Biomedical subjects

L Chao

Publications and source records attributed to L Chao.

At least 199 records · Page 11Linked to original sources

PubMed1987

Identification of tonin in brain and exocrine tissues and in the cell-free translation products encoded by the mRNA of these tissues.

Tissue-specific expression of the esteropeptidase tonin [EC 3.4.99.-] was investigated in rat brain, submandibular gland, pancreas and kidney. Specific polyclonal and monoclonal antibodies to purified rat tonin from the submandibular gland have been developed and characterized and have been purified via a tonin-agarose affinity column. Immunoreactive tonin was measured by a recently developed tonin direct radioimmunoassay using a rabbit tonin antiserum. Resulting tonin levels were found to be 105.27 +/- 2.71 micrograms/mg (of protein) in submandibular gland, 3.18 +/- 0.32 ng/mg in pancreas, 1.35 +/- 0.08 ng/mg in kidney and 0.12 +/- 0.01 ng/mg in brain (means +/- S.E.M.). Western-blot analysis shows that affinity-purified anti-tonin antibody binds to a 32,000-Mr protein from brain and submandibular-gland extracts. The protein, a tonin precursor, was identified from cell-free translation products directly by polyadenylated [Poly(A)+]mRNA species in a wheat-germ system. After the translation products were subjected to immunoprecipitation with affinity-purified tonin antibody, SDS/polyacrylamide-gel electrophoresis of these precipitates revealed two precursors of tonin, with Mr values of 30,000 and 29,000, which are encoded by brain and submandibular-gland mRNA; however, only the 30,000-Mr preprotonin was encoded by pancreas and kidney mRNA. Collectively, the data show that tonin exists in brain, submandibular gland, pancreas and kidney, and can be synthesized by the mRNA of these tissues.

Animals↗

PubMed1987

Identification and characterization of a tissue kallikrein in rat skeletal muscles.

A tissue kallikrein was purified from rat skeletal muscle. Characterization of the enzyme showed that it has alpha-N-tosyl-L-arginine methylesterase activity and releases kinin from purified bovine low-Mr kininogen substrate. The pH optimum (9.0) of its esterase activity and the profile of inhibition by serine-proteinase inhibitors are identical with those of purified RUK (rat urinary kallikrein). Skeletal-muscle kallikrein also behaved identically with urinary kallikrein in a radioimmunoassay using a polyclonal anti-RUK antiserum. On Western-blot analysis, rat muscle kallikrein was recognized by affinity-purified monoclonal anti-kallikrein antibody at a position similar to that of RUK (Mr 38,000). Immunoreactive-kallikrein levels were measured in skeletal muscles which have different fibre types. The soleus, a slow-contracting muscle with high mitochondrial oxidative-enzyme activity, had higher kallikrein content than did the extensor digitorum longus or gastrocnemius, both fast-contracting muscles with low oxidative-enzyme activity. Streptozotocin-induced diabetes reduced muscle weights, but did not alter the level of kallikrein (pg/mg of protein) in skeletal muscle, suggesting that insulin is not a regulator of kallikrein in this tissue. Although the role of kallikrein in skeletal muscle is unknown, its localization and activity in relation to muscle functions and disease can now be studied.

Animals↗

PubMed1987

Tissue kallikrein in rat brain and pituitary: regional distribution and estrogen induction in the anterior pituitary.

