Localization and characterization of IGF-I receptors in fetal and adult human kidneys. Human insulin-like growth factor I (IGF-I) is a growth and differentiating factor produced by various adult and fetal tissues. In the kidney, it has been linked to the proliferative response of renal tubular and glomerular cells, following unilateral or partial nephrectomy, in acromegaly, in diabetes mellitus and in glomerulonephritis. To gain insight into the potential effects of IGF-I in human kidney, a quantitative analysis of IGF-I-binding sites was performed in fetal and adult tissue using 125I-IGF-I. The ligand consistently labelled renal cortex, medulla, and glomeruli while renal vessels were not uniformly marked. The highest affinity of binding sites was found in glomeruli (adult kidneys: Kd 24.7 +/- 5.1 pM; Bmax 5.2 +/- 0.5 fmol/mg tissue equivalent (TE); n = 4; fetal kidneys: Kd 17.0 +/- 2.5 pM; Bmax 4.5 +/- 0.7 fmol/mg TE; n = 4) and cortical tubules, while vessels and renal medulla (adult kidneys: kd 47 +/- 3.9 pM, Bmax 2.6 +/- 0.3 fmol/mg TE; n = 4; fetal kidneys: kd 41.6 +/- 9.2 pM, Bmax 3.5 +/- 0.4 fmol/mg TE; n = 3) had only about half the affinity of binding and a significantly reduced maximal capacity. The strong binding of 125I-IGF-I to glomeruli supports the view that IGF-I may be involved in modulating glomerular structure and function. Fetal renal growth may depend on the action of IGF-I on glomerular cells and tubular epithelia of the kidney.


Insulin-like growth factor I and its variant, des(1-3)IGF-I, improve nitrogen balance and food utilization in rats with renal failure. The growth-promoting effects of IGF-I were examined in rats following partial nephrectomy and compared with the effects of the des(1-3) variant of IGF-I. Four groups of rats were subjected to 5/6 nephrectomy (n = 8 per group) and treated for 7 days with IGF-I (0.9 or 2.2 mg/kg BW/day), des(1-3)IGF-I (0.9 mg/kg/BW/day), or vehicle (0.1 M acetic acid) administered subcutaneously by osmotic pump. A group of vehicle-treated, sham-operated control rats (n = 7) was included. Food utilization was significantly improved in all three peptide-treated groups, by 13-16% compared with the vehicle-treated nephrectomized group. Also, nitrogen balance was enhanced, particularly in the des(1-3)IGF-I group, in which nitrogen excretion was reduced by 24%, with the low- and high-dose IGF-I groups showing 16 and 18% reductions, respectively. Serum urea levels were significantly decreased, by 25%, in the des(1-3)IGF-I group, with 20 and 17% reductions being observed in the low- and high-dose IGF-I groups. Muscle protein degradation was found to be significantly attenuated with des(1-3)IGF-I treatment but was not significantly affected in the two IGF-I-treated groups. While carcass composition was not altered with IGF peptide treatment, absolute mass of protein in the carcass was improved in rats treated with the high dose of IGF-I. These results show that IGF-I or, more particularly, des(1-3)IGF-I may be efficacious in overcoming impaired growth in renal failure.


The rationale for the use of growth hormone or insulin-like growth factor I in adult patients with renal failure. Since the development of recombinant DNA technology, there has been a rapid expansion of research concerning the use of recombinant DNA synthesized human growth hormone (rhGH) for the treatment of clinical disorders. rhGH has been used to treat patients with acute catabolic stress caused by surgery, trauma and sepsis, children with chronic renal insufficiency and impaired growth, patients undergoing maintenance hemodialysis who are malnourished, and individuals on weight reduction diets. These studies indicate that rhGH enhances protein balance in acutely stressed patients and in malnourished maintenance hemodialysis patients, promotes catch-up growth in children with chronic renal failure, and may reduce protein wasting and enhance lipolysis in obese individuals on weight reduction diets. Experimental studies suggest that in addition to enhancing anabolism, rhGH may increase both immune function and the rate of wound healing. Many, but not all, of the effects of rhGH are mediated through insulin-like growth factor I (IGF-I). For example, the hyperglycemic and lipolytic effects of rhGH do not seem to be caused by IGF-I. Animal or human studies suggest that with severe malnutrition or severe sepsis, rhGH treatment may neither increase serum IGF-I levels nor promote anabolism. These observations provide a rationale for administering IGF-I as an anabolic hormone for severely malnourished or septic patients with renal failure. Further studies will be necessary to examine both the short-term and long-term potential benefits and adverse effects of rhGH or rhIGF-I treatment in these conditions.


Increase of circulating insulin-like growth factor-I in chronic renal failure is reduced by low-protein diet. We performed a nutritional trial to assess the variations of circulating insulin-like growth factor-I (IGF-I) in chronic renal failure (CRF). Eight patients suffering from mild renal failure (SCr = 374 +/- 52 mumol/l) were prescribed a standard diet for 1 month followed by 1 month of protein restriction. Mean protein intake was 0.77 and 0.46 g/kg BW/day, mean caloric intake 25 and 24.7 kcal/kg BW/day for the first and the second month, respectively. After each period of diet, nitrogen balances were negative (-1.2 +/- 1.6 and -1.6 +/- 0.9 g/N/day). Despite these low-caloric conditions, mean serum IGF-I level was at the upper level of normal (358 +/- 136 ng/ml) after 1 month of standard protein intake, and statistically reduced (289 +/- 122 ng/ml, p < 0.002) by the low-protein diet. No correlation was observed between serum IGF-I levels and protein, caloric intake, and nitrogen balances for the two periods. Estimation of the IGF-I binding by the ratio of extracted to nonextracted IGF-I value suggested abnormal binding in CRF. This binding was modified by reduced protein intake. In conclusion, larger studies are needed in CRF to assess the significance of IGF-I variations and the IGF-I binding proteins which modulate the bioactivity of this growth factor.


Factors causing catabolism in maintenance hemodialysis patients. Malnutrition is common in maintenance hemodialysis (HD) patients who are frequently anorectic. The protein requirements in HD patients are increased compared with nonuremics. Factors which tend to increase the protein requirements are: low energy intake, metabolic acidosis, leading to enhanced catabolism of branched-chain amino acids in muscle, and dialytic losses of amino acids and glucose. In addition, the HD procedure results in inhibition of muscle and whole body protein synthesis, and induces an inflammatory response (blood membrane interaction, endotoxins) leading to enhanced muscle protein breakdown. The relative importance of the various factors leading to enhanced net protein catabolism in HD patients remains to be elucidated.


Use of bioelectrical impedance for the nutritional assessment of chronic hemodialysis patients. Although malnutrition poses a significant risk to the well-being of chronic hemodialysis patients, their nutritional assessment is usually empirical. We studied body composition by bioelectrical impedance (BEI) prospectively in 39 patients followed for 5-12 months. BEI correctly discriminated between underweight and overweight patients in terms of fat mass (21 +/- 5 vs. 34 +/- 10%; p = 0.002), lean body mass (78 +/- 4 vs. 67 +/- 10%; p = 0.004) and total body water (57 +/- 3 vs. 49 +/- 7%; p = 0.002), respectively. Serial body weights did not correlate with changes in lean body mass (LBM) as measured by BEI. While 28% of patients lost weight, 41% lost LBM. Most striking is the contrast between the patients who showed no change in LBM by BEI and those whose body weight remained neutral (3 vs. 28%). BEI is a most sensitive clinical tool for assessing changes in LBM in hemodialysis patients.


Impaired gut barrier in experimental chronic uremic rats. The intestinal permeability in chronic uremic rats on a high- and a low-protein diet was measured with a mixture of differently sized polyethylene glycols (PEG 400 and PEG 1000). The permeability was assessed by the 24-hour urinary recovery of PEG molecules (range 326-1,162 Da) administrated orally or intravenously. The urinary recovery after oral load, when corrected with the recovery after intravenous administration, was significantly enhanced in the uremic rats. A decreased urinary recovery was observed in the groups on the low-protein feed diet. There was also decreased selectivity towards larger PEGs in uremia. The results thus suggest that (i) the intestinal permeability is increased in chronic uremic rats, and (ii) the intestinal permeability is influenced by a low-protein diet.


Erythrocyte vitamin B1, B2 and B6 in nephrotic syndrome. Vitamin status was evaluated in 33 patients with chronic glomerulonephritis and nephrotic syndrome who had normal glomerular filtration rates. Erythrocyte vitamin B1 and B2 were within the normal reference range. Vitamin B6 was significantly decreased. Long-term, 6-month supplementation with riboflavin, 2 mg/24 h, and pyridoxine HCl, 50 mg/24 h, in 9 patients improved erythrocyte vitamin B2 and B6 levels even though the clinical status and the disease activity of the basic disease persisted or progressed.


