Transgenic mice with increased hexosamine flux specifically targeted to beta-cells exhibit hyperinsulinemia and peripheral insulin resistance.

Hexosamines have been shown to mediate effects of hyperglycemia and so-called "glucose toxicity" in insulin-sensitive tissues. To determine the effects of hexosamines on insulin synthesis and secretion, transgenic mice were created to overexpress the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), specifically in beta-cells. GFA activity in islets of heterozygous transgenic mice was elevated 76% compared with littermate controls. The increased GFA activity led to 1.4- and 2.1-fold increased pancreatic insulin content in 2- and 10-month-old transgenic mice, respectively (P < 0.005). Fasting insulin levels were 1.6-fold higher than in littermate controls (P < 0.05). Hyperinsulinemia was evident despite a 28% reduction in insulin mRNA levels. The fasting glucose levels in the transgenic mice equaled that of controls aged 2-4 months but exceeded that of the controls aged 6-10 months (means +/- SE 6.9 +/- 0.2 vs. 5.9 +/- 0.2 mmol/l, P < 0.001). By 8 months, the males were overweight and mildly diabetic (fasting glucose 8.8 +/- 0.5 mmol/l) despite persistent hyperinsulinemia. Insulin resistance was confirmed in both males and females using the euglycemic-hyperinsulinemic clamp technique; glucose disposal rates decreased by 48% in transgenic mice (P < 0.01). Triglyceride levels did not differ, and free fatty acid levels were lower in the transgenic animals. ATP levels were unchanged in the transgenic islets. We conclude that hexosamine biosynthesis is involved in the regulation of insulin content in beta-cells by glucose. Increased hexosamine flux in the beta-cell results in hyperinsulinemia, insulin resistance, and (in males) mild type 2 diabetes.

Overexpression of glutamine: fructose-6-phosphate amidotransferase in the liver of transgenic mice results in enhanced glycogen storage, hyperlipidemia, obesity, and impaired glucose tolerance.

To examine the effect of increased hexosamine flux in liver, the rate-limiting enzyme in hexosamine biosynthesis (glutamine:fructose-6-phosphate amidotransferase [GFA]) was overexpressed in transgenic mice using the PEPCK promoter. Liver from random-fed transgenic mice had 1.6-fold higher GFA activity compared with nontransgenic control littermates (276 +/- 24 pmol x mg(-1) x min(-1) in transgenic mice vs. 176 +/- 18 pmol x mg(-1) x min(-1) in controls, P < 0.05) and higher levels of the hexosamine end product UDP-N-acetyl glucosamine (288 +/- 11 pmol/g in transgenic mice vs. 233 +/- 10 pmol/g in controls, P < 0.001). Younger transgenic mice compared with control mice had lower fasting serum glucose (4.8 +/- 0.5 mmol/l in transgenic mice vs. 6.5 +/- 0.8 mmol/l in controls, P < 0.05) without higher insulin levels (48.0 +/- 7.8 pmol/l in transgenic mice vs. 56.4 +/- 5.4 pmol/l in controls, P = NS); insulin levels were significantly lower in transgenic males (P < 0.05). At 6 months of age, transgenic animals had normal insulin sensitivity by the hyperinsulinemic clamp technique. Hepatic glycogen content was higher in the transgenic mice (108.6 +/- 5.2 pmol/g in transgenic mice vs. 32.8 +/- 1.3 micromol/g in controls, P < 0.01), associated with an inappropriate activation of glycogen synthase. Serum levels of free fatty acids (FFAs) and triglycerides were also elevated (FFAs, 0.67 +/- 0.03 mmol/l in transgenic mice vs. 0.14 +/- 0.01 in controls; triglycerides, 1.34 +/- 0.15 mmol/l in transgenic mice vs. 0.38 +/- 0.01 in controls, P < 0.01). Older transgenic mice became heavier than control mice and exhibited relative glucose intolerance and insulin resistance. The glucose disposal rate at 8 months of age was 154 +/- 5 mg x kg(-1) x min(-1) in transgenic mice vs. 191 +/- 6 mg x kg(-1) x min(-1) in controls (P < 0.05). We conclude that hexosamines are mediators of glucose sensing for the regulation of hepatic glycogen and lipid metabolism. Increased hexosamine flux in the liver signals a shift toward fuel storage, resulting ultimately in obesity and insulin resistance.

Hexosamines stimulate leptin production in transgenic mice.

