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Non-insulin-dependent diabetic (NIDD) male Wistar rats develop a cardiomyopathy approximately 9 mo after the onset of the diabetic condition. This cardiomyopathy is characterized by reduced contractility, relaxation, cardiac work, and diastolic compliance. Although the basis for these defects is not completely understood, altered cellular Ca2+ regulation appears to play a major role in their development. In both isolated sarcolemmal membrane and cardiomyocytes, significant diabetes-linked defects in Ca2+ metabolism were observed. A small, but significant, decrease in the rate of sarcolemmal ATP-dependent Ca2+ transport of the diabetic heart was observed. Also evident was a major defect in sarcolemmal Na(+)-Ca2+ exchange as determined by reduced Na(+)-dependent Ca2+ transport into vesicles and Na(+)-dependent Ca2+ efflux from 45Ca(2+)-loaded cardiomyocytes from diabetic rats. In isolated cardiomyocytes, it was observed that the relative fluorescence of fura-2 at 502 nm was higher in cells from NIDD hearts, suggestive of a higher cytosolic free Ca2+. Consistent with diabetes-linked defects in Ca(2+)-transporter activities, the accumulation of Ca2+ after depolarization with KCl was greater in the diabetic. This study demonstrates that diabetes-induced defects in Ca2+ movement by the various Ca2+ transporters lead to abnormal cytosolic Ca2+ regulation by the diabetic cardiomyocytes. This observation supports the notion that abnormal Ca2+ regulation contributes to the development of the NIDD cardiomyopathy.

The effect of insulin supplementation on diabetes-induced alterations in the levels of functional mitochondrial anion transport proteins has been determined. The experimental approach consisted of the extraction of the pyruvate, dicarboxylate, and citrate transport proteins from the mitochondrial inner membrane with Triton X-114 using rat liver mitoplasts (prepared from control, diabetic, or insulin-supplemented diabetic animals) as the starting material, followed by the reconstitution of the function of each transporter in a proteoliposomal system. This experimental strategy permitted the quantification of the functional levels of these three transporters in the absence of the complications that arise when such measurements are carried out with intact mitochondria (or mitoplasts). We found that treatment of diabetic rats (i.e., animals that were injected with streptozotocin 3 weeks earlier) on a daily basis with insulin for 3 weeks resulted in a reversal of the diabetes-induced (a) increase in the extractable and reconstitutable total (and specific) transport activities of the pyruvate and dicarboxylate transporters and (b) decrease in the activity of the citrate transporter. These findings indicate that diabetes-induced alterations in the functional levels of mitochondrial anion transport proteins are a direct consequence of the insulin insufficiency that characterizes this disease. Furthermore, this study provides the first demonstration that insulin participates in the regulation of the functional levels of liver mitochondrial anion transport proteins.

A new homeobox gene, HB24, has been isolated from a human B-lymphocyte cDNA library. Northern blot analysis of polyadenylated RNA purified from activated human B cells revealed a single mRNA transcript of approximately 2.3 kb. Two cDNA clones were sequenced and provided 2,250 nucleotides (nt) of DNA sequence information. There is a single methionine codon-initiated open reading frame of 1,458 nt in frame with a homeobox and a CAX repeat, and the open reading frame is predicted to encode a protein of 51,659 daltons. When the homeodomain from HB24 was compared to known mammalian and Drosophila homeodomains it was found to be only moderately conserved, but when it was compared to a highly diverged Drosophila homeodomain, H2.0, it was found to be 80% identical. The HB24 mRNA was absent or present at low levels in normal B and T lymphocytes; however, with the appropriate activation signal HB24 mRNA was induced within several hours even in the presence of cycloheximide. Characterization of HB24 expression in lymphoid and select developing tissues was performed by in situ hybridization. Positive hybridization was found in thymus, tonsil, bone marrow, developing vessels, and in fetal brain. HB24 is likely to have an important role in lymphocytes as well as in certain developing tissues.

Eighty female subjects rated therapy descriptions of a case involving the treatment of inhibited female orgasm in group and individual formats. Half of the treatment descriptions described the patient's husband participating in some of the homework exercises. After each treatment description, subjects completed a series of treatment acceptability ratings (Treatment Evaluation Inventory, Semantic Differential, Credibility Rating Scale) designed to evaluate the alternative treatment dimensions. A case history of primary anorgasmia, representative of actual clients and problems typically seen in sex therapy clinics, was presented to subjects via written material and audiotape players. A clear preference for individual format over group format was consistently found in credibility and acceptability ratings. Inclusion of the identified patient's partner in therapy was not a salient factor in acceptability ratings for the female subjects. Implications of this research paradigm are discussed in light of the current state of the art in sex therapy, and future research needs are identified.

