A histoenzymatic study of rat intrafusal muscle fibres. The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (alpha-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K2-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkali-preincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K2-EDTA preincubation. Following either acetic acid solution or cold and room temperature K2-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K2-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing alpha-GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles.


Heparan sulfate at the surface of HeLa cells. HeLa cells, labeled with Na235SO4, release into the culture medium 35SO4 bound to plasma membrane vesicles next to 35SO4-glycoproteins and free 35SO4. Plasma membrane vesicles, experimentally produced by treatment with formaldehyde, contain 35SO4 and their surface can be stained with high iron diamine. Scanning of chromatograms of the trypsinate from labeled cells demonstrates radioactivity on the spot of heparan sulfate. It is concluded that HeLa cells synthesize heparan sulfate, which is incorporated at the plasma membrane and released by shedding of small vesicles.


Structural and functional analysis of cloned deoxyribonucleic acid containing the trpR-thr region of the Escherichia coli chromosome. Specialized transducing phages containing the thr-trpR region of the Escherichia coli chromosome were derived from a strain with lambda prophage inserted in thr. Cloning of segments of the chromosomal deoxyribonucleic acid of one such lambda thr + trpR+ phage in various plasmid vectors established that a 1.3-kilobase BamHI fragment carried trpR+ intact. Strains with a multicopy plasmid vector containing the BamHI insert produced 20-fold-higher levels of trp aporepressor than did the wild-type strain of Escherichia coli. Similarly, induction of lambda thr + trpR+ lysogens resulted in increased aporepressor levels. The 1.3-kilobase trpR+ BamHI fragment was inserted in either orientation downstream from lambda pLN in a plasmid vector in which transcription from lambda pL was under the control of a temperature-sensitive lambda repressor. Induction established the orientation of transcription of trpR and led to the production of 100-fold-increased levels of trp aporepressor. A presumptive 23,500-dalton trpR+ polypeptide was detected by using lambda pLNtrpR+ plasmid deoxyribonucleic acid in a cell-free transcription-translation system.


Protein-associated lipid of Bacillus stearothermophilus. The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.


Cyanobacterial ribonucleic acid polymerases recognize lambda promoters. We compared the initiation specificities in vitro of deoxyribonucleic acid-dependent ribonucleic acid polymerases purified from two cyanobacteria, Fremyella diplosiphon and Anacystis nidulans, and from Escherichia coli. A restriction fragment made from lambda deoxyribonucleic acid was used as a template. The cyanobacterial and E. coli ribonucleic acid polymerases recognized the same lambda promoters but exhibited different sensitivities to the inhibitor heparin, suggsesting differences in the structure of the initiation complexes.


Physical characterization of ilv-lac fusions. Electron microscopic heteroduplex analysis and comparative restriction digests have been used to characterize lambda p123(209), the complementary pair of phages used in the Casadaban technique of gene fusion. Derivatives of lambda 1(209) constructed to carry fusions of the lac genes to the control regions of the ilvC and ilvEDA operons were also analyzed. These physical maps have provided confirmation of the genetic models for these constructions and physical specifications important in interpreting the behavior of these ilv-lac fusions.


Location of a ColE1 deoxyribonucleic acid region that affects the plaque-forming ability of lambda-ColE1 hybrid bacteriophage. The plaque-forming ability of a hybrid phage between plasmidColE1 and phage lambda carrying amber mutations in genes O and P was inhibited by the presence of ColE1 in suppressor-deficient Escherichia coli cells. ColE1 deoxyribonucleic acid regions concerned with this inhibition were examined by using various deletion and transposon insertion derivatives of ColE1, and it was found that the presence of the deoxyribonucleic acid region extending between 420 and 613 base pairs upstream from the initiation site of ColE1 deoxyribonucleic acid replication (J. Tomizawa, H. Ohmori, and R. E. Bird, Proc. Natl. Acad. Sci. U. S. A. 74: 1865--1869, 1977) was essential for this function.


Suppression of tif-mediated induction of SOS functions in Escherichia coli by an altered dnaB protein. The tif-1 mutation in the Escherichia coli recA gene is known to cause induction of the various "SOS" functions at high temperature, including massive synthesis of the recA protein, lethal filamentation, elevated mutagenesis, and, in lambda lysogens, induction of prophage. It is shown here that the deoxyribonucleic acid initiation mutation dnaB252 suppresses all these manifestations of tif expression. Induction of lambda by ultraviolet irradiation, however, is not affected by the dnaB252 mutation. No similar suppression of tif is observed with other dnaB mutations affecting deoxyribonucleic acid elongation or with other deoxyribonucleic acid initiation mutations at the dnaA and dnaC loci. The fact that an alteration of the dnaB protein specifically suppresses tif-mediated SOS induction implies a role of the replication apparatus in this process, as has been suggested for ultraviolet induction. The induction of lambda is known to proceed via repressor cleavage, presumably promoted by an activated (protease) form of the recA protein. Since lambda induction is normal after ultraviolet irradiation of the tif-1 dnaB252(lambda) strain, tif-mediated induction in this strain may be blocked in a tif-specific step leading to activation of the recA (tif) protein. It is possible that the recA (tif) mutant protein may be directly involved in the replication complex in processes leading to this activation.


Lambda transducing bacteriophage carrying deletions of the argCBH-rpoBC region of the Escherichia coli chromosome. Deletions in the rpoBC region have been transferred to phage lambda and characterized in detail by genetic, structural, and functional tests. We thus extend and confirm knowledge of the organization of this part of the chromosome. The new phages are useful tools for studying the genes for the bacterial transcription and translation machinery.


Deoxyribonucleic acid sequence homologies among bacterial insertion sequence elements and genomes of various organisms. Plasmid and phage deoxyribonucleic acid (DNA) harboring bacterial insertion sequence (IS) elements IS1, IS2, and IS5 were characterized and used as probes to detect homologous sequences in various procaryotic and eucaryotic genomes. The hybridization method used permits the detection of sequences partially homologous to the elements. Hybridization of the IS-containing probes to each other revealed a region of limited homology between IS1 and IS2. Homologous sequences were then detected by computer analysis of the published IS1 and IS2 nucleotide sequences. The homologous sequence contains a tandemly repeated tetranucleotide sequence which resembles the repeated sequence at the hot spot for spontaneous mutations in the lacI gene (P. J. Farabaugh, U. Schmeissner, M. Hofer, and J. Miller, J. Mol. Biol. 126:847-863, 1978). Homology between the IS elements and various genomes was determined by hybridizing labeled DNA containing IS1, IS2, and IS5 sequences to Southern blots of chromosomal DNA cleaved with restriction endonucleases. IS1 and IS5 appear limited to the enteric bacteria, whereas IS2 sequences can also be detected in Pseudomonas putida, Pseudomonas aeruginosa, and Serratia marcescens. Bacteria which appear not to possess extrachromosomal elements, e.g., Caulobacter crescentus, did not show homology with any insertion sequences tested. In addition, sequences homologous to IS1, IS2, or IS5 were not detected in Saccharomyces cerevisiae, Dictyostelium discoideum, or calf thymus DNA.


The effects of linoleate hydroperoxide on respiration and oxidative phosphorylation of rat liver mitochondria. Linoleate hydropepoxide, purified by silica gel chromatography and at concentrations 70-100 nmol/mg mitochondrial protein, activated state 4 respiration and Mg-ATPase activity of mitochondria to levels of 80% and 25%, respectively, of those induced by 300 microM DNP, and completely inhibited oxidative phosphorylation. These effects are the same as those caused by linoleate, but the hydroperoxide caused more rapid degeneration of the activated respiration of mitochondria than linoleate. Further addition of the hydroperoxide induced oligomycin-insensitive Mg-ATPase to a level 3 times that obtained with DNP, accompanied by clearing of the mitochondrial suspension and release of malate dehydrogenase from the matrix. The extent of the effects caused by the methyl ester of linoleate hydroperoxide was much less than by the free acid.


Isolation and characterization of mucopolysaccharides from rat liver mitochondria. Mucopolysaccharides were isolated from rat liver mitochondria which had been labeled with 35S-sulfate. They were prepared from trichloroacetic acid (TCA)-insoluble and -soluble fractions of lipid-free mitochondria. These fractions were digested with pronase exhaustively, and the mucopolysaccharides were recovered in the void volume fractions of gel filtration of the pronase digests on Sephadex G-50, monitored by radioactivity determination. Identification of these mucopolysaccharides was based on electrophoresis on cellulose acetate film using three different media, enzymatic and chemical degradations specific to each type of mucopolysaccharide, using chondroitinases, heparitinase, and nitrous acid. From the TCA-insoluble fraction, chondroitin sulfate A and dermatan sulfate were obtained in a ratio of about 1 : 2, based on 35S-radioactivities, whereas the TCA-soluble fraction yielded chondroitin sulfates A/C, dermatan sulfate, and heparan sulfate in a ratio of about 1 : 3 : 12. The total amount of mitochondrial mucopolysaccharides was about 3 mg/g protein, distributed between the TCA-insoluble and -soluble fractions in a ratio of about 1 : 3.


Demonstration and quantitation of catalytic and noncatalytic bound ATP in submitochondrial particles during oxidative phosphorylation. Techniques are described for studying the labeling of ADP and ATP bound to the ATP synthase complex of beef heart submitochondrial particles catalyzing oxidative phosphorylation. These suffice for measurements of bound nucleotides during the time required for a single turnover, during steady state net ATP synthesis, or under quasiequilibrium conditions of ATP formation and hydrolysis. Results show that the "tightly bound"  ATP associated with isolated submitochondrial particles does not become labeled by medium [32P]Pi rapidly enough to qualify as an intermediate in ATP synthesis. In contrast to chloroplast preparations, little or no bound [32P]Pi committed to ATP formation is present on particles during steady state synthesis. Also, highly active particles synthesizing ATP from [32P]Pi and filtered after EDTA addition have no detectable bound [32P]ATP even though several ATPs have been made per synthase complex. However, under quasiequilibrium conditions membrane-bound ADP and ATP are present whose labeling characteristics qualify them as intermediates in ATP synthesis. In addition, a hexokinase-accessibility approach shows the presence of a steady level of bound ATP. Lack of detection of bound intermediates under other conditions is regarded as reflecting the ready reversibility of oxidative phosphorylation, with consequent facile cleavage of bound ATP and release of bound Pi.


