New insulin replacement technologies: overcoming barriers to tight glycemic control.

New insulin analogues act more quickly, allowing better postprandial glycemic control and making intensive control easier. New methods of delivering insulin, notably inhaled insulin, will soon provide alternatives to painful injections. Improved glucose sensors may eventually make an artificial pancreas possible.

Potentiation of D2-dopamine receptor-mediated suppression of zif 268 by non-competitive NMDA receptor antagonists in reserpinized rats.

Striatopallidal output neurons, which coexpress D2-dopamine receptors and NMDA receptors, are logically a potential site of interaction between corticostriatal glutamatergic input and dopaminergic systems. Recent hypotheses about the etiology of schizophrenia have implicated both excitatory amino acid and dopamine systems. The present study was designed to examine, in vivo, the interaction between D2-dopamine receptors and NMDA receptors in the regulation of the expression of the early immediate genes (IEGs), zif 268 and jun B, in striatopallidal neurons. We tested whether coadministration of NMDA antagonists interacted with the actions of the D2 agonist, quinpirole, on IEG expression following dopamine depletion with reserpine. When rats were pretreated with the non-competitive NMDA receptor antagonists, MK 801 (1 mg/kg) or PCP (20 mg/kg), together with quinpirole, the quinpirole reversal of reserpine induction of zif 268 mRNA was potentiated in all regions examined. MK 801 alone had no significant effect on reserpine induction of zif 268 mRNA. Pretreatment with the competitive NMDA receptor antagonist, CPP (5 mg/kg), did not significantly alter the dose response of zif 268 mRNA expression to quinpirole in any region. There was no significant effect of MK 801 on jun B mRNA expression, either on the response to quinpirole or when administered alone with reserpine. Our findings provide evidence of an interaction between the NMDA receptor channel system and the D2-dopamine system on a molecular level in striatopallidal neurons carrying output from the basal ganglia.

Effects of prenatal cocaine exposure upon postnatal development of neostriatal dopaminergic function.

Pregnant rats were injected twice daily with 20 mg/kg cocaine (or saline) from gestational day 10 to parturition. Brains from offspring were examined with quantitative receptor autoradiography [D1 receptor (D1R), D2 receptor (D2R) and dopamine transporter (DAT)] and quantitative in situ hybridization [D1R mRNA, D2R mRNA, preproenkephalin (PPE) mRNA] for markers of neostriatal dopaminergic function. Prenatal cocaine exposure did not alter postnatal development of striatal D1R sites, but D1R mRNA levels were reduced by a third at days 14 and 35. D2R sites were increased over control in lateral striatum by day 6, and remained elevated through postnatal day 35. Total D2R mRNA was increased over control in both medial and lateral striatum at 7 and 14 days but was equal to control at 35 days. Prenatal cocaine exposure increased DAT density at postnatal days 1 through 5, but reduced it at days 14 and 35; PPE mRNA expression was reduced at days 7, 14 and 35. Many of these results are similar to those found in experimental animals and humans following cocaine withdrawal.

Increased PGE2 from human monocytes isolated in the luteal phase of the menstrual cycle. Implications for immunity?

The reproductive hormones are implicated in the well documented sexual dimorphism in cellular and immune responses. Prostaglandins (PGs) are mediators of the immune response with their concentration and relative amounts being pivotal to their impact. In resident peritoneal macrophages isolated from mice we had previously noted that the cells from females synthesized significantly more PG than males. In these experiments we investigated whether PG metabolism in the human monocyte was influenced by gender and by stage of the menstrual cycle. Monocytes isolated from the female and activated in vitro with LPS produced on average significantly more PG into the medium than the males. Among females, significantly more PG was found in the medium from cells isolated during the luteal phase of the cycle than during the early follicular phase. It was also in this luteal phase in which the female differed substantially from males. We suggest that the in vivo hormonal changes associated with the menstrual cycle modulate monocyte synthesis of PG and other immune modulators such as IL-1. This could be a key to understanding differences in vulnerability between males and females as well as within phases of the cycle, to immune and inflammatory insult.

