Domain motions and quaternary packing of phosphofructokinase-2 from Escherichia coli studied by small angle x-ray scattering and homology modeling

The binding of MgATP and fructose-6-phosphate to phosphofructokinase-2 from Escherichia coli induces conformational changes that result in significant differences in the x-ray-scattering profiles compared with the unligated form of the enzyme. When fructose- 6-phosphate binds to the active site of the enzyme, the pair distribution function exhibits lower values at higher distances, indicating a more compact structure. Upon binding of MgATP to the allosteric site of the enzyme, the intensity at lower angles increases as a consequence of tetramer formation, but differences along higher angles also suggest changes at the tertiary structure level. We have used homology modeling to build the native dimeric form of phosphofructokinase-2 and fitted the experimental scattering curves by using rigid body movements of the domains in the model, similar to those observed in known homologous structures. The best fit with the experimental data of the unbound protein was achieved with open conformations of the domains in the model, whereas domain closure improves the agreement with the scattering of the enzyme-fructose-6-phosphate complex. Using the same approach, we utilized the scattering curve of the phosphofructokinase-2-MgATP complex to model the arrangement and conformation of dimers in the tetramer. We observed that, along with tetramerization, binding of MgATP to the allosteric site induces domain closure. Additionally, we used the scattering data to restore the low resolution structure of phosphofructokinase-2 (free and bound forms) by an ab initio procedure. Based on these findings, a proposal is made to account for the inhibitory effect of MgATP on the enzymatic activity.     

Body size and fecundity of Juxtafabia muliniarum (Brachyura: Pinnotheridae) associated with Saccostrea palmula (Bivalvia: Ostreidae), Costa Rica

Size and fecundity observations of pea crab (Juxtafabia muliniarum) from the paleal cavity of the oyster Saccostrea palmula were made from May 1998 to May 1999. Infestation frequency was 18.52% in a sample of 540 oysters. Of 136 pea crabs, 36% were couples, 60% were single females and 4% were single males. The mean caparace length of J. muliniarum was 5.6 +/- 0.74 mm (range 4.0 to 7.6 mm) for females and 2.71 +/- 0.60 mm (range 1.6 to 4.0 mm) for males. The mean weight was 0.180 +/- 0.084 g (range 0.06 to 0.4 g) for females and 0.011 +/- 0.003 g (range 0.01 to 0.02 g) for males. Ovigerous females (43.75% of all females) were found in all months. The caparace length-fecundity relationship was F = 3904.6 Ln (Lc)--4651.1. The caparace length-weight relationship was P = 6 x 10(-4) Lc3.2122. The mean sex-ratio was 1.0 male: 2.4 females. Saccostrea palmula infected only by females was the dominant group (60.78%). This mollusk is a new host record for the crab.     

Serum prolactin is associated with apoptosis in men with human immunodeficiency virus infection

We examined the in vivo and in vitro production of prolactin (PRL) in 20 untreated HIV-infected men compared to 14 uninfected men and its association with the cell cycle and apoptosis. Compared to uninfected men, the HIV-infected men had: (i) higher fasting serum bioactive (BIO) PRL; (ii) lower serum immunoreactive (RIA) and BIO-PRL responses to intravenous metoclopramide; (iii) greater BIO-RIA PRL ratio both fasting and during intravenous metoclopramide; (iv) lower percentage of non-stimulated PBMC in the G0/G1 phase, but a higher percentage in the S phase, of the cell cycle with normal response to Concanavalin-A; and (v) higher in vitro production of BIO-PRL by non-stimulated PBMC, which was blocked after Concanavalin-A. Fasting serum BIO-PRL positively correlated with the percent of non-stimulated PBMC in S + G2/M phases. The percentage of apoptotic PBMC negatively correlated with CD4+ T lymphocytes and with the area under the serum RIA-PRL curve, but positively correlated with the area under the curve for the BIO/RIA ratio. These results suggest that in these HIV-infected men: (i) a diminished dopaminergic tone may exist, as an adaptive mechanism attempting to survive; and (ii) BIO-PRL may participate as a cofactor in the stimulation of T-cell proliferation.     

Ligand-dependent structural changes and limited proteolysis of Escherichia coli phosphofructokinase-2

An isoform (rhesus UGT1A01) orthologus to the human UGT1A1 was cloned and sequenced from female rhesus monkey liver cDNA using primers designed from the human nucleotide sequences. Open reading frame analysis of the PCR-generated product encodes a 533-amino acid protein with a proposed 27-residue signal peptide. Nucleotide sequence comparison of rhesus UGT1A01 to other rhesus UGT1A isoforms detected a single-transition mutation at nucleotide 1520 (T-->C), resulting in a neutral F to S substitution at position 507. Rhesus UGT1A01 was greater than 99 and 95% identical to cynomolgus UGT1A01 and human UGT1A1, respectively. The rhesus UGT1A01 was expressed in HK-293 cells for functional analysis. Catalytic activity of UGT1A01 was determined with 7-hydroxy-4-(trifluoromethyl)-coumarin and more specific human UGT1A1 substrates (1-naphthol, beta-estradiol, 17 alpha-ethinylestradiol, and bilirubin). Expression of UGT1A01 protein was also detected by a Western blot utilizing a polyclonal antibody developed against the human UGT1A family.     