Factors affecting chronic renal failure progression: results from a multicentre trial. The Northern Italian Cooperative Study Group. In order to evaluate the prognostic factors concerning the rate of progressive deterioration of renal function, we made an inductive analysis of the behaviour of 456 patients in a multicentre, formal prospective trial aimed at clarifying the possible role of protein restriction in retarding the progression of chronic renal insufficiency (CRI). The main clinical and laboratory findings in patients whose plasma creatinine (PCr) levels doubled in comparison with baseline randomization values or who needed dialysis within 24 months after onset of the study were compared with those of the other patients. In addition, independently of the assigned diet, we tested the main variables that might affect CRI progression (sex, systolic and diastolic blood pressure, change in body weight, hematocrit, calcium-phosphate product, proteinuria, protein catabolic rate, total cholesterol and triglycerides). We used multiple regression analyses and also plotted the mean values of these parameters in each patient against an estimate of the deterioration of chronic renal failure based on the difference between the final and the initial reciprocal of the PCr and the creatinine clearance (CCr) levels. A descriptive analysis of the behaviour of PCr in the three CRI groups and in the four underlying diseases groups was made. PCr levels at entry, underlying disease and proteinuria were prognostic factors for CRI progression. The increase in PCr was 0.0102 mg/dl/month in patients with nephrosclerosis, 0.0203 mg/dl/month in interstitial nephropathy, 0.0360 mg/dl/month in glomerulonephritis and 0.0704 mg/dl/month in polycystic kidney disease.(ABSTRACT TRUNCATED AT 250 WORDS)


Multicentre randomized study on the effect of a low-protein diet on the progression of renal failure in childhood: one-year results. European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood. After an adaptation period of 6 months, 200 children (mean age 10.3 years; 138 boys and 62 girls) were stratified according to their renal diseases and the progression of renal failure during the adaptation period and randomized for low protein intake amounting to the WHO safe levels for age (0.8-1.1 g/kg/day) or free protein intake. Energy intake of both groups should be near the WHO recommendations. Compliance was controlled by written dietary diaries and urea-N excretion. 101 children had been randomized for the diet group and 99 for the control group. 165 of these patients finished their first year after randomization. Mean protein intake was 126% of the recommendations in the diet group vs. 187% in the control group while energy intake was similar in both groups (85 vs. 90%). SDS for height at start and 1 year later did not change (diet: -0.9 vs. -1.1; control: -0.9 vs. -0.9). Weight gain was not different. Only the stratification for two groups according to the progression of renal failure ('nonprogressive' and 'progressive' diseases) was used in the analyses. Progression of renal disease during the adaptation period proved to be a significant prognostic factor. Mean loss of GFR/year was similar in patients with 'nonprogressive' diseases in the control group (-1.1 ml) and the diet group (-1.3 ml). In patients with 'progressive' diseases mean loss of GFR was significantly higher in the diet group (-6.5 ml) than in the control group (-4.0 ml).(ABSTRACT TRUNCATED AT 250 WORDS)


Amino acid solutions for CAPD: rationale and clinical experience. Protein-calorie malnutrition is present in a sizable proportion of dialysis patients. In CAPD patients, constant glucose absorption from dialysate may displace other calorie sources, such as protein, and may suppress the appetite, thus contributing to malnutrition. Use of amino acids in place of glucose as the osmotic agent has been studied extensively. Ultrafiltration and small-molecule clearance similar to that with glucose can be achieved with amino acid solutions, but nitrogenous waste produced by amino acids limits the extent to which they can replace hypertonic glucose. Side effects of CAPD with amino acids appear to be minor and easily manageable. Most studies have found at least some nutritional benefit of amino acid solutions in addition to that of lowering the glucose load. Short-term studies of amino acid solutions for dialysis indicate that they may improve protein nutrition in malnourished CAPD patients.


Effect of glucocorticoids and extracellular pH on protein metabolism in cultured cells. Chronic renal failure (CRF) is complicated by metabolic acidosis and muscle wasting. Protein degradation (PD) in skeletal muscle is accelerated in rats with CRF and correction of uremic acidosis returns PD to normal. Experimentally induced acidosis in normal rats accelerates PD and requires an intact adrenal axis. To investigate mechanisms of pH-induced changes in protein metabolism, BC3Hl myocytes and LLC-PK1 renal epithelial cells were studied. Low extracellular pH increases PD in myocytes but does not change PD in LLC-PK1 cells. In both types of cells, intracellular pH changes predictably as extracellular pH is varied. Exogenous glucocorticoids (GC) do not alter PD in either cell line, but inhibit protein synthesis in BC3Hl myocytes. Since extracellular pH stimulates PD only in BC3Hl myocytes and since LLC-PK1 cells may not possess GC receptors, we can compare and contrast the effects of pH and GC on protein metabolism to study the role of GC in acid-stimulated proteolysis.


Therapeutic value of recombinant human growth hormone in children with chronic renal failure. In chronic renal failure (CRF), serum growth hormone (GH) levels are elevated due to a reduced renal metabolic clearance. Although the concentration of circulating GH is increased in CRF, insensitivity to GH is noted. This is mainly due to a decreased production of hepatic insulin-like growth factor (IGF)-I and an accumulation of its binding protein (IGFBP-3), both resulting in a low concentration of free active IGF-I. Treatment with recombinant human (rh) GH in doses of about 30 IU/m2/week, increases the serum concentration of IGF-I, normalizes somatomedin (IGF) bioactivity and leads to catch-up growth. The anabolic effects of rhGH are seen from an increase in muscle mass and a reduction of fat mass. rhGH treatment is also able to compensate for the growth-depressing effects of corticosteroids in animal studies and in children after renal transplantation.


Effects of dietary protein on eicosanoid production and on the activity of related enzymes in glomeruli and tubules. Glomeruli obtained from rats fed a high-protein diet for 8 weeks produce increased amounts of eicosanoids due to greater activity of both PE-specific phospholipase A2 and cyclooxygenase. Eicosanoid synthesis in response to angiotensin II is also greater in glomeruli from rats fed a high-protein diet when compared to rats fed a low-protein diet. There was also increased synthesis of eicosanoids in medullary/papillary tubules but not in proximal cortical tubules of rats fed a high-protein diet. This appears to be due to increased activity of PC-specific phospholipase A2 and cyclooxygenase in medullary/papillary tubules.


Effect of dietary protein on glomerular proteinase activities. Recent studies suggest that proteolytic enzymes are involved in the degradation of extracellular matrix components of the renal glomerulus. In the present study, the effects of feeding 3 different protein diets on glomerular cysteine proteinase and metalloproteinase activities to healthy rats for 6 weeks were examined. The diets contained 5, 20, or 60% casein and were made isocaloric by starch. On sacrifice, the glomeruli were isolated by differential sieving. Proteolytic activities were measured using fluorogenic substrates and were expressed per glomerular DNA content. Body weight was virtually unchanged by the amount of protein ingested, whereas kidney weight was closely correlated with dietary protein content (5%: 1,625 +/- 324; 20%: 2,110 +/- 326; 60%: 2,705 +/- 910 mg). Activity of cathepsin B, the most abundant cysteine proteinase in the glomerulus, decreased with protein loading (5%: 1,498 +/- 110; 20%: 1,321 +/- 82; 60%: 914 +/- 84 pmol/min/micrograms DNA). The same pattern emerged with cathepsin L (5%: 869 +/- 71; 20%: 846 +/- 70; 60%: 517 +/- 83 pmol/min/micrograms DNA) and cathepsin H (5%: 498 +/- 45; 20%: 478 +/- 55; 60%: 330 +/- 39 pmol/min/micrograms DNA). The differences between the 20 and 60% groups were statistically significant for all 3 cathepsins measured. The intraglomerular activity of the metalloproteinase collagenase declined significantly with the amount of protein ingested (5%: 233 +/- 14; 20%: 189 +/- 13; 60%: 137 +/- 11 microU/micrograms DNA). Gelatinase activity also fell as protein intake increased (5%: 183 +/- 18; 20%: 115 +/- 10; 60%: 94 +/- 11 F/micrograms DNA).(ABSTRACT TRUNCATED AT 250 WORDS)


Fibronectin metabolism in isolated glomeruli from obese Zucker rats. The obese Zucker rat develops non-immune-mediated spontaneous focal glomerulosclerosis. Mesangial matrix expansion is an important initiating hallmark of such glomerular damage and fibronectin is a normal constituent of mesangial extracellular matrix. Using a quantitative method based on enzyme immunoassay, we have assessed the intraglomerular fibronectin content and its degradation in obese Zucker rats and their lean littermates. In the obese Zucker rats the glomerular fibronectin content was significantly higher than in the control animals. Furthermore, proteinase activity against fibronectin was significantly reduced in the glomeruli of obese Zucker rats compared to the control animals. These data demonstrate that in obese Zucker rats there is a glomerular accumulation of fibronectin which we propose is at least partly due to diminished proteolytic digestion. We speculate that this result might indicate a possible mediator of progressive glomerulosclerosis in this animal model.


Effect of renal disease on glomerular proteinases. Until now, little is known about the self-perpetuating mechanism leading to terminal renal failure in chronic renal disease. The common pathological feature of progressive renal insufficiency is focal and segmental glomerulosclerosis. The experimental counterpart of this process is represented by models of streptozotocin diabetes, Adriamycin nephropathy and Goldblatt hypertension. The main initiating hallmark of glomerulosclerosis is an accumulation of glomerular proteins, whose balance is apparently influenced by the activity of glomerular proteinases. In isolated glomeruli of kidneys from experimental animals, total proteinase activity was assayed with an unspecific but sensitive azocasein assay. The activity was significantly reduced in all experimental models at acid and neutral pH when relating enzyme activity to the glomerular protein and DNA content. The demonstration of reduced glomerular proteinase activity in the animal models of glomerulosclerosis could represent a new additional common pathogenetic mechanism. Glomerular protein accumulation could be a result of a synergistic interaction between hemodynamic and biochemical factors; we suggest the latter to be a decrease in glomerular proteinase activity.