Hexosamine flux has been shown to mediate aspects of nutrient sensing in insulin sensitive tissues and has been hypothesized to represent a satiety signal that results in shunting of fuel toward storage as fat. It has been recently reported that in vitro treatment of fat and muscle cells with hexosamines and acute glucosamine infusion in intact rats stimulate leptin secretion. In order to investigate the effects of chronic, physiologic increases in hexosamine flux on leptin we have examined leptin mRNA and serum leptin in mice overexpressing the rate-limiting enzyme for hexosamine synthesis, GFA, in muscle and fat. Increased levels of UDP-N-acetylglucosamine, the principal end-product of the hexosamine pathway were seen in transgenic fat, consistent with the overexpression of GFA. After overnight fasting, the transgenic mice were hyperleptinemic compared to littermate controls (4.5+/-0.5 ng/ml in transgenic, 2.8+/-0.2 in control, p = 0.005) despite equal body weights. In the random-fed state, the leptin levels of control mice increased to 4.1+/-0.5 ng/ml (p = 0.01) whereas the leptin levels in the transgenics did not increase any further (3.7+/-0.4 ng/ml). Leptin mRNA levels were also increased in transgenic fat (2.7+/-0.6 in transgenic compared to 0.8+/-0.2 in control, arbitrary units normalized to actin, p < 0.007). Despite increased leptin, the transgenic animals did not have lower body fat content. We conclude that hexosamine flux in fat regulates leptin synthesis and secretion.

Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione.

Hexosamines have been hypothesized to mediate aspects of glucose sensing and toxic effects of hyperglycemia. For example, insulin resistance results when the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), is overexpressed in muscle and adipose tissue of transgenic mice. The glucose infusion rates required to maintain euglycemia at insulin infusion rates of 0.5, 2, 15, and 20 mU/kg x min were 39-90% lower in such transgenic mice, compared with their control littermates (P < or = 0.01). No differences were observed in hepatic glucose output, serum insulin levels, or muscle ATP levels. Uptake of 2-deoxyglucose, measured under conditions of hyperinsulinemia, was significantly lower in transgenic hindlimb muscle, compared with controls (85.9 +/- 17.8 vs. 166.8 +/- 15.1 pmol deoxyglucose/g x min). The decrease in glucose uptake by transgenic muscle was associated with a disruption in the translocation of the insulin-stimulated glucose transporter GLUT4. Fractionation of muscle membranes on a discontinuous sucrose gradient revealed that insulin stimulation of control muscle led to a 28.8% increase in GLUT4 content in the 25% fraction and a 61.2% decrease in the 35% fraction. In transgenic muscle, the insulin-stimulated shifts in GLUT4 distribution were inhibited by over 70%. Treatment of the transgenic animals with the thiazolidinedione troglitazone completely reversed the defect in glucose disposal without changing GFA activity or the levels of uridine 5'-diphosphate-N-acetylglucosamine. Overexpression of GFA in skeletal muscle thus leads to defects in glucose transport similar to those seen in type 2 diabetes. These data support the hypothesis that excess glucose metabolism through the hexosamine pathway may be responsible for the diminished insulin sensitivity and defective glucose uptake that are seen with hyperglycemia.

Antithyroid effects in vivo and in vitro of vitexin: a C-glucosylflavone in millet.

Millet diets rich in C-glycosylflavones (C-GF) are goitrogenic, and its three most abundant C-GF inhibit in vitro thyroid peroxidase, suggesting that these compounds are the goitrogens in millet. However, proof of a cause and effect relationship between C-GF and goitrogenesis requires a demonstration of in vivo antithyroid activity by the purified isolated compounds. Vitexin, one of the three major C-GF in millet, was used to test this hypothesis. Twenty-four female Wistar rats, divided into groups of six rats each and fed Purina iodine-rich diet (12 micrograms I-/day.rat), were administered acutely by gastrointestinal tube goitrogen-free water (controls), methimazole (0.5 mumol), and vitexin (20 and 80 mumol). 125I (1 microCi) was injected ip 1 h later, and the rats were killed 2 h after the injection. The thyroid glands were removed and analyzed for their content of total 125I and 125I-labeled compounds. Rats given vitexin, in contrast to those receiving methimazole, did not show suppressed thyroid 125I uptake. However, significant inhibition of the coupling mechanism (high 125I-labeled monoiodotyrosine plus diiodotyrosine/125T3 plus T4 ratio and low 125T3 and T4 concentrations) did occur with the highest dose of vitexin. These results provide direct evidence in vivo of C-GF antithyroid activity, strongly supporting the concept that C-GF are the goitrogens in millet.

