Karyotype heterogeneity and unclassified chromosomal abnormalities.

In a departure from traditional gene-centric thinking with regard to cytogenetics and cytogenomics, the recently introduced genome theory calls upon a re-focusing of our attention on karyotype analyses of disease conditions. Karyotype heterogeneity has been demonstrated to be directly involved in the somatic cell evolution process which is the basis of many common and complex diseases such as cancer. To correctly use karyotype heterogeneity and apply it to monitor system instability, we need to include many seemingly unimportant non-specific chromosomal aberrations into our analysis. Traditionally, cytogenetic analysis has been focused on identifying recurrent types of abnormalities, particularly those that have been linked to specific diseases. In this perspective, drawing on the new framework of 4D-genomics, we will briefly review the importance of studying karyotype heterogeneity. We have also listed a number of overlooked chromosomal aberrations including defective mitotic figures, chromosome fragmentation as well as genome chaos. Finally, we call for the systematic discovery/characterization and classification of karyotype abnormalities in human diseases, as karyotype heterogeneity is the common factor that is essential for somatic cell evolution.

Heterogeneity of cell death.

Cell death constitutes a number of heterogeneous processes. Despite the dynamic nature of cell death, studies of cell death have primarily focused on apoptosis, and cell death has often been viewed as static events occurring in linear pathways. In this article we review cell death heterogeneity with specific focus on 4 aspects of cell death: the type of cell death; how it is induced; its mechanism(s); the results of cell death, and the implications of cell death heterogeneity for both basic and clinical research. This specifically reveals that cell death occurs in multiple overlapping forms that simultaneously occur within a population. Network and pathway heterogeneity in cell death is also discussed. Failure to integrate cell death heterogeneity within analyses can lead to inaccurate predictions of the amount of cell death that takes place in a tumor. Similarly, many molecular methods employed in cell death studies homogenize a population removing heterogeneity between individual cells and can be deceiving. Finally, and most importantly, cell death heterogeneity is linked to the formation of new genome systems through induction of aneuploidy and genome chaos (rapid genome reorganization).

Combined multicolor-FISH and immunostaining.

The combination of multicolor-FISH and immunostaining produces a powerful visual method to analyze in situ DNA-protein interactions and dynamics. Representing one of the major technical improvements of FISH technology, this method has been used extensively in the field of chromosome and genome research, as well as in clinical studies, and serves as an important tool to bridge molecular analysis and cytological description. In this short review, the development and significance of this method will be briefly summarized using a limited number of examples to illustrate the large body of literature. In addition to descriptions of technical considerations, future applications and perspectives have also been discussed focusing specifically on the areas of genome organization, gene expression and medical research. We anticipate that this versatile method will play an important role in the study of the structure and function of the dynamic genome and for the development of potential applications for medical research.

The purMN genes of Rhizobium leguminosarum and a superficial link with siderophore production.

We isolated a mutant of R. leguminosarum initially on the basis of reduced production of the siderophore vicibactin on chrome azurol sulfonate (CAS)/agar indicator plates. The mutation was in the purMN operon and the mutant was shown to be an adenine auxotroph and defective for nodulation of peas. The siderophore defect appears to be trivial, being due to diminished growth of the auxotroph on agar-based minimal medium, which contains unknown contaminant(s) that allow it grow poorly. Transcriptional fusions showed that purMN was transcribed at relatively high levels in media containing purines. Expression was enhanced, approximately twofold, if purines were omitted.

Adaptation to hyperoxia in the neonatal rat: kinetic parameters of the oxygen-mediated induction of lung superoxide dismutases, catalase and glutathione peroxidase.

The activities of the enzymes superoxide dismutase, catalase and glutathione peroxidase increase in the lungs of neonatal rats exposed to normobaric hyperoxia. The oxygen-mediated increase in activity of these enzymes, known from previous studies to be an inductive response, was studied in 10- and 25-day-old rats as a function of both oxygen concentration and length of time of exposure to greater than 95% oxygen. In the lungs of 10-day-old rats the increase in superoxide dismutase, catalase, and glutathione peroxidase occurs only at 80% ambient oxygen or greater. In 25-day-old rats a similar pattern of response occurs with pulmonary catalase and glutathione peroxidase. However, unlike the response in 10-day-old rats, pulmonary superoxide dismutase does not increase in oxygen-exposed 25-day-old rats. The time course of enzyme induction was different for 10-day-old rats compared with 25-day-old rats. Exposure of 10-day-old rats to 95+% oxygen resulted in a significant increase in activity of superoxide dismutase after only 4 h when compared with air-exposed control animals. Catalasee and glutathione peroxidase in the same age group increased significantly after 6 h and 12 h of exposure to oxygen, respectively. Maximal levels of superoxide dismutase, catalase and glutathione peroxidase were reached after 6, 12 and 24 h of exposure to hyperoxia, respectively. This level of activity was then maintained throughout the subsequent exposure time up to 96 h. The activity of pulmonary catalase and glutathione peroxidase in 25-day-old rats did not increase significantly after 6 h of exposure to hyperoxia. An apparent plateau of increased activity was reached after 24 h of exposure. As observed with the 3 enzymes in 10-day-old rats, maximal enzyme activities were maintained throughout the subsequent period of oxygen exposure up to 96 h.