Studies were initiated to investigate whether mechanisms exist within mitochondria to repair damage incurred by mitochondrial DNA after exposure to alkylating toxins. A clonal isolate from a rat insulinoma cell line was utilized to measure the formation and repair of alkali-labile sites within the mitochondrial genome after exposure to the alkylating antibiotic streptozotocin. Alkali-labile sites were formed in mitochondrial DNA in a dose-dependent fashion. Eight hours after exposure to the toxin, 55% of the lesions were removed. The level of repair increased to 70% after 24 h. In comparison, only 46% of N7-methylguanines were removed across the entire cellular genome. These studies demonstrate that streptozotocin causes appreciable mitochondrial DNA damage in a dose-dependent manner and provide the first evidence that a repair mechanism for alkali-labile sites is present within the mitochondrion.

The repair of DNA damage induced by methylnitrosourea (MNU) in restriction fragments containing the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells was compared to that in equal size restriction fragments containing a nontranscribed flanking sequence 3' to the DHFR gene or the c-fos gene. Following exposure to 10(-3) M MNU, restriction fragments containing either the DHFR gene or the 3' flanking sequence had similar amounts of alkali labile sites, approximately 2 sites/restriction fragment. Fragments encompassing the c-fos gene had less than 2 breaks/fragment. Twenty-four h after exposure to MNU a consistent, but slight and not statistically significant, difference was seen with more adducts removed from the DHFR gene than the 3' flanking sequence. No repair was detected in the c-fos containing fragments. In addition, the repair of N7-methylguanine in the overall genome was assessed by use of a 32P end-labeling technique. Seventy % of this major alkylation product was repaired after 24 h. These findings establish that repair heterogeneity occurs in Chinese hamster ovary cells after exposure to MNU.

We have cloned a full-length cDNA for the B cell membrane protein CD22, which is referred to as B lymphocyte cell adhesion molecule (BL-CAM). Using subtractive hybridization techniques, several B lymphocyte-specific cDNAs were isolated. Northern blot analysis with one of the clones, clone 66, revealed expression in normal activated B cells and a variety of B cell lines, but not in normal activated T cells, T cell lines, Hela cells, or several tissues, including brain and placenta. One major transcript of approximately 3.3 kb was found in B cells although several smaller transcripts were also present in low amounts (approximately 2.6, 2.3, and 1.6 kb). Sequence analysis of a full-length cDNA clone revealed an open reading frame of 2,541 bases coding for a predicted protein of 847 amino acids with a molecular mass of 95 kD. The BL-CAM cDNA is nearly identical to a recently isolated cDNA clone for CD22, with the exception of an additional 531 bases in the coding region of BL-CAM. BL-CAM has a predicted transmembrane spanning region and a 140-amino acid intracytoplasmic domain. Search of the National Biological Research Foundation protein database revealed that this protein is a member of the immunoglobulin super family and that it had significant homology with three homotypic cell adhesion proteins: carcinoembryonic antigen (29% identity over 460 amino acids), myelin-associated glycoprotein (27% identity over 425 amino acids), and neural cell adhesion molecule (21.5% over 274 amino acids). Northern blot analysis revealed low-level BL-CAM mRNA expression in unactivated tonsillar B cells, which was rapidly increased after B cell activation with Staphylococcus aureus Cowan strain 1 and phorbol myristate acetate, but not by various cytokines, including interleukin 4 (IL-4), IL-6, and gamma interferon. In situ hybridization with an antisense BL-CAM RNA probe revealed expression in B cell-rich areas in tonsil and lymph node, although the most striking hybridization was in the germinal centers. COS cells transfected with a BL-CAM expression vector were immunofluorescently stained positively with two different CD22 antibodies, each of which recognizes a different epitope. Additionally, both normal tonsil B cells and a B cell line were found to adhere to COS transfected with BL-CAM in the sense but not the antisense direction.(ABSTRACT TRUNCATED AT 400 WORDS)

Free radicals produced in chicken bone tissue by 137Cs gamma-rays were measured using electron paramagnetic resonance spectroscopy. The yield of radicals was found to be proportional to the absorbed dose. Additive re-irradiation of previously irradiated bones is the basis of a method to estimate the absorbed dose in radiation-processed foods. The ability of the method to provide accurate dose assessments for a range of doses (0.5-7.4 kGy) is tested here. A linear fit to the data yields reasonable dose estimates for bone irradiated less than 2 kGy, but fails at the higher doses using a linear approximation to the dose response. These data and their implications are discussed.

Depressive syndromes are prevalent among prison inmates. By design, penal institutions attempt to punish, deter, and isolate criminals. The present study represents a beginning step in evaluating treatment interventions which may alleviate depression among incarcerated felons. 10 inmates at a large maximum-security prison were randomly assigned to group cognitive therapy or individual supportive treatment plus brief counseling contacts. Analysis indicated statistical and clinical significance on measures of depressive symptomatology for treatment conditions across the periods of assessment. The findings support the utility of depression-treatment programs for prisoners. Moreover, the treatment modalities are presented along a variety of dimensions which may be useful when considering such interventions at facilities where psychological services are limited.

