UDP-glucuronosyltransferases in human intestinal mucosa

While UDP-glucuronosyltransferases (UGTs) are known to be expressed at high levels in human liver, relatively little is known about extrahepatic expression. In the present study, UGT2B family isoforms involved in the glucuronidation of steroid hormones and bile acids have been characterized in microsomes prepared from jejunum, ileum and colon from six human subjects. Glucuronidation of androsterone and testosterone was highly significant and increased from proximal to distal intestine. In contrast, hyodeoxycholic acid was glucuronidated at a low level in jejunum and ileum and activity was barely detectable in colon. No significant glucuronidation of lithocholic acid was found. Small phenols were glucuronidated with much lower activity than found in liver. High levels of UGT protein were detected with polyclonal anti-rat androsterone- and testosterone-UGT antibodies, whereas UGT2B4, a major hepatic hyodeoxycholic acid-specific UGT, was undetectable using a highly specific anti-human UGT2B4 antibody. Screening for RNA expression by RT-PCR confirmed the absence of UGT2B4 and UGT1A6 and showed expression of UGT2B7, a hepatic isoform shown to glucuronidate androsterone, in all intestinal segments. To our knowledge, the presence of functional androsterone and testosterone directed isoforms in human intestine is a novel finding which supports the idea that the intestinal tract functions as a steroid-metabolizing organ and plays a significant role in steroid hormone biotransformation.     

A PHEX gene mutation is responsible for adult-onset vitamin D-resistant hypophosphatemic osteomalacia: evidence that the disorder is not a distinct entity from X-linked hypophosphatemic rickets

Previous investigators described a kindred with an X-linked dominant form of phosphate wasting in which affected children did not have radiographic evidence of rickets, whereas older individuals were progressively disabled by severe bowing. They proposed that this kindred suffered from a distinct disorder that they referred to as adult-onset vitamin D-resistant hypophosphatemic osteomalacia (AVDRR). We recently identified a gene, PHEX, that is responsible for the disorder X-linked hypophosphatemic rickets. To determine whether AVDRR is a distinct form of phosphate wasting, we searched for PHEX mutations in affected members of the original AVDRR kindred. We found that affected individuals have a missense mutation in PHEX exon 16 that results in an amino acid change from leucine to proline in residue 555. Clinical evaluation of individuals from this family indicates that some of these individuals display classic features of X-linked hypophosphatemic rickets, and we were unable to verify progressive bowing in adults. In light of the variability in the clinical spectrum of X-linked hypophosphatemic rickets and the presence of a PHEX mutation in affected members of this kindred, we conclude that there is only one form of X-linked dominant phosphate wasting.     

Delayed-interval delivery in a quadruplet pregnancy after intrauterine death of a partial molar pregnancy and preterm delivery. A case report

BACKGROUND: Delayed-interval delivery is infrequent in twin gestation and more rare in triplet and quadruplet gestation. Coexistence of a triploid pregnancy with a normal fetus has not previously been reported to have resulted in survival of the normal fetus. CASE: A 26-year-old woman, gravida 2, para 0-0-1-0, was diagnosed with a quadruplet pregnancy. At 16 1/2 weeks' gestation she developed preeclampsia and severe hyperemesis. Ultrasound was consistent with partial molar pregnancy in quadruplet D. Quadruplet D died in utero, and the preeclampsia and hyperemesis resolved. At 19 5/7 weeks, spontaneous rupture of the membranes and preterm labor occurred, and quadruplet A, stillborn female weighing 260 g, was delivered. With the use of antibiotic therapy, tocolysis and bed rest, the remaining two fetuses were maintained in utero until 32 6/7 weeks' gestation, when quadruplet B, a 1,470-g female, and quadruplet C, a 1,700-g female, were delivered. CONCLUSION: This was the first reported case of surviving fetuses coexisting with a partial molar pregnancy. This case was also complicated by preterm delivery and successful delayed-interval birth in a quadruplet pregnancy.     