In vitro measurement of antithyroid compounds and environmental goitrogens.

A specific, sensitive, and reproducible in vitro assay for antithyroid compounds and environmental goitrogens has been used to investigate antithyroid activity (AA) in small samples of water supplying 15 localities in endemic and nonendemic goiter areas of western Colombia. A significant positive correlation was observed between goiter prevalence and AA in water collected from the pipelines of these localities. Samples at the water source showed only borderline significance. No significant correlation was observed in waters between AA and total hardness (ppm) or concentrations of Ca, Mg, sulfates, chlorides, silicates, nitrates, and iodine. AA was also demonstrated by this in vitro assay in well water previously shown experimentally to be goitrogenic and that supplied the endemic goiter district of Candelaria town in western Colombia. In contrast, water from the well supplying the area of lower endemicity was found to possess little AA. These results provide experimental support for epidemiological observations that demonstrate a relationship between the sources of drinking water and goiter prevalence rates, and are consistent with previous findings indicating that organic antithyroid compounds contaminate water supplies in areas where goiter persists despite adequate iodine supplementation.

Thyroid function in neonates from goitrous and nongoitrous iodine-sufficient areas.

We compared thyroid function between newborns from goitrous and nongoitrous localities in which iodine intake has been supplemented since 1955. Cord serum samples were analyzed in 185 infants born during a 9-month period (1986-1987) in 2 goitrous and 1 nongoitrous localities of western Colombia. Urinary iodine was determined in all mothers before delivery. No significant differences were found among neonates of the 3 localities (Kruskal-Wallis test) for the various thyroid hormone values, and all values were within the normal range, although there was a trend in distribution of TSH to higher values in both goiter areas. Thyroid autoantibodies (antithyroglobulin and antithyroid microsomal) were negative in all neonates, and iodine intake, as indicated by urinary iodine, was adequate and similar among the mothers of the 3 groups. Those newborn infants with serum TSH values higher than 20 mU/L were reexamined 5-7 months later. At this time, all infants had lower serum TSH values and their serum free T4 index and T3 values were normal. Gestational age, weight, and height at birth were normal and also equal among the neonates in the 3 localities. These results indicate that neonates from goitrous iodine-sufficient areas have thyroid function similar to that of infants born in nongoitrous areas equally supplemented with iodine, and therefore, they are not more at risk to develop congenital hypothyroidism.

Antithyroid and goitrogenic effects of millet: role of C-glycosylflavones.

Pearl millet [Pennisetum millet (L.) leeke] is the main source of food energy for the rural poor in many areas of the semiarid tropics. Epidemiological evidence suggests that millet may play a role in the genesis of endemic goiter in these areas, and sparse experimental data in rats support this suspicion. This study was undertaken to determine in vivo in rats and in vitro using porcine thyroid slices and a thyroid peroxidase (TPO) assay the goitrogenic and antithyroid effects of millet diets, extracts of millet, and certain pure compounds contained therein. For use in these studies, whole grain millet was progressively dehulled to yield successively four bran and four flour fractions in which direct analyses revealed progressively lower concentrations of C-glycosylflavones. In vivo feeding of bran fraction 1, that richest in C-glycosylflavones, led to a significant increase in thyroid weight and antithyroid effects. Feeding of bran fraction 2, the next richest in C-glycosylflavones, produced similar, but less marked, changes. In vitro studies of 125I metabolism using porcine thyroid slices indicated that extracts of bran fractions 1 and 2 were most potent, producing changes similar to those produced by methimazole (MMI). At a concentration of 60 mumol/L, glucosylvitexin, the major C-glycosylflavone present in millet, had effects comparable to those of 1 mumol/L MMI. Similarly, in studies of porcine TPO, extracts of bran fraction 1 caused pronounced (85%) inhibition of enzyme activity, and progressively less inhibition was induced by extracts of bran fractions 2, 3, and 4. Overall, the TPO-inhibiting activities of the various millet fractions closely correlated with their C-glycosylflavone concentrations. Three C-glycosylflavones present concentrations. Three C-glycosylflavones present in millet, glucosylvitexin, glycosylorientin, and vitexin, also inhibited TPO activity. Thus, in vivo and in vitro studies revealed that millet diets rich in C-glycosylflavones produce goitrogenic and antithyroid effects similar to those of certain other antithyroid agents and small doses of MMI. We conclude that in areas of iodine deficiency in which millet is a major component of the diet, its ingestion may contribute to the genesis of endemic goiter.

