Crystal structure of recombinant triosephosphate isomerase from Bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three-dimensional structures points to the importance of hydrophobic interactions

The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2-phosphoglycolate was determined by X-ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR. Twofold averaging and solvent flattening was applied to improve the quality of the map. Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the "closed" conformation in either subunit. The crystallographic R-factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 A for 248 C alpha atoms and have average temperature factors of 18.9 and 15.9 A2, respectively. In both subunits, the active site Lys 10 adopts an unusual phi, psi combination. A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B. stearothermophilus TIM. Although the ratio Arg/(Arg+Lys) is higher in B. stearothermophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stearothermophilus TIM. Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family. Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B. stearothermophilus enzyme. However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B. stearothermophilus TIM compared to all five other known TIM structures.     

Differential distribution of tyrosine hydroxylase fibers on small and large neurons in layer II of anterior cingulate cortex of schizophrenic brain

A series of recent postmortem investigations of the anterior cingulate cortex in schizophrenic brain have suggested that there may be a loss and/or impairment of inhibitory interneurons in layer II. It has been postulated that changes of this type could secondarily result in a relative increase of dopaminergic inputs to GABAergic interneurons. To test this hypothesis, an immunoperoxidase technique was developed to extensively and reliably visualize tyrosine hydroxylase-immunoreactive (TH-IR) varicose fibers in human postmortem cortex. This method has been applied to the anterior cingulate (ACCx; Brodmann area 24) and prefrontal (PFCx: Brodmann area 10) cortices from a cohort of 15 normal control and 10 schizophrenic cases. The number of TH-IR varicosities in contact with large neurons (LN), small neurons (SN), and neuropil (NPL) was blindly analyzed using a computer-assisted microscopic technique. There was no significant difference in density of TH-IR varicosities in apposition with either LN or SN cell bodies observed in either ACCx or PFCx of schizophrenics when compared to normal controls. The density of varicosities was significantly reduced in NPL of layers V and VI in ACCx, but 2 neuroleptic-free cases did not show this change, suggesting that these decreases of TH-IR varicosities may be related to antipsychotic effects on corticostriatal projection cells in this region. When the density of TH-IR varicosities on SNs was compared to that observed on LNs, both groups showed a higher density on SNs. In ACCx, this pattern was much more pronounced for the schizophrenic group, particularly in layer II where the density on SNs was three times higher than that for LNs (P = 0.01). Unlike the changes in layer V, this latter change in layer II showed no relationship to neuroleptic exposure. There was a positive correlation between age and the density of TH-IR varicosities on SNs of layer II in ACCx; however, the patients were younger than the controls and would have been expected to show a lower density, rather than a higher one, if age considerations had accounted for the group differences. Overall, the results reported here suggest that there are no gross differences in the distribution of TH-IR varicosities in various laminae of the dorsolateral prefrontal cortex. In the anterior cingulate region, however, there may be a significant shift in the distribution of TH-IR varicosities from large neurons to small neurons that occurs selectively in layer II of schizophrenic subjects. Using size criteria, the majority of small neurons are likely nonpyramidal, while the majority of large neurons are predominantly pyramidal in nature. Taken together with other accumulating evidence of preferential abnormalities in this lamina of the cingulate region, the findings reported here are consistent with a model of schizophrenia in which a subtle "miswiring" of ventral tegmental inputs may result in a relative, though not absolute, hyperdopaminergic state with respect to an impaired population of GABAergic interneurons.     

Natural history of coronary atherosclerosis using quantitative angiography in men, and implications for clinical trials of coronary regression. The Harvard Atherosclerosis Reversibility Project Study Group

Previous studies of the natural history of coronary disease generally relied on estimates of percent stenosis derived from visual assessment of the coronary angiogram. In a study of 26 patients, serial quantitative angiography was performed 3 years apart to determine changes in both absolute measurements of the luminal diameter and relative percent stenosis. Initially, the mean minimal diameter of 74 coronary obstructions was 1.94 +/- 0.09 mm, the mean "normal" reference diameter was 3.06 +/- 0.11 mm, and the mean percent stenosis was 37%. At follow-up, there was a mild reduction of 0.12 +/- 0.04 mm (6%) in the minimal diameter (p < 0.005), and an increase in percent stenosis to 39% (p = 0.03). The average diameter of 85 arterial segments without a focal obstruction either initially or at follow-up showed mild but significant progression (-0.11 +/- 0.04 mm; p = 0.02). Using a minimal change of 0.27 mm in arterial diameter as a categoric variable, progression occurred in 26% of 74 arterial segments, no significant change in 65%, and regression in 9%. The only significant determinant of disease progression was the initial severity of disease. Obstructed arteries with a larger initial minimal diameter and presumably milder disease progressed more rapidly than did those with a smaller diameter (r = -0.42; p = 0.0002). There was no effect of age on the rate of progression (r = 0.02; p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance liquid chromatographic method for resolving the enantiomers of mexiletine and two major metabolites isolated from microbial fermentation media