DNA damage and repair were studied in a DNA fragment containing the insulin gene after treatment of cells with methylnitrosourea. For these studies, two clonal isolates from the same rat insulinoma cell line which differ in that the insulin gene is transcribed in one (RINr 38) and is silent in the other (RINr B2) were utilized. Both the determination of immunologically reactive insulin released and the expression of insulin mRNA were used to verify that the gene was transcribed in the RINr 38 cells and not in the RINr B2 cells. Repair kinetics for the removal of alkali-labile sites were comparable across the entire genome in the RINr 38 and RINr B2 cells as determined using alkaline sucrose gradient sedimentation and a 32P end-labeling assay for the quantitation of N7-methylguanine. Quantitative DNA blot analysis was utilized to assess the formation and repair of alkali-labile sites within the restriction fragment containing the insulin gene. Alkali-labile sites appeared to be formed equally within the restriction fragment containing the insulin gene in both the RINr 38 and RINr B2 cells. However, at 24 h, 60% of the lesions were removed from the fragment in the RINr 38 cells, where the gene was transcribed, compared to the removal of only 20% in the RINr B2 cells, where the gene was silent. Thus, it appears that alkali-labile sites induced by exposure to methylnitrosourea are repaired more efficiently in the DNA fragment containing the insulin gene when it is actively transcribed.

The effect of streptozotocin-induced diabetes on the levels of functional mitochondrial anion transport proteins has been determined. The experimental approach utilized for these studies consisted of the extraction of each of four mitochondrial anion transport proteins from rat liver mitoplasts (isolated from diabetic and control animals) with the nonionic detergent Triton X-114, followed by the functional reconstitution of each transporter in a liposomal system via the freeze-thaw-sonication technique. This approach permitted the quantification of transporter function without the complications that occur when such measurements are carried out with intact mitochondria (or mitoplasts). We found that experimental diabetes caused an increase in the extractable and reconstitutable specific (and total) transport activities of the pyruvate and dicarboxylate transporters, a decrease in the activity of the citrate transporter, and no significant change in the activity of the phosphate transporter relative to control values. An examination of the time course of the appearance of changes in the reconstitutable activities of the pyruvate and citrate transporters following the injection of streptozotocin revealed differences. Thus, whereas the activity of the pyruvate transporter displayed the most pronounced increase (193%) 1 week following streptozotocin injection and then subsequently declined from this peak and plateaued at later times (99% and 96% increases at 3 and 8 weeks, respectively), the activity of the citrate transporter progressively decreased with time (31-51% decreases at 1-8 weeks). We suggest that the observed diabetes-induced changes in mitochondrial anion transporter function are predictable on the basis of diabetes-induced alterations in the activities of enzymes that constitute metabolic pathways to which these transporters either supply substrate or remove product. Furthermore, we speculate that mitochondrial anion transport proteins may be regulated in coordination with the enzymes of such associated metabolic pathways.

The MSZ diabetic male mouse represents one of the most useful tools available to researchers interested in analyzing the consequences of insulin dependent diabetes in male mice. In contrast to the high mortality induced by single high doses of SZ, protracted administration of smaller SZ dosages yields a more stable diabetic condition. Moreover, in insulitis prone strains such as BKs, the model allows "synchronization" of beta cell destruction such that the inflammatory events occur on a predictable timescale. The MSZ-diabetic mouse represents a diabetic condition in which the primary etiopathologic effect is produced by an environmental toxin, and not by a genetically programmed loss of tolerance to beta cell specific antigens. In this regard, etiopathogenesis in the MSZ model is quite distinct from that underlying autoimmune type I diabetes in humans, NOD mice, and BB rats, and it is probably not appropriate to refer to pathogenesis in the MSZ model as one of "autoimmune insulitis" as has sometimes been done. The fact that insulitis in the MSZ model may not be "autoimmune," but may actually be a normal response to either tissue damage or to beta cells that have been structurally modified by a chemical, makes the model of special interest. Clearly, there is no single cause of insulin dependent diabetes, with disease induction representing a genetic susceptibility interacting with environmental triggers, such as toxins in the diet (including nitrosamines and fungal metabolites) as well as pathogenic viruses. The MSZ model will continue to be actively investigated because of insights it will afford regarding the genetic bases for susceptibility and resistance to diabetogenic environmental toxins. The model will be of further value by contributing to knowledge of the complicated interactions between pancreatic islet cells, other endocrine cells, and leukocytes in maintenance of glucose homeostasis.

A human fibrosarcoma cell line, 8387, produces and secretes multiplication-stimulating activity (MSA)-like polypeptide growth factors, which are associated with a high molecular weight binding protein. The purification of 8387-MSA involved concentration of serum-free media conditioned by 8387 cells, dialysis, and chromatography of the acid-soluble growth-promoting activity on Bio-Gel P-100 in 1 M acetic acid and subsequently on carboxymethylcellulose. The purification was followed by a radioreceptor assay specific for MSA and an assay for growth-stimulating activity. Both activities chromatographed through the purification steps of 8387-MSA. 8387-MSA competes with 125I-labeled rat MSA for binding on rat cells with a specific activity 5 times lower than that of rat MSA and stimulates [3H]-thymidine incorporation into DNA of normal rat fibroblasts at comparable concentrations. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the MSA-competing activity co-migrated with a single band of protein that behaved heterogeneously on discpolyacrylamide gels in the presence of 6 M urea, yielding two bands associated with biological activity. 8387-MSA is a single chain polypeptide with an apparent molecular weight of 14,500.