Transformation of pecan and regeneration of transgenic plants.

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of 'Elliott', 'Wichita', and 'Schley' were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line 'Elliott'6, 3 from 'Schley'5/3, and 3 from 'Wichita'9. Transgenic embryos of 'Wichita'9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.

Postnatal development of D1 dopamine receptors in the medial prefrontal cortex, striatum and nucleus accumbens of normal and neonatal 6-hydroxydopamine treated rats: a quantitative autoradiographic analysis.

The postnatal development of dopamine (DA) D1 receptors in the medial prefrontal cortex (mPFC), striatum (STR) and nucleus accumbens (NAC) of control and perinatally 6-hydroxydopamine (6-OHDA) lesioned rats was examined using quantitative autoradiography of 3H-SCH 23390 binding. D1 receptors are present at one week and increase only slightly to a stable level by 2 weeks in the STR and NAC. Their ontogeny is not altered by intracisternal injection of 6-OHDA 5 days after birth. A biphasic pattern of appearance of D1 receptors was found in the mPFC. D1 receptors are present in the mPFC at 1 week, increase 3-fold by 2-3 weeks, and then decline at 4 and 6 weeks. 6-OHDA lesions do not significantly alter this pattern. At all postnatal ages. D1 receptor binding in the mPFC exhibits a laminar distribution with increased receptor density in deep cortical layers (V, VI) compared to more superficial cortical layers (I, II). Both superficial and deep layers of D1 receptors in the mPFC show similar postnatal developmental patterns. DA turnover rates are consistently about 10-fold higher in frontal pole compared to remainder of forebrain at all postnatal ages. Early 6-OHDA lesions increase DA turnover in forebrain, but lead to a persistent reduction in DA turnover in frontal pole by 2 weeks of age.

Apparent synaptic dopamine deficiency induced by withdrawal from chronic cocaine treatment.

The effects on motor behavior and forebrain dopamine (DA) synaptic function of withdrawal from chronic cocaine treatment were examined with simultaneous activity monitoring and microdialysis in nucleus accumbens. Rats exhibited behavioral sensitization to daily 30 mg/kg i.p. cocaine. After 18 days of daily cocaine and 7 days of withdrawal, dialysate DA and homovanillic acid (HVA) levels were reduced 36-38%, consistent with a synaptic DA deficiency.

Improved efficiency of the walnut somatic embryo gene transfer system.

AnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5-6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos.

Infections in the diabetic host.

The presence of the diabetic state seems to predispose patients to more severe and unusual types of infections. Awareness and early recognition of these sometimes devastating problems, coupled with appropriate medical and surgical treatment and aggressive metabolic control of diabetes, provide the maximum opportunity for healing and recovery.

Modification of guinea pig lung anaphylaxis by central nervous system (CNS) perturbations.

Since several studies indicate that protection from lethal anaphylaxis is mediated by anterior hypothalamic (AH) lesions, we investigated the hypothesis that central nervous system perturbations can modify release of mediators from antigen-challenged sensitized lungs. Three types of perturbations were made in guinea pigs: AH perturbation (electrodes inserted and the current was applied), anterior hypothalamic sham (AS) (electrodes placed as in the AH group but no current was passed), and posterior hypothalamic (PH) perturbation (electrodes placed and the current was applied). A control group was sham operated (electrodes not inserted). Eleven days after the operation, guinea pigs receiving brain perturbations and half the control group were sensitized to the antigen ovalbumin. The other half of the control group received vehicle only (nonsensitized). Twenty-five days after this procedure, lungs were perfused in situ, and the outflows were collected before and after injection of antigen. The perfusates were assayed for immunoreactive prostaglandin and histamine, and the lungs were assayed for cAMP, guanosine monophosphate, and histamine. Release of mediators and changes in lung cyclic nucleotides after perfusion with antigen were significantly greater in all the antigen-sensitized compared to the nonsensitized animals. Within the anaphylactic groups, significant reductions in mediators and in cyclic nucleotides were found in the animals with perturbations of the AH region compared to the CO animals. The time course of mediator release was not altered. The results extend to the biochemical level, the observation that the perturbation of the AH region can markedly modify the anaphylactic response, and indicate that this effect may be due to altered release of mediators.