Toxicity and pharmacokinetics of subconjunctival amphotericin B. An experimental study.

The pharmacokinetics and toxicity of subconjunctival (S/C) amphotericin B (AmB) were evaluated in Dutch-belted rabbits. Following the S/C injection of 1,500 micrograms of AmB, corneal and aqueous levels were determined by bioassay. The highest levels were present in the periphery of debrided corneas at 1 h (90.12 +/- 2.4 micrograms/g). The debrided central cornea contained 30.84 micrograms/g, almost double the amount present in the intact central cornea. These levels were transient; in the central intact cornea only 2.08 micrograms/g could be detected at 2 h. Peak aqueous levels were low (0.95 +/- .24 micrograms/mL in debrided corneas at 1 h). The S/C injection of 1,500 micrograms of AmB in sodium deoxycholate produced a severe inflammatory response in the conjunctiva, episclera, iris, anterior chamber, and superior rectus muscle that persisted 10 days. Injection of sodium deoxycholate alone produced a similar but less severe response.

Bipyridylium herbicide toxicity: effects of paraquat and diquat on isolated rat hepatocytes.

Paraquat (1,1'-dimethyl-4,4'-bipyridylium) and diquat (1,1'-ethylene-2,2'-bipyridylium) are the two most widely used bipyridylium herbicides today. Both compounds, however, have also been found to cause liver damage in animals and man. In this study, isolated rat hepatocytes were used to assess the cytotoxicity of these two compounds. Five indices of cell damage were used to quantitate cytotoxicity: dye (trypan blue) uptake, loss of cell respiration, the extracellular release of lysosomal enzymes, the formation of thiobarbiturate (TBA)-reacting substances, and the oxidation of cellular NADH and NADPH. Diquat was shown to be more toxic than paraquat toward these cells in the first three assay systems. The acute LC50 for diquat was determined to be 80 mM, whereas the acute LC50 for paraquat was estimated to be greater than 1 M. This difference in cell sensitivity could be explained, at least in part, by the observed differences in herbicide uptake rates for these two compounds. Diquat uptake was calculated to be 9.0 +/- 1.1 nmoles/hour/10(6) cells, whereas paraquat uptake was only 5.5 +/- 0.5 nmoles/hour/10(6) cells. The dose-response curves for enzyme release and loss of cell respiration superimposed the lethality curves for both compounds. An oxidative mechanism of cytotoxicity was suggested for diquat by: the establishment of both a concentration-dependent and a time-dependent increase in lipid peroxidation (formation of TBA-reacting substances); complete oxidation of both NADPH and NADH at herbicide levels less than the LC50; and the finding that diquat stimulated glucose oxidation at subtoxic doses.

Field rickets in turkey poults: biochemical findings.

Two clinical cases of field rickets and one of nutritional rickets in turkey poults were studied. Plasma levels of calcium, phosphorus, alkaline phosphatase, and vitamin D metabolites were determined. Concentrations of calcium, phosphorus, and alkaline phosphatase in affected poults were typical of nutritional rickets. The mean plasma calcium concentration in rachitic poults was not significantly different from that in controls. Mean values for plasma phosphorus were 1.5 to 2.4 mg/dl lower in the rachitic poults, and values for alkaline phosphatase activity were 1.3-1.7 times greater. In assays of vitamin D metabolites, mean 24, 25 dihydroxyvitamin D3 values were consistently lower in rachitic poults.

The immunological response of llamas (Lama glama) following experimental infection with Mycobacterium bovis.

