Differential molar heat capacities to test ideal solubility estimations

PURPOSE: Calculation of the ideal solubility of a crystalline solute in a liquid solvent requires knowledge of the difference in the molar heat capacity at constant pressure of the solid and the supercooled liquid forms of the solute, delta Cp. Since this parameter is not usually known, two assumptions have been used to simplify the expression. The first is that delta Cp can be considered equal to zero; the alternate assumption is that the molar entropy of fusion, delta Sf, is an estimate of delta Cp. Reports claiming the superiority of one assumption over the other, on the basis of calculations done using experimentally determined parameters, have appeared in the literature. The validity of the assumptions in predicting the ideal solubility of five structurally unrelated compounds of pharmaceutical interest, with melting points in the range 420 to 470 K, was evaluated in this study. METHODS: Solid and liquid heat capacities of each compound near its melting point were determined using differential scanning calorimetry. Linear equations describing the heat capacities were extrapolated to the melting point to generate the differential molar heat capacity. RESULTS: Linear data were obtained for both crystal and liquid heat capacities of sample and test compounds. For each sample, ideal solubility at 298 K was calculated and compared to the two estimates generated using literature equations based on the differential molar heat capacity assumptions. CONCLUSIONS: For the compounds studied, delta Cp was not negligible and was closer to delta Sf than to zero. However, neither of the two assumptions was valid for accurately estimating the ideal solubility as given by the full equation.     

Spatially singlemode broad-area semiconductor laser with planar external cavity

A broad-area laser diode combined with a planar external waveguide cavity operates in the fundamental mode and reshapes the output emission into a circular 15/spl deg/ beam. A 500 /spl mu/m-long by 40 /spl mu/m-wide laser diode with uncoated facets coupled with the uncoated ModeReShaper (MRS) planar chip has a coupling efficiency of /spl sim/40% and stabilised the fundamental mode at drive currents up to three-times threshold.     

Borrelia burgdorferi sensu lato in Russia and neighbouring countries: high incidence of mixed isolates

A total of 365 isolates of Borrelia burgdorferi sensu lato from 12 major administrative territories of Russia (from St. Petersburg in the west to South Sakhalin in the east) and from the Czech Republic, Estonia, Lithuania, Byelorussia, Moldavia, Ukraine and Kirghizia were identified by analysis of restriction polymorphism of ribosomal rrf-rrl spacer amplicons. The isolates were obtained mainly from ixodes persulcatus and I. ricinus ticks. Other sources included small mammals, human patients and I. trianguliceps ticks. The results showed that B. garinii (two variants) together with B. afzelii circulated throughout the territories studied. The distribution of the variant NT29 of the species B. garinii, the most frequently isolated, was associated with that of I. persulcatus ticks. B. burgdorferi sensu stricto, and the species B. valaisiana and B. lusitaniae (formerly the genomospecies VS116 and PotiB2, respectively) were isolated only from I. ricinus ticks in the western part of the studied territories. None of these three species were found in 327 isolates from Russia where I. persulcatus is the most frequently distributed vector. This work also provides evidence for a high incidence of mixed Borrelia infections within vectors and hosts (9.3% of isolates were mixtures of Borrelia species). A detailed analysis of Borrelia species distribution over the territories studied is presented.     

A novel functional human eukaryotic translation initiation factor 4G

Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.     

