First-in-human phase I study of SOR-C13, a TRPV6 calcium channel inhibitor, in patients with advanced solid tumors.

Introduction This was an open-label, dose escalation (3 + 3 design), Phase I study of SOR-C13 in patients with advanced tumors of epithelial origin. Primary objectives were to assess safety/tolerability and pharmacokinetics. Secondary goals were to assess pharmacodynamics and efficacy of SOR-C13. Methods SOR-C13 was administered IV QD on days 1-3 and 8-10 of a 21-day cycle. Doses were 2.75 and 5.5 mg/kg (20-min infusion) and 1.375, 2.75, 4.13 and 6.2 mg/kg (90-min infusion). Toxicity was assessed by National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Dose limiting toxicity (DLT) was assessed within the first treatment cycle. Tumors were evaluated, using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, after two cycles. Results Twenty-three patients were treated. No drug-related serious adverse events occurred. DLTs occurred in six patients: asymptomatic, drug-related, transient Grade 2 hypocalcemia (4 patients), and unrelated Grade 3 anemia and Grade 3 atrial fibrillation, 1 patient each. Calcium and vitamin D supplementation eliminated further Grade 2 hypocalcemia. One Grade 3 treatment emergent adverse event, urticaria, was definitely related to SOR-C13. Four possibly drug-related, Grade 3 events (alanine aminotransferase and aspartate aminotransferase elevation, headache, and hypokalemia) were observed. Of 22 evaluable patients, 54.5% showed stable disease ranging from 2.8 to 12.5 months. The best response was a 27% reduction in a pancreatic tumor with a 55% reduction in CA19-9 levels at 6.2 mg/kg. Conclusion SOR-C13 was safe and tolerated up to 6.2 mg/kg. The Maximal Tolerated Dose (MTD) was not established. Stable disease suggested antitumor activity.

First Report of Colletotrichum chlorophyti Infecting Soybean Seed in Arkansas, United States.

Colletotrichum chlorophyti was first reported in the United States in 2009 on soybean petioles (Glycine max [L.] Merr.) collected from Alabama, Illinois, and Mississippi (4). This species has not been reported to infect seed, unlike other Colletotrichum spp. (2). From the 2012 growing season, soybean seeds obtained from the National Agricultural Statistics Service representing 151 seed lots from growers' fields in 11 states were assayed by plating them on acidified potato dextrose agar (APDA). Before plating, seeds were surface disinfected by sequential immersion in 50% ethanol for 30 s, 20% commercial bleach for 1 min, two 1 min rinses in sterile distilled water, and kept at 25°C in the dark for 1 week. Infected seeds from one seed lot from Arkansas produced colonies similar to Colletotrichum spp. This seed lot was visually examined and divided into asymptomatic or discolored symptomatic seeds. Because of the limited number of seeds in the seed lot, 20 seeds that asymptomatic and 40 seeds that appeared symptomatic were assayed on APDA as previously described. Asymptomatic seeds did not produce any dark fungal colonies. Among the symptomatic seeds, five appeared to have flecked light gray seed coats with some larger grayish to black and irregular spots where cracks were sometimes formed, and they developed small black fungal masses or became entirely dark on the surface. Five fungal isolates were obtained from these infected seeds. On APDA, the isolates initially produced white to pink smooth-margined colonies, turned black with age, produced no aerial growth, and filled a 9 cm diameter petri dish within 10 days. DNA of one isolate was extracted for PCR and sequencing of the ITS region with ITS1 and ITS4 primers (3). From the BLAST analysis, the sequence was 100% identical to C. chlorophyti isolates, IMI 103806, and CBS 142.79 (Accession Nos. GU227894 and GU227895, respectively). To test for pathogenicity, the fungus was sub-cultured on APDA and eight APDA discs (4 mm diameter) were set into 50 ml potato dextrose broth inside a 250-ml flask and shook at a speed of 100 rpm at room temperature (24 ± 1°C) for 10 days. The mycelium was then weighed, fragmented with a blender, and resuspended in sterile distilled water to a final concentration of ~40 mg/ml. The mycelial suspension was sprayed on soybean seedlings of cv. Williams 82 (two plants/pot) at growth stage V1 to V2 until runoff. The inoculated plants were kept in a moist chamber (>90% relative humidity) for 48 h at 24 ± 1°C in the dark, and then transferred to normal plant growing conditions. At 5 days post-inoculation (dpi), the leaves showed typical symptoms caused by C. chlorophyti, including necrosis on the edge of young leaves and petioles, formation of irregular dark brown lesions, and leaves became scrolled (4). Setose acervuli, curved conidia with tapered ends (21.4 ± 1.1 × 3.8 ± 0.3 µm), and chlamydospores were found on the detached symptomatic leaves after 12 dpi. No perithecia formed. The morphology matched the description of C. chlorophyti (1,4). To our knowledge, this is the first report of C. chlorophyti in Arkansas and the first time that this species has been reported infecting seed of any plant. References: (1) S. Chandra and R. N. Tandon. Curr. Sci. 34:565, 1965. (2) G. L. Hartman et al. Page 13 in: Compendium of Soybean Diseases, APS Press, St. Paul, MN, 1999. (3) T. J. White et al. Page 315 in: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (4) H.-C. Yang et al. Plant Dis. 96:1699, 2012.