On the subunit stoichiometry of the F1-ATPase and the sites in it that react specifically with p-fluorosulfonylbenzoyl-5'-adenosine. During the inactivation of the nucleotide-free F1-ATPase at pH 7.0, by p-fluorosulfonyl[14C]benzoyl-5'-adenosine ([14C]FSBA) in the presence of 20% glycerol, about 4.5 g atoms of 14C are incorporated/350,000 g of enzyme. Isolation of the subunits has shown: (a) over 90% of the incorporated label is associated with the alpha and beta subunits; (b) the amount of label incorporated into the alpha subunit is about 0.5 g atoms/mol which is nonspecifically associated with a number of tyrosine and lysine residues; (c) the amount of radioactivity incorporated into the beta subunit is about 0.9 g atoms/mol which correlates with the degree of inactivation of the enzyme and resides on a single tyrosine residue; (d) up to 2.2 mol of alpha subunit have been isolated from each mole of inactivated enzyme; and (e) about 2 mol of beta subunit have been isolated from each mole of inactivated enzyme. These results account for the incorporation of 4.5 g atoms of 14C which are incorporated/mol of ATPase during inactivation if there are three copies each of the alpha and beta subunit present in the enzyme. It has also been shown that 4-chloro-7-nitrobenzofurazan (NBD-Cl) and FSBA react with different tyrosine residues when they inactivate the ATPase. In addition, it has been shown that the ATPase inactivated with FSBA retains the capacity to bind up to 2.2 mol of [14C]ADP/350,000 g of enzyme.


Tightly bound magnesium in mitochondrial adenosine triphosphatase from beef heart. Tightly bound magnesium was found in soluble, purified ATPase (F1) from beef heart mitochondria in the amount of 1 mol/mol of F1. Iron, zinc, cobalt, manganese, calcium, sodium, copper, and potassium were not tightly bound at stoichiometric levels. Removal of magnesium by chelating agents caused loss of ATPase activity. Removal of tightly bound nucleotide by gel filtration in 50% glycerol- or 60 mM K2SO4-containing buffers did not remove magnesium. Cold dissociation did release magnesium when complete denaturation was accomplished. The results suggest that magnesium is an integral part of F1, that it is required for activity, and that magnesium and nucleotides are tightly bound at separate sites. The idea that the tightly bound nucleotides are not complexed with cations suggests certain structural requirements at their binding sites which might account for the unusual properties of the sites.


Phosphocreatine does not inhibit rabbit muscle phosphofructokinase or pyruvate kinase. Certain phosphocreatine preparations contain a contaminant that inhibits phosphofructokinase and pyruvate kinase assays. The contaminant can be separated from phosphocreatine by anion exchange chromatography. After appropriate purification, phosphocreatine has no effect on phosphofructokinase or pyruvate kinase; thus, there is no evidence that it serves muscle as a regulator of these enzymes. Although the inhibitory preparations of phosphocreatine contain inorganic phosphate and trace amounts of more negatively charged phosphorylated contaminants, the inhibitor is not inorganic phosphate or pyrophosphate. The nature of the inhibitor remains to be determined.


Binding of mitochondrial ribosomal proteins to a mitochondrial ribosomal precursor RNA containing a 2.3-kilobase intron. In Neurospora, the gene encoding the mitochondrial large subunit (25 S) rRNA contains an intervening sequence of approximately 2.3 kilobases. We have recently identified two temperature-sensitive, nuclear mutants (289-67 and 299-9) which are defective in splicing of the 25 S RNA. When grown at the nonpermissive temperature (37 degrees C), the mutants show decreased ratios of 25 S/19 S RNA and accumulate a novel 35 S RNA which appears to be a continuous transcript of the 25 S RNA gene, including the intervening sequence. In the present work, mitochondrial ribonucleoprotein particles present in the 50 S subunit peak from wild type and mutant 299-9 were analyzed by equilibrium centrifugation in CsCl gradients containing 25 mM MgCl2. The results show that 35 S RNA can be isolated as part of a ribonucleoprotein particle associated with nearly all of the large subunit ribosomal proteins. However, the particles appear to be less stable in CsCl gradients and more sensitive to nucleolytic degradation than particles derived from mature large subunits. Our results indicate that binding of ribosomal proteins to 35 S RNA could precede removal of the intron, but that removal of the intron may be required to achieve stable protein binding.


Phosphorylation and dephosphorylation kinetics of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa. Partial reactions of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa were studied by means of a rapid-mixing apparatus. At 21 degrees C, in the presence of 2 mM MgCl2 and 5 microM [gamma-32P]ATP there was a rapid phosphorylation of the enzyme with a pseudofirst order rate constant of 1400 min-1. Addition of the ATP about 120 ms before the MgCl2 increased this rate constant to 4400 min-1. In the absence of MgCl2 there was no phosphorylation. Addition of 4 or 10 mM KCl to the phosphoenzyme which had been formed in the absence of KCl produced a rapid initial rate of dephosphorylation (k = 2600 and 3200 min-1 respectively). An additional slow component of dephosphorylation was observed when unlabeled ATP was added together with the KCl (k = 700 to 900 min-1). At a 4 mM concentration, KCl stimulated the ATPase activity about 9-fold. At higher concentrations, the activity was reduced in parallel with a reduction of the steady state level of phosphoenzyme. Addition of KCl to the enzyme before the addition of ATP plus MgCl2 resulted in a low rate and extent of phosphorylation. KCl appeared to inhibit the phosphorylation at a level preceeding the E.ATP complex.


The effects of kallikrein, plasmin, and thrombin on hog kidney renin. Pretreatment of hog high molecular weight renin for 30 min at 37 degrees C with 0.12 unit of either kallikrein or thrombin significantly increased (p less than 0.001) the amount of angiotensin I formed during subsequent incubations with homologous angiotensinogen. However, the thrombin-treated hog renin had 13 times more activity than the kallikrein-treated enzyme. Aprotinin did not inhibit the kallikrein-mediated activation of renin; the results indicated that aprotinin inhibited renin preferentially. Plasmin (0.25 unit) had little effect on the activity of high molecular weight renin. The molecular weight of hog renin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was not altered after exposure to either kallikrein, thrombin, or plasmin. These results do not exclude the occurrence of a limited proteolytic event or a conformational change beyond the detection of the current method. The data show that the activation of hog high molecular weight renin by thrombin and kallikrein was not associated with the conversion of renin to Mr = 43,000.


Mutants of Escherichia coli that do not contain 1,4-diaminobutane (putrescine) or spermidine. Strains of Escherichia coli K12 have been constructed which do not contain any of the polyamines normally present in a wild type strain, namely, 1,4-diaminobutane (putrescine) and spermidine. This phenotype arises as a consequence of the assembly into these strains of deletion mutations in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). The polyamine-deficient strains grow indefinitely in the absence of polyamines but with a growth rate one-third of that found in the presence of polyamines. These strains can act as hosts for bacteriophages T4, T7, and f2, although the latter phage is poorly adsorbed; they can also maintain F' factors, ColE1 and P1 plasmids, and lysogeny by bacteriophage lambda. In contrast, the production of bacteriophage lambda in the absence of polyamines is strikingly decreased (greater than 99%) either after infection of a nonlysogen or after induction of a lysogen. A polyamine-deficient Hfr strain can transfer its chromosome to a recipient at a normal rate, but the number of recombinants observed in a cross is decreased approximately 300-fold. No such effect is observed when only the F- recipient strain in a cross is polyamine deficient.


Phenylglyoxal modification of cardiac myosin S-1. Evidence for essential arginine residues at the active site. The role of arginine residues in the catalytic activity of cardiac myosin subfragment-1 (S-1) was investigated by selective modification with phenylglyoxal. Incorporation of about 2.8 mol of phenylglyoxal/mol of S-1 decreased Ca2+-ATPase activity about 50%. Gelation of the protein occurred at about 70% inactivation; however, extrapolation to complete inactivation indicated that loss of activity correlated with modification of about 4 arginyls/mol. Partial inactivation of S-1 with phenylglyoxal also decreased MgADP binding markedly. When S-1 was modified in the presence of 5 mM MgADP, only 2 arginyls/mol were blocked and there was almost complete protection against loss of Ca2+-ATPase activity and ability to bind MgADP. Similar protection against inactivation by phenylglyoxal was obtained with MgATP or sodium pyrophosphate, but not with MgAMP or magnesium adenosine. These results suggest that 2 arginyls/myosin head are important for enzymatic activity, possibly serving as attachment points between enzyme and substrate. These essential arginyls were localized to a 17,000-dalton cyanogen bromide peptide from the heavy chain fragment of S-1.


Mapping and cloning of Neurospora crassa mitochondrial transfer RNA genes. We have obtained collections of recombinant Escherichia coli plasmids containing restriction fragments of Neurospora crassa mitochondrial DNA cloned into pBR322. By hybridization of 32P end-labeled total mitochondrial tRNAs and seven different purified tRNAs to restriction digests of mitochondrial DNA and of recombinant plasmids carrying specific restriction fragments, we have located the tRNA genes on the mitochondrial DNA. We have found that the mitochondrial tRNA genes are present in two major clusters, one between the two ribosomal RNA genes and the second closely following the large rRNA gene. Only one of the two DNA strands within these clusters codes for tRNAs. All of the genes for the seven specific purified tRNAs examined--those for alanine, formylmethionine, leucine 1, leucine 2, threonine, tyrosine, and valine--lie within these clusters. Interestingly, the formylmethionine tRNA hybridizes to two loci within one of these gene clusters. We have obtained a fairly detailed restriction map of part of this cluster and have shown that the two "putative" genes for formylmethionine tRNA are not arranged in tandem but are separated by more than 900 base pairs and by at least two other tRNA genes, those for alanine and for leucine 1 tRNAs.