PARODS--a study of parallel algorithms for ordering DNA sequences

Based on assumptions about the pathophysiology of egg-related lesions in the lower reproductive tract, putative indirect disease markers were investigated in vaginal fluids from 54 Malawi adolescent girls and women infected with S. haematobium. These women received a careful gynecological examination during which biopsies were taken from the cervix, and, if present, also from suspicious lesions in the vagina and the vulva. If the biopsies, either in wet crushed preparations or in histological sections, contained eggs the patients were considered to have female genital schistosomiasis (FGS; n = 33). The remainder (n = 21) were classified as having urinary schistosomiasis only. Eosinophil cationic protein (ECP), a cytotoxic granule protein of eosinophils, neopterin, a second messenger molecule generated during the activation of macrophages, and IgA as an indicator of local B-cell activation were quantitatively determined in vaginal fluid. To clarify the origin of ECP, this protein was also looked for in histological sections by an immunohistochemical method. In order to explore whether such disease markers can be detected after absorption to a tampon-like material, ECP and IgA were also assessed after elution from a non-porous, polypropylene fibre web impregnated with vaginal fluid. The concentration of ECP in vaginal fluid and the degree of immunohistochemical staining in histological sections were significantly higher in patients with FGS than in women with urinary schistosomiasis only. The amount of ECP detected in histological sections correlated to the number of eggs/mm2 of compressed genital tissue (rho = 0.36, P = 0.02), and the concentration of ECP in vaginal fluid correlated to the concentration of neopterin as well as to that of IgA (rho = 0.52, P = 0.004 and rho = 0.37, P = 0.02, respectively). Median neopterin concentration in vaginal fluid was also higher in the FGS group, but the difference was not statistically significant. ECP could also be detected in eluates from impregnated fibre webs, but the concentration was approximately one power of 10 less than in the original vaginal fluid. These results demonstrate that indicators of immunological mechanisms related to the egg-granuloma might be useful as indirect disease markers for women with FGS if assessed in vaginal washings or swab eluates.     

Development of the hypothalamic vasopressin system and nephrons in Meriones shawi during ontogenesis

This study has evaluated the development of the hypothalamic vasopressin system and nephrons of the kidney in desert rodents, Meriones shawi, which effectively retain water by excretion of highly concentrated urine. The vasopressin system was studied immunocytochemically at the 18th fetal day, at the 2nd, 13th, 27th postnatal days and in adulthood. The kidneys were investigated at the 2nd, 13th postnatal days and in adulthood using microdissection technique. Occasional vasopressin-immunoreactive neurons were observed as early as the 18th fetal day, only in the paraventricular nucleus. From the 2nd postnatal day onwards, vasopressin neurons increased progressively in number, being mainly concentrated in the supraoptic and paraventricular nuclei, as well as in the ventral retrochiasmatic region. Transient neuronal populations were also observed at the 13th postnatal day in the lateral preoptic area and anterior hypothalamic nucleus. Apart from the neurons, the glandular cells of the tuberal lobe showed immunostaining from the 18th fetal day, the first age studied, until the 13th postnatal day. The fibers of differentiating vasopressin neurons grew towards the circumventricular/neurohemal organs, terminating in the organum vasculosum of the lamina terminalis and the lateral ventricles as early as the 18th fetal day, as well as the third ventricle, the posterior lobe and the external zone of the median eminence between the 2nd and 13th postnatal days. The kidney in 2-day-old Meriones comprised nephrons at different stages of development from an S-shaped body to well-differentiated nephrons. At the 13th postnatal day, as in adulthood, the nephrons were well differentiated and characterized by long, thin loops descending to different levels of papilla. Thus, according to our morphological data the hypothalamic vasopressin neurons and nephrons in the kidney of Meriones reach the definitive state by the end of the 2nd postnatal week.     

Variations in dietary fat and cholesterol intakes modify antioxidant status of SHR and WKY rats

The effects of varying dietary fat saturation [butter (B), beef tallow (BT)] or polyunsaturation [(n-6) soybean oil (SBO), (n-3) menhaden oil (MO)] and cholesterol content (0.05 and 0.5 g/100 g) on systolic blood pressure (SBP), plasma lipids and tissue antioxidant status were investigated in 14-wk-old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. Varying dietary fat composition for 9 wk had no influence on SBP in either SHR or WKY rats. Rats fed MO diets exhibited smaller (P < 0.05) body weight gains, lower (P < 0.05) feed efficiency ratios and lower (P < 0.05) plasma cholesterol concentrations than those fed the B, BT and SBO diets. Significant (P < 0.05) interactions for animal strain x cholesterol intake and animal strain x fat source were noted for serum cholesterol concentrations. SHR exhibited higher (P < 0.05) RBC and liver catalase (CAT), and heart and liver superoxide dismutase (SOD) activities similar to those of WKY rats. The lower (P <0.01) RBC, heart and liver glutathione peroxidase (GSH-Px) activities observed in SHR coincided with higher (P <0.01) glutathione reductase (GSSG-Red), compared with WKY rats. Dietary cholesterol intake had no effect on RBC, heart and liver total sulfhydryl concentration or GSH-Px activities, but increased (P <0. 001) liver GSSG-Red. Feeding MO resulted in lower (P <0.001) RBC and heart GSH-Px activities. In contrast, feeding B and BT resulted in lower GSH-Px in liver. The significant (P < 0.01) animal strain x fat source interaction obtained for liver GSH-Px activity indicated that SHR responded differently to polyunsaturated fatty acid feeding than their WKY counterparts. Diet-induced changes in tissue antioxidant status were tissue specific and did not affect the development of hypertension in SHR.     

