Topical Tacrolimus 0.1% for treatment of cutaneous microcystic lymphatic malformations.

Microcystic lymphatic malformations as described in the international literature form a subgroup of low-flow congenital vascular malformations (VM) resulting from irregular embryological development. Microcystic lesions normally manifest as an accumulation of lymph- and blood-filled vesicles that, when externalized, cause skin maceration with consequent pain and potential infection resulting in the impairment of the patient's quality of life. There is no consensus on a standardized algorithm nor clear guidelines for successful treatment of this type of lymphatic malformation, and treatment options employed often result in ambivalent and transient outcomes with a high rate of recurrence. The topical formulation of tacrolimus is a well-known FDAapproved anti-T cell agent that was recently identified as a potent activator of ALK1, which is involved in several processes and functions including angiogenesis. We investigated if topical administration of tacrolimus may be an effective therapy for directly targeting cutaneous microcystic lymphatic malformations as a complement to systemic treatment. The study enrolled four patients with cutaneous microcystic lymphatic malformations: three male (ages: 13,15,18) and one female (age: 30). Two of the patients presented lesions on their backs, one patient on the left hand and one on the left lower limb. All four patients received treatment with topical tacrolimus 0.1% twice a day for 10 weeks on a previously selected area for application. Weekly clinical follow-ups were conducted along with close physician-patient contact. All patients displayed a satisfactory response after treatment. Lymphorrhea and bleeding were stopped in all cases and the esthetic aspect of lesions improved in two patients. To date, all patients presented no clinically significant changes to the size or extension of the lesion. Topical tacrolimus treatment is a promising and reasonable option for microcystic lymphatic malformations. Our results encourage further exploration in larger populations with the consideration that it is a safe and effective alternative or complementary therapy to systemic treatment.

DNA determinants of structural and regulatory variation within the murine beta-glucuronidase gene complex.

The murine beta-glucuronidase (GUS) gene complex, [Gus], encompasses the GUS structural element, Gus-s, and a set of regulatory elements which serve to modulate Gus-s expression. Three common GUS haplotypes representing virtually all inbred strains of laboratory mice have been compared with respect to GUS mRNA sequence. Results of such comparisons revealed sequence variations which target the location of one of the GUS regulatory elements to sequences within Gus-s and which account for known electrophoretic and heat stability differences among GUS allozymes of the three common GUS haplotypes.

Overlapping transcriptional units on the same strand within the murine beta-glucuronidase gene complex.

We have identified and partially characterized a complex transcriptional unit within the murine beta-glucuronidase gene complex on chromosome 5. On the same strand and within the first intron of the beta-glucuronidase structural gene, Gus-s, we observe an RNA polymerase II promoter motif. That sequences within this carefully defined region can promote RNA polymerase II transcription is supported by results of in vitro transcriptional runoff assays and by expression of a linked reporter gene in both cultured cells and transgenic mice. Results of RNA blot hybridization and S1 nuclease protection studies reveal a 2.2-kilobase processed liver transcript which is initiated just downstream of the promoter motif and sharing little, if any, sequence with the 2.7-kilobase beta-glucuronidase mRNA. Both RNA species are found in liver where beta-glucuronidase is known to be expressed in all cell types. To our knowledge, this is the first description of eukaryotic mRNAs from overlapping transcription units which share the same strand yet exhibit little, if any, sequence similarity. A possible regulatory relationship between these overlapping structural genes is discussed.

Complete sequence and organization of the murine beta-glucuronidase gene.

The murine beta-glucuronidase structural gene (Gus-s) has been isolated from a BALB/cJ sperm DNA bacteriophage library and its nucleotide sequence established. The gene is organized into 12 exons comprising 17.5% of the 14,009 base pair (bp) region spanning the interval between transcription initiation and the putative site of polyadenylation. A TATA box sequence, embedded within a GC-rich region, is found 28 bp upstream from the transcription initiation site. Eleven members of the B1 family and eight members of the B2 family of murine repetitive elements were identified within Gus-s and 2440 bp of flanking sequence. Other novel sequences found within Gus-s, including a (AC)19 homocopolymer tract within intron 3 and a 23 base pair complex direct repeat within intron 9, are presented and discussed.

The complete nucleotide sequence of murine beta-glucuronidase mRNA and its deduced polypeptide.