Combination Treatment with Antigen-Specific Dual-Sized Microparticle System Plus Anti-CD3 Immunotherapy Fails to Synergize to Improve Late-Stage Type 1 Diabetes Prevention in Nonobese Diabetic Mice.

Type 1 diabetes (T1D) pathophysiology, while incompletely understood, has in part been attributed to aberrant presentation of self-antigen plus proinflammatory costimulation by professional antigen-presenting cells (APCs). Therapies targeting dendritic cells (DCs) offer an avenue to restore antigen-specific tolerance by promoting presentation of self-antigen in an anti-inflammatory or suppressive context. Here, we describe a subcutaneously administered, dual-sized biodegradable microparticle (MP) platform that includes phagocytosable (∼1 µm) and nonphagocytosable (∼30 µm) MPs to deliver pro-tolerogenic factors both intra- and extracellularly, as well as the T1D-associated autoantigen, insulin, to DCs for amelioration of autoimmunity. This MP platform resulted in increased recruitment of DCs, suppressive skewing of DC phenotype with diminished expression of CD86 and MHC-II, increased regulatory T cell (Treg) frequency, and upregulated expression of the checkpoint inhibitor programmed cell death protein 1 (PD-1) on T cells. When administered concomitantly with anti-CD3 antibody, which provides transient T cell depletion while preserving Treg populations, in 12-week-old nonobese diabetic (NOD) mice, regulatory immune populations persisted out to 20 weeks of age; however, combination anti-CD3 and dual-sized MP (dMP) therapy failed to synergistically inhibit diabetes onset.

Design and synthesis of a peptide having chymotrypsin-like esterase activity.

A peptide having enzyme-like catalytic activity has been designed and synthesized. Computer modeling was used to design a bundle of four short parallel amphipathic helical peptides bearing the serine protease catalytic site residues serine, histidine, and aspartic acid at the amino end of the bundle in the same spatial arrangement as in chymotrypsin (ChTr). The necessary "oxyanion hole" and substrate binding pocket for acetyltyrosine ethyl ester, a classical ChTr substrate, were included in the design. The four chains were linked covalently at their carboxyl ends. The peptide has affinity for ChTr ester substrates similar to that of ChTr and hydrolyzes them at rates approximately 0.01 that of ChTr; total turnovers greater than 100 have been observed. The peptide is inhibited by ChTr specific inhibitors and is inactive toward benzoyl arginine ethyl ester, a trypsin substrate. The peptide is inactivated by heating above 60 degrees C, but recovers full catalytic activity upon cooling and lyophilization from acetic acid.

Pituitary receptor site blockade by a gonadotropin-releasing hormone antagonist in vivo: mechanism of action.

Administration of a potent gonadotropin-releasing hormone (GnRH) antagonist [Nac-L-Ala1,pCl-D-Phe2,D-Trp3,6]GnRH as a single subcutaneous injection to castrated adult male rats reduced, by more than 90 percent, both serum luteinizing hormone concentrations and specific pituitary GnRH receptor binding. This effect persisted for 24 hours. The dissociation rate of the antagonist from pituitary membrane homogenates was fourfold slower than the dissociation rate of a potent agonist. The prolonged in vivo inhibition of pituitary GnRH receptor binding and luteinizing hormone secretion by the GnRH antagonist may be mediated by the slower dissociation rate of the antagonist from its specific pituitary membrane receptor site.

