Hypoglycaemia is reduced with use of inhaled Technosphere® Insulin relative to insulin aspart in type 1 diabetes mellitus.

AIM: To evaluate the effect of final HbA1c levels on the incidences of hypoglycaemia in participants with type 1 diabetes treated with inhaled Technosphere® Insulin or subcutaneous insulin aspart, reported in alignment with the International Hypoglycaemia Study Group recommendations. METHODS: In the randomized, phase 3, multicentre AFFINITY-1 study, adults (N = 375) who had type 1 diabetes for ≥ 12 months and an HbA1c level of 58-86 mmol/mol (7.5-10.0%) were randomized to receive basal insulin plus either inhaled Technosphere Insulin or subcutaneous insulin aspart. This was a post-hoc regression analysis on a subset (N = 279) of the randomized AFFINITY-1 cohort for whom baseline and end-of-treatment HbA1c values were reported. Primary outcome measures were incidence and event rates for levels 1, 2 and 3 hypoglycaemia, respectively defined as blood glucose levels of ≤ 3.9 mmol/l, < 3.0 mmol/l or requiring external assistance for recovery. RESULTS: Participants treated with Technosphere Insulin experienced statistically significantly fewer level 1 and 2 hypoglycaemic events and a lower incidence of level 3 hypoglycaemia than participants treated with insulin aspart. The lower rate of hypoglycaemia with Technosphere Insulin was observed across the range of end-of-treatment HbA1c levels. Technosphere Insulin was associated with higher rates of hypoglycaemia 30-60 min after meals, but significantly lower rates 2-6 h after meals. CONCLUSIONS: Participants using Technosphere Insulin experienced clinically non-inferior glycaemic control and lower hypoglycaemia rates across a range of HbA1c levels compared with participants receiving insulin aspart. ClinicalTrials.gov: NCT01445951.

The role of intelligence in phenylketonuria: a review of research and management.

Phenylketonuria (PKU) results in profound intellectual disability in untreated individuals and more subtle cognitive deficits in individuals treated early and continuously. The assessment of intellectual functioning has been an important outcome variable and the focus of extensive research. Since the implementation of neonatal PKU screening programs in the 1960s, research on intellectual functioning in individuals with PKU has played a significant and positive role in guiding therapy and improving results. This is a literature review examining the relationship between intellectual outcome and treatment parameters including initiation of treatment, duration of treatment, and blood phenylalanine (Phe) levels from infancy through adulthood. While current PKU treatment practices have eliminated severe neurological and cognitive impairment, evidence suggests that intellectual functioning, although typically within the average range when PKU is treated early and continuously, may not be maximized under the current definition of well-controlled PKU, which is based on blood Phe levels. Future research assessing intellectual and neurocognitive outcome in PKU will enhance the development of new treatment strategies.

Sensitivity to ionising radiation of transformed human cells containing mutant ras genes.

Measurement of colony forming ability following exposure to gamma-rays has been performed on human retinoblasts transformed with either adenovirus 5 or 12 early region 1 DNA, adenovirus early region 1A plus activated N- or H-ras DNA or SV40 DNA. In contrast to recently reported results (M.D. Sklar, 1988, Science, 239, 645-647), we found no general correlation between transformation with activated ras and increased radiation resistance. Similarly, there was no correlation between D0 values and the level of expression of ras p21 in transformed human retinoblasts as determined by liquid competition assay. Indeed, cell lines with very similar D0 values had ras contents varying by up to one hundred fold. Cell lines transformed with SV40 DNA were generally less sensitive to ionising radiation than adenovirus and/or ras transformants, but even so the variation in sensitivity within these encompassed the whole spectrum of values obtained for the ras transformants. It may be interesting to note, however, that two out of the three ras transformants which were least sensitive to gamma-rays were cell lines expressing the highest levels of p21.

Conformational changes occurring in N-ras p21 in response to binding of guanine nucleotide and metal ions probed by proteolysis performed under controlled conditions.

Variations in susceptibility to proteolysis by trypsin and chymotrypsin have been used as indicators of conformational changes taking place in N-ras p21 in response to ligand binding. It has been observed that changes occur in undenatured protein, rendering it more resistant to degradation, in the presence of divalent cations such as Mg2+ and Ca2+ (suggesting direct binding of metals to the polypeptide) and even more markedly in the presence of GDP and/or Mg2+ GDP. Monovalent cations (Na+ or K+) cannot substitute for Mg2+ or Ca2+. Some capacity to bind guanine nucleotide is also retained by p21 treated with 7 M urea, as evidenced by increased resistance to proteolytic degradation, but the ability to bind divalent cations is irreversibly lost following denaturation. Protein prepared under denaturing conditions from a eukaryotic source, however, never regains the resistance to proteolysis shown by the bacterial p21 indicating irreversible changes in secondary and tertiary structure produced under these conditions.

Attention deficit disorder and the mediating effect of age on academic and behavioral variables.