Aminopeptidase A (= angiotensinase A) in human progressive renal disease. To elucidate factors possibly involved in adaptive cell responses due to progressive deterioration of glomerular structure and function, the activity of angiotensinase A (ATA), a glycoprotein of the glomerular tuft and of the proximal tubule, was analyzed on human kidney sections applying quantitative image analysis. Based on the software the exclusive glomerular expression of ATA was assessed in normal controls and 34 kidneys of patients with various stages of renal insufficiency (1.3-12 mg/dl serum creatinine). The data from diseased kidneys revealed a reduced total ATA-positive glomerular compartment, a larger area of remaining glomeruli, and a rise of the ATA-negative renal interstitium. Irrespective of the progression of the disease, hypertrophic glomeruli of remnant nephrons exhibited a high and homogeneous expression of ATA. Survival of glomeruli in patients with chronic renal failure correlated with the capability of glomerular cells to synthesize ATA at an increased rate, which may prevent angiotensin II-mediated growth effects, the accumulation of extracellular matrix proteins, etc., thus escaping final glomerulosclerosis.


Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides. DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5' terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3' terminus. Increasing annealing temperatures from 15 degrees to 70 degrees C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3'-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.


The SEG1 element: a new DNA region promoting stable mitotic segregation of plasmids in the zygomycete Absidia glauca. A series of new vectors for the model zygomycete Absidia glauca was constructed on the basis of the structural neomycin resistance (Neor) gene controlled by the promoter of the gene for elongation factor 1 (TEF). In order to select for transformed colonies with a stable Neor phenotype, spores from primary transformants were pooled and grown for two sporulation cycles under non-selective conditions. Southern blot analysis of DNA from single spore isolates originating from independent transformant pools allowed the identification of two autonomously replicating plasmids. Retransformation of Escherichia coli and restriction analysis of the two plasmids provided evidence for spontaneous in vivo insertion of a new DNA element (SEG1) from the A. glauca genome. The inserted regions in both plasmids are essentially identical and do not represent repetitive DNA. Compared with other autonomously replicating vectors, these SEG1-containing plasmids are mitotically extremely stable and are passed on to the vegetative spore progeny of a retransformed A. glauca strain. We assume that SEG1 contains structural elements involved in partitioning and stable segregation of plasmids. For the construction of stable transformants of A. glauca, the SEG1 element may be regarded as a major breakthrough, because stabilization of transformed genetic traits by integration is difficult to achieve in all mucoraceous fungi and all known replicating plasmids are mitotically unstable.


G:C-->T:A and G:C-->C:G transversions are the predominant spontaneous mutations in the Escherichia coli supF gene: an improved lacZ(am) E. coli host designed for assaying pZ189 supF mutational specificity. Escherichia coli K12 strain KS40 and plasmid pKY241 were designed for easy screening of supF mutations in plasmid pZ189. KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 (lacZ(am)CA7020). Using in vitro mutagenesis, an amber mutation was introduced into the cloned gyrA structural gene, of E. coli, to give pKY241, a derivative of pACYC184. When KS40 containing pKY241 (designated KS40/pKY241) is transformed with pZ189, nalidixic acid-resistant GyrA protein is produced from the chromosomal gyrA gene and wild-type GyrA protein from pKY241 because of the suppression of the gyrA amber mutation by supF. It is known that the wild-type, otherwise nalidixic acid-sensitive, phenotype is dominant over the nalidixic acid-resistant phenotype. Thus, KS40/pKY241 gives rise to nalidixic acid-sensitive colonies when it carries a pZ189 plasmid with an active supF suppressor tRNA. If the supF gene on the plasmid carries an inactivating mutation then KS40/pKY241 will form nalidixic acid-resistant colonies. By using this system, the spontaneous mutational frequency of the supF gene on pZ189 was calculated to be 3.06 x 10(-7) per replication. Among 51 independent supF mutations analyzed by DNA sequencing, 63% were base substitutions, 25% IS element insertions, 9.6% deletions and 1.9% single-base frameshifts. The base substitutions included both transversions (84.8%) and transitions (15.2%), the largest single group being G:C to T:A transversions (45.4% of the base substitutions). These results demonstrate that the KS40/pKY241 system we have developed can be used to characterize the DNA sequence changes induced by mutagens that give very low mutational frequencies.


High frequency, heat treatment-induced inactivation of the phosphinothricin resistance gene in transgenic single cell suspension cultures of Medicago sativa. One descendant of the Medicago sativa Ra-3 transformant T304 was analysed with respect to the somatic stability of the synthetic phosphinothricin-N-acetyltransferase (pat) gene which was used as a selective marker and was under the control of the 5'/3' expression signals of the cauliflower mosaic virus (CaMV) gene VI. In order to quantify gene instability, we developed a system for culturing and regenerating individual cells. Single cell suspension cultures derived from T304 and the ancestral non-transgenic M. sativa cultivar Ra-3, were established. The cells were regenerated into monoclonal calli. In transgenic calli, the phosphinothricin (Pt)-resistance phenotype was retained after more than 2 months of non-selective growth. In contrast, up to 12% of the suspension culture cells grown under nonselective conditions and at constant temperature (25 degrees C) lost the herbicide-resistance phenotype within 150 days. Surprisingly, a heat treatment (37 degrees C), lasting for 10 days, during the culture period resulted in an almost complete (95%) loss of the Pt resistance of the suspension culture cells. However, the frequency of cell division was identical in cultures grown under normal and heat treatment conditions. A biochemical test revealed that no phosphinothricin-N-acetyltransferase activity was present in heat treated, Pt-sensitive cells. The resistance level of the Pt-sensitive transgenic cells was equivalent to that of the wild-type cells. A PCR analysis confirmed the presence of the pat gene in heat treated, Pt-sensitive cells. From these results it is concluded that the Pt resistance gene was heat-inactivated at a high frequency in the M. sativa suspension cultures.


In vivo studies on the effects of immunity genes on early lytic transcription in the Halobacterium salinarium phage phi H. We have studied in vivo the effects of putative immunity genes on the expression of an early lytic gene of the Halobacterium salinarium phage. phi H. We transformed an H. salinarium host with DNA coding for a putative repressor gene, the transcript of which has been designated T6. We show that, in vivo, this gene specifically shuts off production of the early lytic transcript T4. A construct carrying the DNA transcribed as T4, but without its putative repressor binding sequences, shows T4 transcription enhanced to a level comparable to that observed in lytic growth of mutant phages capable of growing on immune H. salinarium strains. This transcript is insensitive to the action of the T6 product. The product of this 'unrepressed' T4 transcript is able to complement in trans the repressed T4 on superinfecting phi H-sensitive phages, allowing these to grow on a strain containing the repressor gene. It has, however, no effect on the production of repressor. We also mapped the start and end points of two other transcripts, T9 and T10, which are expressed only in the lysogenic state by cells immune to superinfection by phage, cloned the coding DNA and used it to transform H. salinarium. This DNA, though transcribed by the transformants, has no detectable effect on the cells, which remain susceptible to phage infection.


The developmentally regulated Aspergillus nidulans wA gene encodes a polypeptide homologous to polyketide and fatty acid synthases. The Aspergillus nidulans wA gene is required for synthesis of a green pigment present in the walls of mature asexual spores (conidia); wA mutants produce colorless (white) conidia. We determined the transcriptional structure and DNA sequence of the wA gene. wA consists of 5 exons separated by short (40-60 bp) introns. The processed transcript has the potential to encode a protein consisting of 1986 amino acid residues. The predicted WA polypeptide showed extensive sequence similarities with bacterial and fungal polyketide synthases and vertebrate fatty acid synthases, particularly within conserved active sites. Properties of the yellow conidial wall pigment intermediate suggest that it is a polyketide rather than a fatty acid. It is therefore likely that wA encodes all or part of a polyketide synthase involved in the formation of this pigment intermediate.


Structure and expression of the phosphoglycerate kinase (Pgk) gene of Drosophila melanogaster. The gene that encodes phosphoglycerate kinase in Drosophila melanogaster (Pgk) has been isolated and characterized. There is a single copy of Pgk in the Drosophila genome located at cytogenetic position 23A1-2. Transcripts of Pgk are 1.6 kb long and are found during development with a profile similar to the expression pattern of other genes of the glycolytic pathway. There are substantial amounts of maternal transcript in early embryos which decline in abundance until mid-embryogenesis when transcript levels increase; levels remain high, during larval stages, fall during pupariation and rise again at emergence. The nucleotide sequence of the Pgk gene reveals two small introns, one of which is at a position identical to the site of an intron found in Pgk genes from other organisms. The Pgk gene has no TATA box in the region of transcription initiation and has multiple transcription initiation sites that are closely spaced within 110 nucleotides of the translation start site.


Towards a physical map of the fertility genes on the heterochromatic Y chromosome of Drosophila hydei: families of repetitive sequences transcribed on the lampbrush loops Nooses and Threads are organized in extended clusters of several hundred kilobases. The understanding of structure and function of the so-called fertility genes of Drosophila is very limited due to their unusual size--several megabases--and their location on the heterochromatic Y chromosome. Since mapping of these genes has mainly been done by classical cytogenetic analyses using a small number of cytologically visible lampbrush loops as the sole markers for particular fertility genes, the resolution of the genetic map of the Y chromosome is restricted to 3-5 Mb. Here we demonstrate that a substantially finer subdivision of the megabase-sized fertility genes in the subtelomeric regions of the Y chromosome of Drosophila hydei can be achieved by a combination of digestion with restriction enzymes having 6 bp recognition sequences, and pulsed field gel electrophoresis. The physical subdivision is based upon large conserved fragments of repetitive DNA in the size range from 50 up to 1600 kb and refers to the long-range organization of several families of repetitive DNA involved in Y chromosomal transcription processes in primary spermatocytes. We conclude from our results that at least five different families of repetitive DNA specifically transcribed on the lampbrush loops nooses and threads are organized as extended clusters of several hundred kb, essentially free of interspersed non-repetitive sequences.


beta-Galactosidase of Kluyveromyces lactis (Lac4p) as reporter of gene expression in Candida albicans and C. tropicalis. Vectors containing fusions of the Candida albicans ACT promoter to heterologous genes were constructed and transformed into a C. albicans host strain. beta-Galactosidase (Lac4p) activity was detected in transformants carrying an ACT fusion to the Kluyveromyces lactis LAC4 gene, while fusions to the Escherichia coli lacZ gene and to other heterologous genes were not expressed. Lac4p was also produced by C. tropicalis transformants carrying the ACT/LAC4 fusion. Plasmids in transformed C. albicans strains were present either as free multimers in high copy number or, more frequently, integrated into the genome in low copy number yielding high and low LAC4 mRNA and Lac4p expression levels, respectively. Lac4p-expressing transformants of C. tropicalis, but not of C. albicans, were able to utilize lactose as sole carbon source. An ACT/LAC4 fusion was not differentially expressed during the yeast and hyphal growth phases of C. albicans, indicating that the ACT promoter is not regulated during morphogenesis. These results define the first reporter gene system for convenient monitoring of gene expression in Candida species.