Engineering immunomodulatory biomaterials for type 1 diabetes.

A cure for type 1 diabetes (T1D) would help millions of people worldwide, but remains elusive thus far. Tolerogenic vaccines and beta cell replacement therapy are complementary therapies that seek to address aberrant T1D autoimmune attack and subsequent beta cell loss. However, both approaches require some form of systematic immunosuppression, imparting risks to the patient. Biomaterials-based tools enable localized and targeted immunomodulation, and biomaterial properties can be designed and combined with immunomodulatory agents to locally instruct specific immune responses. In this Review, we discuss immunomodulatory biomaterial platforms for the development of T1D tolerogenic vaccines and beta cell replacement devices. We investigate nano- and microparticles for the delivery of tolerogenic agents and autoantigens, and as artificial antigen presenting cells, and highlight how bulk biomaterials can be used to provide immune tolerance. We examine biomaterials for drug delivery and as immunoisolation devices for cell therapy and islet transplantation, and explore synergies with other fields for the development of new T1D treatment strategies.

Apolipoprotein A and B (Sf 100-400) metabolism during bezafibrate therapy in hypertriglyceridemic subjects.

This study describes the effects of bezafibrate, an analogue of clofibrate, on the plasma lipid and lipoprotein profiles of 11 hypertriglyceridemic subjects and on their metabolism of apolipoproteins A-I, A-II, and B. The major action of the drug was to lower plasma triglyceride (by 58%; P less than 0.01). This was accompanied by a reduction in the level of very low density lipoprotein apoprotein B (Svedberg units of flotation [Sf] 60-400), whose mean residence time in the plasma fell threefold (from 3.4 to 1.0 h). Synthesis of the B protein in this fraction was not significantly altered, so the drug acts to accelerate the transit of very low density lipoprotein particles down the delipidation cascade. The metabolism of very low density lipoprotein remnant apoprotein B (Sf 12-100) changed little in response to treatment, although we detected a 30% increment (P less than 0.05) in the plasma concentration of this fraction. The mean residence time of these remnant particles in the plasma did not correlate with that of Sf 100-400 very low density lipoprotein apoprotein B, nor was this parameter altered by the drug. The most consistent and significant perturbation seen in the Sf 0-12 fraction (low density lipoprotein) was a reduction in the fractional catabolism of its apoprotein B moiety (26%; P less than 0.05). In those subjects who were grossly hypertriglyceridemic and who responded well to treatment, the level of this protein rose substantially owing to a combined increase in its synthesis and a reduction in its catabolism. In the group as a whole, high density lipoprotein cholesterol rose 13% (P less than 0.02), and detailed examination showed that this was associated with a small but significant increment in the plasma concentration of the high density lipoprotein subfraction 2. High density lipoprotein subfraction 3 also rose on the average, but this was not a consistent feature in all patients. The plasma concentrations and turnovers of the A proteins (A-I and A-II) were not significantly altered by bezafibrate therapy.

Evidence for renal kinins as mediators of amino acid-induced hyperperfusion and hyperfiltration in the rat.

This study examined the role of tissue kallikrein and kinins in renal vasodilation produced by infusion of amino acids (AA). In rats fed a 9% protein diet for 2 wk, intravenous infusion of a 10% AA solution over 60-90 min reduced total renal vascular resistance and increased glomerular filtration rate (GFR) by 25-40% and renal plasma flow (RPF) by 23-30% from baseline. This was associated with a two- to threefold increase in urinary kinin excretion rate. Acute treatment of rats with aprotinin, a kallikrein inhibitor, resulted in deposition of immunoreactive aprotinin in kallikrein-containing connecting tubule cells and inhibited renal kallikrein activity by 90%. A protinin pretreatment abolished the rise in urinary kinins and prevented significant increases in GFR and RPF in response to AA. In a second group of rats pretreated with a B2 kinin receptor antagonist, [DArg Hyp3, Thi5,8 D Phe7]bradykinin, AA infusion raised urinary kinins identically as in untreated controls, but GFR and RPF responses were absent. Aprotinin or the kinin antagonist produced no consistent change in renal function in rats that were not infused with AA.AA-induced increases in kinins were not associated with an increase in renal kallikrein activity. Notably, tissue active kallikrein level fell 50% in AA-infused rats. These studies provide evidence that kinins generated in the kidney participate in mediating renal vasodilation during acute infusion of AA.