Dietary n-3 fatty acids and arthritis.

We have evidence that dietary fish oil (FO) decreases severity of collagen-induced arthritis (CIA), changes the fatty acid composition of macrophage (M phi) membrane phospholipids, decreases M phi synthesis of prostaglandins (PGs), changes chemotactic ability of M phi s, and affects metabolism of acute phase proteins. Gender also has pronounced effects on susceptibility to CIA and M phi prostaglandin profiles. The mechanisms by which dietary n-3 fatty acids may act to alleviate symptoms of CIA, as well as interactions of dietary n-3 and n-6 fatty acids and gender are discussed. We suggest that the ability of FO diets to influence favourably the course of chronic inflammatory diseases is mediated via alterations in n-6 fatty acid metabolism and that intrinsic differences in n-6 fatty acid metabolism may account not only for our reported gender differences in incidence and severity of CIA, but also the well-documented sexual dimorphism in immune/inflammatory responses in general.

Inhibition of ethylene biosynthesis by salicylic Acid.

Salicylic acid inhibited ethylene formation from ACC in self-buffered (pH 3.8) pear (Pyrus communis) cell suspension cultures with a K(1) (app) of about 10 micromolar after 1 to 3 hours incubation. Inhibition appeared noncompetitive. Among 22 related phenolic compounds tested, only acetylsalicylic acid showed similar levels of inhibition. Inhibition by salicylic acid was inversely dependent on the pH of the culture medium and did not require a continuous external supply of salicylate. When compared to known inhibitors of the ethylene forming enzyme, cobalt, n-propyl gallate, and dinitrophenol, inhibition by salicylic acid most closely resembled that by dinitrophenol but salicylic acid did not produce the same degree of respiratory stimulation. Results are discussed in terms of other known effects of salicylic acid on plants, pH-dependency, and the possible influence of salicylic acid on electron transport.

A fish oil diet reduces the severity of collagen induced arthritis after onset of the disease.

We have previously reported that compared to a corn oil diet a fish oil diet (5% by weight) fed to B10R.III mice before the induction of collagen induced arthritis markedly reduced disease severity. In this study we determine whether a fish oil diet could reduce the severity of collagen induced arthritis if begun after the arthritis was clinically apparent. Mice were initially fed either a fish oil or corn oil diet and immunized with bovine type II collagen 4 weeks later. At the onset of collagen-induced arthritis, half of the corn oil fed mice were switched to fish oil and arthritis assessed on a weekly basis. Four weeks after the diet change until killing 5 weeks later, the mice switched to fish oil developed much less severe arthritis than the corn oil fed controls. Thus the severity index of corn oil fed mice ranged between 9.4 and 7.1; the severity index of fish oil fed mice was between 6.8 and 4.3 while the mice switched to fish oil ranged between 7.2 and 5.6. Analysis of peritoneal macrophages 13 weeks after immunization showed that macrophages from fish oil fed mice incorporated eicosapentaenoic acid into phospholipids and produced less arachidonate products than corn oil fed mice. There was no difference between macrophages obtained from mice switched from corn oil to fish oil and those maintained on fish oil with respect to fatty acid composition of membrane phospholipids or prostaglandin profile. These results suggest that arthritis severity may be modulated after the onset of CIA by altering the PG profile of macrophages present at inflammatory sites.

Dietary fish oil modulation of macrophage amyloid P component responses in mice.