We have detected tissue kallikrein and kallikrein mRNA in various brain regions with a kallikrein direct RIA and with nucleic acid hybridization using a kallikrein cDNA probe. In the direct RIA, rat urinary kallikrein-like activity was found in the pituitary and pineal glands, hypothalamus, cerebral cortex, cerebellum, and brain stem. Pituitary and pineal gland kallikrein concentrations were significantly higher than those in other regions. Only in pituitary was there a significant difference in tissue kallikrein concentration according to sex, with glands from female rats showing levels 4-fold higher than those from male rats. Kallikrein mRNAs were detected in all of the regions and were about 4-fold higher in female than in male pituitary gland. Northern blot analyses show sex dimorphism of pituitary kallikrein mRNA, similar in size to submandibular gland and kidney mRNA. In castrated male rats, whole pituitary kallikrein content was reduced to 50% of the control value and increased 1.7-fold with testosterone replacement and 18-fold with 17 beta-estradiol treatment. Neither T4 nor cortisol affected whole pituitary kallikrein levels in the castrated male rat, but testosterone decreased pituitary kallikrein in normal female rats by 35%. When anterior pituitary or neurointermediate lobe extracts were separately examined, immunoreactive kallikrein was 10.2- and 1.3-fold higher respectively, in female than in male rat lobes. Estradiol benzoate (30 micrograms/kg) administration increased kallikrein levels 90- and 22-fold, respectively, in the anterior pituitary of gonadectomized male and female rats, while it increased by only 40-50% kallikrein levels in the male and female neurointermediate lobe. In dot blot analysis, kallikrein mRNA levels were increased 5-fold by 17 beta-estradiol in the whole pituitary of castrated male rats. In the cytoplasmic dot hybridization analysis, estradiol benzoate treatment increased kallikrein mRNA levels 54-fold in the anterior pituitary of ovariectomized rats. The data show that a tissue kallikrein indistinguishable thus far from a urinary kallikrein is widely distributed in brain and pituitary and that levels of enzyme and mRNA are comparable in certain central sites. Kallikrein levels in the anterior and neurointermediate pituitaries are differentially regulated by estrogen.

Animals↗

PubMed1987

Sperm and seminal plasma antigens from autoimmune men induce immunological infertility.

Adult male rabbits were immunized with normal saline (controls), sperm extracts from 2 autoimmune men, seminal plasmas from the same autoimmune men, sperm extract from a fertile nonautoimmune man, and seminal plasma from the same fertile nonautoimmune man. All the sperm donors were free from infections. Rabbits immunized with fertile men's sperm extract and seminal plasma had significantly elevated postimmunization hemagglutinating but not cytotoxic sperm antibodies and reproduced normally. Rabbits immunized with autoimmune men's sperm and seminal plasma antigens developed high titers of cytotoxic and hemagglutinating sperm antibodies in their serum and seminal plasma and their reproduction was markedly reduced. Their sera and seminal plasma reduced motility of sperm from a normal donor. The immune responses were confirmed by electron microscopic immunocytochemistry. This technique revealed membrane-bound endogenous IgG on sperm from only those rabbits immunized with sperm extracts from autoimmune men. These antisera reacted against a protein in the 58,000 D range; antisera to fertile man's sperm extract reacted against three proteins with molecular weights of 15,000, 18,400, 25,000, and 44,000 D, as judged by Western blot. Rabbit antisera to seminal plasma from autoimmune men reacted against several proteins; additionally, it detected two proteins with 43,000 and 68,000 molecular weight detected by antiserum to fertile man's seminal plasma. Sperm and seminal plasma antigens from autoimmune men are different in their elicited immunogenic responses from those of fertile nonautoimmune men. These responses are relevant to infertility.

Animals↗

PubMed1986

Identification of a new tissue-kallikrein-binding protein.

We have identified a tissue-kallikrein-binding protein in human serum and in the serum-free culture media from human lung fibroblasts (WI-38) and rodent neuroblastoma X glioma hybrid cells (NG108-15). Purified and 125I-labelled tissue kallikrein and human serum form an approximately 92,000-Mr SDS-stable complex. The relative quantity of this complex-formation is measured by densitometric scanning of autoradiograms. Complex-formation between tissue kallikrein and the serum binding protein was time-dependent and detectable after 5 min incubation at 37 degrees C, with half-maximal binding at 28 min. Binding of 125I-kallikrein to kallikrein-binding protein is temperature-dependent and can be inhibited by heparin or excess unlabelled tissue kallikrein but not by plasma kallikrein, collagenase, thrombin, urokinase, alpha 1-antitrypsin or kininogens. The kallikrein-binding protein is acid- and heat-labile, as pretreatment of sera at pH 3.0 or at 60 degrees C for 30 min diminishes complex-formation. However, the formed complexes are stable to acid or 1 M-hydroxylamine treatment and can only be partially dissociated with 10 mM-NaOH. When kallikrein was inhibited by the active-site-labelling reagents phenylmethanesulphonyl fluoride or D-Phe-D-Phe-L-Arg-CH2Cl no complex-formation was observed. An endogenous approximately 92,000-Mr kallikrein-kallikrein-binding protein complex was isolated from normal human serum by using a human tissue kallikrein-agarose affinity column. These complexes were recognized by anti-(human tissue kallikrein) antibodies, but not by anti-alpha 1-antitrypsin serum, in Western-blot analyses. The results show that the kallikrein-binding protein is distinct from alpha 1-antitrypsin and is not identifiable with any of the well-characterized plasma proteinase inhibitors such as alpha 2-macroglobulin, inter-alpha-trypsin inhibitor, C1-inactivator or antithrombin III. The functional role of this kallikrein-binding protein and its impact on kallikrein activity or metabolism in vivo remain to be investigated.

