hMG with timed intercourse in a "good prognosis" group of patients.

The efficacy of presacral neurectomy for the relief of midline dysmenorrhea.

The present study was undertaken to evaluate prospectively the efficacy of presacral neurectomy for the treatment of midline dysmenorrhea. All patients had moderate to severe dysmenorrhea and stage III-IV endometriosis. Of the patients undergoing presacral neurectomy (N = 17), only two had a recurrence of pain. The remainder of the patients undergoing presacral neurectomy remain pain-free at 42 months of follow-up. Of the patients undergoing resection of endometriosis but not presacral neurectomy (N = 9), none received relief of midline pain. Relief of lateral pain, back pain, and dyspareunia was variable in both groups. Our findings corroborate previous retrospective studies showing that presacral neurectomy is highly effective in the treatment of dysmenorrhea. We speculate that the most common reasons for failure of presacral neurectomy are inappropriate selection of patients and incomplete resection of the presacral nerve plexus.

Vulvar congenital dysplastic angiopathy.

Congenital dysplastic angiopathy is a syndrome consisting of vascular angiomata, congenital varicosities, and trophic changes of the soft tissue and the skeleton. Frequently referred to as Klippel-Trenaunay or Klippel-Trenaunay-Weber syndrome, it rarely affects the female genitalia. An 18-year-old woman underwent evaluation and treatment for Klippel-Trenaunay syndrome with vulvar involvement. Preoperative evaluation included consultation with pediatric surgeons, gynecologic surgeons, and an interventional radiologist as well as individual and family psychological counseling. Attention to intraoperative detail resulted in minimal operative blood loss and preservation of normal anatomy. A postoperative hematoma was treated aggressively with surgical evacuation and drainage. Six-month follow-up revealed functionally and cosmetically normal vulvar anatomy.