PURPOSE: This Phase I trial tests the ability of a new hyperthermia device, the transrectal ultrasound probe, to heat the prostate gland, and evaluates the toxicity of transrectal ultrasound hyperthermia (TRUSH) given with concurrent standard external beam irradiation in the treatment of locally-advanced adenocarcinoma of the prostate. METHODS AND MATERIALS: Between June, 1990 and August, 1991, 14 patients with American Urological Society Stage C2 or D1 adenocarcinoma of the prostate were treated with TRUSH concurrently with standard external beam radiotherapy to the prostate. Twenty-two heat treatments were delivered in 14 patients; 8 patients received two TRUSH procedures, each separated by 1 week. Patient age ranged between 53-86 (mean: 72) years. Three patients had well-, 6 patients had moderately-, and 5 patients had poorly-differentiated adenocarcinoma of the prostate. Karnofsky status ranged from 70-90. Standard radiotherapy to the prostate and periprostatic tissues was delivered using a four-field approach with 1.8-2 Gy daily fractions delivered 5 x/week to a total dose of 67-70 Gy calculated to the minimum tumor volume. TRUSH was delivered after transperineal placement of multipoint thermometry probes by a urologist, under transrectal ultrasound guidance. Two to three thermocouple probes containing seven sensors each were placed in the prostate in an attempt to sample temperatures throughout the gland. The sensor depth from the rectal wall ranged from 5-25 mm. RESULTS: Thirty-six percent of all sensors were heated above 42.5 degrees C averaged over 30 min; and all patients had at least some sensors within the prostate heated to temperatures > or = 42.5 degrees C. The average temperature of all sensors of all sensors (T(ave) +/- s.d.) over all treatments, however, was only 41.9 degrees C +/- 0.9 degrees C over 30 min. The maximum temperature for normal tissues outside the gland was 41.1 degrees C +/- 1.3 degrees C. Treatments have been well-tolerated with few complications. Tolerance has been "good" in 17/22, "fair" in 3/22, and "treatment limiting" in 2/22 treatments secondary to position intolerance and/or pain. There has been one episode of hypotension related to narcotic administration and three episodes of rapidly resolving pain during hyperthermia treatment. Mild hematuria has occurred in 5/22, and moderate hematuria has occurred in 2/22 transperineal thermometer catheter placements. CONCLUSION: In conclusion, TRUSH is well-tolerated and has great potential for consistently heating the prostate gland. We anticipate that further equipment modifications will improve our ability to heat the entire prostate to temperatures > 42.5 degrees C.     

Infant feeding practices among nursing personnel in Malaysia

Concerned by the alarming decline in breast feeding in the urban and rural areas of Malaysia, we conducted a critical review of infant feeding practices among nursing personnel from representative centres using questionnaires. It was found that although 75% of mothers breast-fed their babies at birth only 19% did so at 2 months and 5% at 6 months respectively. Chinese mothers initiated breast feeding less frequently compared with Indian or Malay mothers. The prevalence of breast feeding was higher among lower category nurses, lower income groups and those from health centres. Decision for breast feeding was based in most instances on conviction derived from reading, lectures or advice from relatives. The vast majority of mothers listed "work' as the main reason for termination of breast feeding followed by "insufficient breast milk' and satisfactory past experience with bottle feeding. The ramifications of these findings and measures to improve the prevalence and duration of breast feeding are discussed.     

Intervention for improving the coproparasitological diagnosis in the Province of Las Tunas, Cuba, 1993-1994

Control of quality of coproparasitological diagnosis was carried out in the Province "Las Tunas", between January and March 1993, It was found that 72% of coproparasitological microscopists had deficiencies related to the diagnosis, due to several causes: The microscopists did not receive any periodical training, as they did not participate at the parasitological section; the microscopists had no manual of reference; they had less than ten years of experience; they did not know the concentration technics that were established in the country. Between July 1993 and January 1994 two Medical Parasitology Courses were imparted. In July 1994 another quality control was performed. 80% of evaluated microscopists obtained good results. Through this study was established the National Parasitology Quality System Control that supports the National Intestinal Parasites Control Program.     

Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis

We show that a single clinical isolate of the human opportunistic pathogen Pseudomonas aeruginosa (strain PA14), which previously was shown to be pathogenic in mice and plants, also kills Caenorhabditis elegans. The rate of PA14-mediated killing of C. elegans depends on the composition of the agar medium on which PA14 is grown. When PA14 is grown on minimal medium, killing occurs over the course of several days and is referred to as "slow" killing. When PA14 is grown on high-osmolarity medium, killing occurs over the course of several hours and is referred to as "fast" killing. Several lines of evidence, including the fact that heat-killed bacteria are still capable of fast but not slow killing of C. elegans, indicate that fast and slow killing occur by distinct mechanisms. Slow killing involves an infection-like process and correlates with the accumulation of PA14 within worm intestines. Among 10 PA14 virulence-related mutants that had been shown previously to affect pathogenicity in plants and mice, 6 were less effective in killing C. elegans under both fast- and slow-killing conditions, indicating a high degree of commonalty among the P. aeruginosa factors required for pathogenicity in disparate eukaryotic hosts. Thus, we show that a C. elegans pathogenicity model that is genetically tractable from the perspectives of both host and pathogen can be used to model mammalian bacterial pathogenesis.     