Carrier Proteins↗

PubMed1986

Age and hormonal dependence of tonin levels in rat submandibular gland as determined by a new direct radioimmunoassay.

A simple and sensitive direct radioimmunoassay for tonin (EC.3.4.99.-) has been developed. This assay incorporates a modified and convenient poly(ethylene glycol) technique for separation of free from bound tonin. A rabbit antiserum in a final dilution of 1:160,000 was used and the purified tonin was labelled with 125I by using a lactoperoxidase method. It detects 20 pg of immunoreactive tonin per tube. Serial dilutions of rat submandibular gland extracts showed complete parallelism with tonin standard curves. No cross-reactivity with rat tissue kallikrein was seen. Intra- and inter-assay errors were 3.2 and 5.6%, respectively. Using this assay, immunoreactive tonin was detected in the rat submandibular gland as early as 3 weeks after birth (body wt. approximately 50-60 g). Tonin levels are shown to be dependent on age and sex with significantly higher levels in male than in female rats. Castration results in decrease of tonin levels and 17 alpha-methyltestosterone replacement reversed the level to higher than the sham-operated control rats. Cortisol treatment increased, but thyroxine or oestradiol had no effect, on tonin levels in the submandibular gland of castrated rats. This newly developed radioimmunoassay can now be used to measure low levels of tonin in various tissues and body fluids to address questions about its regulation and functional significance.

Aging↗

PubMed1986

Sex dimorphism and hormonal regulation of rat tissue kallikrein mRNA.

The regulation of rat tissue kallikrein mRNA levels was investigated by RNA filter hybridization using a kallikrein cDNA probe and by in vitro translation of isolated RNA. The synthesis of a 37 kDa polypeptide has been hybrid-arrested by the tissue kallikrein cDNA in cell-free translation assays directed by submandibular mRNA and the quantitation of this protein was used as a measure of translational activity of kallikrein mRNA. In Northern blot analyses, relative kallikrein mRNA levels were found to be 60-fold higher in submandibular gland than in pancreas and 20-fold higher than in kidney. In addition, it was noted that kallikrein mRNA is differentially regulated in tissues from male and female animals. Rat submandibular gland kallikrein mRNA is 2-times more abundant in male animals as in females. This trend is reversed in kidney, where the kallikrein mRNA in females is twice that in male animals. Relative submandibular gland kallikrein mRNA levels were found to be responsive to hormonal manipulation as measured by both Northern blotting and cell-free translation assays. Castration of male animals resulted in a decrease of kallikrein mRNA which could be partially restored by treatment with thyroxine and almost completely restored by testosterone treatment. The submandibular gland kallikrein mRNA in normal female rats was increased up to 30% by administration of testosterone or thyroxine. Adrenalectomized female rats showed a decrease in submandibular kallikrein mRNA which was significantly increased by testosterone, thyroxine, aldosterone and cortisol, with the largest effect being a 2- to 2.4-fold increase in cortisol-treated rats. These results indicate that hormones regulate tissue kallikrein synthesis in the rat submandibular gland, at least in part, by altering kallikrein mRNA accumulation.

Adrenalectomy↗

PubMed1986

Immunological identification of rat tissue kallikrein cDNA and characterization of the kallikrein gene family.