Examination of four newly synthesized 2-chloroethylnitrosoureas in comparison with BCNU, CCNU, MeCCNU, chlorozotocin and hydroxyethyl-CNU in preterminal rat leukemia L 5222. The newly synthesized nitrosoureas 1-(2-chloroethyl)-1-nitroso-3-(methylenecarboxamido)-urea (Acetamido-CNU), 1-(2-chloroethyl)-1-nitroso-3-(4-morpholino)-urea (Morpholino-CNU), 1-(2-chloroethyl)-1-nitroso-3-(1-piperidino)-urea (Piperidino-CNU), and 2-[3-(2-chloroethyl)-3-nitrosoureido]-ethylmethanesulfonate (Ethylmethanesulfonato-CNU) were compared in their chemotherapeutic activity against preterminal rat leukemia L 5222 with BCNU, CCNU, MeCCNU, Chlorozotocin, and Hydroxyethyl-CNU. With respect to the dose range effecting a median survival time of more than 90 days, MeCCNU was superior to the other substances. Chlorozotocin, on the other hand, was the only substance which achieved no cures in this experimental arrangement. From the four newly introduced substances the water-soluble substances Acetamido-CNU and Morpholino-CNU were approximately comparable to CCNU with regard to the dose range effecting a median survival time of greater than 90 days. Piperidino-CNU and Ethylmethanesulfonato-CNU also effected cures; however, only Piperidino-CNU in one dosage effected a median survival time of greater than 90 days.


Characterization of gastric mucosal membranes. X. Immunological studies of gastric (H+ + K+)-ATPase. Gastric mucosal homogenates from hog were fractionated by differential and density gradient centrifugation and free-flow electrophoresis. The two major membrane fractions (FI and FII) thus obtained are distinct both enzymically and in terms of transport reactivity. This heterogenicity extends to their antigenic activity. Purified antibodies which were raised against the K+-ATPase-containing H+ transport fraction FI were of two types: inhibitory and non-inhibitory. Inhibitory antibodies reduced the K+-ATPase activity by approximately 80% and the K+-p-nitro-phenylphosphatase activity by approximately 40% in a concentration-dependent manner, while the small Mg++-dependent component of the enzyme activity was unaffected. Antibodies inhibiting the K+-ATPase also inhibited H+ transport. These antibodies did not cross-react with the other major membrane fraction isolated by free-flow electrophoresis, FII, and gave a single band on rocket immunoelectrophoresis. Antibodies against this FII fraction also did not react with the K+-ATPase and were heterogeneous, giving at least four bands with rocket immunoelectrophoresis and inhibiting both the 5'-nucleotidase and Mg++-ATPase of this fraction. Immunofluorescent staining of tissue sections showed that the FI was derived from the parietal cell of gastric tissue and was localized to the supranuclear area of the cell. Staining of isolated rat gastric cell suspensions by FI antibodies confirmed the selectivity of the antibody and showed a polar, plasma membrane localization. FII antibodies also largely stained the parietal cells in tissue sections. In the 16 hog tissues tested, FI antibodies cross-reacted only with gastric fundus, thyroid and weakly with thymus. Immunoelectronmicroscopy showed that FI antibodies reacted strongly with the secretory membrane at the apical cell surface of the parietal cells and at the secretory canaliculi, weakly with the apical surface of the zymogen cell, and not with the basal-lateral surface of the cells. Thus, the protontranslocating ATPase is localized in the parietal cells and in the region postulated to be the site of acid secretion.


Poor mixed lymphocyte reaction stimulatory capacity of leukemic B cells of chronic lymphocytic leukemia patients despite the presence of Ia antigens. The human Ia-like antigens, selectively expressed on B lymphocytes, are now recognized to be closely associated with, or identical to, the gene products of the major histocompatibility complex responsible for stimulation in the mixed lymphocyte reaction. The leukemic B lymphocytes of patients with chronic lymphocytic leukemia express these antigens very well. In the present study they were readily detected by several techniques utilizing both allo- and heteroantisera. However, the leukemic B cells from most patients were found to be extremely poor stimulating cells in the mixed lymphocyte reaction. This was particularly apparent when comparisons were made on a B-cell basis with isolated normal B lymphocytes. Leukemic cell death, abnormal kinetics of leukemic cell-mediated stimulation, and serum or cellular suppressor factors do not appear to explain these findings. Studies comparing cells from a leukemic patient with those of her HLA identical sibling and results of mixed lymphocyte reactions between normal and leukemic subjects discordant for D-region-associated Ia antigens ruled out genetic explanations for the differences observed. Experiments with normal peripheral blood mononuclear cells depleted of T cells and monocytes exclude the quantitative deficiency of monocytes which is found in the peripheral blood of most leukemic patients as an explanation. The present results with chronic lymphocytic leukemia cells indicate that the mere expression of the Ia-like antigens by cell populations does not render them effective stimulators. The accumulated evidence obtained indicate that abnormalities, particularly of membrane function and metabolism, known to occur in chronic lymphocytic leukemia lymphocytes may be involved in the poor stimulatory capacity of the leukemic B cells.


Studies of immune functions of patients with systemic lupus erythematosus. T-cell subsets and antibodies to T-cell subsets. Antibodies to T cells present in the plasma of patients with active systemic lupus erythematosus (SLE) plus complement are able to eliminate concanavalin A-induced suppressor function for the proliferative responses of T cells to allogeneic lymphocytes (MLR) and of B cells to pokeweed mitogen (PWM). Such antibodies were found to be effective in eliminating suppressor function only when T cells were treated before activation; there was no effect when treatment was performed after activation. These studies indicate that the antibodies preferentially interact with a T cell necessary for the generation of suppressor cells, rather than with mature, activated suppressor cells. Studies of individual SLE patients indicate that the same defects observed in SLE T cells were induced in normal T cells by plasma from that patient. Such observations suggest that many T-cell defects associated with active SLE may not be intrinsic T-cell abnormalities, but, rather, secondary effects of anti-T-cell antibodies. Studies of the T-cell subpopulations responsible for suppression of the MLR and PWM responses indicate that only T gamma cells (T cells bearing receptors for the Fc portion of immunoglobulin [Ig]G) acted as precursors of suppressor cells for the MLR, whereas both T gamma and T non-gamma cells (T cells not bearing receptors for the Fc portion of IgG) could be activated to suppress the PWM response. Consistent with this observation, SLE anti-T-cell antibodies that preferentially killed T gamma cells preferentially eliminated suppressor cells for the MLR.


Effect of fasting and streptozotocin diabetes on insulin binding and action in the isolated mouse soleus muscle. To investigate whether skeletal muscle is resistant to insulin in insulinopenic states, insulin binding and biological effects on glucose utilization were studied in isolated soleus muscles from 24- or 48-h-fasted mice and from streptozotocin-diabetic mice. Both 48-h fasting and diabetes led to an increase in insulin binding at insulin concentrations <3.4 nM. In both states, submaximal concentrations of insulin were also more effective in stimulating muscle 2-deoxyglucose uptake and glycogen synthesis, and in activating glycogen synthase. This resulted in a two- to fourfold leftward shift in the insulin dose-response curves in muscles from both groups compared with control. No change in insulin binding or biological effects was detected in muscles from 24-h-fasted mice. Maximal insulin effectiveness on 2-deoxyglucose uptake and glycolysis was either unchanged or only slightly enhanced in 48-h-fasted mice and in diabetic animals, compared with controls. Maximal insulin effects on glycogen synthesis and glycogen synthase activation were unaltered by fasting or diabetes. Basal glucose uptake and glycolysis were similar in all groups of mice. In conclusion, when soleus muscles from 48-h-fasted mice and from diabetic mice are compared with controls it can be observed that, (a) at low insulin concentrations insulin binding is increased and insulin effectiveness in stimulating glucose transport and metabolism is enhanced; (b) biological responses to maximally effective insulin concentrations are either unaltered or slightly increased; (c) basal rates of glucose transport and metabolism are essentially unaltered. These results indicate that in insulinopenic states soleus muscle is not insulin resistant in vitro but is hypersensitive to low concentrations of insulin, and normally responsive to maximally effective doses of the hormone.


Suppression of immune complex vasculitis in rats by prostaglandin. Immune complex-induced vascular damage can be markedly suppressed by treatment of rats with either prostaglandin (PG)E1 or its stable derivative, 15-(S)-15-methyl PGE1, but not with PGF2 alpha. In addition, PGD2 and PGE2 also show suppressive effects. The PGE1 derivative is considerably more effective than PGE1 and shows potent anti-inflammatory activity even after oral administration. Suppression of the vasculitis reaction is reflected by a greatly diminished increase in vasopermeability, indicating little or no vascular damage. In suppressed animals, the infiltration of neutrophils is greatly reduced, and those leukocytes that have appeared at tissue sites fail to show phagocytic uptake of immune complexes. In suppressed animals, the skin sites nevertheless show deposits of immune complexes and C3 fixation in vascular walls. Neutrophils harvested from the blood of rats treated with PGE1 show depressed responsiveness in chemotaxis and in enzyme secretion after incubation with chemotactic peptide. These studies indicate that certain PG have potent anti-inflammatory activity, which may be related to their effects on leukocytes.


Induction of suppressor activity in the autologous mixed lymphocyte reaction and in cultures with concanavalin A. T lymphocytes that are activated in the autologous mixed lymphocyte reaction (MLR) have suppressor activity. Concanavalin A (Con A) augments the suppressor activity generated in cultures containing both T and non-T lymphocytes and can induce suppressor activity in T-lymphocyte preparations that contain too few (10%) non-T cells to generate a significant autologous MLR. However, when such T-lymphocyte preparations are further depleted of adherent cells and contain less than 2% non-T cells, Con A fails to induce suppressor activity. These findings support the concept that an autologous MLR may play an important role in generation of suppressor cells by Con A.


Importance of hepatic portal circulation for insulin action in streptozotocin-diabetic rats transplanted with fetal pancreases. The importance of the hepatic portal circulation in the response to insulin was assessed in streptozotocin-diabetic rats transplanted with syngeneic fetal pancreases. Partial reversal of diabetes was accomplished by transplantation of two or three fetal pancreases beneath the capsule of the kidney; complete reversal followed shunting of the venous drainage from the transplants to the liver. Plasma glucose after streptozotocin of 509+/-31 mg/dl (mean+/-SEM) fell after transplantation to 395+/-23 and after the shunt to 143+/-5 mg/dl. Urine volume fell from 84+/-4 to 50+/-5 ml/d and then to normal (17+/-1 ml/d) after the shunt. Glucose excretion which was 8.1+/-0.3 g/d after streptozotocin fell after transplantation to 4.8+/-0.3 g/d and after the shunt completely disappeared from the urine. The disappearance rate of glucose injected into the circulation, which was 0.50+/-0.07%/min in untreated diabetes, increased to 1.39+/-0.38%/min after transplantation and to 2.52+/-0.31%/min after the shunt, not different from normal controls (2.79+/-0.25). Plasma immunoreactive insulin (IRI) was below normal (25-35 muU/ml) and unresponsive to glucose in untreated diabetic rats. After transplantation IRI levels ranged from 73-223 muU/ml and there was no rise after glucose injection. After the shunt both the basal IRI (36+/-5 muU/ml) and the peak response to glucose at 10 min (58+/-7 muU/ml) were the same as in normal controls (42+/-4 and 62+/-7 muU/ml, respectively). The fall in IRI after the shunt is explained by increased extraction of insulin passing into the liver and also diminished secretion. After removal of the transplants plasma glucose and urine values returned almost to pretransplant levels. Secretion of insulin by transplanted pancreases into the liver enhances the effectiveness probably by increased extraction and action and reveals the importance of the normal route for insulin delivery.


