Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and cholesterol-HDL in patients with dysplasia of the uterine cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 19 female subjects with cervical dysplasia and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion and HDL- and total cholesterol by enzymatic colorimetry. Plasma mean values of vitamin A and HDL-cholesterol were lower (P less than 0.05 and P less than 0.01, respectively) than in the control group. On the contrary total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the severity of the dysplasia and vitamin A plasma levels.     

Insulin regulates MAP kinase phosphatase-1 induction in Hirc B cells via activation of both extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK)

Previously, we have reported that insulin induces the expression of the dual-specificity tyrosine phosphatase Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and that this may represent a negative feedback mechanism to regulate insulin-stimulated MAP kinase activity. In this work, the mechanism of regulation of MKP-1 expression by insulin was examined, particularly the role of the MAP kinase superfamily. Inhibition of the ERK pathway attenuated insulin-stimulated MKP-1 mRNA expression. Expression of dominant negative molecules of the JNK pathway also abolished insulin-stimulated MKP-1 expression. However, inhibition of p38^MAPK activity by SB202190 had no effect on insulin-stimulated MKP-1 induction. Simultaneous inhibition of the ERK and JNK pathways abolished the ability of insulin to stimulate MKP-1 expression, however, this combined inhibition was neither additive nor synergistic, suggesting these pathways converge to act on a common final effector. In conclusion, induction of MKP-1 mRNA expression in Hirc B cells by insulin requires activation of both the ERK and JNK pathways, but not p38^MAPK.     

Glu-857 moderates K+-dependent stimulation and SCH 28080-dependent inhibition of the gastric H,K-ATPase.

The rabbit H,K-ATPase alpha- and beta-subunits were transiently expressed in HEK293 T cells. The co-expression of the H,K-ATPase alpha- and beta-subunits was essential for the functional H,K-ATPase. The K+-stimulated H,K-ATPase activity of 0.82 +/- 0.2 micromol/mg/h saturated with a K0.5 (KCl) of 0.6 +/- 0.1 mM, whereas the 2-methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080)-inhibited ATPase of 0.62 +/- 0.07 micromol/mg/h saturated with a Ki (SCH 28080) of 1.0 +/- 0.3 microM. Site mutations were introduced at the N,N-dicyclohexylcarbodiimide-reactive residue, Glu-857, to evaluate the role of this residue in ATPase function. Variations in the side chain size and charge of this residue did not inhibit the specific activity of the H,K-ATPase, but reversal of the side chain charge by substitution of Lys or Arg for Glu produced a reciprocal change in the sensitivity of the H,K-ATPase to K+ and SCH 28080. The K0.5 for K+stimulated ATPase was decreased to 0.2 +/-.05 and 0.2 +/-.03 mM, respectively, in Lys-857 and Arg-857 site mutants, whereas the Ki for SCH 28080-dependent inhibition was increased to 6.5 +/- 1.4 and 5.9 +/- 1.5 microM, respectively. The H,K-ATPase kinetics were unaffected by the introduction of Ala at this site, but Leu produced a modest reciprocal effect. These data indicate that Glu-857 is not an essential residue for cation-dependent activity but that the residue influences the kinetics of both K+ and SCH 28080-mediated functions. This finding suggests a possible role of this residue in the conformational equilibrium of the H,K-ATPase.     

Plasma values of vitamin A and its binding proteins (retinol binding proteins and prealbumin), carotene, total cholesterol and HDL-cholesterol in subjects with breast carcinoma

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 33 subjects with breast cancer and compared to those of a group of healthy subjects previously described. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Mean plasma values of vitamin A, carotene and HDL-cholesterol were lower (P less than 0.01) than in the control group, the same applies to the RBP and PA mean levels (P less than 0.05). On the contrary, the mean value of total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related to RBP and PA. No significant statistical correlation was found between clinical stage and vitamin A plasma levels.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and HDL-cholesterol in patients with carcinoma of the cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol of 40 subjects with carcinoma of cervix uteri were determined and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Plasma mean values of carotene, vitamin A, RBP and HDL-cholesterol were lower (P less than 0.01) than in the control group, and the same applies to the PA mean plasma level (P less than 0.05). On the contrary, total cholesterol mean value of the patients resulted to be higher (P less than 0.01) than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the clinical stage and the histological grading and vitamin A plasma levels.     