Muscle quality. II. Effects Of strength training in 65- to 75-yr-old men and women

To determine the effects of strength training (ST) on muscle quality (MQ, strength/muscle volume of the trained muscle group), 12 healthy older men (69 +/- 3 yr, range 65-75 yr) and 11 healthy older women (68 +/- 3 yr, range 65-73 yr) were studied before and after a unilateral leg ST program. After a warm-up set, four sets of heavy-resistance knee extensor ST exercise were performed 3 days/wk for 9 wk on the Keiser K-300 leg extension machine. The men exhibited greater absolute increases in the knee extension one-repetition maximum (1-RM) strength test (75 +/- 2 and 94 +/- 3 kg before and after training, respectively) and in quadriceps muscle volume measured by magnetic resonance imaging (1,753 +/- 44 and 1, 955 +/- 43 cm3) than the women (42 +/- 2 and 55 +/- 3 kg for the 1-RM test and 1,125 +/- 53 vs. 1,261 +/- 65 cm3 for quadriceps muscle volume before and after training, respectively, in women; both P < 0.05). However, percent increases were similar for men and women in the 1-RM test (27 and 29% for men and women, respectively), muscle volume (12% for both), and MQ (14 and 16% for men and women, respectively). Significant increases in MQ were observed in both groups in the trained leg (both P < 0.05) and in the 1-RM test for the untrained leg (both P < 0.05), but no significant differences were observed between groups, suggesting neuromuscular adaptations in both gender groups. Thus, although older men appear to have a greater capacity for absolute strength and muscle mass gains than older women in response to ST, the relative contribution of neuromuscular and hypertrophic factors to the increase in strength appears to be similar between genders.     

Direct photoaffinity labeling of the Kir6.2 subunit of the ATP-sensitive K+ channel by 8-azido-ATP

ATP-sensitive potassium channels are under complex regulation by intracellular ATP and ADP. The potentiating effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We determined whether ATP directly interacts with a binding site on the Kir6.2 subunit to mediate channel inhibition by analyzing binding of a photoaffinity analog of ATP (8-azido-[gamma-32P]ATP) to membranes from COS-7 cells transiently expressing Kir6.2. We demonstrate that Kir6.2 can be directly labeled by 8-azido-[gamma-32P]ATP but that the related subunit Kir4.1, which is not inhibited by ATP, is not labeled. Photoaffinity labeling of Kir6.2 is reduced by approximately 50% with 100 microM ATP. In addition, mutations in the NH2 terminus (R50G) and the COOH terminus (K185Q) of Kir6.2, which have both been shown to reduce the inhibitory effect of ATP upon Kir6.2 channel activity, reduced photoaffinity labeling by >50%. These results demonstrate that ATP binds directly to Kir6.2 and that both the NH2- and COOH-terminal intracellular domains may influence ATP binding.     

Sexual function following high energy microwave thermotherapy: results of a randomized controlled study comparing transurethral microwave thermotherapy to transurethral prostatic resection

PURPOSE: We evaluate changes in sexual function in patients treated with high energy transurethral microwave thermotherapy compared to transurethral resection of the prostate. MATERIALS AND METHODS: A total of 147 patients randomized to undergo transurethral microwave thermotherapy or transurethral resection of the prostate were asked to complete a self-administered questionnaire evaluating sexual function before, and 3 and 12 months after treatment. The questionnaire dealt with such items as social status, libido, quality of erection, ejaculation and overall satisfaction of sexual functioning. RESULTS: There was a statistically significant improvement of micturition in both groups. The improvement in the transurethral prostatic resection group was significantly better than in the transurethral microwave thermotherapy group. Antegrade ejaculation occurred at 3 months following treatment in 27% of the transurethral prostatic resection group compared to 74% of the transurethral microwave thermotherapy group and at 1 year in 37 and 67%, respectively. Significantly more patients undergoing transurethral prostatic resection (36%) had changes in sexual function compared to the transurethral microwave thermotherapy group (17%). The transurethral microwave thermotherapy group was more satisfied with the sex life. Of these patients 55% graded sex as very satisfying compared to 21% in the transurethral prostatic resection group. The severity of symptoms was not correlated with sexual function in this study. In general, older patients had sexual dysfunction more often, while younger patients had pain during sexual activities more frequently. CONCLUSIONS: Although clinically less effective, high energy transurethral microwave thermotherapy is a better therapeutic option than surgery for patients who want to preserve sexual function. In particular ejaculation is often preserved after transurethral microwave thermotherapy while there is significant deterioration following transurethral prostatic resection. In general, older patients have greater sexual dysfunction.