Leucine and serine induce mecillinam resistance in Escherichia coli. We have previously shown that resistance to the beta-lactam mecillinam in Escherichia coli can be brought about by a high ppGpp pool, as observed under conditions of partial amino acid starvation and RelA-dependent induction of the stringent response. We show here that our E. coli wild-type strain, which is sensitive to mecillinam on minimal glucose plates, becomes resistant in the presence of L-leucine or L-serine (or cysteine, which inactivates the antibiotic). The resistance, which is not a transient effect and does not depend on the physiological state of the cells when plated, is specific for mecillinam and is reversed by the presence of isoleucine and valine in the medium. At least in the case of serine, the resistance is RelA-dependent. We conclude that the presence of leucine and serine in the growth medium cause partial starvation for isoleucine/valine, leading to induction of the stringent response and concomitant resistance to mecillinam.


Restoration of the wild-type locus in an RuBP carboxylase/oxygenase mutant of Synechocystis PCC 6803 via targeted gene recombination. The interaction between homologous DNA sequences, distant from each other in the chromosome, was examined in the cyanobacterium Synechocystis PCC 6803. Most of the rbcL gene encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) was duplicated in the genome by a targeted insertion of a 3'-truncated gene copy into the psb A-I locus. Both rbcL genes, in the psb A-I region and at the rbc locus, were non-functional; The former due to the 3' truncation, and the latter due to a deletion in the 5'-region (creating a 5' truncation) and a mutation associated with an insertion of the Rhodospirillum rubrum rbc gene, yielding a high-CO2-requiring mutant ('cyanorubrum'). The 3' and the 5' truncated rbcL genes were linked to chloramphenicol and kanamycin resistance markers, respectively. Decreasing the kanamycin selective pressure concomitantly with exposure of the double resistance mutant to air, resulted in air-growing colonies. Analysis of their genomes, Rubisco proteins, and their ultrastructure revealed: 1) Reconstitution of a full-length cyanobacterial rbcL gene at the rbc locus; 2) simultaneous synthesis of the cyanobacterial (L8S8) and R. rubrum (L2) enzymes in meroploids containing both mutated and reconstituted rbcL genes; 3) reappearance of carboxysomes. Our results indicate extensive recombinatorial interactions between the homologous sequences at both loci leading to reconstitution of the cyanobacterial rbcL gene.


Lysis protein T of bacteriophage T4.

Lysis protein T of bacteriophage T4. Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage lambda is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of lambda (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18,000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.


Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha. Dihydroxyacetone synthase (DAS) and methanol oxidase (MOX) are the major enzyme constituents of the peroxisomal matrix in the methylotrophic yeast Hansenula polymorpha when grown on methanol as a sole carbon source. In order to characterize their topogenic signals the localization of truncated polypeptides and hybrid proteins was analysed in transformed yeast cells by subcellular fractionation and electron microscopy. The C-terminal part of DAS, when fused to the bacterial beta-lactamase or mouse dihydrofolate reductase, directed these hybrid polypeptides to the peroxisome compartment. The targeting signal was further delimited to the extreme C-terminus, comprising the sequence N-K-L-COOH, similar to the recently identified and widely distributed peroxisomal targeting signal (PTS) S-K-L-COOH in firefly luciferase. By an identical approach, the extreme C-terminus of MOX, comprising the tripeptide A-R-F-COOH, was shown to be the PTS of this protein. Furthermore, on fusion of a C-terminal sequence from firefly luciferase including the PTS, beta-lactamase was also imported into the peroxisomes of H. polymorpha. We conclude that, besides the conserved PTS (or described variants), other amino acid sequences with this function have evolved in nature.


Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures. Genetic transformation of cereals by direct DNA delivery via microprojectile bombardment has become an established procedure in recent years. But the derivation of functional transgenic plants, especially in wheat, is still problematic, mainly due to low efficiency of DNA delivery and the reduced regeneration capability of microprojectile-bombarded tissue. We focussed on these two aspects and found that the regeneration of scutellar calli of wheat can be rendered highly efficient and considerably accelerated by a liquid culture phase in screen rafts. We also found that the expression of a reporter gene following DNA delivery by microprojectile can be improved by maintaining the scutellar calli in 0.25 M mannitol before and after bombardment, by bombardment in the presence of silver thiosulfate and Ca(NO3)2 (rather than CaCl2) and by the elimination of spermidine from the DNA/microprojectile mixture. A protocol that includes all these features leads to several-fold higher transient expression of the reporter gene than have previously published procedures.


SCM4, a gene that suppresses mutant cdc4 function in budding yeast. The gene SCM4 encodes a protein which suppresses a temperature-sensitive allele of the cell division cycle gene CDC4 in Saccharomyces cerevisiae. SCM4 was cloned on a 1.8 kb BamHI fragment of yeast genomic DNA in the high copy-number vector pJDB207, which results in a 50- to 100-fold increase in the level of the 700 nucleotide SCM4 transcript in vivo. The SCM4 gene encodes a 20.2 kDa protein of 187 amino-acids with a clear tripartite domain structure in which a region rich in charged residues separates two domains of largely uncharged amino acids. Although the apparent allele specificity of cdc4 suppression suggests that the CDC4 and SCM4 proteins interact, disruption of SCM4 demonstrates that the gene product is not essential for mitosis or meiosis; however, it may be a member of a family of related, functionally redundant proteins.


Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4. The vitopine Ti plasmid pTiS4 of Agrobacterium vitis has an unusual T-DNA organization. The pTiS4 oncogenes, localized by screening selected pTiS4 clones for growth-inducing activity, are localized on three T-DNAs, whereas in all other characterized Ti plasmids one or two T-DNAs are found. The nucleotide sequences and predicted amino acid sequences of the pTiS4 oncogenes set them apart from the corresponding genes from other Ti or Ri plasmids. The oncogenes induce the same type of reaction on various test plants as the well-known pTiAch5 oncogenes but the pTiS4 ipt gene induces considerably more shoots than its Ach5 homologue. We have also identified the gene coding for vitopine synthase as well as a vitopine synthase pseudogene. Both sequences show homology to the octopine synthase gene. In terms of both nucleotide sequence and overall organization, the pTiS4 T-DNAs appear to be only distantly related to previously characterized T-DNAs.


The pso4-1 mutation reduces spontaneous mitotic gene conversion and reciprocal recombination in Saccharomyces cerevisiae. Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain. Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination. The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant. However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination. Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation. Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.


Characterization of the secretion efficiency of a plant signal peptide in Bacillus subtilis. The ability of the Bacillus subtilis secretion machinery to interact with a heterologous signal peptide was studied using a plant (wheat alpha-amylase) signal peptide. The plant signal peptide was capable of mediating secretion of Escherichia coli alkaline phosphatase and B. amyloliquefaciens levansucrase from B. subtilis. This secretion was dependent on the plant signal peptide, as deletion of five amino acids from the hydrophobic core resulted in a block of secretion. Attempts to improve the efficiency of the plant signal peptide in B. subtilis were made by increasing the length of the hydrophobic core from 10 to 16 residues by insertion of 2, 4, 5 or 6 amino acids. None of the alterations improved the secretion efficiency relative to the wild-type plant signal peptide.


Characterization of the amdA-regulated aciA gene of Aspergillus nidulans. The structure, function and regulation of the acetate inducible aciA gene of Aspergillus nidulans was analysed. The aciA locus was mapped to chromosome 1 at a position where no acetate inducible gene has been previously located. The nucleotide sequence of aciA was determined, the structures of two transcripts were determined and the sequences of the polypeptide products of the gene were deduced. Construction of an aciA loss-of-function mutant was achieved via insertional inactivation, but it did not reveal a phenotype for an aciA- strain. The larger polypeptide, AciA, was found to have a putative dinucleotide cofactor binding site. Acetate inducibility of aciA was found to be dependent on the amdA regulatory gene. Use of a 5' deletion series of an aciA--lacZ fusion and an in vivo regulatory protein binding (titration) assay allowed the region required for amdA activity to be localized to a 124 bp segment 5' to aciA. A 13 bp region of sequence similarity was observed between aciA and the coregulated amdS gene in the regions required for amdA-mediated regulation of these genes. This sequence may have a role in amdA regulation of the two genes.