Cell-mediated cytotoxic responses of spleen cells from mice bearing primary, chemically induced tumors. Sarcomas were induced in C57BL/6 mice by using 3-methylcholanthrene. Spleen cells taken from these mice bearing primary, chemically induced tumors and from matched control mice were assessed for the capacity to generate cell-mediated cytotoxic cell activity after in vitro sensitization. Spleen cells from tumor-bearing mice generated strong cell-mediated cytotoxic responses against alloantigens and antigens on syngeneic cells.


Cell-mediated lympholysis by human maternal and neonatal lymphocytes: mother's reactivity against neonatal cells and vice versa. Maternal reactivity in cell-mediated lympholysis (CML) against cells from her own child is, in average, half of the maternal reactivity against unrelated adult cells. This finding remains the same when cells from a newborn or from an older child are used, suggesting that the reduced maternal reactivity is based rather on the one haplotype identity between the mother and child than on the occurrence of specific maternal tolerance. Consistently, CML-capacity of the child, directed against cells of own mother, is half of the control values, again independently of the child's age.


Sex-related immunocompetence of BALB/c mice. I. Study of immunologic responsiveness of neonatal, weanling, and young adult mice. The responses of lymphoid cells from the thymus, lymph nodes, and spleen of male and female BALB/c mice were evaluated to determine if sex-related variations in immune expression could be found. Immunologic assays used included blastogenic responses to mitogens, mixed lymphocyte responses, and direct and indirect measurement of plaque-forming cells against soluble and particulate antigens. The results indicated that responses of spleen cells from young adult female mice were higher than those of males in all comparative tests. Little or no differences between the sexes were observed in the mitogenesis of lymph nodes and thymuses. Newborn mice did not demonstrate the sex-associated immune differences. Among the weanling mice slight differences between male and female spleen cells responsiveness to mitogenic agents were observed.


Lyt phenotypes of responding cells in secondary alloantigen responses. Lymphocytes responding in a "secondary" MLC-CML system, after in vitro sensitization, apparently fall into two classes with regard to their Lyt phenotype. First, are the cells that form the majority of the proliferating cells after restimulation with either I or K + I differences, which are Lyt 1-2, and second, are Tc that are Lyt 1-2+. The Lyt 1-2- proliferating cells are not cytotoxic and are lysed by treatment with anti-Thy 1.2 serum in the presence of C.


Regulation of the murine IgE antibody response. I. Characterization of suppressor cells regulating both persistent and transient responses and their sensitivity to low doses of X-irradiation. Anti-ovalbumin (OA) IgE antibody responses were measured in B6D2F1 mice as a function of time and antigen dose. One hundred to 200 microgram of OA in Al(OH)3 elicited transient responses, whereas 1 to 10 microgram of OA in Al(OH)3 elicited persistent anti-OA IgE responses of high titer. T cells isolated from the spleens of mice mounting either a persistent or a transient response strongly suppressed primary anti-DNP IgE responses in unirradiated recipient mice that were immunized with DNP-OA in Al(OH)3; it was, therefore, concluded that suppressor T cells (Ts cells) were activated during both the persistent and transient IgE responses. Nevertheless, in the present study it was not possible to completely rule out the contention that IgG antibodies may also have been suppressing the IgE response. With a modified adoptive transfer system, it was shown that these Ts cells were sensitive to low doses (250 R) of x-irradiation. The suppressive activity of long-term OA primed cells was also shown to be markedly enhanced when cultured for 24 hr with soluble OA; this finding was interpreted to indicate the presence of memory suppressor cells.


The Qa-2 antigen on lymphocyte subpopulations. Mixed lymphocyte culture and cell-mediated lympholysis. Treatments of spleen cells from Qa-2+ strains with Qa-2 antiserum plus complement (C) have revealed that the Qa-2 antigen is present on restricted functional lymphocyte subpopulations. Anti-Qa-2 plus C reduced the mixed lymphocyte culture response and inhibited the generation of cytolytic effector cells. This treatment, however, did not affect cytolytic effector cells once they were generated.


[The topographical localization of spinal motoneurons of the rat and its numerical alternation in regard to development (author's transl)]. The topographical localization of spinal motoneurons innervating the forelimb muscles of the rat and its numerical alternation in regard to development was observed by HRP (horseradish peroxidase) method. HRP method is a histochemical technique to trace the cellular origin of axon terminal net work. When HRP is injected into the muscles, the enzyme is taken up by nerve endings and transported retrogradely by the axoplamic flow to the original cell body, and does not transported hematogenously and does not cross the synapse. The spinal motoneurons were found in spindle-shape longitudinal cell columns according to its innervation. The motoneurons supplying the biceps brachii muscle were located in the dorso-lateral nucleus on the anterior horn of the 4th cervical segment, and extended downward to the ventral nucleus in 8th cervical segment, forming a spindle-shape cell column. The thick spindle-shape cell column consisting of cells which innervated the triceps brachii muscle lied in the ventro-medial nucleus of the 6th, 7th and 8th cervical segment. The neurons innervating the flexor carpi ulnaris muscle were located in 7th and 8th cervical segment, but few in number compared to those of the biceps and triceps. The motoneurons innervating the biceps brachii muscle decreased in number with increasing age according to exponential curve. The total number of HRP-labeled neurons in two week old rat ranged from 2,932 to 2,017 and markedly decreased during first eight weeks, in eight week old rat ranged from 1,637 to 603. After this, they gradually decreased to 312 in 50 weeks on an average, where the number corresponded to about 13% of those of two week old rat. The data indicate that ventral horn has a great deal of excess neurons which has a distinct neuromuscular connection as suggested by Prestige, in the developmental stage of Xenopus laevis. In young rats (ten days to two weeks of age), HRP-labeled neurons were also present in the ventral horn on the non-injected side. These large and multigonal contralateral HRP-labeled neurons constituted a characteristic feature of spinal motoneurons, and distributed through the same segment as those on injected side. The total number of HRP-labeled neurons within non-injected side ranged from 563 to 197 and corresponded from one-fifth to one-tenth of that in the injected side. In five weeks or elder rat, no HRP-labeled neurons could be observed in non-injected side. This indicates that in immature rats the muscles are innervated bilaterally by the spinal nerve, since HRP is transported by retrograde axoplasmic flow and does not cross the synapse, and the bilateral motor innervation has disappeared with maturation. The bilateral motor innervation is therefore, presumed to be an immature mode of innervation limited to the early stage of life. Although bilateral motor innervation following the nerve injury in the limb at the early stage of life has been confirmed by ourselves, the significance of the immature mode of innervation is still hard to explain.


Erythropoiesis and artificial blood substitution with a perfluorocarbon-polyol. Rats were perfused with Fluosol-43, a perfluorotributylamine-pluronic F-68 emulsion, to produce hematocrits of about 6% and the mechanisms of blood cell replacement were studied. The packed RBC volume and number of RBCs per cubic millimeter increased gradually as the time after perfusion was completed until reaching the preperfusion level at about 1 week. The WBCs per cubic millimeter, which were not as severely depleted because of their vast storage compartment, increased to preperfusion levels by 24 hr and continued to increase through 7 days. Plasma did not show significant Ep activity until 48 to 72 hr after perfusion was concluded. Thus, although 90% of the RBCs were removed, elevated erythropoietin activity was not detected immediately because the perfluorocarbon compound replaced the capacity of the RBCs as an oxygen and carbon dioxide carrier.


Experimental IgA nephropathy.

Experimental IgA nephropathy. An animal model for IgA immune complex nephritis was developed. IgA immune complexes formed in vitro with an IgA anti-dinitrophenyl (DNP) derived from MOPC-315 plasmacytoma, and dinitrophenylated bovine serum albumin (DNP-BSA) produced mild focal glomerulonephritis in mice. Similar, but more severe pathological changes were produced with complexes formed in vivo either in normal mice or MOPC-315 tumor-bearing mice. In contrast to the focal nature of the PAS-positive glomerular lesions observed by light microscopy, immunofluorescent examination revealed IgA deposits in all glomeruli. This discrepancy between immunofluorescent and histopathologic findings as well as the distribution of the immune complexes within the affected glomeruli, are some of the features which bear resemblance between this experimental model and human IgA nephropathy. Fixation of complements by DNP-BSA-IgA immune complexes, formed in vitro or in vivo, was shown to occur in the glomeruli of mice with IgA immune complex nephropathy. The pattern of C3 glomerular deposits was similar to that of IgA. However, complement proved to be nonessential for complex deposition. This conclusion is based on the observation that decomplemented mice, although showing no deposition of C3 in their glomerulus, developed glomerular immunohistological changes similar to those observed in experimental mice that were not decomplemented. Polymeric IgA was observed to be critical for renal deposition of complexes and induction of nephritic histological changes. In contrast, monomeric IgA immune complexes failed to produce glomerular deposits. This finding raises the possibility that secretory IgA, which is predominantly polymeric, may play a role in human IgA-associated glomerulonephritis.


Investigation of the structure of the blue copper protein from Rhus vernicifera stellacyanin by 1H nuclear magnetic resonance spectroscopy. The 270-MHz 1H nuclear magnetic resonance spectra of Cu(II), Cu(I), and apo-stellacyanin are reported and compared. The data indicate that little conformational change occurs on reduction of the protein or on removing the copper ion. In the aromatic region of the spectra of the holoprotein, resonances associated with two freely titrating histidines are observed. Two additional sharp resonances are observed in the spectra of the apostellacyanin which are tentatively assigned to additional histidines. This result requires that not more than two histidines can be ligands since there are only four histidines in the whole protein. The absence of methionine has been reported and is one of the possible causes for the difference between stellacyanin and the other copper blue proteins. A comparison of these data with those available for other blue copper proteins, in conjunction with the sequence information, leads to a proposed structure for the copper site in stellacyanin.