Quantitative evaluation by computer-based video densitometry of film response to different isotopes and fluorographic treatments

Previous investigations of fluorographic methods have generally reported the minimum level of radioactivity that can be detected for a given length of exposure or the amount of radioactivity needed to reach a given optical density value. Even when the full characteristic curve is given, comparisons with other published values are difficult or, if different lengths of exposures are used, impossible. Computer-based video densitometry was used to evaluate different fluorographic methods. Sections of polyacrylamide gels with uniform distributions of either 35S or 125I radioactivity were embedded with different fluors and used to expose X-ray film for various lengths of time. Due to its compatibility with algorithms used in densitometry, the effectiveness of various treatments was evaluated by fitting relevant parameters to a modified Hill-type equation. The two parameters that were affected by treatments were the amount of radioactivity needed to half-saturate the film (ED50, or the midpoint) and the slope at the midpoint. The relationship between length of exposure and film density was investigated. Since the ED50 values for both 35S and 125I were found to vary in a linear fashion with respect to 1/time, one can determine the ED50 for lengths of exposure not reported. A unique feature of this method of evaluating effectiveness of different treatments, therefore, is that it is possible to compare films that were exposed for different lengths of time. The method of presenting data used here overcomes many of the difficulties in comparing different fluorographic and film treatments, is compatible with the needs of computer-based densitometry, and is suggested as a useful way in which to present the relationship among isotopes, fluors, and films.     

A dry lab for medical engineers?

Post-operatory wound infections are a very uncommon finding after thyroidectomy. For these reasons international guidelines do not routinely recommend systemic antibiotic prophylaxis. The benefits of this antibiotic prophylaxis is not supported by clinical evidence in the literature. We have conducted a multicentric randomized double-blind trial on 500 patients who had undergone thyroidectomy for goitre or thyroid carcinoma. The 500 patients enrolled in the study (mean age 47 years) were randomized in two subgroups of 250 patients. 250 patients were treated with standard antibiotic prophylaxis with sulbactam/ampicillin 1 fl (3 gr.) 30 min before surgery. No antibiotic prophylaxis was instituted in the remainder 250 patients. Our RCT showed that prophylactic antibiotic treatment is not beneficial in patients younger than eighty years old, with no concomitant metabolic, infective and hematologic disease, with no cardiac valvulopathies, not under steroidal or immunosuppressive treatment, and not severely obese. Our study should be regarded only as a preliminary RCT, and should be followed by a study in which a larger number of patients should be enrolled so that statistically significant data can be obtained.     

Packing interactions between transmembrane helices alter ion selectivity of the yeast Golgi Ca2+/Mn2+-ATPase PMR1

PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions.     

Ex vivo and in vivo biological effects of a truncated form of the receptor tyrosine kinase stk when activated by interaction with the friend spleen focus-forming virus envelope glycoprotein or by point mutation

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope protein, gp55, which interacts with the erythropoietin (Epo) receptor complex, causing proliferation and differentiation of erythroid cells in the absence of Epo. Susceptibility to SFFV-induced erythroleukemia is conferred by the Fv-2 gene, which encodes a short form of the receptor tyrosine kinase Stk/Ron (sf-Stk) only in susceptible strains of mice. We recently demonstrated that sf-Stk becomes activated by forming a strong interaction with SFFV gp55. To examine the biological consequences of activated sf-Stk on erythroid cell growth, we prepared retroviral vectors which express sf-Stk, either in conjunction with gp55 or alone in a constitutively activated mutant form, and tested them for their ability to induce Epo-independent erythroid colonies ex vivo and disease in mice. Our data indicate that both gp55-activated sf-Stk and the constitutively activated mutant of sf-Stk induce erythroid cells from Fv-2-susceptible and Fv-2-resistant (sf-Stk null) mice to form Epo-independent colonies. Mutational analysis of sf-Stk indicated that a functional kinase domain and 8 of its 12 tyrosine residues are required for the induction of Epo-independent colonies. Further studies demonstrated that coexpression of SFFV gp55 with sf-Stk significantly extends the half-life of the kinase. When injected into Fv-2-resistant mice, neither the gp55-activated sf-Stk nor the constitutively activated mutant caused erythroleukemia. Surprisingly, both Fv-2-susceptible and -resistant mice injected with the gp55-sf-Stk vector developed clinical signs not previously associated with SFFV-induced disease. We conclude that sf-Stk, activated by either point mutation or interaction with SFFV gp55, is sufficient to induce Epo-independent erythroid colonies from both Fv-2-susceptible and -resistant mice but is unable to cause erythroleukemia in Fv-2-resistant mice.