A tissue kallikrein cDNA was identified by direct immunological screening with affinity-purified anti-rat tissue kallikrein antibody from a rat submandibular cDNA library constructed with the expression vector pUC8. Sequence analysis of the kallikrein cDNA revealed an encoded protein 97% homologous to the partial amino acid sequence of rat submandibular kallikrein. This cDNA was used to hybrid-select kallikrein-specific RNA from submandibular gland. Translation of the hybrid-selected RNA in a cell-free assay system resulted in the production of a 37 kDa peptide representing the preproenzyme. In addition, hybrid-selection of RNA under less stringent conditions showed cross-hybridization with other submandibular gland mRNA species. In correlation with these results, analysis of rat genomic DNA showed extensive hybridization, suggesting a family of closely related kallikrein-like genes. Consequently, a Charon 4A rat genomic library was screened for kallikrein genes by hybridization with rat tissue kallikrein cDNA. Thirty-four clones were isolated and found to be highly homologous by hybridization and restriction enzymes analyses. Fourteen unique clones were identified by restriction enzyme site polymorphisms within DNA segments which hybridized to the kallikrein cDNA probe and it was estimated that at least 17 different kallikrein-like genes are present in the rat. Sequence and structural analysis of one of the genomic clones revealed a gene structure similar to that of other serine proteinases. Comparison of the partially sequenced exon regions of the gene with the sequence of rat tissue kallikrein cDNA reveals 89% identity when aligned for the greatest homology. However, the genomic sequence predicts termination codons in all three translational reading frames, implying that this gene is nonfunctional, i.e., a pseudogene. Comparison of the rat genomic sequence to a kallikrein-like gene from the mouse reveals extensive preservation of exons, less identity within introns and no significant homology between extragenic regions.

Animals↗

PubMed1986

Active kallikrein, preprokallikrein, and kallikrein-inhibitor complex.

Active kallikreins isolated from various exocrine and endocrine tissues were identified by a monoclonal antibody in Western blot analyses to be approximately 38,000 dalton proteins. Kallikreins isolated from rat pancreas, kidney, submandibular gland, brain, spleen and urine were indistinguishable with respect to molecular weight and immunological characteristics. Preprokallikreins were synthesized in a cell-free translation system directed by mRNAs and immunoprecipitated by affinity-purified kallikrein antibody. Analysis of the precipitates by SDS-polyacrylamide gel electrophoresis revealed a approximately 37,000 dalton polypeptide in kidney, brain and submandibular gland translation products. This 37,000 dalton kallikrein precursor was hybrid-arrested by a kallikrein cDNA encoding tissue kallikrein which was isolated from a rat submandibular gland cDNA library. The immunoprecipitates of products directed by pancreatic mRNA showed a major protein with Mr of approximately 30,000. An endogenous approximately 92,000 dalton component in rat urine and kidney was also identified by a monoclonal antibody to tissue kallikrein and represents a kallikrein-inhibitor complex. These results indicate that tissue kallikreins can be initially synthesized as 37,000 or 30,000 dalton prepropeptides and then converted into a 38,000 dalton active form by proteolytic processing and glycosylation. The active kallikrein is capable of binding to an inhibitor to form a 92,000 dalton complex.

Animals↗

PubMed1986

Characterization of rat kallikrein-like multigene family and its expression in the submandibular gland.

A cDNA clone encoding rat tissue kallikrein was isolated from a submandibular cDNA library. The kallikrein cDNA clone was used as a probe to analyze the complexity of the kallikrein-like gene family and its expression. The results indicate that rat kallikrein-like genes identified with this probe belong to a very large and highly homologous multigene family. A number of these genes, perhaps as many as a dozen or so, are expressed in the submandibular gland.

Animals↗

PubMed1986

Kallikrein localization in rat brain by immunohistochemistry.

Using an antibody-peroxidase bridge technique with either polyclonal antibodies or a specific monoclonal antibody, immunoreactive kallikrein was localized in all three lobes of the pituitary, in ependymal cells lining the third ventricle, and in cell bodies of the following hypothalamic neuronal nuclei: supraoptic, arcuate, paraventricular and ventromedial.

Animals↗

PubMed1986

A monoclonal antibody to rat tissue kallikrein: use in biochemical and immunohistochemical studies.

A monoclonal antibody (V4G6) to rat tissue kallikrein (EC 3.4.21.35) has been developed and characterized. This clone showed no cross-reactivity with rat tonin, rat esterase A or human urinary kallikrein in either radioimmunoassay or enzyme-linked immunosorbent assay. The monoclonal antibody used in the direct radioimmunoassay detects purified rat urinary kallikrein in a range of 0.32 to 40 ng per tube. The displacement curves for rat submandibular gland, pancreatic and kidney extracts and urine were parallel with the standard curve of purified rat urinary kallikrein. Analysis of immunoprecipitates from [14C] DFP labeled submandibular gland extract with SDS-polyacrylamide gel electrophoresis, demonstrates that this antibody recognizes only one 38,000 dalton serine protease while polyclonal antiserum identifies multiple species. Using this specific monoclonal reagent, tissue kallikrein was localized immunohistochemically in the granular convoluted tubules and striated ducts of the rat submandibular gland and in the acinar cells of the rat pancreas. The results showed that the monoclonal antibody (V4G6) can specifically recognize a single kallikrein in the tissue extracts without cross-reacting with other kallikrein-related serine proteases. This monoclonal antibody can be used as a specific reagent for quantitation, identification and immunohistochemical studies of tissue kallikrein.