Antagonist, partial agonist and antiproliferative actions of B-9870 (CU201) as a function of the expression and density of the bradykinin B1 and B2 receptors.

BACKGROUND AND PURPOSE: A bradykinin (BK) B2 receptor (B2R) antagonist, B-9870 (CU201), has been proposed to behave as a 'biased agonist' at B2Rs and to exert anti-neoplasic effects. It was unclear whether these effects were determined by the activation of B2Rs by the drug. B-9870 was evaluated for antagonism or stimulation of several responses mediated by the rabbit B2R or B1 receptor (B1R); its anti-proliferative activity was also characterized. EXPERIMENTAL APPROACH AND KEY RESULTS: B-9870 was an insurmountable B2R antagonist in the rabbit jugular vein contractility assay, but a partial agonist in HEK 293 cells expressing the rabbit B2R or a green fluorescent protein (GFP) conjugate of the latter (ERK1/2 phosphorylation, [Ca2+]i, [3H]-arachidonate release, endocytosis). The agonist-like effects of B-9870 were inhibited by the B2R antagonist LF 16.0687 and absent in untransfected cells. In addition, B-9870 was a surmontable antagonist of the rabbit B1R in the aorta contractility assay, and blocked Lys-des-Arg9-BK-induced ERK1/2 phosphorylation in HEK 293 cells expressing a fluorescent B1R conjugate. B-9870 inhibited the growth of MDA-MB-231 cells. The latter effect was not influenced by B1R or B2R antagonists and was not apoptotic. MDA-MB-231 cells expressed a small population of B2Rs but no B1Rs; they responded to BK (small calcium transients) and B-9870 behaved as an antagonist. CONCLUSION AND IMPLICATIONS: B-9870 is a dual B1R and B2R antagonist with confirmed stimulating effects at the B2R in high expression systems only. Its cell type-specific anti-proliferative effect occurs at a high concentration, independently from kinin receptors and apoptosis.

Long chain fatty acids inhibit and medium chain fatty acids activate mammalian cardiac hexokinase.

We investigated the effect of non-esterified fatty acids (FAs) on bovine heart hexokinase (type I: ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). Long chain FAs (C14 to C20) inhibited the enzyme in a way that correlated positively with both the chain length and the degree of unsaturation. Medium chain FA with 12 or less carbons activated hexokinase in a chain length dependent manner with the greater activation shown by laurate. The activation constant of laurate was 91.5 microM with a maximal activation of 60.3%. Oleate caused a maximal decrease in specific activity of 25% with an inhibition constant of 79 microM. Using the fluorescent probe cis-parinarate, we found a saturable binding site with K(d) of 3.5 microM. Oleate competed the fluorescent probe from the protein with a K(d) of 1.4 microM. Medium chain FAs did not compete the probe from HK. The binding of fatty acid to the protein appears to be entropically driven as indicated by an Arrhenius analysis (DeltaS=+231.6 J mol(-1) deg(-1)). The presence of oleate significantly increased the K(ATP)(m) from 0.47 mM to 0.89 mM while the K(glucose)(m) in the presence of the FA (0.026+/-0.003 mM) was not significantly different from the control (0.014+/-0.004 mM). A decrease in V(max) values in the presence of oleate indicated that a mixed allosteric inhibition was operating.

Fatty acid binding to rat liver fatty acid-binding protein is modulated by early glycolytic intermediates.

Fatty acid binding to rat liver fatty acid binding protein in the presence of glycolytic metabolites and at different pH (optimal 7.2) and ionic strength was studied. Binding decreased logarithmically with ionic strength. Glucose and glucose-6-phosphate increased fatty acid binding significantly with K0.5 within physiological ranges while glucose-1-phosphate and phosphate ion caused no effect.

Differential and coordinate responses of the human genes encoding the heat stable alkaline phosphatases to cAMP and sodium butyrate in the choriocarcinoma line JEG-3.