Isolation of purine auxotrophic mutants of Lactococcus lactis and characterization of the gene hpt encoding hypoxanthine guanine phosphoribosyltransferase. Five purine auxotrophic mutants of Lactococcus lactis were isolated. L. lactis was capable of converting adenine, guanine and hypoxanthine to AMP, GMP and IMP, respectively, indicating the existence of adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HGPRT) activities. A 1.3 kb DNA fragment from L. lactis was cloned by complementation of the hpt mutation in Escherichia coli. Introduction of this fragment into L. lactis resulted in an increase in HGPRT activity. In vitro transcription and translation analysis showed that the fragment coded for a polypeptide with M(r) of 22,000. The nucleotide sequence of this hpt gene was determined.


A umuDC-independent SOS pathway for frameshift mutagenesis. The chemical carcinogen N-acetoxy-N-2-acetylaminofluorene induces mainly frameshift mutations, which occur within two types of sequences (mutation hot spots): -1 frameshift mutations within contiguous guanine sequences and -2 frameshift mutations within alternating GC sequences such as the NarI and BssHII restriction site sequences. We have investigated the genetic control of mutagenesis at these sequences by means of a reversion assay using plasmids pW17 and pX2, which contain specific targets for contiguous guanine and alternating GC sequences, respectively. Our results suggest that mutations at these hot spot sequences are generated by two different genetic pathways, both involving induction of SOS functions. The two pathways differ both in their LexA-controlled gene and RecA protein requirements. In the mutation pathway that acts at contiguous guanine sequences, the RecA protein participates together with the umuDC gene products. In contrast, RecA is not essential for mutagenesis at alternating GC sequences, except to cleave the LexA repressor. The LexA-regulated gene product(s), which participate in this latter mutational pathway, do not involve umuDC but another as yet uncharacterized inducible function. We also show that wild-type RecA and RecA430 proteins exert an antagonistic effect on mutagenesis at alternating GC sequences, which is not observed either in the presence of activated RecA (RecA*), RecA730 or RecA495 proteins, or in the complete absence of RecA as in recA99. It is concluded that the -1 mutation pathway presents the same genetic requirements as the pathway for UV light mutagenesis, while the -2 mutation pathway defines a distinct SOS pathway for frameshift mutagenesis.


Organization of specific DNA sequence elements in the region of the replication origin and matrix attachment site in the chicken alpha-globin gene domain. The distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site (MAR) of the alpha-globin gene domain was investigated. The fragment was shown to contain a CR1-type repetitive element and two stably bent DNA sequences. One of them colocalizes with the previously described MAR element and with the recognition site for a proliferating-cell-specific, DNA-binding protein. The melting pattern of a set of subfragments of the region proved to be non random. No correlation between the distribution of readily melting sequences and bent DNA was found. The possible importance of curved, low-melting and repetitive DNA sequences for the organization of the upstream boundary of the alpha-globin gene domain and the function of the replication origin is discussed.


Effect of T-DNA configuration on transgene expression. T-DNA vectors were constructed which carry a beta-glucuronidase (gusA) gene fused to the promoter of the nopaline synthase (nos) gene and the 3' end of the octopine synthase (ocs) gene. This reporter gene was cloned at different locations and orientations towards the right T-DNA border. For each construct, between 30 and 60 stably transformed calli were analysed for beta-glucuronidase activity. Depending on the T-DNA configuration, distinct populations of gusA-expressing calli were obtained. Placing the reporter gene in the middle of the T-DNA results in relatively low expression levels and a limited inter-transformant variability. Placing the gene with its promoter next to the right border led to an increase in both the mean activity and the variability level. With this construct, some of the calli expressed the gusA gene at levels four to five times higher than the mean. In all these series, at least 30% of the calli contained reporter gene activities that were less than half of the mean expression level. Separating the gusA gene from the right T-DNA border by an additional 3'-untranslated region, derived from the nos gene, resulted in an increase in the mean expression to a level almost four times higher than that of constructions carrying the reporter gene in the middle of the T-DNA. Moreover, the number of transformants with extremely low activities decreased by at least 50% and this resulted in significantly lower inter-transformant variability independently of the orientation of the reporter gene on the T-DNA.


Apical secretion and association of the Drosophila yellow gene product with developing larval cuticle structures during embryogenesis. The yellow (y) gene of Drosophila melanogaster is required for the pigmentation of larval and adult cuticle structures. The deduced y protein sequence includes two putative N-linked glycosylation sites and a putative signal peptide, suggesting that it might be a secreted molecule. Consistent with the characteristics of a secreted protein, our in vitro translation studies using RNA synthesised from the y cDNA demonstrate that the nascent y polypeptide is a preprotein that cotranslationally translocates into the endoplasmic reticulum (ER) membrane and becomes glycosylated. The N-terminal peptide is cleaved from the preprotein between the two alanine residues at positions 21 and 22, to release the final product into the lumen of the ER. Antibodies raised against the y polypeptide detect the protein starting at 13 h post-fertilization in epidermal cells and in the cuticle structures secreted by them that later become pigmented; in addition, yellow protein is detected in the cuticle structures associated with Keilin's organs. The embryonic beta-galactosidase staining pattern of a transgene, bearing a construct in which expression of the lacZ gene is driven by the y promoter, is also described and is similar to that of the y protein. Our results indicate that the y gene product is an apically secreted protein which becomes an immobilised structural component of the pigmented cuticle.


XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida. The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to beta-galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229-->Ile) and Val274 (Asp274-->Val) substitutions over the N-terminal His41 (Arg41-->His) substitution, and the intraallelic dominance of Thr45 (Arg45-->Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88-->Leu) and Arg256 (Pro256-->Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Val288 (Asp288-->Val), and the double mutant was susceptible to activation by benzoates.(ABSTRACT TRUNCATED AT 250 WORDS)


Isolation of DNA markers linked to a beet cyst nematode resistance locus in Beta patellaris and Beta procumbens. In cultivated beet no useful level of resistance of the beet cyst nematode (BCN) Heterodera schachtii Schm. has been found, unlike the situation in wild species of the section Procumbentes. Stable introgression of resistance genes from the wild species into Beta vulgaris has not been achieved, but resistant monosomic additions (2n = 18 + 1), diploids of B. vulgaris with an extra alien chromosome carrying the resistance locus, have been obtained. Here we describe a new series of resistant monosomic fragment addition material of B. patellaris chromosome 1 (pat-1). We further describe the cloning of a single-copy DNA marker that specifically hybridizes with a monosomic addition fragment of approximately 8 Mb (AN5-90) carrying the BCN resistance locus. This marker and another fragment-specific, single-copy DNA marker probably flank the BCN locus on the addition fragment present in the AN5-203 material, which is approximately 19 Mb in size. Furthermore, several specific repetitive DNA markers have been isolated, one of which hybridizes to AN5-90 and also to DNA from a smaller DNA segment of Beta procumbens, present in line B883, carrying a BCN resistance locus introgressed into the B. vulgaris genome. This suggests that the specific repetitive marker is closely linked to the BCN locus.


The DAC2/FUS3 protein kinase is not essential for transcriptional activation of the mating pheromone response pathway in Saccharomyces cerevisiae. The DAC2/FUS3 gene of Saccharomyces cerevisiae, which encodes a CDC28/cdc2-related protein kinase, is essential both for the arrest of cell division induced by mating pheromones and for cell fusion during conjugation. To elucidate the role of the DAC2 gene product in the pheromone response pathway, I determined the nucleotide sequence of the DAC2 gene and characterized two types of deletion mutants of the DAC2 gene. Here, I show that the DAC2 gene is identical to the FUS3 gene and that dac2/fus3 deletion mutants respond to mating pheromones by activating transcription. Therefore, the DAC2/FUS3 gene is not essential for transcriptional activation in the pheromone response pathway. The DAC2/FUS3 protein kinase has a positive role in cell fusion during sexual conjugation.


Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae. A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu-) was transformed by pRC2312 to Leu+ at a frequency of 1.41 x 10(5) colonies per microgram DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura+ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 x 10(3) per microgram DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per microgram DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15 +/- 3 per haploid genome in S. cerevisiae and 2-3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7-12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade- strains of both C. albicans and S. cerevisiae to Ade+. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.


The regulatory gene nit-2 of Neurospora crassa complements a nnu mutant of Gibberella zeae (Fusarium graminearum). The nnu mutant of Gibberella zeae (=Fusarium graminearum) is unable to catabolize many of the nitrogen sources utilized by its wild-type parent, and may have suffered a mutation in the major nitrogen regulatory locus. Transformation of this mutant with the major nitrogen regulatory gene from Neurospora crassa, nit-2, restored the wild-type phenotype, thus confirming that the nnu mutation is in the major nitrogen regulatory locus of G. zeae. Our results are consistent with the premise of conservation of the structure of regulatory factors and suggest the possibility that functional DNA homologues of this regulatory element occur across a broad range of ascomycetous fungi.


Evolution and environment in the Hominoidea. Between 10 and 20 million years ago, a variety of hominoid primates lived in Africa, Europe and Asia. The question of which of these, if any, lie closest to the ancestries of humans and modern apes remains a lively source of debate. Recent fossil discoveries, though, shed light on the environments in which the various groups of hominoid emerged and, it is hoped, on their evolution. But the lack of a hominid fossil record before about 5 million years ago--and any fossil record for the African apes--is still a frustrating barrier.