Hexosamines regulate leptin production in human subcutaneous adipocytes.

The hexosamine biosynthetic pathway has recently been proposed as a mechanism through which cells "sense" nutrient flux to regulate leptin release. This study was undertaken to examine the regulation of leptin production by hexosamines in human adipocytes. Adipose tissue UDP-N-acetylglucosamine, an end product of hexosamine biosynthesis, was elevated 3.2-fold, and ob messenger ribonucleic acid was elevated 2-fold in the sc adipose tissue of 17 obese [body mass index (BMI), 41.3+/-12.0 kg/m2; age, 31+/-5 yr] subjects compared to 14 lean (BMI, 23.4+/-1.6 kg/m2; age, 33+/-11 yr) subjects. Serum leptin was increased 2.7-fold in the obese subjects. A significant positive relationship was found between adipose tissue UDP-N-acetylglucosamine and BMI (Spearman correlation = 0.576; P = 0.0007) and between UDP-N-acetylglucosamine and serum leptin (Spearman correlation = 0.4650; P = 0.0145). Treatment of isolated sc adipocytes with 1 mmol/L glucosamine, an intermediate product in UDP-N-acetylglucosamine biosynthesis, increased leptin release 21.4+/-17.6% (mean +/- SD) over control (P = 0.0365) and 74.5+/-82.8% over control (P = 0.0271) in adipocytes from lean (BMI, 23.2+/-1.6 kg/m2; n = 6) and obese (BMI, 55.4+/-13.0 kg/m2,; n = 9) subjects, respectively, by 48 h of culture. Inhibition of UDP-N-acetylglucosamine biosynthesis with 6-diazo-5-oxo-norleucine reduced glucose-stimulated leptin release from cultured adipocytes 21.8+/-32.4% (P = 0.0395; n = 12) and ob gene expression 19.9+/-18.9% (P = 0.0208; n = 8) by 48 h of treatment. These findings suggest that hexosamine biosynthesis regulates leptin production in human adipose tissue.

Antithyroid effects in vivo and in vitro of babassu and mandioca: a staple food in goiter areas of Brazil.

Babassu (Orbignya phalerata), a palm-tree coconut fruit, mixed with mandioca (Manihot utilissima) is the staple food of people living in the endemic goiter area of Maranhao in Brazil, where goiter prevalence among schoolchildren was still 38% in 1986 despite an adequate iodine intake in most of the population. Therefore, the question arose as to whether or not the ingestion of babassu alone or mixed with mandioca contributed to the persistence of endemic goiter in this area of Brazil. In this investigation we examined the potential antithyroid effects of babassu and mandioca by means of in vivo studies in Sprague-Dawley rats, in vitro studies in porcine thyroid slices and using a purified porcine thyroid peroxidase (TPO) system. Samples of various edible parts of babassu and mandioca flour were homogenized and extracted in goitrogen-free water (GFW) for in vivo experiments, and in methanol (100 g/l), GFW or 0.06 mol/l phosphate buffer (pH 7.0) for in vitro experiments. The edible parts of babassu produced significant in vivo antithyroid effects (p < 0.05- < 0.001) in rats on a high iodine intake (14 micrograms I- day-1.rat-1), as well as distinct and reproducible antithyroid and anti-TPO activities in both in vitro systems, their action being similar to that of the thionamide-like antithyroid drugs propylthiouracil and methimazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of the 10-kD antigen structural gene in mycobacteria by using the polymerase chain reaction.

The structural gene encoding the 10-kD antigen from Mycobacterium tuberculosis was amplified by the polymerase chain reaction. The 297-base-pair (bp) product was detected among 45 strains representing 14 mycobacterial species, but was absent from 11 species related to the mycobacteria. The gene was localized to a approximately 2000-bp SstII restriction fragment of the organisms' chromosomes.

DNA hybridization studies of a nucleotide sequence homologous to transposon Tn1545 in the "Minnesota" strain of multiresistant Streptococcus pneumoniae isolated in 1977.

Nucleotide sequences homologous to the transposon-specific region and the tetM determinant mediating tetracycline resistance of the multiresistance transposon Tn1545, originating in Streptococcus pneumoniae, were detected by hybridization in the first multiresistant pneumococcus reported in the United States. The sequences were localized to a common 25-kb EcoRI restriction fragment of the chromosome.