Sentinel lymph node biopsy for melanoma

Serum levels of leptin are decreased in underweight AN patients and increase with weight restoration. To assess the relationship of decreased leptin levels with other hormonal abnormalities in AN and to evaluate the possible role of increasing leptin levels, alone or in combination with other hormones, in the resumption of menses that accompanies weight gain, we studied cross-sectionally sixty-five consecutively enrolled AN patients. Subjects were divided in three groups: (I) underweight and amenorrheic; (II) weight-recovered but still amenorrheic; and (III) weight-recovered and eumenorrheic women. Patients in group I had decreased BMI, serum leptin, estradiol (E2), insulin-like growth factor 1 (IGF-1) and urinary growth hormone (GH) levels and increased sex hormone-binding globulin (SHBG) levels, compared to AN patients in groups II and III. Moreover, although no differences in leptin levels or BMI were observed between amenorrheic and eumenorrheic weight-recovered patients (groups II and III), free E2 and GH levels were higher (P<0.02) in weight-recovered, eumenorrheic women. Thus, it appears that leptin is a necessary, but not a sufficient, factor for the resumption of menses in AN patients.     

Applications of S-layers

The wealth of information existing on the general principle of S-layers has revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from many organisms are capable of recrystallizing as closed monolayers onto solid supports at the air-water interface, on lipid monolayers or onto the surface of liposomes. Particularly their repetitive physicochemical properties down to the subnanometer scale make S-layers unique structures for functionalization of surfaces and interfaces down to the ultimate resolution limit. The following review focuses on selected applications in biotechnology, diagnostics, vaccine development, biomimetic membranes, supramolecular engineering and nanotechnology. Despite progress in the characterization of S-layers and the exploitation of S-layers for the applications described in this chapter, it is clear that the field lags behind others (e.g. enzyme engineering) in applying recent advances in protein engineering. Genetic modification and targeted chemical modification would allow several possibilities including the manipulation of pore permeation properties, the introduction of switches to open and close the pores, and the covalent attachment to surfaces or other macromolecules through defined sites on the S-layer protein. The application of protein engineering to S-layers will require the development of straightforward expression systems, the development of simple assays for assembly and function that are suitable for the rapid screening of numerous mutants and the acquisition of structural information at atomic resolution. Attention should be given to these areas in the coming years.     

Locus coeruleus modulates thalamic nociceptive responses via adrenoceptors

This study investigated the parafascicular (PF) neuronal nociceptive responses and their modulation following electrical stimulation of the locus coeruleus (LC) and intrathecal (i.t.) or intracerebroventricular (i.c.v.) administration of two alpha-adrenoceptor antagonists, the alpha2-antagonist, yohimbine, and the alpha1-antagonist, prazosin. The main results were as follows: (1) the nociceptive evoked discharges in PF neurons were suppressed by preceding stimulation of LC; (2) the suppressive effect of LC stimulation on PF neurons was replaced by a facilitatory effect following pretreatment of i.t. yohimbine in 14 units tested, while i.t. prazosin failed to alter the LC-induced suppression, even when the prazosin dose was doubled; (3) i.c.v. pretreatment with prazosin strengthened the suppressive effect of LC stimulation on PF neurons; (4) i.c.v. norepinephrine (NE) administration induced, in PF neurons, a biphasic response to noxious stimulation; an early, brief (about 10 min) inhibitory effect followed by a late, long-lasting facilitatory effect; and (5) i.c.v. pretreatment of yohimbine or prazosin prevented the inhibitory or facilitatory responses released by NE, respectively. These results provide evidence that: (1) the LC-descending projections exhibit a suppressive effect on nociceptive transmission at the spinal level through alpha2-receptors; and (2) the LC-ascending projections exhibit dual effects, facilitatory and inhibitory, at the medial thalamus (PF) level through alpha1- and alpha2-receptors, respectively.     