Llamas were experimentally infected with Mycobacterium bovis in order to evaluate the axillary skin test and the ELISA as diagnostic procedures for tuberculosis in llamas (Lama glama). Six llamas were given a single intratracheal challenge with 1 of 2 doses of a recent field isolate of M. bovis and 2 llamas were left as noninfected controls. This resulted in a progressive disease in some animals with 1 mortality as early as 68 d post-infection (PI). The tuberculin skin test, at the axillary site, was positive in 4 of 5 infected llamas at 80 d PI. At 143 d PI, all 3 surviving lamas were positive, including the one which had not responded at 80 d PI. The application of skin and serological tests throughout the course of this experiment adds support for the need to further evaluate the skin test and its anamnestic effect on serodiagnosis since serological responses were generally not observed in the absence of skin testing or antibiotic treatment. The wide variation in M. bovis antigens recognized by the serological response would indicate that a diagnostic panel should include multiple antigens such as MPB70 and lipoarabinomannan (LAM). While skin testing or serology alone may be of limited value to diagnose tuberculosis in llamas, together they may offer an enhanced potential for immunodiagnosis of tuberculosis.

Free fatty acid-induced hypocalcemia in food-deprived dairy cattle.

Food deprivation in 6 dairy cows caused significant increases (P greater than F = 0.0054) in serum free fatty acid concentrations, but did not cause clinically important decreases in serum ionized calcium concentration. Several determinants, including blood pH, serum albumin, and serum bicarbonate were studied as factors that could potentially alter calcium ion concentration. Although several changes were significant, they were small and were not considered to be clinically important in the context of calcium-ion binding. It was concluded that free fatty acid binding of calcium ion did occur, but that such binding did not contribute significantly to the etiopathogenesis of calcium responsive midlactational downer cow syndrome.

Inherited, chronic, progressive hepatic degeneration in Bedlington terriers with increased liver copper concentrations: clinical and pathologic observations and comparison with other copper-associated liver diseases.

One hundred nineteen hepatic tissue samples from 117 Bedlington Terriers were divided into 6 groups depending on the severity of histopathologic hepatic changes. Group 0 comprised dogs with microscopically normal livers. Group I dogs had copper-positive, lipofuscin-containing lysosomes present in centrilobular hepatocytes. Microfoci of hepatic necrosis, in addition to the increased numbers of the copper-positive, lipofuscin-containing lysosomes in centrilobular and periportal hepatocytes, were present in group II dogs. Group III dogs had more copper-positive, lipofuscin-containing lysosomes present translobularly and morphologic changes consistent with chronic active hepatitis. Mixed micro- or macronodular cirrhosis and translobular presence of copper-positive, lipofuscin-containing lysosomes characterized group IV dogs. Dogs in group V had massive hepatic necrosis and morphologic changes that were consistent with the changes in group III and IV dogs. Histochemical staining for copper was useful in making the microscopic diagnosis of this disease and was shown to be necessary in early diagnosis (group I) when other clinical and pathologic values associated with this syndrome were not consistently abnormal. Copper histochemical stains varied in sensitivity. Timm's silver sulfide was more sensitive for copper than was rubeanic acid, which was more sensitive than rhodanine staining. The brown pigment associated with the copper in the lysosomes was shown to be lipofuscin pigment with the aid of histochemical staining with orcein, Prussian blue, periodic acid-Schiff, and acid-fast stains together with fluorescent microscopy (excitation maxima: 365 nm; emissions: 420 + nm). Since these were positive only in later stages of the hepatic disease, they were not especially useful in its early diagnosis. The severity of the histopathologic hepatic changes was shown to increase with age and was associated with increasing hepatic copper concentration. These observations illustrate that this inherited, chronic hepatic degeneration in the Bedlington Terrier is progressive. Clinical chemical tests were diagnostically useful only in later stages of the disease. Alanine transaminase activity was of most value, but was not always abnormal, even when severe hepatic damage was present. Clinical signs of hepatic disease were seen in dogs in groups III, IV, and V. Death due to hepatic failure occurred only in dogs in groups III, IV, and V. Hemosiderin was present in increased amounts in the liver, bone marrow, spleen, and lymph nodes of affected Bedlington Terriers, indicating that a possible defect in iron metabolism and/or an increase in RBC turnover existed.(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical evaluation of commercial reagent strips for detection of significant bacteriuria in dogs and cats.

The reagent strip technique of quantitative urine culture was a satisfactory screening test for the detection of significant bacteriuria in dogs and cats. The reagent strip technique was found to be of almost equal sensitivity as the calibrated loop technique in identifying significant bacteriuria. Significant bacteriuria was detected in 38 of 148 urine samples evaluated by the calibrated loop technique. Of these 38 urine samples, significant bacteriuria was detected in 35 by the commercial test. False-negative results were obtained with reagent strips in 1 urine sample which had significant bacteriuria by the calibrated loop technique. The other 2 samples had bacterial counts of suspicious significance (10,000/ml of urine) by the reagent strip technique. False-positive results were obtained with the reagent strip technique in 3 urine samples; however, all 3 urine samples had bacterial counts (10,000/ml of urine) of suspicious significance when evaluated by the calibrated loop technique. The reagent strip technique was not satisfactory for the examination of patients with gross hematuria. The nitrite test portion of the commercial test did not detect significant bacteriuria in all samples and, therefore, was not suitable as a screening test for significant bacteriuria in dogs and cats. The nitrite test may have been inhibited by ascorbic acid, a metabolite which normally may exist in the urine of these animals.