To determine if rheumatoid arthritis (RA) patients have an increased frequency of Epstein-Barr virus (EBV) shedding into saliva, throat washings were collected from 59 patients with RA and 64 healthy adult controls. EBV in filtered samples was detected by transformation of human umbilical cord lymphocytes. EBV was detected in 27% (16/59) of throat washings of RA patients compared to 11% (7/64) of control samples. This difference is significant (p less than 0.05). RA patients on steroids had a frequency of positivity of 43% (10/23) compared to 17% (6/36) in patients not on steroids. No correlation was demonstrated between steroid dose received by the patient and detection of EBV in throat washings. Parotid fluid samples were collected from 18 RA patients and 11 healthy controls and were tested for EBV by the transformation bioassay. EBV was detected in 18% (2/11) of samples from normal controls, but samples from RA patients were uniformly negative.

The distribution of net photosynthetic activity of leaves was measured in a mixed grass (Setaria sphacelata var. sericea)-legume (Desmodium intortum) pasture stand using a method based on concurrent measurement of the rate of CO2 exchange, and (14)CO2 dosing followed by rapid harvesting according to height strata. Comparisons were also made between plots which differed in the period of regrowth following defoliation.The usual superiority of leaf net photosynthetic rates of a C4 grass, compared with C3 legume leaves, was found in the upper, well illuminated strata. These rates were, however, much lower than those usually described for horizontally exposed leaves, primarily because leaves in the pasture stand were inclined to the horizontal. At greater depth in the canopies, the superiority of rates in the grass was less evident, and consequently the relative contributions of grass and legume to canopy photosynthesis became more dependent on their leaf area indices.Attention is drawn to the relative simplicity of the method for examining the contribution of leaves, which may differ according to species or position in the canopy, to productivity of the whole stand.

Epidemiological observations suggest that environmental factors play a role in the pathogenesis of insulin dependent diabetes mellitus (1). Several chemicals have been identified as specific beta cell toxins (2-4). We report here studies to determine the feasibility of using monolayer cultures of pancreatic beta cells from neonatal rat to screen potential diabetogenic chemicals. Cytotoxicity was monitored both by phase microscopy and the release of insulin into the culture medium. In comparative studies, cellular protein and release of 51chromium (51Cr) were measured after addition of test compounds to cultures of fibroblasts derived from pancreatic tissue. The nitrosoamides 1 methyl-l-nitrosourea (MNU), 1,3 bis (2-choroethyl) nitrosourea (BCNU), chlorozotocin (CLZ), and the beta cell toxin, streptozotocin (SZ), were examined. CLZ and SZ were more toxic to pancreatic beta cells than to fibroblasts. In contrast, MNU and BCNU damaged both beta cells and fibroblasts at identical concentrations. These results suggest that in vitro techniques can be used to identify chemicals that selectively injure beta cells. Although SZ-induced toxicity was ameliorated with addition of nicotinamide to cultures of beta cells, nicotinamide did not prevent damage caused by CLZ. This observation indicates different mechanisms of drug-induced cytotoxicity.

Nucleic acid sequences homologous to a single-stranded [(3)H]DNA transcript prepared from a baboon type C virus replicating in dog thymus cells can be readily detected in the cellular DNA of several Old World monkeys (baboon, patas, African green, and two species of macaques-rhesus and stumptail). These results demonstrate that primates other than the baboon also contain endogenous type C viral genes. With the hybridization conditions employed (S(1) nuclease, 65 C), no homologous sequences were detected in DNA from human or New World monkey tissues. Of various nonprimate tissues examined, only domestic cat cellular DNA was partially homologous to the baboon virus [(3)H]DNA transcript. In reciprocal experiments, [(3)H]DNA transcripts of RNAs from endogenous cat viruses (RD-114/CCC group) show a significant partial homology with cellular DNA from Old World primates (baboon, patas, and rhesus monkey). The partial homology between type-C-related information in the DNA of domestic cats and various Old World monkeys suggests the possibility of horizontal transmission between the progenitors of these animals at some point in evolution. No nucleic acid sequences homologous to [(3)H]DNA transcripts prepared from type C viruses isolated from tumor tissue of a woolly monkey and a gibbon ape could be detected in any primate tissue DNA examined; however, a partial nucleic acid homology was found between woolly monkey and gibbon ape type C viral [(3)H]DNA and normal mouse cellular DNA.