Selective effects of vibration on monosynaptic and late EMG responses in human soleus muscle after stimulation of the posterior tibial nerve or a tendon tap. In normal subjects it was possible to evoke tendon and Hoffman reflexes which were followed by late EMG responses with a latency of 150-250 ms after the reflex stimuli. Analysis of the covariations of reflexes and late responses revealed that muscle spindle sensitivity and strength of the preceding twitch are not essential factors in determining the occurrence of the late responses as opposed to excitability changes within the spinal cord. Inhibition of monosynaptic reflexes and facilitation of late EMG responses to vibration indicate a difference in central pathways. A polysynaptic pathway may be involved in the late responses.


Echocardiographic abnormalities in infants of diabetic mothers. In order to evaluate the presence of myocardial hypertrophy and/or abnormalities of postnatal cardiovascular adaptation, echocardiograms were performed on 34 infants of diabetic mothers. Based on cardiopulmonary assessment, the IDM were divided into three groups: Group I with congestive heart failure predominating: Group II with respiratory distress predominating: Group III asymptomatic. Hypertrophy of the interventricular septum and of the walls of left and right ventricles was frequently present in IDM: this change was most notable in association with clinical CHF. Six IDM, four of whom were found to have CHF, had additional echocardiographic signs of subaortic stenosis. All IDM had normal indices of left ventricular performance, despite the presence of CHE. In IDM with respiratory distress, the right ventricular pre-ejection period to ventricular ejection time ratio was elevated, suggesting an abnormality of the transitional pulmonary circulation. Poor maternal diabetes control and maternal systemic hypertension were closely correlated with evidence of myocardial hypertrophy in the infants.


The natural history of hypertrophic cardiomyopathy in infants of diabetic mothers. Eleven infants of diabetic mothers with hypertrophic cardiomyopathy have been followed for 30 to 40 months. All infants presented with cardiorespiratory distress and were found to have disproportionate septal hypertrophy on echocardiogram. Cardiac catheterization was done in four infants; three had significant subaortic obstruction. One infant had remarkable improvement after treatment with propranolol. Two infants who received digoxin did poorly and responded favorably to cessation of therapy. The natural history of HCM-IDM appears to be benign, with a resolution of symptoms within two to four weeks and a resolution of septal hypertrophy within two to 12 months. Most of the infants need only supportive care; if pharmacologic intervention is deemed necessary, propranolol appears to be the drug of choice. The natural history of this entity is that of spontaneous regression of symptoms and septal hypertrophy irrespective of therapy.


Fibromuscular dysplasia of renal arteries: an important cause of renovascular hypertension in children. Fibromuscular dysplasia of renal arteries was the cause of hypertension in four consecutive children with renal artery stenosis. Two were asymptomatic, the third had had hypertension for seven years but had not been treated, and the fourth, a 9-month-old infant, presented with cardiac failure. Heart enlargement and left ventricular hypertrophy were present in all. Rapid sequence urograms demonstrated a smaller kidney and delayed appearance and disappearance of the contrast medium on the affected side in all. Angiograms showed left RAS in all. Peripheral plasma renin activity was elevated in only three of the four patients. Antihypertensive and diuretic drugs were not very effective therapeutically. Ischemia of the ipsilateral kidney probably prevented normal growth and led to shrinkage of the kidney in one patient. Following nephrectomy the BP has remained normal without any therapy for 24 to 64 months. With normalization of BP, accelerated growth ensued, the cardiomegaly regressed and the hypertensive retinopathy resolved. These patients demonstrate that: (1) FMD is an important cause of RAS. (2) the well-known radiologic feature of FMD, the beaded appearance, is usually not seen in children. (3) control of BP leads to normalization of linear growth, usually impaired in severe hypertension, and (4) target organ complications such as cardiomegaly, LVH, and hypertensive retinopathy are reversible in one to 10 months.


Autogenetic inhibition of motoneurones by impulses in group Ia muscle spindle afferents. 1. Inhibitory post-synaptic potentials evoked by adequate stimulation of group Ia muscle spindle afferents of homonymous and synergistic muscles and by selective electrical stimulation of tendon organ afferents were analysed in motoneurones of triceps surae and plantaris. 2. Selective activation of Ia afferents was verified to occur with brief stretches of triceps surae and plantaris 35 micrometer or less in amplitude with an initial muscle tension of 5 N; stretches of 30--35 micrometer were estimated to activate 80--90% of Ia afferents in these muscles. Under the same conditions the lowest thresholds for group Ib tendon organ afferents were about 40 micrometer. 3. Stretches less than or equal to 30 micrometer evoked i.p.s.p.s in 80% of triceps surae and plantaris motoneurones; lowest thresholds for evoking i.p.s.p.s wef triceps surae and plantaris motoneurones; lowest thresholds for evoking i.p.s.p.s were 10 micrometer or less. However, such low thresholds for stretch-evoked i.p.s.p.s, lower than the thresholds for activation of Ib afferents, were found mainly in spinalized, unanaesthetized (after decerebration) or lightly anaesthetized animals. The latencies of these i.p.s.p.s indicated disynaptic and trisynaptic coupling between Ia afferents and motoneurones. The i.p.s.p.s were evoked (i) from the homonymous and synergistic muscles stretched together, (ii) from the homonymous muscles alone and (iii) from the synergistic muscles alone. 4. Control experiments showed that i.p.s.p.s could be evoked by stretches sub-threshold for discharging motoneurones, thus showing that those i.p.s.p.s were not mediated by Renshaw cells. The stretch-evoked i.p.s.p.s disappeared after sectioning the nerves from the corresponding muscles, further excluding their mediation by afferents other than group Ia afferents from thf stretched muscle. 5. In order to selectively activate tendon organ afferents, thresholds for excitation of Ia afferents by electrical stimuli were increased to a level above the threshold for Ib afferents by prolonged muscle vibration (Coppin, Jack & MacLennan, 1970). I.p.s.p.s evoked by stimuli near threshold for Ib afferents appeared with latencies indicating disynaptic coupling. Later (trisynaptic) components of Ib i.p.s.p.s required somewhat stronger stimuli. 6. Amplitudes of Ia i.p.s.p.s evoked by muscle stretches activating about 80% of muscle spindle afferents were compared with amplitudes of Ib i.p.s.p.s due to less than 50% of tendon organ afferents of the same muscles. The Ia i.p.s.p.s were much smaller (16--35%) than the Ib i.p.s.p.s. The amplitudes of such Ia and Ib i.p.s.p.s constituted about 10 and 25--66%, respectively, of the maximal i.p.s.p.s evoked by electrical stimulation of all group I afferents. 7. We conclude that inhibition of motoneurones may be evoked from Ia muscle spindle afferents from homonymous and synergistic muscles as well as from Ib tendon organ afferents...


Unique leukemic non-T/non-B lymphoid cell lines (REH and KM-3): absence of MLR-S and presence of suppressor cell activity for normal T-cell response. The present study unequivocally demonstrates that leukemic non-T/non-B lymphod cells from three cell lines (NALL-1, NALM-6 and NALM-16) possess a strong stimulating capacity in "one-way" mixed lymphocyte reaction (MLR-S), while leukemic cells from two non-T/non-B cell lines (REH and KM-3) possess no MLR-S. It is speculated that leukemic non-T/non-B lymphoid cells with MLR-S may represent less differentiated leukemic B cells and leukemic non-T/B lymphoid cells without MLR-S may represent less differentiated leukemic T cells. The REH or KM-3 cells without MLR-S also act as suppressor cells on normal T lymphocyte response to mitogen and allogeneic cells by secreting a potent suppressor activity. The MOLT-4 leukemic T lymphoid cells with no MLR-S, on the other hand, do not act as suppressor cells on T lymphocyte response. The soluble factor(s) secreted by the REH or KM-3 cell line is non-toxic to T lymphocytes and heat-sensitive. A significant suppression of T lymphocyte response is still observed, even when the active material is only present for one hour prior to the addition of PHA or it is added several days after the beginning of cultures. The biological and physico-chemical nature of this active material has not been defined. Further studies are currently in progress for biological and physico-chemical characterization and isolation of the active material.


Characteristics of peritoneal lymphocytes from New Zealand Black and normal mice autoreactive for mouse erythrocytes. New Zealand Black mice spontaneously develop autoimmune hemolytic anemia. Peritoneal cavity lymphocytes from NZB mice include a high proportion of cells that bind autologous erythrocytes. These cells produce ani-erythrocyte antibody that can be demonstrated when target erythrocytes are treated with bromelin. Such autoreactive cells are also present in normal mice, though in much smaller numbers. Aging and sex influence the peritoneal anti-erythrocyte response to bromelin-treated mouse erythrocytes. Older mice generally produce more rosettes and plaques than younger animals, and females have higher responses than males. During short-term culture in the absence of antigen, the response to bromelin-treated mouse erythrocytes increases. By contrast, the response to untreated mouse erythrocytes decreases during the same culture conditions. The in vitro response to bromelin-treated mouse erythrocytes by New Zealand Black peritoneal lymphocytes is suppressed by a cell that binds to untreated erythrocytes. The enhanced response to bromelin-treated mouse erythrocytes in culture may reflect the loss of this suppressor cell.