Probing the role of cysteine residues in the EcoP15I DNA methyltransferase

Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.     

Adenylosuccinate synthetase of the yeast Saccharomyces cerevisiae: purification and properties

Motor and sensory nerve conduction velocities (MNCV and SNCV) were reduced in the sciatic nerve of rats after 4 weeks of untreated streptozotocin-induced diabetes, and declined further during the following 4 weeks. Treating diabetic rats with the novel peptide HP228 had no effect on the decline of MNCV after the first 4 weeks of diabetes but attenuated the decline in SNCV. HP228 treatment also prevented any further decline in MNCV or SNCV between weeks 4 and 8 of diabetes. Consequently, at the conclusion of the study, the nerve conduction velocities (NCVs) in treated rats were significantly (both P < .001) higher than in untreated diabetic rats. Reduced nerve homogenate Na+,K+-adenosine triphosphatase (ATPase) activity in diabetic rats was significantly (P < .05) increased by HP228 but remained significantly (P < .05) lower than in untreated controls. HP228 treatment also reduced nerve Na+,K+-ATPase activity of control rats compared with untreated controls (P < .05). There was no effect of HP228 on the hyperglycemia, nerve polyol accumulation, myo-inositol depletion, reduced nerve laser Doppler blood flow, thermal hypoalgesia, or reduced mean axonal caliber in diabetic rats or on any of these parameters in control rats. These data demonstrate that a novel peptide may protect against the slowing of nerve conduction in prolonged diabetes and that the mechanism of action is unrelated to aldose reductase inhibition, prevention of nerve ischemia, or axonal atrophy. HP228 may prove a potential therapeutic agent for the treatment of prolonged diabetic neuropathy.     

Accumulation of co-localised unesterified cholesterol and neutral lipids within vacuolised elastin fibres in athero-prone areas of the human aorta

To investigate whether there are alterations of elastin fibres in the arterial intima at the pre-atherosclerotic stage, grossly normal areas of human thoracic aorta were taken soon after death from 13 healthy trauma victims whose ages ranged from 16 to 40 years. Two areas were compared: atherosclerosis-prone (AP) areas localised to the dorsal aspect of the aorta along the rows of intercostal branch origins, and atherosclerosis-resistant (AR) areas from the ventral aorta. Electron microscopic analysis combined with cytochemical staining was applied. Unesterified cholesterol was identified using the filipin-staining technique while neutral lipids were visualised by the OTO-technique. Intimal features were studied by combining the filipin-staining and the OTO-technique. Electron microscopical examination showed that in both AR and AP areas, some elastin fibres in the intima were vacuolised. Unesterified cholesterol was found to be predominantly localised in the musculoelastic layer, in particular, inside the vacuolised elastin fibres. This localisation was seen in all 13 AP areas studied in contrast to the AR areas where it was observed in only four of 13 aortas studied (P < 0.0005, chi2-test). Accumulation of neutral lipids inside vacuolised elastin fibres was found in five out of 13 AP areas but was not observed in any of the AR areas (P=0.01, chi2). A combination of the filipin-staining and OTO-techniques showed that some deposits of neutral lipids and unesterified cholesterol within vacuolised elastin fibres were independently located from each other, but more frequently, neutral lipids were co-located with unesterified cholesterol. The present observations indicate a difference between AP and AR intimal areas which, in particular, relates to the structure of elastin fibres in the musculoelastic layer. The observations suggest that alterations of the extracellular matrix are involved in the trapping and retention of cholesterol and neutral lipids within the intima at an early stage in the development of atherosclerotic lesions.