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Biomedical subjects

S K Harlander

Publications and source records attributed to S K Harlander.

At least 19 recordsLinked to original sources
PubMed1996

Breaking through the glass ceiling: an industrial perspective.

Many companies have recognized that the workforce of the future will be significantly more diverse than in the past. A substantial number of these new employees will be women. Industry must create an environment that attracts the best, brightest, and most talented women and provide an environment that empowers them to reach their full potential. Barriers to advancement, such as the real or perceived "glass ceiling," inhibit the individual and ultimately negatively affect the company. Successful companies will recognize that gender diversity is a critical business strategy and implement aggressive programs to assist women in breaking through the glass ceiling. Gender diversity initiatives should include examination of quality of life issues, training and development, organizational policies and practices, and management accountability. Mentoring programs clearly play an important role in gender diversity programs. Personal mentoring experiences in academic and corporate environments, as well as a pilot mentoring program at Land O'Lakes, will be discussed.

Animal Husbandry↗
PubMed1993

Construction of first-generation lactococcal integrative cloning vectors.

Using a randomly-cloned, HindIII-digested, chromosomal fragment from Lactococcus lactis subsp. lactis LM0230, first-generation lactococcal integrative cloning vectors were developed. Through dideoxy DNA sequence analysis, the cloned chromosomal DNA fragment was determined to be 1026 base pairs. Southern hybridization studies demonstrated applicability of the integrative vector to other strains of L. lactis and L. lactis subsp. cremoris. Identification of a single NruI site near the middle of the chromosomal fragment allowed insertion of the erythromycin (Em)-resistance (eryr) gene obtained from L. lactis IL1837. Integration of the eryr gene into the L. lactis LM0230 chromosome was achieved by a Campbell-like recombination. The nisin (Nis)-resistance (nisr) gene from L. lactis IL1904 was inserted into the NruI site in a separate clone and integration into the L. lactis LM0230 chromosome was achieved via a replacement recombination event following electroporation of the linearized nisr fragment flanked by the cloned chromosomal DNA. Transformants grown in the absence of either Em or Nis for > 200 generations and subsequently transferred to various concentrations of the selectable agent confirmed the stability of the integrated genes. Further studies involving the Nis-resistant (NisR) transformant suggested that the integrated nisr gene may be amplifying within the host chromosome.

Base Sequence↗
PubMed1993

Comparative effects of exogenous lactase (beta-galactosidase) preparations on in vivo lactose digestion.

Microbial-derived beta-galactosidase (beta-gal) enzyme preparations improve in vivo lactose digestion and tolerance through enhanced gastrointestinal digestion of lactose. Three different beta-gal preparations, Lactogest (soft gel capsule), Lactaid (caplet), and DairyEase (chewable tablet) and placebo were fed to lactose maldigesters with either 20 g or 50 g of lactose to compare the efficacy of these products and to further establish a dose-response relationship for use. All enzyme preparations dramatically reduced both the peak and total breath hydrogen production when fed with milk containing 20 g of lactose. Four capsules of Lactogest, two caplets of Lactaid, or two tablets of DairyEase (each treatment containing approx 6000 IU) reduced total hydrogen production significantly (P < 0.05) below that observed with two capsules of Lactogest (containing approx 3000 IU) in a stoichiometric manner. Symptoms were significantly (P < 0.05) less severe with all the beta-gal products. In contrast, with 50 g of lactose in water, peak and total hydrogen production was modestly, but not significantly reduced by the enzyme treatment. Furthermore, symptom scores for bloating, cramping, nausea, pain, diarrhea, and flatus were not different between treatments and the control. The 50-g lactose dose appeared to overwhelm the ability of either 3000 or 6000 IU of beta-gal to assist significantly with lactose digestion. Results from these studies demonstrate the relative equivalency of chewable, caplet, and soft-gel beta-gal products, based on IUs of enzyme fed.

Administration, Oral↗
PubMed1991

Strains and species of lactic acid bacteria in fermented milks (yogurts): effect on in vivo lactose digestion.