Arthritis-susceptible B10.RIII mice, maintained on either fish oil (FO) or corn oil (CO) diets (5% by weight), and amyloid-susceptible CBA/J mice fed chow diets were given 20 micrograms purified LPS by i.p. injection. Both strains of mice responded to LPS with a 20- to 30-fold increase in plasma amyloid P component (AP) levels. There were no differences in the response between males and females or between FO and CO treatment groups. The data demonstrated that cultured peritoneal macrophages (M phi) respond to LPS stimulation with increased secretion of AP. In contrast to plasma AP levels, the MO response to LPS stimulation, as measured by production of AP, was influenced by both gender and diet. Although M phi from both male and female mice on the CO diet and male mice on the FO diet responded similarly, those from female mice on the FO diet secreted only 25 to 35% as much AP as did the other three groups. There were no dietary effects on the LPS-induced serum amyloid A protein response nor was there detectable serum amyloid A protein produced by the M phi. These results demonstrate that unstimulated, resident peritoneal M phi secrete AP as a normal constituent and in increasing amounts in response to LPS stimulation.

Randomized controlled trial of topical hyperbaric oxygen for treatment of diabetic foot ulcers.

The effect of 2 wk of topical hyperbaric oxygen (THO) treatment on the healing of diabetic foot ulcers without associated gangrene was evaluated in a prospective, controlled, and randomized manner in 28 patients. There were 12 patients in the THO group (group 1) and 16 in the control group (group 2). Clinical management of the two patient groups was similar except for THO treatment in the group 1 patients. Clinical parameters, including age, sex, baseline fasting serum glucose levels, duration of diabetes mellitus, duration of foot ulcers, presence of peripheral neuropathy or arterial insufficiency, and evidence of osteomyelitis as determined by radiographs and/or radionuclide scans, were comparable in both groups of patients. No statistical differences (Student's t test) were seen in the number of microorganisms isolated from curettage cultures of the base of the ulcer at days 0, 7, and 14 of the study between groups 1 and 2. In contrast to previous studies, there was a paucity of anaerobic microorganisms isolated from these foot ulcers without associated gangrenous changes. Ulcer areas were estimated by multiplying the maximum width by the maximum length in millimeters at days 0, 7, and 14. Analysis of variance and Student's t test revealed progressive significant reductions in the ulcer areas in both groups when days 0, 7, and 14 were compared and in ulcer depths in both groups when days 0 and 14 were compared. However, such ulcer size changes did not differ statistically between the control and THO groups. A trend toward slower healing was observed in the THO group. Healing of diabetic foot ulcers was not accelerated by THO in this study.

A fish oil diet retards experimental amyloidosis, modulates lymphocyte function, and decreases macrophage arachidonate metabolism in mice.

Several recent reports have shown that diets in which the only source of fat was fish oil can modify the course of diseases with an inflammatory or immune component. In these experiments we examined the effect of a fish oil diet on experimental amyloidosis in mice. In most azocasein-treated mice, amyloid deposits were found in the spleen, varying from a trace to wide and contiguous perifollicular bands. The spleens of mice receiving fish oil had significantly less amyloid than did spleens of mice fed corn oil. A marked increase in spontaneous blastogenesis that occurred in azocasein-treated mice on corn oil was suppressed in azocasein-treated mice on fish oil. Substitution of the unsaturated fatty acids of corn oil with those of fish oil was also found to modify the prostaglandin profile of macrophages. This altered profile may change cellular immune function and/or enhance the processing of serum amyloid A to retard the induction of secondary amyloidosis in mice.

Striatal D1- and D2-dopamine receptor sites are separately detectable in vivo.