Epidemiologic typing of Enterobacter sakazakii in two neonatal nosocomial outbreaks.

Two unrelated hospital outbreaks of Enterobacter sakazakii, involving meningitis, bacteremia, and colonization of neonates, were investigated. In each of these outbreaks, E. sakazakii was isolated from both patients and dried infant formula. In previous outbreaks, the source and mode of transmission of E. sakazakii in neonatal infections was not determined. In this study, we used a combination of typing methods (plasmid analysis, antibiograms, chromosomal restriction endonuclease analysis, ribotyping, and multilocus enzyme electrophoresis) to evaluate the isolates from each outbreak as to their relatedness. The typing results differed among outbreaks, but in each one, patient and formula isolates shared the same typing pattern. The only exceptions were disk antibiograms, which often varied among colonies selected from each of the isolates. Plasmid analysis, chromosomal restriction endonuclease analysis, ribotyping, and multilocus enzyme electrophoresis all were effective as epidemiological typing methods for E. sakazakii, especially when used in combination. By using this typing scheme, we have confirmed that E. sakazakii from intrinsically contaminated dried infant formula was the source of neonatal infection.

Antithyroid and goitrogenic effects of coal-water extracts from iodine-sufficient goiter areas.

Goiter in iodine-sufficient areas has been linked to water-borne goitrogens in watersheds and aquifers rich in coal and shale. In the present study, the potential antithyroid and goitrogenic effects of coal-water extracts (CWE) were investigated in vivo in rats after chronic and acute oral administration of CWE, and in vitro by a thyroid peroxidase (TPO) enzyme system. CWE was prepared by continuous extraction of ground (40 mesh) Appalachian coal with goitrogen-free water (GFW). Female Buffalo rats fed on Purina iodine-rich diet (12 micrograms I-/day/rat), were given ad lib CWE (50 mg/ml; approximately 20 mL/day/rat) or GFW (controls) for 2 months. At the end of the experiment, 125I 1 microCi, was injected i.p. and 4 h later the thyroid glands were removed, weighed, and analyzed histologically and for total 125I and 125I-labeled compounds. Rats on CWE had larger thyroid glands [7.2 +/- 0.3 mg/100 g (mean +/- SE) vs 5.0 +/- 0.5 controls; p < 0.005] with distinct histological changes of smaller thyroid follicles, some with columnar epithelium, and with more dense colloid than in controls, and had significant inhibition of the coupling mechanism for production of thyroid hormones [125MIT + DIT/125T3 + T4: 5.1 +/- 0.2 vs 3.9 +/- 0.1 controls, p < 0.005; and 125T3 + T4 (%): 10.6 +/- 0.3 vs 12.6 +/- 0.4 controls, p < 0.005]. Female Sprague-Dawley rats under the same conditions as Buffalo rats were given acutely by GI tube 2 mL of CWE (5 g/mL) or GFW (controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular identification of mutations associated with anti-tuberculosis drug resistance among strains of Mycobacterium tuberculosis.

BACKGROUND: Understanding the etiologic organism, antimicrobial resistance mechanisms, and transmission of multidrug-resistant tuberculosis (MDR-TB) can be of great value in optimizing strategies to control and prevent its development and transmission. METHODS: One hundred and fifty-five Mycobacterium tuberculosis complex isolates from patients with pulmonary tuberculosis (TB) in Cairo, Egypt were studied. In vitro drug susceptibility testing against rifampin (RIF), isoniazid (INH), streptomycin (SM), ethambutol (EMB), and pyrazinamide (PZA) was performed. Resistance was studied by the standard agar proportion method. Single strand conformation polymorphism (SSCP) and DNA sequence analysis were used to detect mutations in the genes that encode resistance to rpoB, katG, rpsL, and embB. RESULTS: Among 155 consecutive M. tuberculosis isolates, 25 (16.1%) were MDR-TB; 13 of these were from newly diagnosed untreated cases, 12 were from re-treated cases, and none of the MDR-TB isolates had matching IS6110 fingerprints. Among the MDR-TB isolates, rpoB mutations were found in 76% of RIF-resistant isolates, katG mutations were found in 47.1% of INH-resistant isolates, rpsL mutations were found in 55.6% of SM-resistant isolates, and embB mutations were found in 36.4% of EMB-resistant isolates. CONCLUSIONS: No major differences were found in the frequencies of mutations or types of amino acid substitution between newly diagnosed untreated cases and re-treated cases. The high prevalence of MDR-TB at this hospital underscores the need for continuous monitoring of strains and antimicrobial resistance.