Carboxyhemoglobin concentrations in normal cattle.

Percentage carboxyhemoglobin (HbCO) values were determined in 29 cows and 24 bulls in Minnesota during the summer of 1973. Results indicated that, in normal cattle, the HbCO value was 0.3%. Individual and daily variations in these values were recorded in 2 calves.

Blood concentrations of beta hydroxybutyrate in clinically normal Holstein-Friesian herds and in those with a high prevalence of clinical ketosis.

Statistical parameters necessary for the comparison of herd blood beta hydroxybutyrate (HB) concentration means with HB concentrations in the general population were determined in 11 clinically normal herds of Holstein-Friesian cows. These parameters were then used to compare clinically normal cows from 2 Holstein-Friesian herds experiencing a high prevalence of clinical ketosis with the normal population. Blood HB concentration means were compared with plasma glucose concentration means for their relative value as predictors of clinical ketosis prevalence within a herd. Blood HB concentration values were distributed approximately log normal and were directly related to amount of milk production. Herds with a high prevalence of clinical ketosis had blood HB log means higher (P less than 0.01) and glucose means lower (P less than 0.05) than did the normal population. Herd blood HB log means should be determined on 7 or more animals within specified production groups. When these means exceed the normal population log mean by more than 1 SD, susceptible cows in the herd may be at increased risk for the development of clinical ketosis. Blood HB log means may be of greater value than are plasma glucose concentration means for the prediction of herd ketosis prevalence.

Influence of ration composition and energy balance on blood beta-hydroxybutyrate (ketone) and plasma glucose concentrations of dairy cows in early lactation.

The effect of ration composition, with respect to concentrate, crude protein, and methionine hydroxyanalog content, on blood beta-hydroxybutyrate and plasma glucose concentrations was assessed in Holstein cows every 2 weeks over the first 6 weeks of lactation. The correlation of these metabolites with estimated energy balance, and the effects of these ration variables on this correlation were studied. High concentrate diets (60% of dry matter) compared with low concentrate diets (40% of dry matter) increased mean plasma glucose values and reduced mean blood beta-hydroxybutyrate concentration. Variation in crude protein and methionine hydroxyanalog supplementation did not affect metabolite concentration. The correlations between blood beta-hydroxybutyrate and energy balance and between plasma glucose and energy balance were weak and subject to the influence of variation in ration composition. It was concluded that plasma glucose and blood beta-hydroxybutyrate concentrations cannot be used as valid indicators of energy balance. However, it did appear that blood beta-hydroxybutyrate might be used as an indicator of the relative glucogenic potential of dairy rations and that blood concentrations of this metabolite could potentially be used to adjust factors in the ration which influence glucose availability to the cow.

Serum selenium concentrations and glutathione peroxidase activities in cattle grazing forages of various selenium concentrations.

In cows from 15 dairy herds (n = 210), serum selenium (Se) concentrations ranged from 0.021 to 0.789 microgram/ml, whereas 0.05 to 0.40 microgram/ml is the reported range for adequate serum Se concentrations in cattle. Serum Se concentrations of dairy cattle appeared to follow a geographic distribution pattern. On the basis of herd mean serum Se concentrations, adequate serum Se concentrations were found in cattle from only 1 of 5 herds grazing forage in the geographic area classified as Se deficient for cattle. Adequate mean serum Se concentrations were found in cattle from 4 of 5 herds located in geographic areas described as having variable forage Se concentrations (Se-marginal areas). Of the 10 herds from these 2 areas, there were only 2 herds in which 95% of the cattle had serum Se concentrations in the Se-adequate range (0.05 to 0.40 microgram/ml). In 2 selected neighboring farms in the Se-deficient area, cattle in 1 herd had adequate serum Se concentrations and cattle in the other herd had less than adequate serum Se concentrations (less than 0.05 microgram/ml). Therefore, more cattle are at risk of developing Se-deficiency disease than is commonly believed and forage of neighboring farms may have different Se concentrations. Serum Se concentrations (up to 0.789 microgram/ml) correlated with glutathione peroxidase enzyme activity; this serum Se concentration (0.789 microgram/ml) is approximately 6.2 times higher than previously reported in dairy cattle. Therefore, RBC glutathione peroxidase activity may be useful in determining the diagnosis of chronic Se toxicosis.

