Unique effects of energy versus estrogen deficiency on multiple components of bone strength in exercising women.

Many female athletes are energy and/or estrogen deficient, but the independent effects on bone health have not been isolated. Energy deficiency was detrimental at the tibia while estrogen deficiency was detrimental at the radius. Nutrition must be considered alongside menstrual recovery when addressing compromised bone health in female athletes. INTRODUCTION: The purpose of this study was to describe volumetric bone mineral density (vBMD), bone geometry, and estimated bone strength in exercising women (n = 60) grouped according to energy status (energy replete (EnR: n = 30) vs. energy deficient (EnD: n = 30)) and estrogen status (estrogen replete (E2R: n = 33) vs. estrogen deficient (E2D: n = 27)), resulting in four distinct groups: EnR + E2R (n = 17), EnR + E2D (n = 13), EnD + E2R (n = 16), EnD + E2D (n = 14). METHODS: Energy status was determined using the ratio of measured to predicted resting energy expenditure (mREE/pREE). Estrogen status was based on self-reported menstrual status confirmed by daily evaluation of urinary estrone-1-glucoronide (E1G), pregnanediol glucuronide (PdG), and luteinizing hormone (LH). Eumenorrheic women were considered E2R, amenorrheic women were E2D, and oligomenorrheic women were categorized based on history of menses in the past year. Bone was assessed using peripheral quantitative computed tomography (pQCT). RESULTS: EnD women exhibited lower total vBMD, trabecular vBMD, cortical area, and BSI at the distal tibia and lower total vBMD, smaller cortical area and cortical thickness, and larger endosteal circumference at the proximal tibia compared to EnR women (p < 0.042). E2D women had lower total and cortical vBMD, larger total and trabecular area, and lower BSI at the distal radius and lower cortical vBMD at the proximal radius compared to E2R women (p < 0.023). Energy and estrogen interacted to affect total and trabecular area at the distal tibia (p < 0.021). CONCLUSIONS: Efforts to correct energy deficiency, which in turn may promote reproductive health, are warranted in order to address the unique contributions of energy status versus estrogen status to bone health.

Two tandemly arrayed genes encode the (histidine) secretory acid phosphatases of Leishmania donovani.

Leishmania donovani promastigotes constitutively secrete a glycosylated and phosphorylated acid phosphatase activity. This secretory acid phosphatase (SAcP) was purified from L. donovani culture supernatants and amino-acid sequence was obtained from both the N-terminus and a tryptic peptide fragment derived from the isolated protein. A polymerase chain reaction (PCR)-based strategy, using degenerate oligo primers designed from the amino-acid sequence data, identified two single-copy, tandemly arrayed open reading frames (ORFs) capable of encoding the L. donovani SAcP (SAcP-1, 2052 bp and SAcP-2, 2124 bp). Both SAcP-1 and -2 were shown to be actively transcribed by L. donovani promastigotes by reverse transcription (RT) and PCR amplification. The deduced amino-acid sequences of SAcP-1 and SAcP-2 show high conservation to each other in four regions: a 23-amino-acid signal peptide; a catalytic domain containing several potential N-linked glycosylation sites; a Ser/Thr-rich repeat region containing multiple potential phosphorylation sites and a common C-terminus. Within the catalytic domain, the L. donovani SAcPs possess two conserved consensus sequences characteristic of histidine acid phosphatases (AcPs). Furthermore, antisera to native L. donovani SAcP immunoprecipitated in vitro transcription/translation products of both SAcP-1 and SAcP-2. Cumulatively, these data indicate that the acid phosphatase activity constitutively secreted by L. donovani promastigotes is composed of two (histidine) AcP isoforms that are encoded by SAcP-1 and SAcP-2, respectively.

Multiple cysteine proteinases of the pathogenic protozoon Tritrichomonas foetus: identification of seven diverse and differentially expressed genes.

The cattle protozoan parasite Tritrichomonas foetus has multiple forms of cysteine proteinases. To investigate their diversity, PCR and reverse transcriptase PCR were used to isolate genomic DNA and cDNA fragments, respectively, encoding different cysteine proteinases. Seven genes have been identified, TFCP3-6 from amplification of genomic DNA and TFCP7-9 from amplification of cDNA. Comparison of the predicted amino acid sequences indicates that the T. foetus enzymes are cathepsin-L-like rather than cathepsin-B-like in structure. However, there is considerable diversity among the proteinases. TFCP7 and TFCP8 are most similar to one another (78% identity), while TFCP3 and TFCP9 are the least closely related (30% identity). All but one of the genes are single-copy, the exception being TFCP3, which was present in multiple copies in one of the three isolates examined. Single transcripts were detected for each of the seven genes. TFCP8 was expressed at the highest levels, while transcripts for TFCP4 were only just detectable. In T. foetus F2, the strain from which the genomic DNA and mRNA were isolated, transcripts of the five other genes were present at intermediate levels. When two other isolates were compared with F2, differences in the expression of individual genes were apparent, with either one or two of them not expressed. In spite of these differences the major cysteine proteinases detected in the three isolates using substrate-SDS-PAGE appeared identical. The data show that the multiplicity of cysteine proteinases in T. foetus is due, in part at least, to the presence of multiple genes and that some of the genes encode cysteine proteinases which are not among the high-activity enzymes detected previously.