Seventy-nine children, independently diagnosed as having attention deficit disorder (ADD) with or without hyperactivity and ranging in age from 6 years, 0 months, to 12 years, 11 months, were administered the Wechsler Intelligence Scale for Children-Revised (WISC-R), the Wide Range Achievement Test (WRAT), and the Child Behavior Checklist (CBC). Multiple regression analyses indicated a significant multiple correlation between age and achievement variables (R = 0.42, p = 0.003) and age and behavioral (CBC) variables (R = 0.55, p = 0.01). Post hoc analyses indicated that much of this variance was accounted for by a significant negative association between age and Full Scale IQ-WRAT Arithmetic difference scores, and significant positive correlations between age and scores on the Social Withdrawal and Uncommunicative scales from the CBC. A nonparametric chi-square analysis indicated that older children with ADD are significantly more likely (p = 0.037) than younger ADD children to have a discrepancy of 15 or more points between IQ and math achievement scores. Results suggest that older ADD children are more likely than younger ADD children to experience academic and socioemotional difficulties.

The relationship between continuous performance tasks and neuropsychological tests in children with attention-deficit hyperactivity disorder.

We examined the relationship between measures of sustained attention and impulsivity, as obtained by computerized continuous performance tasks of the Gordon Diagnostic System and a battery of intellectual, achievement, and neuropsychological tests. Subjects were 119 boys (between the ages of 6 yr., 0 mo. and 12 yr., 11 mo.), diagnosed with Attention-deficit Hyperactivity Disorder, using DSM-III or DSM-III--R criteria. Only two measures, the number of correct responses for Vigilance and Distractibility tasks, correlated consistently with other measures (e.g., intellectual measures, the WRAT--R Arithmetic subtest, Beery Test of Visual and Motor Integration, and various sensory-motor variables from the Halstead-Reitan Neuropsychological Battery). The results suggest a unique contribution of continuous performance tasks in the measurement of attention, in a population of children with ADHD, which is not assessed by more traditional tests.

Enhanced expression of p53 in human cells infected with mutant adenoviruses.

The expression of p53 in human cells infected with wild-type (wt) and mutant adenoviruses has been examined. With wt Ad5 and Ad12, and Ad12 viruses carrying lesions in the E1A or the 19K E1B genes, there was a pronounced decrease in level of p53 during the course of infection. However, when cells were infected with mutant viruses which did not express the larger E1B proteins (Ad12 dl620 and in602 and Ad5 dl338 and pm381) the concentration of p53 increased markedly to levels comparable to those seen in adenovirus transformed cells. This increase in level of p53 correlated closely with the advent of E1A expression. Infection with Ad5 dl355 (which carries a lesion in the E4 gene) also resulted in an increase in p53 expression. We have concluded that these results can be explained on the basis of the known ability of E1A to stabilize p53 and of the E1B 58K:E4 34K protein complex to regulate mRNA metabolism during viral infection, although large increases in expression of p53 or any other cellular proteins following infection with these viruses have not previously been reported. It is suggested that the high concentrations of p53 could explain the inability of 54K and 58K negative mutants to transform cells in culture. In cells infected with dl355 both the Ad5 E1B 58K protein and p53 were located in the nucleus. It was shown by coimmunoprecipitation experiments that these proteins formed a complex which was stable in the presence of high concentrations of NaCl. The interaction of the Ad12 E1B 54K protein and p53 has also been demonstrated in Ad12 E1-transformed cells by immunoprecipitation experiments. These data, taken in conjunction with previous results, have suggested that increased expression of p53 is unrelated to complex formation with the larger Ad E1B proteins.

Effects of MK-801 on recovery from sensorimotor cortex lesions.

Histologic evidence suggests that drugs acting as noncompetitive antagonists at the N-methyl-D-aspartate receptor can have beneficial or pathologic effects on central nervous system neurons. In the present experiments we examined the effects of MK-801 on recovery of behavioral function after unilateral lesions in the rat somatic sensorimotor cortex. In the first experiment, rats with unilateral sensorimotor cortex lesions were given either MK-801 (1 mg/kg) or saline 12-16 hours after surgery. Additional injections were given on postoperative days 2, 4, and 6. Behavioral tests measured somatosensory asymmetries (i.e., bilateral tactile stimulation tests) and forelimb placing. After creation of sensorimotor cortex lesions, rats showed an ipsilateral somatosensory bias and an impairment in placing the contralateral forelimb. Rats treated with MK-801 recovered slightly faster than saline-treated animals as measured by a bilateral tactile stimulation test (p less than 0.05). In contrast, there was no significant difference between the groups in the recovery of forelimb placing. In a second experiment, rats with sensorimotor cortex lesions were treated with a single injection of MK-801 after behavioral recovery. Twenty hours after the MK-801 injection, rats with sensorimotor cortex lesions showed a reinstatement of the placing deficits. The impairment endured for at least 7 days after injection. These behavioral data support the idea that MK-801 can have either beneficial or detrimental effects when administered after brain damage.