Synovial membrane changes after experimental transection of the cranial cruciate ligament in dogs.

Degenerative joint disease and inflammation of the synovial membrane were produced in the left stifle of 16 dogs by severing the cranial cruciate ligament. Arthrotomy only was performed on the right stifle. Synovial membrane from these joints was histologically examined at 1, 2, 8, and 13 weeks after surgical operation. Similar tissue was obtained from 4 healthy dogs for comparison. Inflammatory changes in the synovium of the left stifle progressed with time and were prominent at 8 weeks postoperatively; subsynovial fibrosis was greatest at 13 weeks. Inflammation of the synovial membrane and subsynovial tissue was characterized by synovial cell hypertrophy and hyperplasia, plasma cell and lymphocyte infiltration, and increased vascularization of the subsynovial region.

Effects of prolonged distention of retention catheters on the urethra of dogs and cats.

The effects of inflation of a balloon-tipped catheter in the distal part of the urethra of clinically healthy male and female dogs and cats were evaluated. Tissues were evaluated at 1 and 14 days after balloon inflation. Macroscopic and microscopic lesions were limited to the distal part of the urethra occupied by the inflated balloon in animals which were euthanatized 1 day after balloon inflation. The lesions were characterized by submucosal urethral hemorrhage. This investigation indicates that balloon inflation for 15 minutes duration in the distal part of the urethra of dogs and cats results in a mild reversible inflammatory reaction.

Complications of retrograde contrast urethrography in dogs and cats.

Urinary bladder lesions varying histologically from a focal hemorrhagic cystitis to a diffuse transmural fibrinonecrotic cystitis were encountered in 11 of 14 dogs and cats euthanatized 1 day after retrograde urethrography. Focal-to-diffuse hemorrhagic urethritis was histologically encountered in 14 of 14 dogs and cats euthanatized 1 day after retrograde urethrography. Lesions in the urinary bladder and urethra were not reversible within 14 days and were evident histologically in 10 of 15 urinary bladders and 11 of 15 urethras of dogs and cats euthanatized 14 days after retrograde urethrography. During urethrography, iatrogenic mural leakage of contrast media into the bladder wall or bladder wall rupture was encountered radiographically in 6 of 15 dogs and 3 of 14 cats. However, macroscopic evidence of bladder rupture was encountered in only 1 male dog at necropsy. Immediately after retrograde urethrography, macroscopic hematuria occurred in 12 of 15 dogs and 9 of 14 cats. Microscopic hematuria was not encountered in dogs or cats euthanatized 14 days after urethrography. Pyuria was encountered less frequently and was present in only 2 cats euthanatized 14 days after urethrography. One day after urethrography, urinary tract infection characterized by urine cultures with greater than 10(5) microbes/ml was encountered in 4 of 29 dogs and cats. All animals with urinary tract infection were female dogs or cats. At day 14, only 1 female cat had a positive urine culture. Seemingly, the technique of retrograde urethrography was the cause of the encountered lesions.

Influence of modified protein diets on electrolyte, acid base, and divalent ion balance in dogs with experimentally induced chronic renal failure.

The influence of a maintenance diet (44.4% dry weight protein) and 2 reduced protein diets (8.2 and 17.2% dry weight protein) on acid-base, electrolyte, and divalent ion balance of dogs with experimentally induced chronic renal failure was studied for 40 weeks. Moderate-to-severe hyperchloremic metabolic acidosis developed in dogs fed the 8.2% protein diet, but not in the groups fed the 17.2% and 44.4% protein diets. Serum sodium and potassium concentrations were not altered significantly by the diets. Mean serum calcium concentrations increased significantly in the 3 groups after renal failure was induced; however, serum calcium concentration generally did not exceed the normal range. Dogs fed reduced protein diets had smaller serum magnesium and phosphorus concentrations than did the dogs fed the maintenance diet. Microscopic alterations in bone morphology consistent with canine renal osteodystrophy were not detected after 40 weeks of renal failure in dogs fed the 17.2% and 44.4% protein diets. Examination of skull radiographs of the dogs prepared 40 weeks after renal failure was induced revealed that the lamina dura dentes were visible in all groups. It was concluded that the 8.2% protein diet was of therapeutic benefit in management of altered divalent ion metabolism in dogs with chronic renal failure. However, the high frequency of concomitant acidosis indicated that appropriate alkalinization therapy should be included with this diet. Adverse effects of feeding the 17.2% protein diet were not detected. However, it was less effective than the 8.2% protein diet in minimizing development of hypermagnesemia and skeletal changes consistent with renal osteodystrophy.