Identification and molecular cloning of four cysteine proteinase genes from the pathogenic protozoon Trichomonas vaginalis.

The parasitic protozoon Trichomonas vaginalis produces multiple forms of cysteine proteinase (CP). The molecular basis for this has now been examined by cloning DNA fragments encoding CPs. Using generic degenerate oligonucleotide primers based on two well-conserved regions within the central region of all eukaryotic CPs, several polymerase chain reaction fragments were isolated from T. vaginalis genomic DNA and shown to encode different CPs. One fragment with a well-represented sequence was used as a general probe to screen a T. vaginalis cDNA library at moderate stringency and five different cDNA clones were isolated. Preliminary sequencing showed that they encoded similar but distinct CPs. In the process of confirming the 5' end of one of these cDNA clones using RACE-PCR (rapid amplification of cDNA 5' ends-polymerase chain reaction), an additional sequence encoding a different CP was identified. The corresponding clone (TvCP3) and the three longest clones from the library screen (TvCP1, TvCP2 and TvCP4) were characterized further. TvCP1 and TvCP2 were full-length and TvCP3 and TvCP4 were apparently slightly less than full-length. Comparison of the predicted amino acid sequences of the four clones showed that TvCP1 and TvCP4 are related (72% identity). TvCP2 is closer to TvCP1 (60%) and TvCP4 (65%) than is TvCP3, which has 53%, 59% and 56% identity to TvCP1, TvCP2 and TvCP4, respectively. Comparison with the sequences of other known CPs indicated that the T. vaginalis gene products all belong to the cathepsin L/cathepsin H/papain branch of the papain superfamily. The TvCP1, TvCP2 and TvCP4 sequences are related (38-45% identity) to those of CP2 of Dictyostelium discoideum, human cathepsin L, three CPs from lobster and CPs from black gram, oilseed rape and rice (oryzains alpha and beta). TvCP3 shows less identity to the other eukaryotic CPs but is most similar to D. discoideum CP2 (38%). The four predicted amino acid sequences share some features distinct from the majority of CPs, which suggests they might have had a common evolutionary origin. The most striking feature of sequences TvCP1, TvCP2 and TvCP3 is the apparent lack of a pre-sequence (signal sequence) for TvCP1 and very short pre-sequences for TvCP2 and TvCP3. Southern analysis indicated that the organization of the genes corresponding to the TvCP cDNAs differs. The TvCP1, TvCP2 and TvCP3 genes are single-copy, whereas the TvCP4 gene appeared to be multiple-copy.(ABSTRACT TRUNCATED AT 400 WORDS)

The oxidative response of rabbit peritoneal neutrophils to leishmanias and other trypanosomatids.

Luminometry has been used to measure the respiratory burst of rabbit peritoneal neutrophils that is elicited by different forms and species of Leishmania and Herpetomonas. Mid-log phase and metacyclic promastigotes of L. major evoked large responses; that due to metacyclics was lower and slower, but they also bound in smaller numbers than mid-log phase cells. Promastigotes of L. mexicana mexicana also stimulated a large respiratory burst whereas amastigotes elicited little or none. Leishmania donovani promastigotes and culture forms of H. muscarum muscarum and H. m. ingenoplastis all evoked large responses by neutrophils. There was, however, very little response to L. mexicana mexicana promastigotes, L. donovani promastigotes or H. muscarum muscarum when they were added in large numbers. This 'inhibition' was not apparent with L. major.

Interaction of leishmania metacyclics with macrophages.

Metacyclics of L. major and putative metacyclics of L. m. mexicana survived better in explanted murine macrophages than promastigotes from mid-log phase cultures. The latter forms, however, attached in greater numbers to macrophages and, in the case of L. major, also more became intracellular. Only a small percentage of the macrophages infected with amastigotes exhibited a respiratory burst (as detected by nitroblue tetrazolium (NBT) reduction), whereas this occurred with most of the macrophages infected with either metacyclic or non-infective promastigotes of L. major. Approximately half of the macrophages infected with L. m. mexicana promastigotes reduced NBT. The results suggest that avoidance of the oxygen metabolite arm of the host cell's microbicidal activity is not the main survival strategy for metacyclics entering macrophages. Metacyclics of L. major, however, were found to be less sensitive than mid-log phase promastigotes to hydrogen peroxide and also human serum; properties which may aid their survival.

Biochemical characteristics of the metacyclic forms of Leishmania major and L. mexicana mexicana.

Metacyclic forms of Leishmania major and putative metacyclics of L. mexicana mexicana were found to occur in abundance in stationary phase cultures. These forms have been compared in several ways with promastigotes from mid-log phase cultures and, in the case of L. m. mexicana, amastigotes. Metacyclics are smaller, contain less protein and appear more active than other promastigotes. Both forms of promastigote respire at a high rate in the absence of exogenous substrate. The free amino-acid contents of the various forms of the two species have been analysed. They differ in detail but alanine was the major amino acid in all cases. The isoenzyme content of the different forms differed significantly. That of the putative metacyclics of L. m. mexicana was in several respects more similar to amastigotes than promastigotes, suggesting that the form is pre-adapted for life in a mammal. Metacyclics of L. major apparently did not divide in culture but transformed back over a period of 48 h to mid-log phase cells. The results provide further detail of the molecular differences between mid-log phase and metacyclic promastigotes and confirm that metacyclics are a distinct form in the life-cycle.