Human heat stable alkaline phosphatases are encoded by two closely related genes: the PLAP-1, which specifies the term placental enzyme, and the PLAP-2, which is expressed primarily in germ cells. In the choriocarcioma line JEG-3, 8-Br-cAMP induced the accumulation of the mRNA of both genes, while sodium butyrate induced the accumulation of PLAP-2 transcripts only. Each agent increased the transcription rate of one or both of the genes, as assayed by run-on transcription. In transfection of JEG-3 cells with PLAP promoters fused to the firefly luciferase gene, the activity of the PLAP-2 promoter (but not PLAP-1) was induced with sodium butyrate, while both promoters were induced by 8-Br-cAMP. Inducibility of the PLAP-2 promoter by 8-Br-cAMP was still observed when the promoter was shortened to -103, leaving intact a sequence resembling a cAMP response element. The extent of transcriptional activation by either agent was not sufficient to explain the accumulation of PLAP mRNA. These studies suggest that both transcriptional and posttranscriptional processes are involved in the induction of the PLAP-1 and PLAP-2 gene in JEG-3 cells.

A fluorometric study of the interaction of bradykinin with lipids.

The interaction of bradykinin (BK) with lipids has been followed by steady-state fluorescence measurements. Addition of either cerebroside sulfate (CS) or phosphatidylinositol (PI), solubilized with the nonionic surfactant C12E8, to BK or its analogue [Gly6]-BK enhances the relative fluorescence intensity of peptide emission at 288 nm. Fluorometric titration of the peptide with lipid has been used to quantitate the interactions in terms of stoichiometry and equilibrium constant. Job's method of continuous variation for the BK-CS interaction gave a stoichiometry of 1:2 for the complex. The value of the equilibrium constant, K, for the interaction of either BK or [Gly6]-BK with CS is 1.5.10(4) M-1. The BK-PI interaction is weaker; K = 5.0.10(3) M-1. Although electrostatic forces no doubt play a major role in these interactions, measurements on the model peptide Gly-Phe-Gly indicate that the phenylalanine residues of BK are disposed in the hydrophobic environment provided by the lipid-C12E8 mixed micelle. 13C-NMR measurements on [99% 13C alpha-Gly6]-BK show that there is no change in its cis/trans ratio upon interaction with CS. The increase in the relative fluorescence intensity of BK accompanying its cooperative interaction with sodium dodecyl sulfate (SDS) implicates the role of hydrophobic forces in this interaction as well. These results bear on the interpretation of the changes in circular dichroism (CD) of BK caused by SDS.

Low-density lipoprotein metabolism in mice with soft tissue tumours.

This study examines the potential value of low-density lipoprotein (LDL) as a vehicle for directing cytotoxic drugs to tumour cells in mouse model systems. Control and MAC 13 tumour-bearing NMRI mice were injected with tracer doses of 125I-labelled native and cyclohexanedione-modified 131I-labelled LDL. 18 h later the animals were killed and the radioactivities assimilated by various tissues were measured relative to plasma activity at the time of death. These values were used to calculate specific tissue receptor-mediated LDL uptake. All tissues expressed receptors but the liver and adrenal gland were particularly active. In tumour-inoculated animals, the neoplastic lesions were second only to liver in their net assimilation of LDL. CFLP mice bearing virus-induced parotid adenomata gave results similar to those obtained in NMRI animals. In order to improve the selectivity of LDL assimilation we attempted to downregulate LDL receptors in the liver and adrenal gland by administration of the bile acid sodium taurocholate or by subcutaneous injection of hydrocortisone sodium succinate. These manoeuvres together reduced uptake of the lipoprotein into both organs without affecting tumour activity.

The association between diabetic retinopathy and skeletal muscle capillary basal lamina thickening corrected for the influence of age and duration of diabetes.

The association between an objective measure of diabetic retinopathy and skeletal muscle capillary basal lamina thickness was examined in a group of 30 male insulin-treated diabetic subjects, mean age (+/- SD) 44.6 +/- 13.2 yr, duration of diabetes 21.2 +/- 11.2 yr, % ideal body weight (% IBW) 106 +/- 11%. In addition, muscle capillary basal lamina width was measured in a group of 18 nondiabeitc men, mean age 40.7 +/- 16.3 yr and % IBW 118 +/- 23%. The muscle capillary width of the diabetic subjects was significantly greater than that of the nondiabetic group (P less than 0.01), but the values of the two overlapped considerably. In the diabetic group, there was a significant association of basal lamina width with age (P less than 0.01) but not with duration of diabetes. The association between extent of retinopathy and muscle capillary basal lamina width was not strong. The findings of the study do not therefore support the use of an estimate of muscle capillary basal lamina thickness as a single representative measure of diabetic microangiopathy.