Exposure of the isolated, glucose-perfused rat heart to buffer containing 0.4 mM tolbutamide resulted in significant changes in both energy metabolism and myocardial contractility. In the nondiabetic, tolbutamide mediated only small increases in mechanical function at low atrial filling pressure, but this effect increased with increasing preload. By contrast, the stimulation of mechanical function resulting from exposure of the diabetic heart to tolbutamide was independent of preload. As a result, the tolbutamide-mediated, positive inotropic effect in the diabetic heart was greater at lower, but not higher, preload values than the effect in the nondiabetic. Moreover, the changes in energy metabolism initiated by tolbutamide were considerably larger in the diabetic. The most prominent effect was the mobilization of glycogen by tolbutamide in the diabetic, which was considerably greater than that observed in the nondiabetic. The drug also enhanced glucose utilization. The net effect of sulfonylurea exposure was to shift from preferential use of fatty acids as an energy source for contraction to use of glucose. Since the most prominent effect of the drug in the diabetic was the stimulation of glycogenolysis, it is concluded that tolbutamide can dramatically alter the metabolism of a tissue without acting through insulin.

The present investigation utilized a unique, variable ratio schedule of reinforcement (the "bathroom game") to treat a 10-year-old encopretic male. Dependent measures included confirmed incidents of (a) soiling and (b) appropriate bowel movements monitored across an ABAB design (Baseline 1, "Bathroom Game 1", Baseline 2, "Bathroom Game 2") with one-year follow-up. During "bathroom game" conditions, contingent monetary rewards were provided for non-instances of soiling and appropriate bowel movements. Such rewards were progressively and systematically leaned-out over the course of treatment on a pre-determined variable ratio schedule. Results indicated a clear demonstration of functional control and clinically significant treatment effects during both experimental periods. These findings are discussed with regard to the positive features of the "bathroom game" procedure and recommendations are made for future investigations in the area.

Two newly synthesized nitrosoureido sugars have been evaluated for their antitumor activity and diabetogenic potential in a number of in vitro and in vivo preclinical tumor model systems. 2-Amino-2-deoxy-N'-methyl-N'-nitrosoureido-1,3,4,6-tetra-O-acetyl-alpha- D- mannopyranose (MAZ), a lipophilic mannosamine derivative, and ethyl-6-deoxy-3,5-di-O-methyl-6-(3-methyl-3-nitrosoureido)-alph a- D-glucofuranoside (EDOMEN or CGP 6'809), were both found to inhibit L1210 leukemia cell growth in vitro by 50% at approximately 5.0 X 10(-5) M. At these concentrations, little effect was noted immediately on L1210 cell radiolabeled precursor incorporation; however, at higher concentrations, EDOMEN inhibited [3H]leucine and [3H]mannose incorporation, while MAZ specifically decreased L1210 cell [3H]thymidine and [3H]leucine incorporation. Inhibition of Lewis lung carcinoma and B16 melanoma cell growth by 50% in vitro was achieved at higher concentrations of these agents (10(-4) to 10(-3) M). Since the currently available nitrosoureido sugars, streptozotocin and chlorozotocin, have been observed by us to be diabetogenic, EDOMEN and MAZ were evaluated for their specific toxicity to rat pancreatic beta-cells in vitro. Cytotoxicity in beta-cell cultures was monitored both by phase-contrast microscopy and the release of insulin into the culture medium. beta-Cells were found to be 10-fold more sensitive to the toxic effects of MAZ than were pancreatic fibroblasts. EDOMEN, on the other hand, did not damage beta-cells preferentially and therefore was not considered diabetogenic. Both MAZ and EDOMEN had moderate activity as antileukemic agents in mice. At 50 mg/kg/day i.p. for 5 days, MAZ increased the life span of female DBA/2J mice with L1210 leukemia by over 50%. Similarly, doses of EDOMEN at 125 to 250 mg/kg/day i.p. for 5 days increased L1210 leukemic life span by nearly 60%. At these doses, no effect of MAZ was observed on primary Lewis lung carcinoma growth or life span of tumor-bearing C57BL/6 mice. EDOMEN, however, increased life span in Lewis lung carcinoma mice by up to 33% and caused an apparent antimetastatic effect. These studies indicate that EDOMEN may have enhanced value as a cancer chemotherapeutic agent due to its therapeutic effectiveness, lack of diabetogenic potential, and other favorable formulation properties (water solubility) as compared with other clinically available nitrosoureas.

Both streptozotocin and chlorozotocin, the 2-chloroethyl analogue of streptozotocin, are diabetogenic chemicals in rodents. Although these chemicals are similar structurally, they appear to act on pancreatic B cells via different mechanisms. In studies here, damage and repair of DNA after exposure of an insulin-secreting cell line to streptozotocin and chlorozotocin were assessed by nucleoid sedimentation and alkaline elution. Equitoxic concentrations of streptozotocin and chlorozotocin caused significant single-strand breakage of DNA (p less than 0.005). These lesions were repaired in a time-dependent manner, with most repair completed by 24-h post-exposure to chemicals. Additionally, chlorozotocin caused DNA-DNA and DNA-protein crosslinks in insulinoma cells. When proteinase K was included in the crosslinking assay, a substantial proportion of the chlorozotocin-associated crosslinks proved to be DNA interstrand in nature. Analysis of the amount of interstrand crosslinking in insulinoma cells after exposure to chlorozotocin for 1 h showed that formation of interstrand crosslinks was slow. Increasing amounts appeared over a 24-h period. These results suggest that the formation of irreversible DNA interstrand crosslinks may be a critical factor in cytotoxicity and diabetogenicity caused by chlorozotocin.