Diffusion kinetics in the Pd/Cu(001) surface alloy.

We use atom-tracking scanning tunneling microscopy to study the diffusion of Pd in the Pd/Cu(001) surface alloy as a function of temperature. By following the motion of individual Pd atoms incorporated in the surface, we show that Pd diffuses by a vacancy-exchange mechanism. We measure an activation energy for the diffusion of incorporated Pd atoms of 0.88 eV, which is in good agreement with our ab initio calculated energy of 0.94 eV.

Overexpression of wild-type p53 and c-Myc in human fetal cells transformed with adenovirus early region 1.

The expression of p53 in a large panel of adenovirus (Ad) 2/5- and 12-transformed human, rat, and mouse cells has been examined. In all cases, in the absence of the larger Ad E1B protein, the level of p53 is very low. In human and rat cells when the Ad 12 E1B 54K polypeptide is expressed, p53 is much more abundant, although this is not the case in Ad 12 E1-transformed mouse cells. We conclude that expression of p53 is determined by virus serotype, host cell type, and viral proteins expressed. p53 in Ad 12 E1-transformed human cells is wild type but has an extended half-life. Stabilization is not through protein-protein interaction with the Ad E1B protein. The level of expression of c-Myc is also elevated in Ad-transformed human cells but this does not correlate with the presence of the E1B protein or with p53. However, Northern blot analysis indicates a direct correlation between mRNA and protein levels. We conclude that c-Myc is regulated at the transcriptional level, whereas p53 is regulated at the post-translational level in adenovirus transformants.

A novel protein expressed in mammalian cells undergoing apoptosis.

Human and rodent cells undergoing apoptosis were observed to express high levels of a novel 45,000 M(r) protein. The protein, which we have termed apoptosis specific protein (ASP), was found in Burkitt lymphoma (BL) cells and in adenovirus-transformed human and rat embryo cells induced into apoptosis by a variety of stimuli, including serum deprivation, exposure to the Ca2+ ionophore, ionomycin, treatment with inhibitors of macromolecular synthesis (cycloheximide and actinomycin D), and cold shock. In BL cells treated with apoptotic stimuli, expression of the oncoprotein Bcl-2 was found to both protect from apoptosis and prevent expression of ASP. ASP was not detected either in viable cells or in cells dying passively by necrosis. Laser scanning confocal microscopy showed high levels of ASP in the cytoplasm of cells displaying the chromatin condensation and fragmentation patterns typical of apoptosis. Retention of ASP was observed even when DNA was no longer detectable, and two-color immunofluorescence staining indicated that the protein primarily colocalized with, but was clearly distinct from, non-muscle actin. These findings, together with the observation that biochemical extraction of ASP was only possible under conditions which caused solubilization of the cytoskeleton, leads us to conclude that ASP forms part of, or at least strongly associates with, a modified cytoskeleton unique to cells undergoing apoptosis. While elucidation of its function will require further work, ASP constitutes a powerful marker for the diagnosis and quantitation of apoptosis in vivo and in vitro.

The high levels of p53 present in adenovirus early region 1-transformed human cells do not cause up-regulation of MDM2 expression.

The cellular protein MDM2 can bind to the tumor suppressor gene product p53 and abrogate its transcriptional activity. In addition, p53 can regulate expression of the mdm2 gene. We and others have previously shown that p53 is present at high levels in adenovirus-transformed cells which express the larger E1B protein. In view of these observations the expression of MDM2 in a panel of adenovirus transformed human cell lines has been examined. Two major species (98K and 80K) were detected, together with a number of minor species of higher and lower molecular weight. While there was little variation in levels of 98K protein between cell lines, appreciable differences in the expression of the 80K component were apparent. There was no correlation between MDM2 and p53 expression in any of the adenovirus transformants, nor with the viral proteins expressed. The pattern and level of MDM2 detected was similar to that seen in human tumor cell lines and in human fetal tissue. Northern blot analysis suggested that MDM2 expression was regulated at the transcriptional level. Stable interactions were observed between p53 and MDM2 in the adenovirus-transformed cell lines and in Ad5 E1 HEK 293 cells a ternary complex of p53, MDM2, and the Ad5 E1B 58K protein was demonstrated. In view of the lack of correlation between the level of p53 and MDM2 in adenovirus E1-transformed cells, the capacity of p53 to cause transcriptional activation was assessed using transfected CAT constructs linked to p53 responsive elements. p53 transcriptional activity was similar in all of the cell lines examined and did not correlate with protein expression. It is concluded, on the basis of all of these data, that the high concentrations of p53 found in adenovirus transformants are not transcriptionally active and have no influence on MDM2 expression. However, when expression of p53 was increased following infection with mutant adenoviruses, which do not express the larger E1B proteins, there was an appreciable increase in p53 transcriptional activity and in the levels of all of the MDM2 components.