Atherogenesis in transgenic mice expressing human apolipoprotein(a). Elevated plasma levels of the lipoprotein Lp(a) are associated with increased risk for atherosclerosis and its manifestations, myocardial infarction, stroke and restenosis (for reviews, see refs 1-3). Lp(a) differs from low-density lipoprotein by the addition of the glycoprotein apolipoprotein(a), a homologue of plasminogen that contains many tandemly repeated units which resemble the fourth kringle domain of plasminogen, and single homologues of its kringle-5 and protease domain. As plasma Lp(a) concentration is strongly influenced by heritable factors and is refractory to most drug and dietary manipulation, the effects of modulating it are difficult to mimic experimentally. In addition, the absence of apolipoprotein(a) from virtually all species other than primates precludes the use of convenient animal models. Here we show that transgenic mice expressing human apolipoprotein(a) are more susceptible than control mice to the development of lipid-staining lesions in the aorta, and that apolipoprotein(a) co-localizes with lipid deposition in the artery walls.


Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production. Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.


Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons. Neurons develop a highly polarized morphology consisting of dendrites and a long axon. Both axons and dendrites contain microtubules and microtubule-associated proteins (MAPs) with characteristic structures. Among MAPs, MAP2 is specifically expressed in dendrites whereas MAP2C and tau are abundant in the axon. But the influence of MAP2, MAP2C and tau on the organization of microtubule domains in dendrites versus axons is unknown. Both MAP2 and tau induce microtubule bundle formation in fibroblasts after transfection of complementary DNAs, and a long process resembling an axon is extended in Sf9 cells infected with recombinant baculovirus expressing tau. We have now expressed MAP2 and MAP2C in Sf9 cells in order to compare their morphology and the arrangement of their microtubules to that found in Sf9 cells expressing tau. We report here that the spacing between microtubules depends on the MAP expressed: in cells expressing MAP2, the distance is similar to that found in dendrites, whereas the spacing between microtubules in cells expressing MAP2C or tau is similar to that found in axons.


The spatial arrangement of cones in the primate fovea. The retinae of Old World primates contain three classes of light-sensitive cone, which exhibit peak absorption in different spectral regions. But how are the different types of cone arranged in the hexagonal mosaic of the fovea? This question has often been answered with artists' impressions, but never with direct measurements. Staining for antibodies specific to the short-wave photopigment has revealed a sparse, semiregular array of cones; but nothing is known about the arrangement of the more numerous long- and middle-wave cones. Are they randomly distributed, with chance aggregations of one type, as Hartridge postulated in these columns nearly 50 years ago? Or do they exhibit a regular alteration, recalling the systematic mosaics seen in some non-mammalian species? Or, conversely, is there positive clumping of particular cone types, as might be expected if local patches of cones were descended from a single precursor cell? We have made direct microspectrophotometric measurements of patches of foveal retina from Old World monkeys, and report here that the distribution of long- and middle-wave cones is locally random. These two cone types are present in almost equal numbers, and not in the ratio of 2:1 that has been postulated for the human fovea.


A cell line that can induce thymocyte positive selection. The thymus positively selects thymocytes that bear T-cell receptors which recognize antigen presented by self major histocompatibility complex (MHC) proteins. Positive selection is usually driven by MHC products on radiation-resistant cortical epithelial cells. It is unknown whether positive selection is mediated by all thymic epithelial cells or by some specialized subsets. Here we introduce an H-2b-expressing thymic epithelial cell line into the thymuses of lethally irradiated H-2k animals reconstituted with H-2b/k F1 BM or fetal liver cells. I-Ab-restricted T cells are found in these animals, demonstrating that selection occurs on the introduced epithelial cells.


Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. Hydrolysis by phospholipase C (PLC) of phosphatidylinositol 4,5-bisphosphate is a key mechanism by which many extracellular signalling molecules regulate functions of their target cells. At least eight distinct isozymes of PLC are recognized in mammalian cells. Receptor-controlled PLC is often regulated by G proteins, which can be modified by pertussis toxin in some cells but not in others. In the latter cells, PLC-beta 1, but not PLC-gamma 1 or PLC-delta 1, may be activated by members of the alpha q-subfamily of the G protein alpha-subunits. An unidentified PLC in soluble fractions of cultured human HL-60 granulocytes is specifically stimulated by G protein beta gamma subunits purified from retina and brain. Identification of a second PLC-beta complementary DNA (PLC-beta 2) in an HL-60 cell cDNA library prompted us to investigate the effect of purified G protein beta gamma subunits on the activities of PLC-beta 1 and PLC-beta 2 transiently expressed in cultured mammalian cells. We report here that PLC-beta 1 and PLC-beta 2 were stimulated by free beta gamma subunits and that PLC-beta 2 was the most sensitive to beta gamma stimulation. Thus stimulation of PLC by beta gamma subunits is isozyme-selective and PLC-beta 2 is a prime target of beta gamma stimulation. Activation of PLC-beta 2 by beta gamma subunits may be an important mechanism by which pertussis toxin-sensitive G proteins stimulate PLC.


Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C. The activation of heterotrimeric G proteins results in the exchange of GDP bound to the alpha-subunit for GTP and the subsequent dissociation of a complex of the beta- and gamma-subunits (G beta gamma). The alpha-subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free G beta gamma complex. G beta gamma has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the G beta gamma complex can act with G alpha subunit to modulate the activity of different isoforms of adenylyl cyclase. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified G beta gamma is found to activate one of them. Here we report that in co-transfection assays G beta gamma subunits specifically activate the beta 2 and not the beta 1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of G beta gamma from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).


Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. The mammalian shc gene encodes two overlapping, widely expressed proteins of 46 and 52K, with a carboxy-terminal SH2 domain that binds activated growth factor receptors, and a more amino-terminal glycine/proline-rich region. These shc gene products (Shc) are transforming when overexpressed in fibroblasts. Shc proteins become phosphorylated on tyrosine in cells stimulated with a variety of growth factors, and in cells transformed by v-src (ref. 2), suggesting that they are tyrosine kinase targets that control a mitogenic signalling pathway. Here we report that tyrosine-phosphorylated Shc proteins form a specific complex with a non-phosphorylated 23K polypeptide encoded by the grb2/sem-5 gene. The grb2/sem-5 gene product itself contains an SH2 domain, which mediates binding to Shc, and is implicated in activation of the Ras guanine nucleotide-binding protein by tyrosine kinases in both Caenorhabditis elegans and mammalian cells. Consistent with a role in signalling through Ras, shc overexpression induced Ras-dependent neurite outgrowth in PC12 cells. These results suggest that Shc tyrosine phosphorylation can couple tyrosine kinases to Grb2/Sem-5, through formation of a Shc-Grb2/Sem-5 complex, and thereby regulate the mammalian Ras signalling pathway.


Spliced leader RNAs from lower eukaryotes are trans-spliced in mammalian cells. Exon sequences present on separate RNA molecules can be joined by trans-splicing in trypanosomatids, Euglena, and in the nematode and trematode worms. Trans-splicing involves an interaction between a 5' splice site present in a spliced leader RNA and a 3' splice site located near the 5' end of pre-messenger RNAs. In vitro trans-splicing of artificial mammalian pre-mRNAs has been reported, but the efficiency of splicing appears to depend on sequence complementarity between the two substrates. There has been speculation that some natural pre-mRNAs can be trans-spliced in mammalian cells in vivo, but alternative interpretations have not been ruled out. Here we show that spliced leader RNAs can be accurately trans-spliced in mammalian cells in vivo and in vitro. Both nematode and mammalian 3' splice sites can function as acceptors for trans-splicing in vivo. These results reveal functional conservation in the splicing machinery between lower eukaryotes and mammals, and they directly demonstrate the potential for trans-splicing in mammalian cells.


The earliest Acheulean from Konso-Gardula. Konso-Gardula is a palaeoanthropological area discovered by the 1991 Palaeoanthropological Inventory of Ethiopia in the southern Main Ethiopian Rift. The Konso-Gardula sediments span the period about 1.3-1.9 million years ago. They contain rich Acheulean archaeological occurrences. Vertebrate fossils include early Homo.


Mitotic domains in the early embryo of the zebrafish. At the midblastula transition in the zebrafish, three, and only three, spatially separate mitotic domains arise with distinctive cycle lengths and rhythms. As in Drosophila and at about the equivalent stage, the mitotic domains reflect the fate map, but they do so only very crudely: two are extraembryonic and the third forms the entire embryo. The domains appear not to subdivide during gastrulation, when the germ layers form and when cells probably commit to their eventual fates. The domains may signal specification of morphogenesis rather than cell fate, because, shortly after they appear, each assumes a different role during epiboly, the first morphogenetic movement of the embryo. During meroblastic cleavage, and continuing in the early blastula, zebrafish blastomeres divide rapidly and synchronously. At the time of the tenth cleavage, the beginning of the midblastula transition, the cell cycle lengthens, and, as in Xenopus and Drosophila, cycle length comes under nucleocytoplasmic control (D.A.K. and C.B.K., manuscript in preparation). This nucleocytoplasmic control seems to be maintained during cycle lengthening in the next 2 or 3 cycles, comprising a midblastula transition period. We now show that functionally distinct subsets of cells that arise during this period have reproducibly different mitotic cycle lengths.


Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer. An inherited deficiency of beta-glucuronidase in humans, mice and dogs causes mucopolysaccharidosis VII (Sly syndrome), a progressive degenerative disease that reduces lifespan (to an average of 5 months in mice) and results from lysosomal storage of undegraded glycosaminoglycans in the spleen, liver, kidney, cornea, brain and skeletal system. Bone marrow transplantation in mutant mice provides a source of normal enzyme ('cross-correction'), which substantially improves the clinical condition and extends the average lifespan to 18 months. Gene therapy by transfer of a beta-glucuronidase gene into mutant haematopoietic stem cells is an alternative approach, but it is not known whether the low expression of vector-transferred genes in vivo would be sufficiently effective. Here we show that retroviral vector-mediated transfer of the gene to mutant stem cells results in long-term expression of low levels of beta-glucuronidase which partially corrects the disease by reducing lysosomal storage in liver and spleen.