Unusual modification of bacteriophage Mu DNA. Bacteriophage Mu DNA was labeled after induction in the presence of [2-(3)H]adenine or [8-(3)H]adenine. Both Mu mom(+).dam(+) DNA and Mu mom(-).dam(+) DNA have similar N(6)-methyladenine (MeAde) contents, as well as similar frequencies of MeAde nearest neighbors. Both DNAs are sensitive to in vitro cleavage by R.DpnI but resistant to cleavage by R.DpnII. These results indicate that the mom(+) protein does not alter the sequence specificity of the host dam(+) methylase to produce MeAde at new sites. However, we have discovered a new modified base, denoted A(x), in Mu mom(+).dam(+) DNA; approximately 15% of the adenine residues are modified to A(x). Although the precise nature of the modification is not yet defined, analysis by electrophoresis and chromatography indicates that the N(6)-amino group is not the site of modification, and that the added moiety contains a free carboxyl group. A(x) is not present in Mu mom(+).dam(+) or Mu mom(-).dam(+) phage DNA or in cellular DNA from uninduced Mu mom(+).dam(+) lysogens. These results suggest that expression of the dam(+) and mom(+) genes are required for the A(x) modification and that this modification is responsible for protecting Mu DNA against certain restriction nucleases. Mu mom(+).dam(-) DNA and Mu mom(-).dam(-) DNA contain a very low level of MeAde (ca. 1 MeAde per 5,000 adenine residues). Since the only nearest neighbor to MeAde appears to be cytosine, we suggest that the methylated sequence is 5'... C-A(*)-C... 3' and that this methylation is mediated by the EcoK modification enzyme.


Electrophoretic characterization of intracellular forms of bacteriophage phi X174 DNA: identification of novel intermediate of altered superhelix density. The replication cycle of bacteriophage phi X174 DNA has been analyzed by agarose gel electrophoresis. The electrophoretic behavior of the predominant species of parental and progeny DNA molecules formed between 5 and40 min after infection was deduced and quantitated. Migration through 1.4% agarose at 5 and 10 V/cm resolved all known viral DNA species as well as fragments of host chromosomal DNA. Among parental replicative form(RF) molecules synthesized, 1 to 3% were full length linear duplexes (RFIII) and approximately 65% were closed circular duplexes (RFI). Most of the input viral strands remained in a duplex structure throughout the period of infection studied here. Among progeny molecules, RFIII was not readily detected unless viral DNA synthesis was inhibited by chloramphenicol. Late in infection, 20% of the progeny RF were found to exist as form I dna. in addition, approximately 1% of the viral DNA was found as unit length linear single strands. Electrophoretic analysis of RF DNA after controlled denaturation suggests the existence of four populations of closed circular RF: (i) molecules of native superhelix density (RFI); (ii) a population of molecules of altered topological linking number, alpha, differing in increments of one superhelical turn (tau) between tau values of 0 and approximately -31; (iii) a superimposed population of topological isomers which under electrophoresis conditions have mean tau value (tau) equal to +5; and (iv) a population of "complexed" molecules with a reduced number of superhelical turns due to their association with single-stranded DNA and RNA. Complexed parental molecules isolated from cells infected at high multiplicity released FRI and homologous single-stranded DNA upon denaturation and are postulated to be intermediates in genetic recombination. Complexed RF DNA isolated from cells infected at low multiplicity release native supercoils upon reaction with RNase H and are observed by electron microscopy to contain displacement loops. Such molecules are likely intermediates in transcription. Our results are consistent with a structure of complexed RFI involving a partially triple-stranded helix in which a covalently closed circular duplex molecule contains a reduced number of superhelical turns due to the unwinding produced by base pairing between one strand of the supercoil and an associated homologous single strand of DNA or RNA.


Sacral signal tracing: the electrophysiology of the bulbocavernosus reflex. We studied 52 men with electrophysiologic testing of the bulbocavernosus reflex to determine reflex latency. The bulbocavernosus reflex was demonstrated to be a segmental polysynaptic reflex with cross-over in the sacral spinal cord. Excellent correlation was obtained between prolonged latency and clinical evidence of neuropathy. The examination may be useful clinically to evaluate patients suspected to have sacral cord lesions or pudendal neuropathy (for example impotence, chronic back pain and lesions of the cauda equina and peripheral nerves).


The flow rate nomogram: I. Development.

The flow rate nomogram: I. Development. In normal individuals the flow rate depends on the initial bladder volume in a non-linear fashion. A flow rate nomogram taking this relationship into account was developed as an aid in the interpretation of urinary flow rate data. With this approach excellent differentiation of normal from obstructed individuals was achieved. In addition, the variability in a single individual's flow rate over time was estimated to be relatively small and, thus, uroflowmetry may be used to identify changes in outflow resistance after medical or surgical therapy.


Angiocardiograms and hemodynamics in idiopathic cardiomyopathy, with reference to histology of biopsied ventricular myocardium. Cardiac catheterization, angiocardiography and ventricular muscle biopsy were performed in forty patients with idiopathic cardiomyopathy and included 21 cases of hypertrophic cardiomyopathy and 19 cases of congestive cardiomyopathy. 1) Cardiac catheterization revealed normal cardiac index and stroke index in both types, although there was a slight tendency toward decrease in cases of CCM. HCM showed slightly elevated right ventricular end-diastolic pressure and left ventricular end-diastolic pressure with a high incidence of atrial kick. CCM showed an elevated mean pulmonary artery, mean pulmonary wedge and left ventricular end-diastolic pressure. 2) Angiocardiographic findings revealed that in HCM left ventricular end-diastolic volume as well as left ventricular end-systolic volume, ejection fraction, meanVcf and MNSER were within normal range, and left ventricular anterior wall thickness, left ventricular mass and shortening of short axis in systole were increased. In CCM left ventricular end-diastolic volume and end-systolic volume increased, and ejection fraction, meanVcf, MNSER were decreased. The left ventricular anterior wall thickness was normal, and the left ventricular mass was smaller compared to the volume. The shortening of long and short axes in systole was slight. Left ventricular asynergy and mitral regurgitation occurred frequently. Coronary cineangiograms revealed normal patterns in both types. 3) Histological findings revealed hypertrophy of myofibers, degenerative changes, i.e. scarcity of myofibrils, deformity of nucleus and vacuolization of myocardial fibers, and collagen proliferation in both types. 4) No definite relationship was seen between parameters of left ventricular function and the findings of biopsied left ventricular muscle except for increase in wall thickness which might be apparently due to hypertrophy of the myocardial fibers.


Immunologic abnormalities in melanoma-prone families. Sixty members of 4 families prone to cutaneous malignant melanoma (CMM) and a genetically determined precursor nevus syndrome underwent extensive immunologic evaluation. The most consistent finding was a diminished in vitro response to pooled alloantigens in the one-way mixed leukocyte culture (MLC) and a tendency to low T-lymphocyte and B-lymphocyte levels. When compared to controls, low B-lymphocyte levels and reduced MLC responses were found not only in family members with CMM and/or precursor nevi but also in unaffected blood relatives and spouses. The genesis of the immune dysfunction and its possible relationship to melanoma pathogenesis remain to be clarified.


Deposition of immune complexes in the ovarian follicle of rabbits with experimental chronic serum sickness. I. Immunopathology. In the present study, deposition of antigen-antibody complexes in the ovarian follicles of the rabbit is described. Forty-one rabbits were immunized with multiple daily injections of bovine serum albumin. Twenty-two rabbits developed systemic chronic serum sickness. The ovaries of rabbits with systemic chronic serum sickness, those of the immunized rabbits that did not develop systemic chronic serum sickness, and those of the nonimmunized rabbits were studied by light, electron, and immunofluorescence microscopy. It was found that granular deposits of bovine serum albumin, rabbit IgG, and C3, presumably as antigen-antibody complexes, were frequently present in the zona pellucida of secondary and tertiary follicles, and in the corpora atretica of rabbits with systemic chronic serum sickness. The oocytes showed an increased number of vacuoles and phagosomes containing electron-opaque material. These observations may contribute to the study of immunologic mechanisms in the pathophysiology of the female reproductive system.


Relational meaning encoded in the two-word utterances of stage 1 Down's syndrome children. This study explored the early two-word utterances from four Down's syndrome children to determine if they encode the same relational meanings as children developing language normally. Nine semantic categories were established to classify the subject's two-word constructions. Absolute and proportional frequencies of relational types were then used to analyze the relational meanings. The results reveal that this classification system accounts for a combined 79% of the two-word utterances expressed by these Down's subjects. It is suggested that Down's children demonstrate as much diversity in their use of relational meanings as normals at the same linguistic stage. These findings are discussed with respect to what Down's children know about the world as they begin to produce two-word combinations.


Experience with the extracardiac conduit. A review is made of all Mayo Clinic cases wherein 468 patients have received 516 extracardiac conduits in the repair of congenital heart defects. All patients had complex defects, which are classified in 10 basic diagnostic categories. The early mortality rate (which ranged from 4% to 49%, according to diagnostic group) averaged 25%, improving with experience. The postoperative complication rate was 70%. In 1% of operations, compression of the conduit was encountered at chest closure. The conduits placed in 333 patients contained a porcine valve. The conduit diameter averaged 22 mm. At completion of the operation, the mean gradient across the conduit was 22.8 mm Hg. At late study this mean gradient remained at 26 mm Hg in patients who had received the currently employed porcine-valved conduit. The average ratio of intraoperative postrepair ventricular (RV/LV) systolic pressures was 0.68. It was lower among the group who were to survive but was not ov prognostic value in individual cases. Among patients who survived the postoperative period, the late mortality rate (based on a mean of 3.6 years' follow-up) averaged 3.5% per year. Of the long-term survivors, 18% have required reoperation, primarily for replacement of calcified aortic homograft conduits used in the earlier part of the experience. Only 0.8% of porcine-valved conduits have required reoperation. Unrestricted life-styles were possible for 90% of survivors.


Changes in membrane structure and function during chick embryonic erythropoiesis. We found considerable differences in the pattern of membrane proteins as well as in the relative amounts of individual components in isolated chick red blood cell membranes during the course of embryonic development. Of special interest in the increase in the relative amounts of two major polypeptides, band 3 and 3.1 (MW 100,000 daltons) with increasing age of the cells. With respect to functional studies, we found that the magnitude of sulfate influx decreases with increasing age of the embryo. Furthermore, the activity of ouabain-sensitive ATPase increases with increasing age (2.5-day embryo to adult). In addition, both the basal and the fluoride-stimulated adenylate cyclase activities decrease as the embryo age increases, whereas the enzyme sensitivity to epinephrine increases with increasing age of the embryo.