Variable arginine vasopressin levels in neonatal congestive heart failure.

Arginine vasopressin levels in 17 neonates with cardiac disease were compared with control levels in 10 healthy newborn infants. Infants with congestive heart failure who were free of left ventricular outflow tract obstruction had a mean level of 80 +/- 18 pg/ml, which was significantly greater than the mean control level (p less than 0.001). Infants with congestive heart failure and left ventricular outflow tract obstruction had a mean vasopressin level of 3 +/- 0.7 pg/ml, which was lower than the mean control level of 6 +/- 0.7 pg/ml (p less than 0.05). The data suggest that impaired forward flow to high pressure sinoaortic and ventricular baroreceptors is necessary for vasopressin release in congestive heart failure. In left ventricular outflow tract obstruction with heart failure these receptors may be impaired or absent, leading to decreased vasopressin release. Low plasma arginine vasopressin may adversely affect circulatory homeostasis.

Helix propensities of basic amino acids increase with the length of the side-chain.

Helix formation in a 17-residue alanine-lysine peptide and analogous peptides with specific lysine --> X substitutions, where X is 2,3-diamino-L-propionic acid, 2, 4-diamino-L-butyric acid or L-ornithine, have been examined using circular dichroism measurements. The dependence of helix content on X, its position in the sequence, and the number of lysine --> X substitutions are reasonably well described by using the Lifson-Roig theory modified to include N-capping, without explicitly considering charge-helix dipole interactions. The helix propensities for these basic amino acids increase with the length of the side-chain in the rank order 2,3-diamino-L-propionic acid < 2,4-diamino-L-butyric acid < ornithine < lysine. This parallels the increase in helix propensities with side-chain length of other polar and charged amino acids.

Position effect on apparent helical propensities in the C-peptide helix.

A search has been made for position effects on apparent helix propensities when another amino acid is substituted for alanine in the C-peptide helix of ribonuclease A. Three internal alanine residues (Ala4, Ala5, Ala6) are used as sites for substitution. Five amino acids, Glu, His, Arg, Lys and Phe, are substituted singly in individual peptides at each of these three positions, and the pH profiles of helix content for the substituted peptides have been determined. The effect of using an acetyl or a succinyl amino-terminal-blocking group has also been determined for each substitution. A strong position effect is found at Ala5: the helix content of the substituted peptide is significantly higher for substitution at position 5 than at positions 4 or 6 in almost all cases. The reason for the position 5 effect is unknown. The results also show that electrostatic interactions often influence substitution experiments, and they provide data on the variability of substitution experiments made with a natural sequence peptide.

Incipient Genome Differentiation in Gossypium. III. Comparison of Chromosomes of G. HIRSUTUM and Asiatic Diploids Using Heterozygous Translocations.

Hybrids between upland cotton (G. hirsutum, genome constitution 2A(h)D(h)) and either A-genome or D-genome diploid species exhibit 26 paired and 13 unpaired chromosomes at metaphase I. The A(h) and D(h) genomes are therefore considered homoeologous with those of the respective diploids. Previous studies, nevertheless, revealed a low level of ("incipient") differentiation between D(h) and various diploid D genomes. The diploid A genomes have been regarded as more closely homologous to A(h) on the basis of low preferential pairing and autotetraploid segregation ratios in allohexaploids.-The present study addressed the following questions: Are the diploid A genomes differentiated from A(h) in meiotic homology? If so, is the differentiation manifested equally by all 13 chromosomes or is it localized in certain chromosomes?-Three diploid A-genome lines representing G. herbaceum and G. arboreum were hybridized by in ovulo culture of embryos (1) with a standard line of G. hirsutum, which differs from G. herbaceum by two and from G. arboreum by three naturally occurring reciprocal translocations involving chromosomes 1-5, and (2) with six lines homozygous for experimental translocations involving chromosomes 6, 7, 10, 11, 12 and 13. Chiasma frequencies in hybrids were compared with those in appropriate G. hirsutum controls. In every comparison overall chiasma frequencies were slightly lower in the hybrids. Therefore A(h) appears to be differentiated from the diploid A genomes. No localized differentiation was detected in chromosomes marked by experimental translocations. The differentiation may be localized mainly in chromosomes 4 and 5.