Bone and haematopoietic defects in mice lacking c-fos. The proto-oncogene c-fos is the cellular homologue of v-fos originally isolated from murine osteosarcoma. Fos protein is a major component of the AP-1 transcription factor complex, which includes members of the jun family. Stable expression of c-fos in mice has been demonstrated in developing bones and teeth, haematopoietic cells, germ cells and in the central nervous system. It has been proposed that c-fos has an important role in signal transduction, cell proliferation and differentiation. We have previously demonstrated that overexpression of c-fos in transgenic and chimaeric mice specifically affects bone, cartilage and haematopoietic cell development. To understand better the function of c-fos in vivo, we used gene targeting in embryonic stem cells to generate cells and mice lacking c-fos. Here we report that heterozygous fos +/- mice appear normal, although females exhibit a distorted transmission frequency. All homozygous fos -/- mice are growth-retarded, develop osteopetrosis with deficiencies in bone remodelling and tooth eruption, and have altered haematopoiesis. These data define the c-Fos protein as an essential molecule for the development of specific cellular compartments.


[Molecular biological techniques in the diagnosis of tropical parasitic diseases]. Recent advances in the development of molecular biological techniques have resulted in their supplementary application for improved diagnosis of tropical parasitic diseases. The main areas of interest are the production of recombinant antigens for immunodiagnosis, and the detection of parasites by hybridization of nucleic acids and by DNA amplification (PCR) in vitro.


Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section. Motoneurons innervating the skeletal musculature were among the first neurons shown to require the presence of their target cells to develop appropriately. But the characterization of molecules allowing motoneuron survival has been difficult. Ciliary neurotrophic factor prevents the death of motoneurons, but its gene is not expressed during development. Although the presence of a neurotrophin receptor on developing motoneurons has suggested a role for neurotrophins, none could be shown to promote motoneuron survival in vitro. We report here that brain-derived neurotrophic factor can prevent the death of axotomized motoneurons in newborn rats, suggesting a role for this neurotrophin for motoneuron survival in vivo.


Brain-derived neurotrophic factor rescues developing avian motoneurons from cell death. During normal vertebrate development, about half of spinal motoneurons are lost by a process of naturally occurring or programmed cell death. Additional developing motoneurons degenerate after the removal of targets or afferents. Naturally occurring motoneuron death as well as motoneuron death after loss of targets or after axotomy can be prevented by in vivo treatment with putative target (muscle) derived or other neurotrophic agents. Motoneurons can also be prevented from dying in vitro and in vivo (Y.Q.-W., R.W., D.P., J. Johnson and L. Van Eldik, unpublished data and refs 7, 13, 14) by treatment with central nervous system extracts (brain or spinal cord) and purified central nervous system and glia-derived proteins. Here we report that in vivo treatment of chick embryos with brain-derived neurotrophic factor rescues motoneurons from naturally occurring cell death. Furthermore, in vivo treatment with brain-derived neurotrophic factor (and nerve growth factor) also prevents the induced death of motoneurons that occurs following the removal of descending afferent input (deafferentation). These data indicate that members of the neurotrophin family can promote the survival of developing avian motoneurons.


Cost reductions. Part 1: An operations improvement process. Today's nursing leaders are commonly expected to reduce labor costs. This two-part series provides a framework for achieving significant cost reductions in the nursing labor budget. Part One describes a process that has been used to identify and implement cost savings in varied hospital settings. Part Two will address organizational culture issues that can hamper or support cost savings.


Nursing involvement in hospital governance: 1990 and 1995. The integration of nursing into organizational governance structures has been advocated to improve practice and enhance the quality of patient care. In 1990, department of nursing involvement in hospital governance occurred primarily through the activities of the CNE. Projections for 1995 suggest that both staff RNs and CNEs will be more involved in governing health care organizations.


RN orientation: cost and achievement analysis. A cost analysis for orienting registered nurses in a traditional manner was initiated prior to a performance-based system. A 15-month study in a 772-bed midwestern hospital tracked the cost of all human and material resources used and determined a level of achievement for each individual in the sample.


Estimating costs of underusing advanced practice nurses. A model for estimating the cost of underutilizing nurses in advanced practice is proposed. Numerical requirements for implementing the model are detailed and examples are provided.


Balancing the pressure ulcer cost and quality equation. Pressure ulcers are a serious national health concern impacting cost of care, reimbursement, and quality of life. National estimates show 1.7 million patients annually develop pressure ulcers with associated health care costs of $8.5 billion. Sixty percent of patients develop these pressure ulcers while in acute care hospitals. Assessment of institution prevalence rates, patient risk profile, standardization of therapy protocols, and increased staff awareness can positively impact the balance between cost and quality patient outcomes.


[Sexual abuse of children: the necessity of medical intervention]. Recently the Dutch Health Council published a report on the medical aspects of sexual abuse of children. The questions underlying this report reflect an attempt to arrive at hard indices of sexual abuse in children through the assessment of physical symptoms. For the medical assessment, the authors refer to the Dutch translation of an English report entitled Physical signs of sexual abuse in children, published by The Royal College of Physicians. The hazards connected with the exclusive focus on physical symptoms are recognized, and the authors stress that medical examination should be part of a much broader assessment of the child and his/her family. However, instead of confining themselves to assessment issues of physical symptoms, the authors add a rather meagre account of behavioural and emotional factors relevant to the diagnosis of sexual abuse of children. Unfortunately, this approach seems to aggravate the problems underlying misdiagnosis rather than to increase diagnostic accuracy and improve adequate care for sexually abused children.


[Sexual violence observed in family practice]. In 11 general medical practices which are part of the 'Huisartsen Peilstation Groningen', the nature and extent were investigated of cases of sexual violence previously unknown to the GP. Questions also investigated were who took the initiative to bringing up this subject, and whether the results yield guidelines for better detection of and attending to sexual violence. The GPs registered these items during 1990 and 1991 after the first contact with any patient about a case of sexual violence (as defined by Draijer) previously unknown to them. The number of registered cases was 64 (corresponding to 17 per 10,000 patients), half of which concerned adults with sexual violence problems in their childhood. Offenders, nature and extent of the violence did not differ from descriptions in the literature. No cases were reported of still continuing violence. The 11 GPs differed significantly in the number of registered cases. The extent to which GP or patient took the initiative to bringing up the subject was not related to the number of cases registered by the GP. Patients referred and those not referred did not differ significantly in nature or extent of the sexual violence. Few follow-up appointments were made with non-referred patients. On the basis of these results, two items were formulated for better detection of and attending to sexual violence problems: stimulating the GP to an open, inviting attitude as well as active continuation of questioning in the case of complaints with a signal value; and making a follow-up appointment for non-referred patients.


[The relationship between job appreciation and absenteeism studied with the use of a simple questionnaire]. In the period February 1986-January 1988 a questionnaire study was carried out among 455 men aged 39-55 years working for the Netherlands Post Office to determine whether a correlation exists between job appreciation and absenteeism. Job appreciation was estimated by means of seven questions concerning: pleasure at work, fatigue after work, job satisfaction, mental stress due to the work, tension in contact with colleagues, tension in contacts with superiors and the physical exertion of working. Answers were scored on a 7-point scale. It was found that a particular score area exists that is correlated with significantly more absenteeism. The results apply to groups, not to individuals.


[An outbreak of mostly extrapulmonary tuberculosis in a family practice]. An outbreak of mainly extrapulmonary tuberculosis (TB) in a group of about 550 patients with rheumatoid arthritis (RA) is described. These patients had been attending the practice of a former general practitioner who treated cases of rheumatoid arthritis with phenylbutazone and steroids. The number of diagnosed TB cases was 55. Six cases had a contagious lung localisation. The possible sources of the outbreak were analysed. Both a visit on a same day as a sputum positive patient (chi 2-trend: 20.4; p < 0.001) and the administration of steroids (odds ratio (OR): 36.2; 95% confidence interval (CI): 8.8-313) were independent risk factors. There also appeared to be a relationship between TB and RA (OR: 4.4; 95%-BI: 2.2-9.1). Exogenous re(infection) and endogenous reactivation are possible causes of this outbreak.


[Dystrophia myotonica and pregnancy].

[Dystrophia myotonica and pregnancy]. We describe the maternal and neonatal complications of pregnancy in two patients with myotonic dystrophy. The disease leads to an increased spontaneous abortion rate, hydramnios, prolonged first and second stages of labour, retained placenta, postpartum haemorrhages and anaesthetic sensitivity in the mother. The neonatal problems are caused by the congenital form of the disease. The major clinical features of congenital myotonic dystrophy are bilateral facial weakness, hypotonia, neonatal distress, feeding difficulties, talipes, tent-shaped mouth, mental retardation and delayed motor development. Relatives of a known myotonic dystrophy patient should be advised to let themselves be examined for this disease. If the disease is diagnosed, information should be given regarding possibilities for prenatal diagnosis. Pregnancy in myotonic dystrophy patients should be monitored by a gynaecologist. Labour has to take place in a hospital with intensive care facilities for mother and child.