Neutralization of conservative charged transmembrane residues in the Na+/glucose cotransporter SGLT1

Our goal was to identify pairs of charged residues in the membrane domains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helices 6-8, express the mutants in oocytes, measure [14C]-alphaMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. alphaMDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D294A/K321A. This suggested that K321 did not form salt bridges with D273 or D294 and that E225 was not involved in salt-bridging. Neutralization of K321 dramatically changed the Na+ uniport and Na+/glucose cotransport kinetics. The maximum rate of uniport in K321A increased 3-5-fold with a decrease in the apparent affinity for Na+ (70 vs 3 mM) and no change in apparent H+ affinity (0.5 microM). The change in Na+ affinity caused a +50 mV shift in the charge/voltage (Q/V) and relaxation time constant (tau)/voltage curves in the presteady-state kinetics. The presteady-state kinetics in H+ remained unchanged. The lower Na+ affinity resulted also in a 200-fold increase in the apparent K0.5 for alphaMDG and phlorizin. Replacements of K321 with alanine, valine, glutamine, arginine, or glutamic acid residues changed the steady-state kinetics in a similar way. Therefore, we suggest that K321 determines, directly or indirectly, (i) the rate and selectivity of SGLT1 uniport activity and (ii) the apparent affinities of SGLT1 for Na+, and indirectly sugar in the cotransport mode.     

Nociceptive c-fos expression in supraspinal areas in avoidance of descending suppression at the spinal relay station

The number and distribution of Fos-like-immunoreactive neurons in different supraspinal brain areas induced by formalin injection into one hindpaw was estimated in rats with transected dorsal half of the spinal cord at the thoracic level in an attempt to avoid most of the descending modulatory actions. The results showed that: (i) after spinal lesion, the peripheral noxious inputs, going up mainly through the ventral spinal cord, elicited a more widespread and densely located Fos-like-immunoreactive neurons in subcortical areas, many of them showed no Fos expression when noxious stimulation was given in rats with intact spinal cord; (ii) at the same time, a small number of subcortical areas, such as the lateral ventroposterior thalamic nucleus and dorsal raphe nucleus, exhibited no significant increase of nociceptive Fos-like immunoreactive neurons after spinal lesion as compared to that with intact spinal cord; and (iii) there appeared a prominent expansion of cortical areas with densely located Fos-like-immunoreactive neurons in spinal-lesioned rats as compared with the limited labelled areas in the control group with intact spinal cord. These results indicate that: (i) in avoiding the spinally descending modulatory mechanisms, more widespread supraspinal and cortical neurons will be recruited and activated in response to the noxious stimulation; and (ii) the descending systems exert differential actions on the spinal targets which project nociceptive signals to different supraspinal regions. The implication of these facts is discussed.     

Imaging and tissue biodistribution of 99mTc-labeled adenovirus knob (serotype 5)

Hepatic sequestration of systemically administered adenoviral vectors reduces the number of viral particles available for delivery to other tissues. The biological basis of this phenomenon was investigated using a new in vivo technique which permitted imaging in real time. Recombinant adenovirus serotype 5 knob (Ad5K) was radiolabeled with the gamma-emitter 99mTc (half-life = 6 h). Scatchard analysis of the 99mTc-Ad5K showed specific, high-affinity binding to U293 cells (Kd = 1.4 +/- 0.5 nM), demonstrating that the radiolabeling process had no effect on receptor binding. In vivo dynamic imaging with an Anger gamma camera revealed that the liver binding followed an exponential rise to maximum, with a measured 100% extraction efficiency. Initially, the liver binding capacity was 3.1 +/- 0.4 micrograms Ad5K, equivalent to approximately 17,000 Ad5K molecules per liver cell. Liver binding was blocked by preincubation of Ad5K with neutralizing anti-Ad5K antibody; a 50% reduction in liver uptake was demonstrated by imaging. Unlabeled Ad5K was more effective in blocking liver uptake of 99mTc-Ad5K, whereas irrelevant unlabeled Ad3K had no effect. Imaging data for the liver uptake studies were in agreement with biodistribution determined by removing and measuring tissues. These data demonstrated that in vivo imaging is a sensitive tool for measuring changes to liver tropism. Similar imaging techniques can be applied to adenovirus vectors to measure specific targeting for gene therapy.