Lactose in yogurt with live bacteria is better tolerated than lactose in other dairy foods, partly because of the activity of microbial beta-galactosidase (beta-gal), which digests lactose in vivo. To evaluate the ability of different strains and species of lactic acid bacteria to digest lactose in vivo, yogurts (containing mixtures of strains of Streptococcus salivarius subsp thermophilus and Lactobacillus delbrueckii subsp bulgaricus) and fermented milks (containing individual species of S thermophilus, L bulgaricus, L acidophilus, or Bifidobacterium bifidus) that varied in microbial beta-gal activity were produced. Selected products were fed to healthy people who cannot digest lactose, and breath hydrogen production was monitored. All yogurts dramatically and similarly improved lactose digestion, regardless of their total or specific beta-gal activity. The response to fermented milks varied from marginal improvement with B bifidus milk to nearly complete lactose digestion with L bulgaricus milk. The results suggest that total beta-gal was not the limiting factor in promoting lactose digestion, perhaps because of a limited rate of intracellular substrate transport.

Adult↗
PubMed1991

Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys.

Total cellular DNA from 28 strains of Listeria monocytogenes isolated from food implicated in food-borne illness and from patients with listeriosis was digested with the restriction endonucleases HindIII, HaeIII, and EcoRI. Following agarose gel electrophoresis, the fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for classifying L. monocytogenes strains, and the resulting subtypes were compared with serotyping and multilocus enzyme electrophoresis classification schemes. A total of 15 distinct and identical groups were obtained when genomic DNA was digested with either HindIII or HaeIII. The most discriminating enzyme for ribotyping of strains was EcoRI, which divided the 28 strains of L. monocytogenes into 6 ribotype groups. DNA fingerprinting and ribotyping differentiated L. monocytogenes from other Listeria spp., including L. ivanovii, L. welshimeri, and L. innocua as well as the lactic acid bacteria Lactococcus lactis subsp. lactis and subsp. cremoris. L. monocytogenes strains isolated from four independent food-borne illness incidents were analyzed by all typing methods. Patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis. DNA fingerprinting was the only method capable of differentiating these strains, or conversely, of proving relatedness of patient-product pairs of isolates. This method was a relatively simple, sensitive, reproducible, and highly discriminating method for epidemiological tracking of L. monocytogenes implicated in food-borne illness.

Animals↗
PubMed1989

Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media.

The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample.

Culture Media↗
PubMed1989

Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation.

To apply recombinant DNA techniques for genetic manipulation of the industrially important lactococci, an efficient and reliable high-frequency transformation system must be available. High-voltage electric pulses have been demonstrated to enhance uptake of DNA into protoplasts and intact cells of numerous gram-negative and gram-positive microorganisms. The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit (BTX Transfector 100; BTX, Inc., San Diego, Calif.). Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 microseconds to 1 s). Transformation efficiencies of 10(3) transformants per microgram of DNA were obtained when dense suspensions (final concentration, 5 x 10(10) CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30 degrees C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.

Culture Media↗
PubMed1989

Preliminary characterization of a food-borne multiple-antibiotic-resistant Salmonella typhimurium strain.

Plasmid characterization studies were conducted on a Salmonella typhimurium strain isolated from pasteurized milk and from a symptomatic patient during the 1985 Illinois salmonellosis outbreak. This strain (Hf) was reported to possess an unusual plasmid profile which distinguished it from all Salmonella strains isolated in the United States prior to 1984. Antibiotic susceptibility testing revealed that the strain was resistant to tetracycline, erythromycin, clindamycin, sulfisoxazole, sulfadiazene, triple sulfa, cefoperazone, streptomycin, mezlocillin, piperacillin, carbenicillin, penicillin, ampicillin, and kanamycin. Plasmid analysis revealed that the strain possessed four plasmids with sizes of approximately 158, 98, 10.2, and 6.0 kilobase pairs (kb). Successive transfer at 43 degrees C led to increased antibiotic sensitivity in 75.5% of the isolates screened. Electroporation and calcium chloride treatment were each used to transform plasmid-free Escherichia coli strains with the plasmid pool from S. typhimurium Hf. Plasmids introduced by transformation ranged in size from 4.4 to 23.2 kb and correlated with resistance to penicillin G, ampicillin, carbenicillin, cephalothin, cefoperazone, cefamandole, mezlocillin, piperacillin, and in some cases, tetracycline and kanamycin. DNA-DNA hybridization experiments localized these resistance genes to a highly duplicated 6.3-kb fragment of the total EcoRI restriction digest of the S. typhimurium Hf plasmid pool.