Localization of messenger RNAs encoding crustacean hyperglycemic hormone and gonad inhibiting hormone in the X-organ sinus gland complex of the lobster Homarus americanus. The localization of messenger RNAs encoding the crustacean hyperglycemic hormone, involved in regulation of carbohydrate metabolism and the gonad inhibiting hormone, which inhibits vitellogenesis, was studied in the eyestalk of the lobster Homarus americanus using complementary RNA probes for in situ hybridization. For the detection of gonad inhibiting hormone messenger RNA, we cloned and sequenced a partial complementary DNA encoding lobster gonad inhibiting hormone and for crustacean hyperglycemic hormone messenger RNA detection an available complementary DNA was used. This approach reveals that there is a frequent but inconsistent cellular co-localization of the two neurohormones. Furthermore, our data show that male lobsters contain an equal number of neuroendocrine gonad inhibiting hormone cells as female lobsters. An additional study, involving the use of in situ hybridization in combination with immunocytochemistry, shows that the synthetic activity of the crustacean hyperglycemic hormone- and gonad inhibiting hormone-producing cells can be followed at the messenger RNA as well as the protein level. This reveals that when strong immunostaining is present, the messenger RNA staining is usually weak or absent and vice versa. In conclusion, the presence of cells, containing only gonad inhibiting hormone messenger RNA or only crustacean hyperglycemic hormone messenger RNA, indicates that lobster crustacean hyperglycemic hormone and gonad inhibiting hormone originate from two different precursors. Co-localization of the two neurohormone messenger RNAs confirms the co-localization at the peptidergic level found by immunocytochemistry and thus these findings were not due to cross-reactions between the two antisera.(ABSTRACT TRUNCATED AT 250 WORDS)


Demyelination, and remyelination by Schwann cells and oligodendrocytes after kainate-induced neuronal depletion in the central nervous system. Excitotoxins are thought to kill neurons while sparing afferent fibers and axons of passage. The validity of this classical conclusion has recently been questioned by the demonstration of axonal demyelination. In addition, axons are submitted to a profound alteration of their glial environment. This work was, therefore, undertaken to reassess axonoglial interactions over time after an excitotoxic lesion in the rat. Ultrastructural studies were carried out in the ventrobasal thalamus two days to 18 months after neuronal depletion by in situ injections of kainic acid. In some cases, lemniscal afferents were identified by using anterograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase from the dorsal column nuclei. Two and four days after kainate injection, numerous dying axons displaying typical signs of Wallerian degeneration were observed in a neuropile characterized by the loss of neuronal somata and dendrites, an increase in number of microglia/macrophages and the disappearance of astrocytes. Ten and 12 days after kainate injection, degenerating axons were no longer observed although myelin degeneration of otherwise unaltered axons was ongoing with an accumulation of myelin remnants in the neuropile. At 16 and 20 days, the demyelination process was apparently complete and axons of different diameters were sometimes packed together. One and two months after kainate injection, the axonal environment changed again: remyelination of large-caliber axons occurred at the same time as reactive astrocytes, oligodendrocytes and numerous Schwann cells appeared in the tissue. Schwann cell processes surrounded aggregates of axons of diverse calibers, ensheathed small ones and myelinated larger ones. Axons were also remyelinated by oligodendrocytes. Horseradish peroxidase-labeled lemniscal afferents could be myelinated by either of the two cell types. After three months, the neuropile exhibited an increase in number of hypertrophied astrocytes and the progressive loss of any other cellular or axonal element. At this stage, remaining Schwann cells were surrounded by a glia limitans formed by astrocytic processes. These data indicate that although excitotoxins are sparing the axons, they are having a profound and complex effect on the axonal environment. Demyelination occurs over the first weeks, accompanying the loss of astrocytes and oligodendrocytes. Axonal ensheathment and remyelination takes place in a second period, associated with the reappearance of oligodendrocytes and recruitment of numerous Schwann cells, while reactive astrocytes appear in the tissue at a slightly later time. Over the following months, astrocytes occupy a greater proportion of the neuron-depleted territory and other elements decrease in number.(ABSTRACT TRUNCATED AT 400 WORDS)


Cytosine arabinoside effects in mouse cerebellar cultures in the presence of astrocytes. Organotypic cerebellar cultures derived from neonatal mice were exposed to recent preparations of cytosine arabinoside that destroyed oligodendrocytes and drastically reduced granule cells, but did not reduce the astrocyte population. The cultures were analysed by light and electron microscopy, and by extracellular electrophysiological recording. Purkinje cells survived in greater numbers than in untreated explants and sprouted excess recurrent axon collaterals that formed heterotypical synapses with Purkinje cell dendritic spines. These changes were similar to those found in earlier studies with a cytosine arabinoside preparation that did reduce the astrocyte population, in addition to destroying oligodendrocytes and granule cells. Results with recent cytosine arabinoside preparations that differed from those obtained previously included astrocytic ensheathment of Purkinje cells and apposition of many unattached dendritic spines, encasement of heterotypical synapses by astroglial processes, a loss of Purkinje cell somatic spines, and a lack of somatic hyperinnervation of Purkinje cells by sprouted recurrent axon collateral terminals. All of these differences were attributed to the presence of adequate numbers of competent astrocytes. Heterotypical synapses formed by sprouted recurrent axon collateral terminals and Purkinje cell dendritic spines were functional, as indicated by cortical inhibition in response to antidromic Purkinje cell activation in the absence of somatic hyperinnervation. These results give further definition to the role of astrocytes in cerebellar development and plasticity.


Autoradiographic localization of binding sites for arginine vasopressin and atrial natriuretic peptide on astrocytes and neurons of cultured rat central nervous system. The cellular localization of binding sites for [125I]arginine vasopressin and [125I]atrial natriuretic peptide was studied in explant cultures of rat spinal cord, brain stem and cerebellum by means of autoradiography. In brain stem cultures, especially in the nucleus of the solitary tract, a great number of neurons revealed binding sites for both peptides. In spinal cord cultures, many neurons of various sizes were labelled by [125I]arginine vasopressin, whereas only a small number of cells showed binding sites for [125I]atrial natriuretic peptide. Neurons in cerebellar cultures revealed little or no binding for the peptides. In addition to neurons, binding sites for [125I]arginine vasopressin and [125I]atrial natriuretic peptide were also observed on glial cells. Simultaneous staining of the cultures with glial fibrillary acidic protein has shown that the labelled cells were glial fibrillary acidic protein-positive and could therefore be identified as astrocytes. Labelling of the cells by [125I]arginine vasopressin and [125I]atrial natriuretic peptide was more intense in spinal cord and brain stem cultures than in cultures of cerebellum, providing evidence for a heterogeneity of astrocytes in different regions of the central nervous system. Binding of both [125I]arginine vasopressin and [125I]atrial natriuretic peptide to neurons and astrocytes could be competed by the unlabelled peptides, suggesting specific binding of the radioligands. Our autoradiographic studies provide good evidence that in addition to neurons, astrocytes also express receptors for arginine vasopressin and atrial natriuretic peptide.


Muscle stiffness and continuous electromyographic activity in old rats; an animal model for spasticity? A mechanomyographic response of the hind foot to passive straightening and bending, as well as an electromyographic activity of the gastrocnemius and tibialis anterior muscles were recorded in old (35-44-month-old) and young female rats. In old rats, spontaneous, tonic electromyographic activity patterns were concurrently observed in both antagonistic muscles; they were low-amplitude, dense tonic activity and continuous, high-amplitude, sparse electromyographic activity. The tonic electromyographic activity was correlated with a decline in the strength and mass of muscles, as well as with motor disturbances, including paresis of the rigidly straightened backward hind legs, dragged behind by an animal. In muscles of old rats, morphological features of a chronic denervation atrophy were found. Baclofen (10 and 15 mg/kg, i.p.) diminished the spontaneous tonic electromyographic activity and potently decreased the whole body muscle tone, whereas Madopar (50 mg/kg of L-DOPA+12.5 mg/kg of benerazide) was ineffective. It is suggested that old rats in which the above-described pathologic alterations are observed might be a useful animal model in the search for basic etiopathological mechanisms of spasticity and similar disturbances found in humans.


Acetylcholinesterase and its association with heparan sulphate proteoglycans in cortical amyloid deposits of Alzheimer's disease. Previous studies have used a sensitive histochemical technique to demonstrate acetylcholinesterase and butyrylcholinesterase within the pathological lesions of Alzheimer's disease. In this study, we used this technique to show that acetylcholinesterase localized in either frozen or fixed neocortical tissue sections is removed after treatment with various glycosaminoglycans, heparinases or proteases. Heparan sulphate, heparinase lyase type I and to a lesser degree, heparin and chondroitin sulphate were effective in solubilizing a large part of the cholinesterase activity. At physiological concentrations, the protease papain or trypsin readily removed activity but collagenase or pronase were relatively less effective. Peptide protease inhibitors and divalent metals did not exhibit any clear effect. The specificity of these observations was shown by inhibition of activity with various anticholinesterases including diisofluorophosphate. Our results suggest that acetylcholinesterase is anchored to and may be released from the heparan sulphate glycosaminoglycans shown to be contained in the lesions. We further suggest that the localization of cholinesterases is closely associated with the accumulation of the glycosaminoglycans in amyloid plaques and neurofibrillary tangles.


Mouse microtubule-associated protein 2 expression in transgenic Drosophila. Full-length mouse microtubule-associated protein 2 complementary DNA inserted between Drosophila heat-shock protein 70 promoter and trailer was introduced in the germ-line of Drosophila melanogaster by P element-mediated transformation. Three transformant lines contained microtubule-associated protein 2 complementary DNA in sense orientation and two in antisense orientation. All sense lines produced microtubule-associated protein 2 after heat-shock in several tissues at low level. In the adult brain, high-level microtubule associated protein 2 expression independent of heat induction occurred in specific neuron types in each sense transformant, suggesting the action of neuron-specific enhancer genes. High microtubule-associated protein 2 levels are expected to interfere with the cells' activity by forming unphysiological cross-links, which may help elucidate the function of the given neurons.