We have characterized in particulate fractions of normal rat striatum the in vivo binding kinetics, binding affinity, and pharmacological profiles of [3H]SCH 23390, a ligand selective for the D1-subtype of dopamine (DA) receptor, and compared these to [3H]spiperone, a ligand classically associated with the D2 DA receptor subtype. The pharmacological specificity of each ligand's in vivo binding is very similar to binding to striatal homogenates in vitro. While similar maximum numbers (Bmax) of striatal binding sites exist in vivo compared to in vitro for both ligands, binding affinities in vivo for both ligands are reduced 125- to 200-fold compared to in vitro. In vivo binding of [3H]SCH 23390 to striatum is not increased by dopamine denervation produced by 6-hydroxydopamine lesions of the nigrostriatal pathway. In vivo binding of [3H]SCH 23390 and [3H]spiperone to striatum is not significantly reduced by increased synaptic concentration of dopamine following D-amphetamine administration. 125I-SCH 23982, the iodinated analogue of SCH 23390, localizes very highly to dopaminergic forebrain areas following i.v. administration. External imaging of mammalian and human brain D1-receptors is potentially feasible with this ligand.

A fish oil diet inhibits amyloid P component (AP) acute phase responses in arthritis susceptible mice.

Amyloid P component (AP) bears close homology with C-reactive protein and behaves as an acute phase reactant in the plasma of mice but not in man. Our aim was to determine whether AP is influenced by diet, gender, and arthritis severity in a murine model of arthritis. B10.RIII mice were segregated according to gender and diet at 8 wk of age: the source of fat was either corn oil, fish oil, or beef tallow (5% by weight). Four weeks later, each mouse was immunized with 100 micrograms fetal bovine type II collagen, and the incidence and severity of arthritis was noted at weekly intervals. AP was measured by competitive ELISA in plasma taken 5 wk and 15 wk after immunization. AP levels were less in fish oil fed males and females. Under all conditions tested AP levels of females were greater than in males. There was a negative correlation between AP levels and the severity of arthritis. We conclude from these data that although AP levels cannot be used as indices of arthritis severity, there are significant dietary and gender effects on AP concentrations as long as 15 wk after immunization with type II collagen.

[3H]spiperone binds selectively to rat striatal D2 dopamine receptors in vivo: a kinetic and pharmacological analysis.

We have determined the kinetic, equilibrium saturation, and pharmacological characteristics of [3H]spiperone ([3H]SPIP) binding to rat brain regional particulate fractions following i.v. injections of [3H]SPIP and compared these parameters to those determined in vitro with traditional ligand-homogenate binding assays. [3H]SPIP binding to rat striatum in vivo and in vitro occurs to a single class of non-interacting binding sites which possess the pharmacological properties of a D2 dopamine (DA) receptor. The potencies of neuroleptic drugs in inhibiting DA receptor-mediated behaviors correlate with their potencies at displacing striatal [3H]SPIP binding in vivo. While striatum possesses a similar density of [3H]SPIP binding sites in vivo (34 pmol/g) and in vitro (31 pmol/g), binding affinity in vivo is about 200 times lower than in vitro. This difference in binding affinities appears to arise from alterations of [3H]SPIP association and dissociation rate constants brought about by tissue homogenization. The implications of our findings for external imaging of DA receptors and studies of DA receptor function in human brain homogenates are discussed.

Gender differences in eicosanoid production from macrophages of arthritis-susceptible mice.

Collagen-induced arthritis (CIA) in rodents is an experimental animal model that shares many clinical and pathologic findings with rheumatoid arthritis in man. Our previous findings suggested that the amelioration of CIA in mice by a fish oil diet was associated with macrophage accumulation and metabolism of eicosapentaenoic acid and a subsequently altered prostaglandin (PG) profile. In these experiments, we examined the role of gender and found that macrophages from female arthritis-susceptible B10.RIII or B10.G mice synthesized more PG and thromboxane than macrophages isolated from the males. Compared with males, female mice had higher circulating anti-type II collagen antibodies but were less likely to develop CIA. Females, especially those on a fish oil diet, developed a much less severe disease than the males. This supports our hypothesis that the type and/or amount of eicosanoid produced from the macrophage may alter the course of experimentally induced arthritis.