Delayed-type hypersensitivity (DTH) in BCG-sensitized mice I. Lack of suppressor T cell activity on DTH to sheep red blood cells. Mice pretreated with an intravenous (i.v.) injection of BCG (BCG-sensitized mice) and then immunized intravenously with a high dose (10(8)--10(9)) of sheep red blood cells (SRBC) 2 weeks later developed strong delayed-type hypersensitivity (DTH) to SRBC, as in mice pretreated with cyclophosphamide (CY) (CY-treated mice) and then immunized with SRBC 2 days later; normal mice given the same dose of SRBC did not show such DTH. The mechanism of this strong DTH to SRBC which developed in BCG-sensitized mice was studied, by comparing it with that in CY-treated mice. The transfer of either whole spleen cells or thymus cells, but not serum, obtained from mice immunized with i.v. injections of 10(9) SRBC 4 days previously (hyperimmune mice) did not suppress either the induction or the expression of DTH to SRBC in BCG-sensitized mice, but suppressed those in CY-treated mice. The suppressor cells were SRBC-specific T cells. Adoptive transfer of DTH to SRBC by spleen cells from either BCG-sensitized mice of CY-treated mice to hyperimmune recipients failed. The adoptive transfer of DTH from BCG-sensitized mice to normal recipients also failed if the spleen cells from hyperimmune mice were cotransferred. Whole body irradiation (600 rad) of mice 2 hr before or after the time of immunization with SRBC reduced significantly DTH to SRBC in both BCG-sensitized and CY-treated mice. It was noticed that the total number of spleen cells in BCG-sensitized mice was 3--4 times larger than that in CY-treated mice. From these results, we conclude that the entity of effector T cells of DTH to SRBC induced in BCG-sensitized mice and in CY-treated mice was not different in terms of susceptibility to suppressor T cells and irradiation, but that the total numbers of effector T cells generated in these mice differed remarkably, resulting in the above-described different responsiveness to suppressor T cells transferred passively.


Down's syndrome, hypothyroidism and diabetes mellitus in an adult. Primary hypothyroidism was diagnosed in a woman aged 33 years, with Down's syndrome and insulin-dependent diabetes mellitus after admission to hospital with hypoglycaemia. The diabetes was stabilized and treatment was commenced with L-thyroxine with a good clinical response. The patient's mother and older sister also had hypothyroidism and all three had no thyroid antibodies. The importance of diagnosing and treating primary hypothyroidism in adult patients with Down's syndrome is stressed even in the absence of thyroid antibodies.


[Electron microscopic study of the pond microflora of the Amu-Dar'ia River delta]. Bacterial forms were studied by electron microscopy in weakly saline lakes of the mesotrophic type in the Amu Darya delta; these forms differed in the character of water nutrition, certain physico-chemical and biological properties. The main population in the lakes represented by Vibrio and rods among which many small forms (0.3--0.5 mu) were found. Long filamentous forms were often encountered. A considerable number of organisms possessed prosthecae, fimbria and various protrustions. Budding bacteria were also detected. Microorganisms with peculiar morphology were most abundant in such places where the total incidence of bacteria was 1x10(6) per litre while water transparency was low.


[Comparative morphological studies of the structure of the articular disk of the temporomandibular joint]. Articular disks (DA) of mandibular articulations of several types of animals of different ages and both sexes (herbivora, carnivora, rodents and omnivora) were studied. Comparative macroscopy emphasized similarities with the DA of humans and other omnivora, as well as some peculiarities. DA is ellyptical in herbivora; its upper surface is divided into two facets by a transversal crest. DA is pinkish and square in carnivora; its posterior part is lacking. DA is half-moonish and white-nacreous in rodents; its upper surface presents a longitudinal crest. Microscopy evidenced some differences: the collagen fibrils are dominating in herbivora, while in carnivora the elastic ones. The rodents present rare elastic fibers; these animals and the ruminants show capillaries in the central part of the DA, while in other animals these are absent. Cells are mainly located in the upper superficial layers of DA. Relationships between form, histological structure and functions, mainly regarding the mastication types, are discussed.


[The ultrastructure of embryonic capillaries in the neuroepithelium of the brain vesicles]. The electronmicroscopic control of the capillaries of the brain vesicles in 5--6-day-old chick embryos revealed an intimate contact between capillary walls and neuroblasts, and on the other hand the presence of a functional endoplasmic reticulum in the endothelial cells. These are only very few vacuoles for protein transport in the cytoplasm of endothelial cells.


Late Eocene of Burma yields earliest anthropoid primate, Pondaungia cotteri. In April 1978, a fragment of a primate lower jaw containing the second and third molar teeth was found in late Eocene exposures of the Pondaung Hills about 1 mile north-west of Mogaung village in northwestern Central Burma. This approximately 40-Myr-old specimen is the first fossil primate found in Burma since the fragmentary remains of the controversial earliest anthropoids Pondaungia cotteri Pilgrim and Amphipithecus mogaungensis Colbert were recovered more than 50 yr ago. The jaw described here is believed to represent further evidence of P. cotteri. Its recovery from undoubled late Eocene exposures coupled with its salient higher primate characters and excellent state of preservation provides the opportunity to substantiate further that the Pondaung primates of Burma are the earliest known record of the Anthropoidea.


Knowledge and technology needed for further development of mental health programmes. Different preventive approaches of mental and neurological disorders are discussed. Special emphasis has been put on the advances of biological research in the elucidation of aetiological and pathogenic mechanisms of major psychoses, such as schizophrenia and affective disorders. Perspectives of genetic and psychopharmacological research are emphasized. Multidisciplinary efforts are needed for the improvement of existing preventive measures.


Familial inverted choreoathetosis.

Familial inverted choreoathetosis. We present a family with infantile onset of progressive choreoathetosis. The movements affected the legs predominantly and also impaired gait. No dementia, seizures, or rigidity was noted. Inheritance was autosomal dominant. This new familial movement disorder can be differentiated easily from other hereditary choreas.


Morphology and physiology of skeletal muscle in aging rodents. Aging in the rodent is accompanied by a progressive loss of skeletal muscle fibers. The muscle twitch also becomes slower, probably as a result of fiber-type conversion from "fast-twitch" to "slow-twitch." The loss of muscle fibers precedes signs of obvious weakness and appears to involve the loss of entire functioning motor units. In the mouse, this loss of motor units correlated with a loss of axons from the innervating nerve trunks.


[Closed abdominal injuries in patients with multiple injuries. Diagnostic considerations on 74 cases]. A diagnostic assessment was made of 74 cases of non-penetrating abdominal wounds observed over the previous 5 years. Laparoscopy performed in 49 polytraumatised patients made a decisive contribution to accurate, early diagnosis, with an indication for emergency surgery in 35 cases (32 visceral lesions and 3 retroperitoneal haematoma), whereas it was clear from the negative finding that surgery was not required in the remaining 14. It is felt that all additional radiological and instrumental diagnostic should be employed, and that careful clinical examination and repeated inspection, preferably by the same surgeon, are important in cases of multiple injury, since the situation may become dramatically worse in the space of a few hours.


Are the endorphins active in clinical pain states? Narcotic antagonism in chronic pain patients. To test the possibility of endorphin release in clinical pain states naloxone was given, alternate with saline, in a double-blind study to 10 patients with chronic neuralgia or low back pain. There was no significant alteration of the levels of spontaneous pain or heat pain thresholds. The results suggest that the endorphin system does not offer protection of any importance in chronic pain.


Bone marrow transplantation (1958-1978) : conditioning and graft-versus-host disease, indications in aplasias and leukemias. Bone marrow transplantation (BMT), which stimulated great hope for treatment of aplasias and leukemias in 1958 following our first success in grafting this tissue, is, after a long period of study and development, experiencing renewed interest since it is now possible to obtain, in case of transplantation with genotypically matched sibling donors, 70% long survival (cures ?) in aplasia (under the condition that the recipient is not sensitized by previous transfusions) and in leukemia (under the condition that the recipient is transplanted in a period of remission and is not sensitized by transfusions). When the patient does not possess any genotypically matched donor, a trial of incompatible bone marrow transplantation after conditioning with antilymphocyte serum is reasonable, since we have obtained good, although unexplained, results with this method, which should be pursued. In any case, these transplants must be done in intensive care units in hemato-oncology departments.


Cellular (T cell) immunity in the human newborn. The cellular immune system of the human newborn, like the rest of the immunologic apparatus, is anatomically intact, antigenically inexperienced, and functionally deficient. The latter is suggested by the newborns' enhanced susceptibility to infection, diminished delayed cutaneous hypersensitivity reactions, and selective abnormalities (when compared to adults) of measures of cellular immunity in vitro. These include impaired proliferative response to ubiquitous antigens, depressed lymphotoxin, migration inhibition factor, and immune interferon production, and diminished cytotoxic reactions including cell-mediated lympholysis. By contrast, other aspects of neonatal T cell function, such as to mitogens or allogeneic lymphocytes, natural interferon and leukocyte inhibition factor production, and number and percentage of E-rosette-forming cell are generally normal. These decreased functional properties may provide an explanation for the newborns' susceptibility to infection and for the occasional occurrence of engraftment of foreign cells from either the mother or from prenatal or neonatal blood transfusion.


Isolation and identification of bacteriophage phi X174 prohead. The morphogenesis of bacteriophage phi X174 has been investigated by using an in vitro DNA synthesizing system. An extract of a B-mutant-infected cell is capable of synthesizing infectious phage in vitro when the B gene function is provided by the addition of an ammonium sulfate fraction of a C-mutant-infected-cell extract. This fraction contains the omega complex, a complex of phage-coded proteins with S = 108; the B-mutant extract does not. The purified omega complex, isolated from the C-mutant extract, caused the synthesis and encapsidation of viral DNA when added to B-mutant extract. The omega complex contains the B protein but it is the intact omega complex that functioned in the in vitro complementation of the B-mutant extract because other fractions containing B protein but no omega complex had little or no complementing activity. The results indicate that the omega complex is the phi X174 prohead. The B protein is not found in either the 132S or 114S phage particles but is an essential component of the prohead. This suggests that it may have a scaffolding function.


Fidelity of replication of phage phi X174 DNA by DNA polymerase III holoenzyme: spontaneous mutation by misincorporation. DNA from phi X174 is replicated in vitro with a fidelity similar to that found genetically. A mutation of TAG leads to TGG may be induced, however, by varying the concentrations of deoxynucleoside triphosphates, with a frequency proportional to [dGTP]2/[dATP]. This complex concentration dependence is consistent with the active participation of a proofreading mechanism that hydrolytically excises mismatched base pairs as they are formed. A simple kinetic analysis predicts that the frequency of misincorporation depends on the ratio of incorrect to correct deoxynucleoside triphosphates times the concentration of the next triphosphate in the sequence to be added. This suggests that spontaneous mutation by misincorporation depends crucially on the composition of the deoxynucleoside triphosphate pool.


Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1. Studies with reagents that crosslink two thiol groups have shown that it is possible to trap nucleotides at the active site of myosin chymotryptic subfragment 1. Subfragment 1 incorporates nearly stoichiometric quantities of [14C]ATP or [14C]ADP in a manner that depends linearly on the extent of inactivation by either N,N'-p-phenylenedimaleimide or Co(II)phenanthroline/[Co(III)(phenanthroline)2CO3]+ complexes. The incorporated radioactive nucleotide is retained after gel filtration, even when the enzyme derivatives are stored in the presence of EDTA or nonradioactive nucleotides (t 1/2 approximately 5 days). The nucleotide incorporated is not covalently bound because HClO4 denaturation allows immediate release of bound nucleotide. The nucleotide retained is ADP because the gamma-phosphate of [gamma-32P]ATP is lost after trapping. Subfragment 1 inactivated as above does not bind the competitive inhibitor adenosine 5'-[beta, gamma-imido]triphosphate, indicating that the active site is blocked. It is proposed that a jawlike nucleotide cleft closes on MgADP or MgATP, which can be locked shut by crosslinking two thiol groups by reaction with N,N'-p-phenylenedimaleimide or cobalt phenanthroline complexes.


Interactions between DNA-bound repressors govern regulation by the lambda phage repressor. The lambda phage repressor binds cooperatively to the three sites in the right operator (O(R)) according to the following pattern. If the DNA is wild type, O(R)1 and O(R)2 are filled coordinately because of interactions between repressor dimers bound to these two sites. Site O(R)3 is filled only at higher repressor concentrations. In contrast, if O(R)1 is mutant, O(R)2 and O(R)3 are filled coordinately because of interactions between repressors bound to these sites. In this case, the affinity of O(R)3 is increased and that of O(R)2 is decreased relative to the wild type. We infer that a repressor dimer bound to the middle site O(R)2 can interact either with another repressor dimer bound to O(R)1 (wild-type case) or, alternatively, with one bound to O(R)3 (mutant O(R)1 case). We argue that these repressor interactions are mediated by protein-protein contacts between adjacent repressor dimers, because the isolated amino-terminal domains of repressor bind to the operator sites noncooperatively. The cro protein of phage lambda, a second regulatory protein, which recognizes the same three sites in O(R) as does repressor, binds non-cooperatively. Experiments performed in vivo show that regulation of gene expression by repressor can be influenced critically by cooperative interactions. We demonstrate that the effect of repressor in a lysogen on the activity of the promoter P(RM) can be changed from activation to repression by deletion of O(R)1. We explain this effect in terms of the alternative cooperative interactions described above.


Homologous pairing in genetic recombination: complexes of recA protein and DNA. recA protein, which is essential for general genetic recombination in Escherichia coli, promotes the homologous pairing of single-stranded DNA with double-stranded DNA to form a D loop. The amount of recA protein required for the reaction was directly proportional to the amount of single stranded DNA and was unaffected by similar variations in the amount of double-stranded DNA. The ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), which was not rapidly hydrolyzed by recA protein, blocked the formation of D loops but promoted the formation of stable complexes of recA protein and single-stranded DNA. These complexes, in turn, bound homologous or heterologous double-stranded DNA and partially unwound it. Because ATP gamma S competitively inhibited the ATPase activity of recA protein (Km/Ki approximately 300), we infer that ATP gamma S binds at a site that overlaps the site for ATP and that the functional complexes formed in the presence of the analog probably represent partial steps in the overall reaction. If the complexes formed in the presence of ATP gamma S reflect natural intermediates in the formation of D loops, recA protein must promote homologous pairing either by moving juxtaposed single-stranded and double-stranded DNA relative to one another or by forming and dissociating complexes reiteratively until a homologous match occurs.


Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. An efficient transformation system has been developed for Neurospora crassa that uses spheroplasts and pVK88 plasmid DNA. pVK88 is a recombinant Escherichia coli plasmid carrying the N. crassa qa-2(+) gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) and is part of the qa gene cluster. The recipient strain carries a stable qa-2(-) mutation and an arom-9(-) mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were selected as colonies able to grow in the absence of an aromatic amino acid supplement. These colonies were qa-2(+) and had normal levels of catabolic dehydroquinase. DNA.DNA hybridization evidence with appropriate labeled probes indicates clearly that in some instances transformation involves the integration of bacterial plasmid sequences together with the qa-2(+) gene into the N. crassa genome. On the basis of genetic, enzyme assay, and DNA hybridization data, at least three types of transformation events can be distinguished: (i) replacement of the qa-2(-) gene by the qa-2(+) gene without any effect on the expression of the other genes in the qa cluster, (ii) linked insertion of a normal qa-2(+) gene accompanied by inactivation of the adjacent qa-4(+) gene, and (iii) insertion of a normal qa-2(+) gene at an unlinked site in the N. crassa genome. This newly integrated qa-2(+) genetic material is inherited in a typical Mendelian fashion. A low level of transformation has also been obtained by using linear total N. crassa DNA. Two such qa-2(+) transformants are unlinked to the qa-2(-) gene of the recipient.


Cloning of the structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12. The gene (ompA) for the major outer membrane protein II* from Escherichia coli K-12 has been cloned on a 5-megadalton EcoRI fragment by using phage lambda as vector. The gene is expressed during the lytic cycle of the recombinant phage and the insoluble membrane-bound protein was detected in phage plaques with a simple radioimmunoassay. Transfer of the EcoRI fragment into plasmid pSC101 and expression in a host lacking protein II* led to overproduction of protein II* and decreased production of two other major outer membrane proteins. Expression of the plasmid pSC101-ompA+ in minicells derived from an ompA minicell-producing strain led to synthesis, at high rates, of this protein and massive accumulation of a second cell envelope protein most likely representing the biosynthetic precursor of protein II*.


Cloning of integrated Moloney sarcoma proviral DNA sequences in bacteriophage lambda. We have identified integrated proviral DNA sequences of m1 and HT-1 isolates of Moloney sarcoma virus (MuSV) in EcoRI digests of transformed mink cell genomic DNA and have cloned these fragments in bacteriophage lambda. Both the lambda-HT1 phage recombinant, containing a 12.3-kilobase MuSV pair (kb) fragment, and the lambda-m1 phage recombinant, containing a 7.0-kb fragment, possess full copies of the sarcoma viruses along with 5' and 3' host flanking sequences. The MuSV proviral DNA sequences, 6.7 kb for HT-1 and 5.2 kb for m1, are colinear by heteroduplex microscopy with the 1.5-kb difference in size accounted for by two approximately equal to 0.8-kb deleted regions in m1. Both integrated viral genomes are terminally redundant and have integrated at the same site in the provirus but at different sites on the host chromosome. The host sequence flanking integrated HT-1 MuSV have been identified as a single EcoRI restriction fragment of 5.6 kb in normal mink cells.


Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes. Glycosaminoglycans were isolated from purified fractions of glomerular basement membranes and partially characterized by chemical analysis and cellulose acetate electrophoresis. Basement membranes were prepared by detergent treatment of rat glomeruli and subjected to digestion with papain and Pronase. Glycosaminoglycans were isolated from the digests by precipitation with cetyl pyridinium chloride and ethanol. Results of cellulose acetate electrophoresis of the isolated glycosaminoglycan fraction revealed the presence of one major and one minor spot. The major spot was identified as heparan sulfate because it comigrated with the heparan sulfate standard and was sensitive to heparinase and to nitrous acid oxidation but insensitive to chondroitinase ABC and to testicular or leech hyaluronidase. The minor spot was tentatively identified as hyaluronic acid based on its migratory behavior and sensitivity to leech and testicular hyaluronidase. The chemical composition of the isolated glycosaminoglycan was typical of that of heparan sulfate (high carbazole/orcinol ratio, high sulfate content, absence of galactosamine). The data support and confirm the cytochemical data obtained previously [Kanwar, Y. S. & Farquhar, M. G. (1979) Proc. Natl. Acad. Sci. USA 76, 1303-1307] demonstrating that heparan sulfate is the only sulfated glycosaminoglycan detectable in the glomerular basement membrane. The present results suggest that in addition to sulfated glycosaminoglycan some nonsulfated glycosaminoglycan (hyaluronic acid) may also be present in the glomerular basement membrane.


Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. The lactose structural genes, without the lactose promoter, have been incorporated into the bacteriophage Mu genome to form a Mu-lac specialized transducing phage. This phage also carries a gene encoding resistance to ampicillin (Ap)[Mu(Ap, lac)]. After infection and upon establishment of lysogeny, the Mu(Ap, lac) genome can integrate into apparently random sites in the Escherichia coli chromosome. When integration occurs within a gene in the orientation of its transcription, the lactose structural genes are so situated that they become expressed solely from the promoter of that gene. Thus, expression of the lactose genes of Mu(Ap, lac) can be used as an assay for transcription of that gene and for functional and mutational studies of gene regulation.


Transcription promotes recA-independent recombination mediated by DNA-dependent RNA polymerase in Escherichia coli. The Rpo-mediated recombination of phage lambda takes place independently of the recA function and is promoted by DNA-dependent RNA polymerase of Escherichia coli [Ikeda, H. & Kobayashi, I. (1977) Proc. Natl. Acad Sci. USA 74, 3932--3936]. The crossovers were particularly frequent to the cIII-N and N-cII regions which are transcribed actively. To determine whether the transcription process required for the recombination is the initiation step or the chain elongation step, we have examined the effect of bacterial rho mutation, which affects transcription termination, on the distribution of crossover points in the lambda phage genome. The crossovers in the cII-S interval took place more frequently in rho mutant strains than in wild-type strains. Analysis of lambda mRNA showed that much more O-P-Q mRNA is synthesized in the rho mutant cells than in the wild-type cells and is largely produced by the readthrough from the PR promotor. These results strongly suggest that the chain elongation in transcription plays an essential role in this recombination. Physical analysis of the recombinant phage DNA showed that this recombination is a legitimate type. Models are presented to explain how the transcription complex can promote this recA-independent recombination.