Coelomocyte locomotion in the sipunculan Themiste petricola induced by exogenous and endogenous chemoattractants: role of a CD44-like antigen--HA interaction

Cell migration is a key event in the invertebrate immuno-defense system. Microbial products like lipopolysacharide (LPS) and formyl-methyl-leucyl-phenylalanine (fMLP) promote cell recruitment to sites of infection. In mammals, complement activation by factors such as zymosan induces C5a production, which influences leukocyte migration. The endogenous factor hyaluronic acid (HA), an extracellular matrix component, also promotes cell migration through its receptor CD44. We evaluated whether coelomocytes from the sipunculan worm T. petricola migrated towards LPS, fMLP, or zymosan treated plasma (ZTP) and if HA was involved in coelomocyte migration and adhesion. We also evaluated if antibodies specific for mouse HA receptor CD44 inhibited any of the effects induced by HA. Using microchemotaxis chambers we found that coelomocytes migrated towards exogenously and endogenously derived chemoattractants. We also observed that HA was a potent chemotactic signal and that coelomocytes adhered strongly to plates coated with LMW-HA but not with HMW-HA. In addition we found that these HA mediated effects were blocked by the monoclonal antibody IM7 directed to mouse CD44, suggesting that a CD44-like cross-reactive antigen might play a role in HA mediated coelomocyte locomotion.     

Characteristic moisture curves and maximum water content of two crop residues

Limited data are available relating water potential () to crop residue water content (), although this relationship is important to study decomposition and moisture retention of the residue layer in no-till systems and other agricultural situations where residues are used. The objectives of this study were (i) to determine the characteristic moisture curves of rye (Secale cereale L.) and clover residues (Trifolium incarnatum L.), and (ii) to determine residue characteristics that can predict maximum water content of crop residues. Air-dried residues were separated into leaves and stems, cut into 0.5 cm length pieces and saturated with distilled water. Pieces of the drained residues were dried to various water contents in the laboratory and then transferred into thermocouple psychrometer chambers. Characteristic moisture functions of the type = a ^–b, where a and b are empirical constants, were fitted to the data. The characteristic moisture curves had a similar shape to that of a Cecil sandy loam soil used as an example; however, while plant residues were able to retain up to 4.3 g H₂O g^–1, the mineral soil retained only 0.22 g H₂O g^–1. Soluble carbohydrate concentration can be used as a practical index to estimate maximum water content of residues, given the good relationship between both variables (R ² = 0.92).     

Multiple mechanisms generate HLA class I altered phenotypes in laryngeal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21

Major histocompatibility complex (MHC) class I loss or downregulation in cancer cells is a major immune escape route used by a large variety of human tumors to evade anti-tumor immune responses mediated by cytotoxic T lymphocytes. Multiple mechanisms are responsible for such HLA class I alterations. However, the precise frequency of these molecular defects has not been clearly determined in tumors derived from specific tissues. To analyze such defects we aim to define the major HLA class I-altered phenotypes in different tumor types. In this paper we report on HLA class I expression in 70 laryngeal carcinomas. We used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies (mAb), and polymerase chain reaction (PCR) microsatellite amplification of previously selected microsatellite markers (STR) located in chromosome 6 and 15. DNA was obtained from microdissected tumor tissues and surrounding stroma to define the loss of heterozygosity (LOH) associated with chromosome 6p21. Our results showed that LOH in chromosome 6 produced HLA haplotype loss (phenotype II) in 36% of the tumors. In addition, HLA class I total loss (phenotype I) was found in 11%; HLA A or B locus downregulation (phenotype III) was detected in 20%; and HLA class I allelic loss (phenotype IV) in 10% of all cases. We sometimes observed two or more associated mechanisms in the same HLA-altered phenotype, such as LOH and HLA total loss in phenotype I. In only 23% of tumors it was not possible to identify any HLA class I alteration. We conclude that the combination of immunohistological techniques and molecular analysis of tumor DNA obtained from microdissected tumor tissues provides a means for the first time of determining the actual frequency of the major HLA class I-altered phenotypes in laryngeal carcinomas.     

Sleep-disordered breathing, pharyngeal size and soft tissue anatomy in children.

We tested the hypothesis that pharyngeal geometry and soft tissue dimensions correlate with the severity of sleep-disordered breathing. Magnetic resonance images of the pharynx were obtained in 18 awake children, 7-12 yr of age, with obstructive apnea-hypopnea index (OAHI) values ranging from 1.81 to 24.2 events/h. Subjects were divided into low-OAHI (n = 9) and high-OAHI (n = 9) groups [2.8 +/- 0.7 and 13.5 +/- 4.9 (SD) P < 0.001]. The OAHI correlated positively with the size of the tonsils (r2 = 0.42, P = 0.024) and soft palate (r2 = 0.33, P = 0.049) and inversely with the volume of the oropharyx (r2 = 0.42, P = 0.038). The narrowest point in the pharyngeal airway was smaller in the high-compared with the low-OAHI group (4.4 +/- 1.2 vs. 6.0 +/- 1.3 mm; P = 0.024), and this point was in the retropalatal airway in all but two subjects. The airway cross-sectional area (CSA)-airway length relation showed that the high-OAHI group had a narrower retropapatal airway than the low-OAHI group, particularly in the retropalatal region where the soft palate, adenoids, and tonsils overlap (P = 0.001). The "retropalatal air space," which we defined as the ratio of the retropalatal airway CSA to the CSA of the soft palate, correlated inversely with the OAHI (r2 = 0.49, P = 0.001). We conclude that 7- to 12-yr-old children with a narrow retropalatal air space have significantly more apneas and hypopneas during sleep compared with children with relatively unobstructed retropalatal airways.     

Detection of Cyclospora cayetanensis in Wastewater

Cyclospora cayetanensis causes diarrheal disease worldwide without a confirmed mode of transmission. Wastewater was examined for the presence of this organism. Oocysts were detected microscopically, and their identity was confirmed by molecular techniques. These findings verify that current techniques can isolate Cyclospora oocysts and suggest that fecally contaminated water may act as a vehicle of transmission.