Animals↗

PubMed1986

An improved method for the measurement of rat tissue kallikrein using a monoclonal antibody which recognizes only active enzyme.

We have used a monoclonal antibody which recognizes active kallikrein to develop a method for measurement of inactive and active kallikrein in rat urine. The inactive kallikrein levels were calculated from the difference between values before and after trypsin pretreatment in the kallikrein direct radioimmunoassay. In this assay, the final dilution of ascitic fluid containing monoclonal antibody was 1:1.6 X 10(7) which gave 35% specific binding to 125I-labelled kallikrein, and the minimal detectable amount was 0.08 ng/tube. When inactive kallikrein was assessed by this method and a kininogenase assay in 23 randomly collected urines, a significant correlation was observed between the values obtained from the two assays (p less than 0.01). The regression line for this relation was similar to that observed between purified active kallikrein concentration and kininogenase activity. The active and inactive kallikrein excretion rates and the inactive/total kallikrein ratio in rats on normal sodium diet were 74.0 +/- 17.3 micrograms/day (m +/- SD), 82.8 +/- 14.4 micrograms/day and 53.1 +/- 7.1%, respectively. This method can now be applied to studies of prokallikrein activation.

Animals↗

PubMed1986

Sensitivity of DNA-repair-deficient strains of Escherichia coli to rifampicin killing.

We have analyzed the role of RNA polymerase in DNA repair using the antibiotic rifampicin which binds specifically to the beta subunit of the enzyme. Several DNA-repair-deficient strains such as recA, uvr, and polA, and their isogenic parents were used for this study. All repair-deficient strains were found to be hypersensitive to rifampicin killing. Compared to the isogenic parent strains, recA strains are about 50 times more sensitive and the polA strain is about 100 times more sensitive to rifampicin killing. UvrA and uvrB strains are slightly more sensitive to rifampicin than the wild-type strains. The hypersensitivity of repair-deficient strains to rifampicin killing is totally abolished by the introduction of rifampicin-resistant mutations into these strains. We have examined the effect of rifampicin on RNA and protein synthesis in repair-deficient and -proficient strains. RNA and protein synthesis were found to be inhibited by rifampicin to the same extent among all the strains tested. The results also show that the resumption of DNA synthesis was significantly disrupted in DNA-repair-deficient strains following drug removal. Taken together these results suggest that RNA polymerase plays an essential role in DNA metabolism and such function may be replaced by polA and recA gene products and to a lesser extend by uvrA and uvrB gene products.

DNA Repair↗

PubMed1986

Molecular evolution, species distribution, and clinical consequences of an endemic aminoglycoside resistance plasmid.

During the first 6 years after appearing in one hospital, a 92-kilobase conjugative plasmid, pBWH1, which encoded resistance to chloramphenicol and sulfonamides and determined TEM-1 beta-lactamase and 2''-aminoglycoside nucleotidyltransferase, underwent a variety of molecular changes. It was most prevalent initially in isolates of Klebsiella pneumoniae, then in isolates of Serratia marcescens, and finally, after nearly disappearing, in isolates of Enterobacter cloacae. Evolutionary changes in the plasmid did not account for its shifts in species distribution, since the original molecule was found in isolates of each species. The late resurgence of pBWH1 occurred after a copy of its original molecule entered a distinctive ornithine decarboxylase-negative strain of E. cloacae, new to the hospital. The resulting transconjugant strain, chromosomally resistant to topical silver salts and to cephalosporins, and with the addition of pBWH1-encoded aminoglycoside resistance, spread in the hospital by causing an outbreak of sepsis in the burn unit, where these were commonly used antibacterial agents. Thus, an endemic plasmid became prevalent in a new host species because one of its genes supplemented the fitness of an uncommon strain of the species for a particular clinical niche.