Relatedness of tetracycline resistance plasmids among species of coagulase-negative staphylococci.

Four isolates of Staphylococcus aureus and 98 isolates of coagulase-negative staphylococci representing six species all obtained from endocervical cultures were examined for antimicrobial susceptibility and for the presence of plasmids. More than 80% of the isolates were susceptible to each of 12 antimicrobial agents tested, whereas only 33% were susceptible to penicillin G, 30% were susceptible to cadmium chloride, and 41% were susceptible to tetracycline. Although no species-related susceptibility or plasmid patterns were detected, 77 isolates contained at least one plasmid and 43 contained a plasmid similar in mass to a 2.7-megadalton tetracycline resistance plasmid previously reported in staphylococci. Association of tetracycline resistance with plasmids of this size in four species was determined from curing experiments. No plasmids homologous with the tetracycline resistance locus of the Escherichia coli plasmid pBR322 were found among 11 isolates examined by DNA hybridization. Homology with a 2.7-megadalton plasmids (pRC701) from an endocervical isolate of S. aureus, however, was apparent for 2.7-megadalton plasmids harbored by six isolates as well as with larger plasmids harbored by three isolates. Restriction analysis revealed that pRC701 shared structural identity with two plasmids of a similar mass from two species of coagulase-negative staphylococci as well as with a previously characterized tetracycline resistance plasmid originating in S. aureus.

Nucleic acid probes in clinical microbiology.

The infectious disease applications of nucleic acid probe have been described. In addition, the basic procedures of nucleic acid probe technology have been discussed, as have the factors affecting implementation of probe technology in diagnostic laboratories. Despite the questions raised, nucleic acid probes will become part of the diagnostic laboratory in the near future. Commercial interests are developing and marketing new probes, reagents, and kits which will expedite the employment of this technology. High-volume reference laboratories will first use probes as part of a battery of tests which will include ELISA and monoclonal antibody methods. In all probability, probes will replace methods: that have proven to be ineffective, difficult, or costly such as culturing for some enteric pathogens and Legionella, that require long incubation periods, such as mycobacteria, or that have high costs and low yields, such as virology.

In vitro antimicrobial inhibition patterns of nutritionally variant streptococci.

Twenty-four isolates of nutritionally variant streptococci, previously categorized as species included among the viridans streptococci, were tested for susceptibility to 12 antimicrobial agents. Minimum inhibitory concentrations for these isolates and for a control group of viridans streptococci with no apparent nutritional deficiencies were determined in two microdilution systems. Pyridoxal hydrochloride, which enhances growth of the nutritional variants, was added to one of these microdilution systems but not to the other. An agar dilution method was also used to test the nutritionally variant isolates. Minimum inhibitory concentrations determined by the three methods compared favorably. Penicillin, nafcillin, methicillin, and clindamycin were less effective in vitro against the nutritional variants than were the other antibiotics tested. Streptomycin, however, was less effective against the control isolates.

A gene probe for TEM type beta-lactamases.

A restriction fragment of plasmid pBR322 bearing the TEM-1 beta-lactamase structural gene was electroeluted from agarose gels after digestion with EcoRI and HinfI. The 1-kilobase fragment was 32P-labeled and used to examine genetic relationships with nucleic acids encoding seven other beta-lactamase classes. The probe hybridized only with TEM-2 and OXA-2 class plasmids.

Physiological characterization of nutritionally variant streptococci.

Twenty-five isolates of nutritionally variant streptococci submitted to the Streptococcus Laboratory of the Center for Disease Control over a 2-year period were tested for growth requirements and for biochemical reactions. After they were recovered from storage in blood at -170 degrees C, all isolates grew within 48 h in both thioglycollate broth and Todd-Hewitt broth supplemented with 0.001% pyridoxal.HCl. They grew better in the latter, even though they all grew on unsupplemented infusion agar, anaerobe blood agar, and chopped meat-glucose medium. Biochemical patterns of the isolates resemble those of five viridans streptococcal species. Two isolates had patterns which did not resemble those of any viridans species. Biochemical reactions obtained with heart infusion broth base biochemicals and carbohydrate fermentation media compared favorably for an overall agreement rate of 86.5% for key tests. Lactic acid and acetic acid were the major fermentation products detected with gas-liquid chromatography.