The complete nucleotide sequence of murine beta-glucuronidase (GUS) mRNA has been compiled from three overlapping cloned cDNAs and a single GUS-specific genomic clone. The sequence is composed of 2455 nucleotides, exclusive of the poly(A) tail. The 5' and 3' untranslated regions contain 12 and 499 bases, respectively, with the open reading frame encoding a polypeptide of 648 amino acids (74.2 kDa), including a 22 amino acid signal sequence. The nucleotide and deduced amino acid sequences of murine GUS are compared to those published for rat and human GUS and the results are presented. Murine GUS also shares amino acid sequence identity with Escherichia coli GUS and beta-galactosidase. The complete sequences of murine GUS mRNA and its deduced polypeptide provide a basis from which to study the mechanisms responsible for the well-characterized variation in GUS expression among inbred mouse strains.

DNA sequence variation within the beta-glucuronidase gene complex among inbred strains of mice.

Tightly linked to the gene that encodes murine beta-glucuronidase (GUS) are three GUS-specific regulatory elements. Together, these elements define the GUS gene complex. Specific alleles of each regulatory element are associated with a specific GUS structural allele. These associations define the three common forms (haplotypes) of the GUS gene complex, designated A, B, and H. As an initial step in defining the DNA determinants of each regulatory element and to develop DNA markers for the common haplotypes, we have identified several DNA variants by blot hybridization analysis of restricted genomic DNA using GUS-specific cDNA probes. Of 30 tested restriction endonucleases, 24 reveal DNA polymorphisms that distinguish B- and H-haplotype DNA from that of the A haplotype. Of these 24, 18 uncover a restriction fragment length polymorphism in which the polymorphic fragment of A-haplotype DNA is 200-300 bp larger than the corresponding fragment of B- or H-haplotype DNA. DNA sequence analysis of this polymorphic region reveals the presence of a short, interspersed repetitive element of the B2 family within A-haplotype DNA which is absent in DNAs of B- or H-haplotype mice. None of the DNA variations revealed by these analyses can be associated at this time with variation in the regulatory or structural properties of GUS among the common haplotypes. Nevertheless, they do provide useful haplotype-specific markers within the GUS gene complex which are of critical importance for DNA transfer experiments in transgenic mice and in cultured cells.

Genes, manganese, and zinc in formation of otoconia: labeling, recovery, and maternal effects.

Published studies indicate that genes and dietary manganese deficiency cause vestibular defects and ataxic behaviors. Manganese deficiency during development causes otoconial defects in mice, rats, guinea pigs, and chicks. Mutant genes cause otoconial defects in mice, mink, and poultry. Manganese supplementation prevents the otoconial defects in some mutant mice and mink. Manganese is essential, before crystallization of the otoconia, for synthesis of mucopolysaccharides and otoconial matrix. Such defects can be induced, after otoconia are crystallized during fetal development, by dietary zinc deficiency and sulfonamide treatment (inhibits carbonic anhydrase, a zinc-requiring enzyme). Manganese and/or zinc supplementation ameliorates otoconial defects in pallid and lethal-milk (zinc-deficient) mice. Studies herein show that: Developing otoconia can be quantitatively labeled with 45 Ca. This may provide a means for studying calcium metabolism in otoconia over a prolonged period of time and for determining the possible effects of diet, drugs, and other factors on otoconia. Otoconial defects, induced after otoconia form in the fetus, were observed in newborn mice, but disappeared by two days after birth. Conditions of the inner ear may contribute to the calcification of otoconia. Manganese and zinc supplementation of pallid mice via acidified drinking water is more effective than dietary supplementation in preventing otoconial defects. The effectiveness of zinc but not of manganese is related to maternal genotype (+/pa vs. pa/pa). The effect of supplementation of the dams with zinc but not with manganese increases over successive litters. These studies indicate the potential for interaction of genes and trace minerals on otoconial formation and maintenance.

Amino acid change associated with the major polymorphic Hinc II site of Oriental and Caucasian mitochondrial DNAs.

The mitochondrial DNAs (mtDNAs) from 116 Oriental and Caucasian blood samples were analyzed for their Hinc II restriction endonuclease cleavage patterns using Southern analysis and 32P human mtDNA probes. Seven distinct patterns were found, all of which could be interrelated by single nucleotide changes. The predominant pattern (mtHinc II-2) was found in 97% of the Caucasians and 73% of the Orientals. This mtDNA morph had one more Hinc II site than did the second most common morph (mtHinc II-1), which was found only in 20% of the Orientals. Three additional patterns were in a single Oriental sample, a fourth in a single Caucasian sample, and a fifth in one member of each population. The polymorphic site that differentiated mtHinc II-1 and mtHinc II-2 was cloned and sequenced. A single nucleotide change was found that created an Hinc II site and changed the amino acid sequence of the URF5 gene. Comparison of these sequences with those of other primates [15] revealed that the Asian mtHinc II-1 and mtHinc II-4 mtDNAs were identical in this region with those of chimpanzees and orangutans. These results suggest that the Asian mtHinc II-1 mtDNA may have been ancestral to other human mtDNAs.