Animals↗
PubMed1989

A method for determining beta-galactosidase activity of yogurt cultures in skim milk.

A method was developed for determining the specific activity of bacterial beta-galactosidase (EC 3.2.1.23) during growth of Streptococcus thermophilus and Lactobacillus bulgaricus in skim milk. Individual and mixed strain cultures of S. thermophilus (St 3642, St14485) and L. bulgaricus (Lb11842, Lb880) were examined for growth (OD at 600 nm and viable cell counts), acid production, and beta-galactosidase activity (expressed as a function of recoverable TCA-precipitable cellular protein). Cultures were inoculated into 10% skim milk (2% inoculum) and incubated at 40 degrees C for 12 h. Aliquots were removed at 2-h intervals and diluted with ice cold EDTA, pH 12. The EDTA chelates calcium and solubilizes milk protein, allowing separation of the bacteria by centrifugation. Cells were then washed twice with 20 mM phosphate buffer and disrupted by sonication. Cell debris and intact cells were removed by centrifugation and the cell-free extract evaluated for beta-galactosidase activity using o-nitrophenyl-beta-D-galactopyranoside as substrate. Specific activities ranged from 0 to 6 units/mg protein. This simple and reproducible method is applicable for enzyme assays and measurement of cellular components where contamination by milk proteins is a potential problem.

Animals↗
PubMed1988

Plasmid-mediated reduced phage sensitivity in Streptococcus lactis KR5.

The phage insensitivity of Streptococcus lactis KR5 was evaluated for its possible linkage to plasmid DNA. This strain possessed plasmids of 40, 29, 26, 21, 16.5, 10.5, 7.8, and 1.5 Mdal. Plasmid curing using novobiocin resulted in derivatives with increased sensitivity to prolate-headed phage, suggesting the involvement of plasmid DNA in phage insensitivity. Transformation of S. lactis LM0230 protoplasts with the KR5 plasmid DNA pool produced transformants containing a plasmid of about 27 Mdal. These erythromycin-resistant transformants were lactose-positive phage-sensitive or were lactose-negative and exhibited a reduced sensitivity to phage. Agarose gel electrophoresis and restriction endonuclease digestion analysis showed the 27-Mdal plasmid band to be composed of two distinct plasmids of 26 Mdal (pBF61) and 29 Mdal (pBF62), which coded for reduced phage sensitivity and lactose-positive phenotypes, respectively. The mechanisms of reduced phage sensitivity encoded by pBF61 included a restriction/modification system and a mechanism that resulted in reduced plaque size independent of incubation temperature. These results further support the involvement of plasmid DNA in the mechanisms for reduced phage sensitivity in dairy streptococci.

Bacteriophages↗
PubMed1984

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA.

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.

Acriflavine↗
PubMed1984

Molecular cloning of the lactose-metabolizing genes from Streptococcus lactis.

Restriction endonucleases and agarose gel electrophoresis were used to analyze plasmid pLM2001, which is required for lactose metabolism by Streptococcus lactis LM0232. The enzymes XhoI, SstI, BamHI, and KpnI each cleaved the plasmid into two fragments, whereas EcoRI and BglII cleaved the plasmid into seven and five fragments, respectively. Sizing of fragments and multiple digestions allowed construction of a composite restriction map. The KpnI fragments of pLM2001 were cloned into the KpnI cleavage site of the vector plasmid pDB101. A recombinant plasmid (pSH3) obtained from a lactose-fermenting, erythromycin-resistant (Lac+ Eryr) transformant of Streptococcus sanguis Challis was analyzed by enzyme digestion and agarose gel electrophoresis. Plasmid pSH3 contained 7 of the 11 KpnI-HindIII fragments from pLM2001 and 5 of the 7 fragments from pDB101. It was determined that a 23-kilobase (kb) KpnI-generated fragment from pLM2001 had been cloned into pDB101 with deletion of part of the vector plasmid. The recombinant plasmid could be transformed with high frequency into several Lac- strains of S. sanguis, conferring the ability to ferment lactose and erythromycin resistance. The presence of pSH3 allowed a strain deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-dependent phosphotransferase system to efficiently ferment lactose. Under conditions designed to maximize curing of plasmid DNA with acriflavin, no Lac- derivatives could be isolated from cells transformed with pSH3. Seven of the 40 Lac+ colonies isolated after 10 transfers in acriflavin were shown to be sensitive to erythromycin and did not appear to harbor plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1980

Streptococcus mutans dextransucrase: effect of cerulenin on lipid synthesis and enzyme production.