Extracts of spinach contain somatostatin (SRIF)-related material (6-80 pg/g wet wt). The SRIF-related material, when purified on HPLC, was recovered as two major mol wt forms; one that eluted with a retention time similar to that of synthetic SRIF-28 and reacted in both N- and C-terminal-specific immunoassays, and a second peak that eluted with a retention time similar to that of SRIF-14 and reacted only in the C-terminal immunoassays. The purified material was active in a sensitive bioassay, and the bioactivity was neutralized in the presence of anti-SRIF antiserum. Since we have previously described the presence of similar material in bacteria, we also tested extracts of the flowering plant Lemna gibba G3, which was grown under sterile conditions. The Lemna extracts also had SRIF-related material (3.0 pg/g wet wt). Since plants are probably derived evolutionarily from unicellular organisms, the presence of SRIF-like material in higher plants gives support for the hypothesis that vertebrate-type peptide hormones have early evolutionary origins.

The initial step in streptozocin (STZ)-induced beta-cell toxicity has been hypothesized to be the alkylation of specific sites on DNA bases. The enzymatic removal of these lesions results in single-strand breaks that over-activate the nuclear enzyme poly(ADP-ribose) synthetase and critically deplete the cell of NAD. Our studies were performed to quantitatively evaluate the extent of DNA damage in beta-cells and correlate this damage with toxicity. Monolayer cultures of neonatal rat beta-cells were used to determine cytotoxicity and DNA damage after exposure to STZ or the aglycone N-methyl-N-nitrosourea (MNU). Toxicity in beta-cells was determined by correlating morphological alterations observed by phase-contrast microscopy with decrements in immunoreactive insulin release. The extent of DNA damage was determined by alterations in nucleoid density and quantitation of N7-methylguanine formation. Toxicity tests revealed that STZ and MNU were not toxic at equimolar concentrations. Streptozocin was toxic at 10(-3) M, whereas only mild toxicity was observed with MNU at 10(-2) M. Surprisingly, however, at equimolar concentrations the two drugs caused comparable DNA-strand breaks as evidenced by their ability to shift the nucleoid migration ratio in neutral sucrose gradients. Additionally, quantitation of N7-methylguanine formation after exposure to equimolar concentrations of the drugs demonstrated that the two alkylated DNA to the same extent. These findings suggest that factors in addition to the activation of poly(ADP-ribose) synthetase must be responsible for the toxicity seen with STZ, because MNU at a nonlethal concentration is capable of causing comparable DNA damage.

Recently, paradoxical techniques have been increasingly employed by behavior therapists. The present paper: summarizes case study paradoxical investigations, reviews paradoxical procedures evaluated via intrasubject replication designs, and offers suggestions for continued use of single-case methodology in paradoxical research. Five major design strategies (reversal, time-series, multiple-baseline, simultaneous treatment and changing criterion) are discussed and examples provided within each area. Specific recommendations are made for a further merging of single-case experimental designs and paradoxical therapies.

Human umbilical cord blood lymphocytes were immortalized by infection with Epstein-Barr virus (EBV) in the presence of various concentrations of dimethyl sulfoxide (DMSO). 13 continuous lymphoblastoid cell lines were established. DMSO did not affect the efficiency of transformation and establishment of these lines. After a period of 2 months in culture, the lines were tested for EBV production by lethal irradiation (6,000 rad) from a 60Co source and subsequent cocultivation with primary umbilical cord lymphocytes. Cell lines transformed in the presence of 2% DMSO yielded transforming activity in 46.6% of test cultures, compared with 9.5% for lines established without DMSO in the medium. These findings imply that a permanent change in the host/virus relationship resulted from a brief exposure (48 h) to DMSO at the time of transformation.

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor beta subunits. The insulin receptor beta subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor beta subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.

Adult rats treated with high doses of streptozocin became progressively more hyperglycemic during the first month of the diabetic condition. Treatment of these rats with the sulfonylurea glyburide halted, and in some cases, reversed this process in a high percentage of the diabetics. Associated with the glyburide-mediated improvement in fasting blood glucose levels was an increase in myocardial glucose utilization and lactate production. The stimulation of myocardial glucose utilization by insulin was greater in glyburide-treated hearts, indicating that the hyperglycemic agent increased insulin responsiveness. The sulfonylurea also partially restored insulin sensitivity to the normal range. In agreement with previous studies, myocardial mechanical function was significantly impaired in the diabetic heart. When treated with glyburide, the severity of the mechanical defect was significantly less. The sulfonylurea also promoted an increase in myosin ATPase activity and a shift in the myosin isozyme pattern in favour of the most active V1 form. These results imply that glyburide therapy can provide benefit to the diabetic heart by improving energy metabolism and promoting a shift in myosin towards the most active form.