Aminoglycosides↗

PubMed1985

Specific identification of tissue kallikrein in exocrine tissues and in cell-free translation products with monoclonal antibodies.

A panel of six mouse monoclonal antibodies (IgG1) has been prepared against purified rat urinary kallikrein (EC 3.4.21.35) and characterized. In radioimmunoassay, the antibody titres of ascitic fluid giving 50% binding to 125I-kallikrein range from 1:2 X 10(3) to 1:1 X 10(6). Antibodies from four of the clones show no cross-reactivity with human urinary kallikrein, rat urinary esterase A or tonin. However, antibodies from a fifth clone cross-react with tonin and, from a sixth, with both urinary esterase A and tonin. Three of the kallikrein affinity-purified monoclonal antibodies inhibited, whereas one of the antibodies stimulated, kallikrein activity. Tissue kallikrein from rat submandibular-gland and pancreatic extracts and urine were labelled with [14C]di-isopropyl phosphofluoridate, immunoprecipitated with each of the six monoclonal antibodies and identified to be 38 kDa proteins, similar in size to purified rat urinary kallikrein. Western-blot analysis shows that 125I-labelled kallikrein monoclonal antibodies (V4D11) bind directly to a 38 kDa protein in submandibular-gland and pancreatic extracts and urine. Cell-free translation products of submandibular-gland polyadenylylated[poly(A)+]mRNA were immunoprecipitated with affinity-purified sheep anti-kallikrein antibodies and three monoclonal antibodies (V4D11, V4G6 and V1C3). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these immunoprecipitates revealed that two kallikrein precursors with Mr values of 37 000 and 35 000 are encoded by submandibular-gland mRNA. The third monoclonal antibody, V1C3, which binds to active kallikrein, did not recognize either precursor form. Collectively, the data show that these monoclonal antibodies comprise a set of powerful and specific reagents for studies of tissue kallikreins.

Animals↗

PubMed1985

Immunological analysis of rat pancreatic prokallikrein activation.

The present study shows that tissue kallikrein is present in rat pancreas as a proenzyme that can be converted by autolysis to a 38 000 Da active enzyme. The activation of pancreatic prokallikrein was examined by direct radioimmunoassay, enzymatic assays, active-site labeling with immunoprecipitation, and Western blot analyses. A monoclonal antibody (V1C3), which binds only active kallikrein, was used in a direct radioimmunoassay to monitor the appearance of the active enzyme. During a 22-h autolysis of pancreatic extract, a time-dependent increase in active kallikrein concentration paralleled the increase of kallikrein activities measured by both TosArgOMe esterase and kininogenase assays. The activation process was further analyzed by labeling the pancreatic extract with [14C]diisopropylphosphorofluoridate [( 14C]DFP) followed by immunoprecipitation with sheep anti-kallikrein antiserum. Pancreatic prokallikrein was not labeled by [14C]DFP; however, upon autolysis, a 38 000 Da active kallikrein can be labeled with [14C]DFP and increase in quantity with time. Western blot analysis, using a monoclonal antibody (V4D11) which recognizes both latent and active tissue kallikreins, identified a 39 000 Da pancreatic prokallikrein prior to autolysis and a 38 000 Da active kallikrein after 7 h of autolysis. The results indicate that the pancreatic prokallikrein exists as a 39 000 Da protein which may be converted to a 38 000 Da active kallikrein, indistinguishable from purified urinary, brain, spleen or submandibular gland kallikrein.

Animals↗

PubMed1985

Resistance to antibiotics in clinical isolates of Klebsiella pneumoniae.

The antibiotic resistance of Klebsiella pneumoniae isolates from 12 medical centers worldwide, over a 1- to 6-year period, were tested. Clinical isolates of K. pneumoniae were resistant to ampicillin and carbenicillin. Resistance to other antibiotics was less frequent with isolates of K. pneumoniae from 5 of 6 US centers than with those from 6 centers outside the US. In nearly all of the centers, resistance to sulfamethoxazole-trimethoprim, gentamicin, tobramycin, or chloramphenicol was more frequent in isolates of K. pneumoniae than in those of Escherichia coli, while the reverse was true for resistance to tetracycline. Resistance to multiple antibiotics declined gradually in isolates of K. pneumoniae at one center, but rose abruptly again with dissemination of a new plasmid.

Ampicillin↗