The effect of the fatty acid synthesis inhibitor cerulenin on growth and dextransucrase (EC 2.4.1.5) production by Streptococcus mutans 6715 was analyzed. Growth was markedly inhibited by less than 1 microgram of the antibiotic per ml. Under conditions where cerulenin did not inhibit amino acid incorporation into protein but did block acetate metabolism into lipid, the production of extracellular dextransucrase was suppressed. Inhibition was not due to a direct effect of the antibiotic on the enzyme or the lack of release of enzyme from the bacterial cell surface. Gel column chromatography demonstrated that enzyme produced in the presence of cerulenin was highly aggregated, similar to the control enzyme. Although the addition of ysophosphatidylcholine to enzyme which had been synthesized in the presence of cerulenin stimulated glucan formation from sucrose, the increase was not greater than that produced with the control enzyme. The differential inhibition of dextransucrase production by cerulenin indicates that enzyme secretion requires the production of lipid and may reflect the mechanism by whch this enzyme is transported from the bacterial cell.

Antifungal Agents↗
PubMed1978

Streptococcus mutans dextransucrase: stimulation of glucan formation by phosphoglycerides.

Lysophosphatidylcholine (LPC) and other phosphoglycerides stimulated water-insoluble and water-soluble glucan production by the Streptococcus mutans 6715 dextransucrase (EC 2.4.1.5). LPC stimulated crude extracellular dextransucrase 1.7-fold, the water-insoluble glucan-producing alpha form of the enzyme 6.5-fold, the water-soluble glucan-producing beta form of the enzyme 2.1-fold, and the cell-associated dextransucrase 2.0-fold. Kinetic studies demonstrated that LPC did not change the K(m) for sucrose of alpha or beta but increased the maximum velocity of the enzymes. The K(m) for LPC of the alpha enzyme was 10(-5) M. LPC from various sources and synthetic preparations of lauroyl-LPC, myristoyl-LPC, and palmitoyl-LPC all stimulated glucan formation. Portions of phosphoglyceride molecules including fatty acids, phosphatidic acid, glycerophosphoric acid, glycerophos-phorylcholine, and choline, when tested individually or in combinations, did not enhance dextransucrase activity. The increased rates of glucan production caused by LPC and primer dextran were additive. Enzyme incubated with LPC before addition of sucrose was stimulated by dextran primer, and, conversely, enzyme treated with dextran was stimulated by addition of LPC with the sucrose substrate. Thus, dextransucrase can be activated by binding of intact phosphoglyceride molecules to a site on the enzyme that is distinct from either the glucosyl donor or glucosyl acceptor (primer) binding sites. Interactions between the S. mutans dextransucrase and amphipathic phosphoglycerides may explain properties of this enzyme which contribute to the cariogenicity of S. mutans.

Dextrans↗
PubMed1978

Streptococcus mutans dextransucrase: stimulation by phospholipids from human sera and oral fluids.

Serum, gingival crevicular fluid, and parotid, submandibular, and labial minor gland saliva from four individuals stimulated glucan formation from sucrose by the Streptococcus mutans strain 6715 dextransucrase (EC 2.4.1.5). At final dilutions of 1:10 all of the fluids stimulated crude enzyme preparations approximately 1.8-fold. The fluids stimulated the purified water-insoluble glucan-synthesizing form of the dextransucrase approximately 3.2-fold and the water-soluble glucan-producing form of the enzyme approximately 2.4-fold. The fluids all contained concentrations of stimulatory material that could be reduced to undetectable levels only after dilutions of greater than 1:1,000. The increased rates of glucan formation caused by the fluids and dextran were additive, indicating that stimulation by the fluids was primarily due to interactions with entities other than glucan primer molecules. In contrast, the elevated levels of glucan formation in the presence of the fluids was not further enhanced by the addition of lysophosphatidylcholine. Lysophosphatidylcholine purified from parotid and submandibular saliva by solvent extraction and thin-layer chromatography stimulated the dextransucrase as effectively as egg yolk lysophosphatidylcholine. Thus, phospholipids normally found in human oral fluids can enhance the activity of an enzyme believed to be directly associated with the cariogenic potential of S. mutans.

Blood↗