Wistar rats were injected with streptozotocin (SZ) at 3 days of age. This maneuver produced a marked glucose intolerance, as determined by intraperitoneal glucose tolerance tests, but plasma fasting and nonfasting glucose values remained at or near normal throughout the 12-mo study period. Hearts obtained from these glucose-intolerant rats exhibited a progressive cardiomyopathy that consisted of both contractile and metabolic abnormalities. Contractile abnormalities were characterized by reductions in aortic output, ventricular pressure, and cardiac work. Associated with these mechanical defects was a decrease in glucose utilization. These abnormalities were not ameliorated by acute exposure to insulin or changes in the work load of the heart. These results demonstrate that, in the rat, a progressive cardiomyopathy results from persistent glucose intolerance in the absence of fasting hyperglycemia. This cardiomyopathy is reminiscent of that described in human noninsulin-dependent diabetes.

High affinity insulin-like growth factor I (IGF-I) receptors are expressed by two human neural derived cell lines, SK-N-SH and SK-N-MC. Specific [125I]IGF-I binding to crude membranes was 23.4% for SK-N-SH and 10.7% for SK-N-MC, with 50% inhibition of binding by unlabeled IGF-I between 0.6-0.7 nM. Scatchard analysis of crude membrane binding was linear, whereas Scatchard analysis after wheat germ agglutinin purification of the receptor became curvilinear. The IGF-I receptor alpha-subunits of SK-N-SH have an apparent Mr of 126K, whereas that for SK-N-MC is 132K. Despite these differences in alpha-subunit structure both cell lines demonstrate IGF-I-induced autophosphorylation of their own beta-subunits as well as specific IGF-I induced tyrosine kinase activity, suggesting normal coupling between the ligand-binding alpha-subunit and the tyrosine kinase-containing beta-subunit. Furthermore, IGF-I stimulated iododeoxyuridine uptake in both SK-N-SH and SK-N-MC in a dose-dependent manner, suggesting that these cells may be used to study the role of IGF-I action on neural tissues.

Hearts isolated from 12-mo non-insulin-dependent diabetic rats exhibited reduced rates of contractility and relaxation. Associated with the abnormality in contractility was a redistribution in myosin isozyme content to the least active V3 form. Defects in myocardial relaxation also occurred concomitantly with impaired handling of calcium. Total tissue calcium content rose 35% in the diabetic hearts. At the same time, the activity of the pump responsible for maintaining normal cytoplasmic calcium levels was reduced. At a free calcium concentration of 2.0 microM, the rates of sarcoplasmic reticular calcium uptake and adenosinetriphosphatase activity of the diabetic hearts were decreased approximately 30%. Diastolic ventricular stiffness increased dramatically. The net result of these abnormalities in calcium metabolism is a significant impairment in mechanical performance of the diabetic heart.

Eight weeks following streptozotocin-induced diabetes mellitus in rats, the sensitivity of adenylate cyclase to dopamine (DA) and norepinephrine (NE) was reduced in homogenates of retina. Furthermore, the activation of adenylate cyclase in cerebral microvessels (capillaries) by NE, 5'-guanylyl imidodiphosphate (alone or with NE) and forskolin was reduced in diabetic rats versus appropriate controls. In diabetic rats enzyme sensitivity to only NE was attenuated in homogenates of cerebral cortex and cortical piaarachnoid. No differences between controls and diabetics were noted with respect to guanylate cyclase or cyclic AMP phosphodiesterases. The damage observed in retina and microvessels may play an important pathogenic role in diabetes-induced blindness and stroke.

A case is reported in which a liver tissue mass, not connected to the liver, was discovered growing in the right thoracic cavity. This mass appeared following trauma sustained to the liver several years earlier, and was noted to increase slightly in size after its initial detection. After removal of the mass, which proved to be benign hyperplastic liver tissue, the patient did well and remained free of symptoms. It is suggested that during the episode of trauma or during the ensuing surgical repair, a fragment of liver tissue was introduced into the thoracic cavity, and that this fragment eventually developed into a hyperplastic nodular liver mass.

The "M" variant of the encephalomyocarditis (EMC) virus causes a diabetes-like disease in some, but not all, strains of mice. The genetic basis for either resistance or susceptibility to the diabetogenic effect of the virus is not known. After infection with EMC, C57BL/6 mice seldom develop hyperglycemia and the insular lesions are subtle. To explore the possible effects of metabolic influences on the viral susceptibility of the islets, we studied C57BL/6 mice that were carriers of the ob gene. After virus inoculation, obese homozygous C57BL/6-ob/ob mice consistently developed hyperglycemia during the acute stages of infection, whereas nonobese littermates did not. Infection induced more severe lesions in the pancreatic islets of obese mice than in islets of the lean littermates. These studies suggest that the functional activity of the beta-cells influences the severity of the viral injury to the beta-cell, and the consequent occurrence of diabetes.

In studies to evaluate possible inhibitors of the B-cell toxin, streptozotocin, the superoxide scavenger, superoxide dismutase, did not prevent or reduce the toxic effects of streptozotocin as determined by loss of insulin secretion from rat pancreatic B cells in monolayer culture. However, 1,1-dimethyl urea, a scavenger of the hydroxyl radical, did afford significant protection. Both scavengers diminished the cytotoxic effects of alloxan. The inhibitors of poly (ADP-ribose) synthetase, 3-aminobenzamide and nicotinamide, also were effective in attenuating alloxan- and streptozotocin-induced B-cell toxicity. Tests of the hydroxyl-scavenging ability of the three streptozotocin antagonists revealed that 3-aminobenzamide, nicotinamide and 1,1-dimethyl urea were effective scavengers of this free radical. Conversely, 1,1-dimethyl urea, although not as potent as 3-aminobenzamide or nicotinamide, was found to inhibit poly (ADP-ribose) synthetase. These data indicate that these chemicals most likely attenuate alloxan-induced toxicity by scavenging the hydroxyl radical and diminish streptozotocin-induced toxicity by inactivation of the poly (ADP-ribose) system.

Hearts isolated from non-insulin-dependent diabetic rats were found to exhibit reduced rates of basal and insulin-stimulated glucose metabolism. Since tissue levels of fructose 1,6-bisphosphate are significantly reduced in the diabetic heart, it was concluded that phosphofructokinase may be inhibited. However, neither glycogen nor glucose 6-phosphate accumulated in the myocyte, indicating that the phosphofructokinase reaction was not a bottleneck diverting substrate away from glycolysis. The other major factor contributing to decreased glycolytic flux in the diabetic heart is the impairment in glucose transport. Both basal and insulin-stimulated transport of 3-O-methyl-D-glucose was 30% less in the diabetic heart. While insulin sensitivity was unaltered in the diabetic rat, insulin responsiveness was decreased, indicating that the impairment in insulin-stimulated hexose transport was caused by a post-receptor defect. The net result of these abnormalities in glucose metabolism is a significant reduction in the rate of ATP synthesis by the diabetic heart.

The D variant of encephalomyocarditis virus is capable of infecting most inbred strains of mice. However, only certain strains are susceptible to the diabetogenic effect of this virus. In order to understand why some inbred strains do not become diabetic, the pathogenesis of infection was studied in diabetes-resistant C57BL/6J mice. It was the purpose of the investigation to ascertain whether specific host defense factors might play a crucial role in the mechanism of resistance. To determine whether perturbations of the immune response would alter the resistance of these animals, mice were treated with a high dose (1.15 mmol/kg body weight) of the T- and B-cell toxin cyclophosphamide prior to infection with the D variant. This treatment did not induce overt diabetes or glucose intolerance in the mice tested 7 days after infection. Based on this finding, it appeared likely that resistance to the D variant is conveyed by some factor other than cell-mediated immunity. A likely candidate to control this viral infection is the interferon system. To investigate this possibility, C57BL/6J mice were infected with the D variant and the concentrations of serum interferon titred at various intervals thereafter. In contrast to previous reports with diabetes susceptible mice, C57BL/6J mice were found to generate a substantial interferon response against this variant, with peak levels found in the serum at 24 h following infection. Additional studies were performed in which mice were treated with antibody to mouse interferon alpha/beta at the time of infection and again 3 days after infection with the D variant.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorozotocin (chlorozocin, CLZ), the 2-chloroethyl analogue of streptozocin (STZ), was evaluated in three species of rodents. The drug is currently being used in phase II chemotherapeutic trials in man, and appears to be effective in the treatment of certain tumors. In our studies, hyperglycemia was induced in hamsters as early as 2 days after a single intraperitoneal (i.p.) injection of 30-60 mg/kg and was most striking at 4 days. Greater concentrations of CLZ (greater than or equal to 50 mg/kg) were required to produce hyperglycemia in CD-1 mice. Degranulation and necrosis of beta cells developed in hamsters and mice, whereas alpha and acinar cells of the pancreas revealed no morphologic changes. Hyperglycemia was not induced in rats at any concentration tested; however, animals showed abnormal carbohydrate tolerance after administration of 100 mg/kg CLZ (LD50 dosage). The nature of damage by CLZ to beta cells was investigated both in vivo and in vitro. Pretreatment of hamsters with nicotinamide (500 mg/kg, i.p.) failed to alter the extent of CLZ-induced beta cell injury and associated hyperglycemia, but decreased the amount of beta cell necrosis and hyperglycemia in animals receiving STZ. The nonmetabolizable sugar, 3-O-methylglucose (3-O-MG), and 3-aminobenzamide, an inhibitor of the nuclear enzyme, polyADPribose synthetase, prevented STZ-associated damage to beta cells in islet cell cultures, but only 3-O-MG reduced CLZ-induced toxicity. Thus, in comparison to STZ, CLZ appears to be a diabetogenic agent with different species specificity and alternative mechanisms of cytotoxicity. The glucose moiety of both drugs appears critical in the induction of beta cell damage.